key: cord- -xcx ic s authors: spector, sheldon l. title: the common cold: current therapy and natural history()()() date: - - journal: j allergy clin immunol doi: . /s - ( ) - sha: doc_id: cord_uid: xcx ic s despite its prevalence, the common cold is complicated and can be difficult to treat, even symptomatically. there is still no cure for the myriad of viruses that cause the common cold. many of the most popular remedies are either ineffective or counterproductive. this paper reviews the causes and course of upper respiratory infections, and discusses treatment options, including a new anticholinergic aqueous formulation for controlling rhinorrhea. (j allergy clin immunol ; : - .) as new data emerge regarding the pathophysiology of upper respiratory infections (uris), we continue to gain new insight into their treatment and possible complications. it has been estimated that the average preschool child experiences six to ten uris, or "colds," per year; the average adult has two to four? the effects of the common cold can be uncommonly disruptive, forcing otherwise normal individuals to miss work, school, or other important activities. individuals who are at increased risk, such as those with bronchitis or asthma, may also experience a life-threatening exacerbation of their underlying conditions. the average annual expenditure for various cold treatments exceeds $ billion in the united states. this statistic becomes even more provocative when we consider that one of every three individuals with a confirmed infection has no apparent symptoms of a cold. viruses that cause colds can be spread through contact with inanimate surfaces, as well as by hand-to-hand contact? seasonal variations in cold patterns have long been recognized. generally there are fewer colds in the warm summer months and more colds during periods of crowding, particularly the fall. upper respirato~ infection seem to be a significant factor in cold epidemiology. folklore is somewhat at odds with modern science over this issue; there are many cultures where youngsters who have a "cold" are dressed sufficiently to keep them warm on the coldest winter night, even when the outside temperature is tropical. colds are caused by a wide variety, of viruses (table i) . the rhinoviruses, which account for more than % of colds in adults, have more than antigenically different types. coronaviruses also appea r to be responsible for a large percentage of colds, but precisely what that percentage is compared with parainfluenza or respiratory syncytial viruses has not yet been well established. certain viruses appear to be more common in children than adults, but in general, viruses appear in roughly the same proportions in both populations. some viruses may be associated with more severe symptoms than others, for example, the exacerbation of asthma seen with respiratory syncytial virus. however, the most significant factor in the severity of a viral infection seems to be the incubation period. various factors are thought to increase susceptibility to uris. there seems to be a relationship between colds and stress. in assessing the differences between symptomatic and asymptomatic individuals with confirmed viral infections, stone found an association similar to a dose-response between psychologic stress and increased risk of acute infectious respiratory illness; the risk involved increased rates of infections rather than frequency of symptoms after infection. smokers are at greater risk than nonsmokers to develop both infections and symptoms after infection. it also appears that certain drugs may increase susceptibility to colds. aspirin and acetaminophen suppress sero-neutralizing antibody response (p < . ) and are associated with increased nasal symptoms and signs. there is a trend towards longer duration of virus shedding with both of these medications. common cold viruses characteristically cause an infection that is self-limited and of short duration. although shedding of rhinovirus has been shown to last weeks in young adults with experimentally induced colds, s, rhinoinfections with coronavirus are usually detected for only a few days. most colds are not associated with cell necrosis or significant mucosal damage, but there may be some sloughing of columnar epithelial cells. ° initially there is an increase in vascular permeability, ~ followed later by glandular secretions, both of which may have implications with regard to the timing and effectiveness of treatment. the constituents of the glandular secretions provide clues to their origins. , i there is an elaboration of inflammatory mediators such as kinins. when these mediators accumulate along with polymorphonuclear cells, there is an increase in nasal symptoms. since bradykinin is a likely mediator, (table ii) . although doyle et al. reported no increase in nasal responsiveness to an infectious trigger in allergic compared with nonallergic individuals, bardin et al. found that patients with allergic rhinitis had more severe colds independent of preinnoculation anti-body. there is also an increased twitchiness of the tracheal bronchial tree following colds. sir william osler has been quoted as saying, "there is just one way to treat a cold, i.e., with contempt." we seem to have made some progress in the treatment of uris since this statement was made, due largely to a better understanding of the pathophysiology of colds, although there is certainly still room for improvement. some treatments used today are better choices than others in terms of pathophysiology. as mentioned previously, both aspirin and acetaminophen may have a detrimental effect on cold treatment, neutralizing antibodies and increasing nasal symptoms. in a spector j allergy clin immunol may study by sperber et al., naproxen did not alter virus shedding or serum neutralizing antibody in experimental rhinovirus cold, but it had a beneficial effect on such symptoms as headache, malaise, myalgia, and cough. oral o~-agonists relieve congestion in many individuals, although their effect is not dramatic. , o topical decongestants may also help; unfortunately, if they are overused they may also be associated with rebound congestion or worsening of symptoms. the role of antihistamines in the treatment of the common cold has been debated. some antihistamines do not seem to be very effective, whereas others may provide mild benefit. antihistamine/ decongestants do not appear to be effective in the treatment of uris in children. , cromolyn sodium and nedocromil have both been studied; they do not cause a worsening of symptoms, but neither do they seem to provide any significant improvement. , apparently menthol cannot be positively demonstrated to provide a beneficial effect? interferon has been used in various studies with negative results. either intranasal or systemic steroids may suppress inflammation during the first days of infection, and would seem to merit further investigation. ° among the many nonpharmacologic therapies, steam has been shown by various authors to provide no beneficial effect; , however, with proper timing, local hyperthermia or sauna may decrease the incidence of colds or provide slight relief. in general, the use of zinc has been disappointing and is associated with side effects. godfrey et al. commented on the poor bioavailability of the older zinc products and found a statistically significant decrease in the duration of colds with their nonchelated formulations. vitamin c may decrease the duration of cold symptoms. , of course, chicken soup is well known to provide benefit in the common cold (as long as it is one's mother's). in answer to the cynics who doubt such an assertion, chicken soup has been demonstrated to improve mucociliary clearance. there are new pharmacologic therapies on the horizon that may prove useful to the physician in the treatment of the common cold. ipratropium bromide nasal spray, an anticholinergic therapy, has the unique property of specifically controlling rhinorrhea in uris, as has been demonstrated in many studies , a novel attempt at antiviral therapy includes blockade of the receptor where the virus attaches. such a treatment may be useful against rhinoviruses that affect one or two common receptors but may not be applicable to less specific viruses. modern research has demonstrated that uris have myriad causes and complex effects. although some time-honored treatments might have limited usefulness, novel attempts at ameliorating the symptoms of a common cold, such as the use of ipratropium bromide nasal spray or specific antiviral receptor therapy, might represent a significant advance. they are based on a better understanding of the pathophysiology of uris. ineffectiveness of recombinant interferon-beta serine nasal drops for prophylaxis of natural colds chemical disinfection interrupt transfer rhinovirus type from environmental surfaces to hands potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus and rhinovirus signs and symptoms in common colds development of common cold symptoms following experimental rhinovirus infection is related to prior stressful life events smoking, alcohol consumption, and susceptibility to the common cold adverse effects of aspirin, acetaminophen, and ibuprofen on immune function, viral shedding, and clinical status in rhinovirus-infected volunteers studies with rhinoviruses in volunteers: production of illness, effect of naturally acquired antibody, and demonstration of a protective effect not associated with serum antibody sites of rhinovirus recovery after point inoculation of the upper airway human ciliated epithelial cells in nasal secretions analysis of nasal secretions during experimental rhinovirus upper respiratory infections microvascular exudative hyperresponsiveness in human coronavirus-induced common cold fireside conference . common cold kinins are generated in nasal secretions during natural rhinovirus colds a study of the efficacy of the bradykinin antogonist, npc , in rhinovirus infections in human volunteers peripheral blood mononuclear cell interleukin- and interferon-gamma production, cytotoxicity, and antigenstimulated blastogenesis during experimental rhinovirus infection effect of rhinovirus infection on cellular immune parameters in allergic and nonallergic subjects increased levels of interleukin- are detected in nasal secretions of volunteers during experimental rhinovirus colds chemotherapy of rhinovirus colds respiratory viruses and exacerbations of asthma in adults computed tomographic study of the common cold physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds middle ear abnormalities during natural rhinovirus colds in adults effect of experimental rhinovirus infection on the nasal response to histamine and cold air challenges in allergic and nonallergic subjects amplified rhinovirus colds in atopic subjects factors affecting the long-term variability of bronchial responsiveness in an adult general practice population adverse effects of aspirin, acetaminophen, and ibuprofen on immune function, viral shedding, and clinical status in rhinovirus-infected volunteers effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial pathophysiology and pharmacotherapy of common upper respiratory diseases evaluation of an alpha agonist alone and in combination with a nonsteroidal antiinflammatory agent in the treatment of experimental rhinovirus colds evaluation of oral terfenadine for treatment of the common cold effectiveness of an antihistamine-decongestant combination for young children with the common cold: a randomized, controlled clinical trial over-the-counter cold medications: a critical review of clinical trials between and the effect of intranasal nedocromil sodium on viral upper respiratory tract infections in human volunteers intranasal sodium cromoglycate in post-catarrhal hyperreactive rhinosinusitis: a double-blind placebo controlled trial the effects of menthol on reaction time and nasal sensation of airflow in subjects suffering from the common cold ineffectiveness of recombinant interferon-beta serine nasal drops for prophylaxis of natural colds ineffectiveness of postexposurc prophylaxis of rhinovirus infection with lowdose intranasal alpha b interferon in families recombinant human interferon-gamma as prophylaxis against rhinovirus colds in volunteers a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection effect of inhaling heated vapor on symptoms of the common cold effect of inhaling heated vapor on symptoms of the common cold local hyperthermia benefits natural and experimental common colds regular sauna bathing and the incidence of common colds zinc gluconate lozenges for common cold: a double-blind clinical trial zinc gluconate and the common cold: a controlled clinical study vitamin c and the common cold does vitamin c alleviate the symptoms of the common cold?: a review of current evidence effects of drinking hot water, cold water, and chicken soup on nasal ¢,~ ~pec'~or ~ucus velocity and nasal airflow resistance therapeutic effects of an anticboinergic-sympathomimetic combination in induced rhinovirus colds a double-blind, placebo-controlled study of the safety and efficacy of ipratropium bromide nasal spray versus placebo in patients with the common cold modification of experimental rhinovirus colds by receptor blockade key: cord- - ubow authors: galván-román, josé maría; rodríguez-garcía, sebastián c.; roy-vallejo, emilia; marcos-jiménez, ana; sánchez-alonso, santiago; fernández-díaz, carlos; alcaraz-serna, ana; mateu-albero, tamara; rodríguez-cortes, pablo; sánchez-cerrillo, ildefonso; esparcia, laura; martínez-fleta, pedro; lópez-sanz, celia; gabrie, ligia; guerola, luciana del campo; suarez, carmen; ancochea, julio; canabal, alfonso; albert, patricia; rodríguez-serrano, diego a.; aguilar, juan mariano; arco, carmen del; santos, ignacio de los; garcía-fraile, lucio; camara, rafael de la; serra, josé maría; ramírez, esther; alonso, tamara; landete, pedro; soriano, joan b.; martín-gayo, enrique; torres, arturo fraile; zurita cruz, nelly daniela; garcía-vicuña, rosario; cardeñoso, laura; sánchez-madrid, francisco; alfranca, arantzazu; muñoz-calleja, cecilia; gonzález-Álvaro, isidoro title: il- serum levels predict severity and response to tocilizumab in covid- : an observational study date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: ubow background covid- patients can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome (ards) requiring invasive mechanical ventilation (imv). since interleukin- (il- ) is a relevant cytokine in ards, the blockade of its receptor with tocilizumab (tcz) could reduce mortality and/or morbidity in severe covid- . objective to determine whether baseline il- serum levels can predict the need for imv and the response to tcz. methods retrospective observational study performed in hospitalized patients diagnosed of covid- . clinical information and laboratory findings, including il- levels, were collected approximately and days after admission to be matched with pre- and post-administration of tcz. multivariable logistic and linear regressions, and survival analysis were performed depending on outcomes: need for imv, evolution of arterial oxygen tension/fraction of inspired oxygen ratio (pao /fio ) or mortality. results one hundred and forty-six patients were studied, predominantly male ( %); median age was years. forty-four patients ( %) required imv, and patients ( %) received treatment with tcz. il- levels> pg/ml was the best predictor for imv (or: . ; p< . ). early administration of tcz was associated with improvement of oxygenation (pao /fio ) in patients with high il- (p= . ). patients with high il- not treated with tcz showed high mortality (hr: . ; p= . ), as well as those with low il- treated with tcz (hr: . ; p= . ). no relevant serious adverse events were observed in tcz-treated patients. conclusion baseline il- > pg/ml predicts imv requirement in patients with covid- and contributes to establish an adequate indication for tcz administration. abstract background covid- patients can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome (ards) requiring invasive mechanical ventilation (imv). since interleukin- (il- ) is a relevant cytokine in ards, the blockade of its receptor with tocilizumab (tcz) could reduce mortality and/or morbidity in severe objective to determine whether baseline il- serum levels can predict the need for imv and the response to tcz. retrospective observational study performed in hospitalized patients diagnosed of clinical information and laboratory findings, including il- levels, were collected approximately and days after admission to be matched with pre-and post-administration the recent exponential increase in cases of severe acute respiratory syndrome caused by coronavirus (sars-cov- ) has led the world health organization to declare a pandemic. the disease, known as coronavirus disease , has pushed national health systems to the brink of collapse, prompting national governments to impose complete population lockdowns in an attempt to slow down the dynamics of infection ( , ) the spectrum of covid- clinical manifestations varies widely, from mild to severe cases of atypical pneumonia, some of them developing acute respiratory distress syndrome (ards), which often requires invasive mechanical ventilation (imv) and is the leading cause of death. it is suggested that the severity of the respiratory disease caused by sars-cov- is largely due to an exacerbated immune response against the virus ( , ). this response has been observed in previous respiratory virus outbreaks and can be also seen in patients treated with chimeric antigen receptor (car) t cell therapy ( - ). pro-inflammatory cytokines such as interleukin (il) and il- are crucial mediators of this process ( , ). in this regard, recent studies have indicated the usefulness of il- serum levels, lymphocyte count, fibrinogen or d-dimer to evaluate the development of ards and its mortality ( - ). however, further evidence is required to support these observations ( ). tocilizumab (tcz), an anti-il- receptor (il- r) antibody, is the only drug currently licensed for the treatment of the cytokine release syndrome (crs) associated with car-t cell therapy ( ). due to the virulence of the current outbreak, its use has been advised in severe cases of . the rationale is to curb the deleterious effects of inflammation, thereby this is a retrospective observational study including consecutive patients with confirmed detection of sars-cov- rna, baseline il- serum level measurement and admitted to the hospital universitario la princesa (hup) with severe to critical covid- ( ), from february th to march rd (flow chart in suppl. fig. surplus sera from laboratory routine determinations were used to assess il- levels, which were retrospectively quantified in duplicate with the human il- quantikine high sensitivity enzyme-immune assay from r&d systems europe ltd. (abingdon, uk). the intra-assay and inter-assay variability were % and %, respectively. therefore, the decision to treat with tcz was based on the aemps criteria, excluding il- > pg/ml, since no information about il- results was available to physicians during the study period. after a preliminary analysis of data by the end of march, il- measurement was included in the baseline assessment of covid- patients. the administration schedule of tcz at the time of the study was a first intravenous infusion of mg/kg (maximum mg) followed by a second one after hours. to analyze whether il- levels can predict disease severity, two main outcomes were considered: need for imv and all-cause mortality. to determine the effect of tcz we analyzed the evolution of pao /fio between both evaluation times. in out of evaluations arterial oxygen tension (pao ) was j o u r n a l p r e -p r o o f unavailable. so, in order to avoid missing data in these relevant outcomes, this parameter was estimated from mean oxygen saturation (sato ) as proposed elsewhere ( ). il- serum levels showed a heterogeneous distribution in patients and in healthy donors (suppl. fig. a ). to improve its representation in figures, this variable was normalized through natural logarithmic transformation (suppl. fig. b ). the procedure to determine the cut-off for high baseline il- is described below. represented as median and iqr and the mann whitney test was used to assess significant differences. variables with a normal distribution were described by mean±standard deviation (sd) and differences between groups were assessed with student's t-test. qualitative variables were described as counts and proportions and or fisher´s exact test was used for comparisons. correlation between quantitative variables was analyzed using the pearson correlation test. to estimate the % confidence interval of correlation coefficients we used the ci command of stata. to determine whether il- serum levels were able to discriminate between: i) covid- patients vs. healthy donors; ii) patients requiring imv vs. those that did not; or iii) patients treated with tcz vs. not treated, receiver operating characteristic (roc) analysis was performed using the roctab command. each cut-off point was selected based on the best trade-off values between sensitivity, specificity and the percentage of patients correctly classified. positive and negative likelihood ratios and roc curves were also obtained. to determine the variables associated with the need for imv, we performed a multivariable logistic regression analysis that was first modeled by adding all the variables with a p value lower than . in the bivariable analysis, namely total lymphocyte count, d-dimer, ldh, pao /fio , copd, obesity, hypertension, c-reactive protein, and il- (high vs low). the final model was reached with backward stepwise removal of variables with p-value higher than . , and using wald tests to demonstrate that each model was better than its previous iteration. next, we performed a multivariable analysis using generalized linear models nested by patient and visit (xtgee command) in which the dependent variable was pao /fio . this approach allowed us to identify which variables influenced the evolution of pao /fio . the first model included all variables with a p value < . in the bivariable analysis, namely hypertension, baseline radiological pattern, ldh, total lymphocyte count, baseline c-reactive protein, imv. after that, through backward stepwise approach, we obtained the best model as described above. then, to assess the role of il- as predictor of tcz effect on pao /fio , the composite variable il- /tcz (low il- /no tcz, low il- /early tcz, low il- /late tcz, high il- /no tcz, high il- /early tcz and high il- /late tcz) was forced in the model. one hundred and forty-six patients were included; their main demographic and clinical characteristics are shown in table fig. a and b). no significant correlation was found between pao and il- serum levels at baseline (r= - . [ % ci: - . to . ]; p= . ; figure a) , likely due to a higher oxygen supply in the most severe cases; in fact, serum il- levels showed a significant negative correlation with pao /fio ( figure b ; r= - . [ % ci: - . to - . ]; p< . ), meaning that higher levels of il- at baseline were associated with lower pao /fio . in this regard, forty-four patients ( %) required imv at some point during their hospitalization. as expected, these patients showed significantly worse pao /fio levels than those not requiring imv (p< . ; table ). in addition, they showed increased leukocytes, total lymphocytes, il- , c-reactive protein (crp), and procalcitonin (pct) showing a higher inflammatory status than those not requiring imv (p≤ . except p= . for crp and p= . for lymphocyte count; table ). furthermore, a baseline il- above pg/ml (henceforth high il- ) discriminated patients requiring imv with % se and % sp. auc was . ( figure c ; lr+ . , lr- . ). a logistic regression model, adjusted for copd and baseline white blood cell count, also showed that high baseline il- was a predictive biomarker for imv (or: . ; % ci: . to . ; supp table ). even before physicians were aware of the potential value of il- serum levels as a predictor of severe disease, those patients with high il- were more frequently treated with tcz ( figure d; auc . ; pg/ml as cut-off showed se %, sp %, lr+ . , lr- . ), although with less accuracy than for imv. the median time from the beginning of symptoms to tcz treatment was days (iqr: - . ). therefore, we considered early tcz when the treatment was applied before days of disease duration and late tcz after this cut-off. as a consequence of il- r blockade with tcz, a significant trend toward higher il- serum levels after administration of the drug was observed ( since relevant differences were observed between patients treated and not treated with tcz, we used a multivariable analysis to determine which variables influenced the evolution of pao /fio at the short-term. baseline pao /fio and radiological pattern, hta, ldh and crp levels and total lymphocyte blood count significantly explained variation in pao /fio (suppl table ). adjusted by these confounders, the best pao /fio evolution was achieved in j o u r n a l p r e -p r o o f patients with low il- not requiring tcz treatment ( figure c first dot on the left; reference group in analysis showed in suppl table ), likely because they were the less severe patients. patients with low il- that due to their bad evolution were prescribed with tcz showed a significant worsening of pao /fio ( figure c , nd and rd dots; suppl table ). a similar evolution was observed in patients with high il- and late tcz treatment, whereas those with high il- not treated or treated early with tcz showed no significant differences compared with the reference group ( figure c ; suppl table ). role in the regulation of d-dimer production; or ii) more likely, d-dimer production has a slower kinetics than crp or other acute phase reactants, since in patients not treated with tcz a similar rise in d-dimer levels was observed (data not shown ). apart from the novelty and the immediate clinical utility of these findings, a drawback of our study is its retrospective and observational nature involving mainly very severe cases in the group of treatment with tcz. a stricter selection of a control group through a propensity score strategy was unfeasible, since once the physicians were aware of il- measurement they focused their efforts on treating with tcz those patients with the highest il- levels. therefore, prospective studies should be carried out to confirm our observations. in addition, there is some controversy about the reliability of pao /fio as an outcome for improvement of lung damage, especially in patients with imv. however, our population was a mix of patients with imv and non-imv, so despite the many factors that could interfere with pao /fio , we considered it the most objective outcome for patients' assessment. and p (line below the box) before (grey boxes) and after (white boxes) treatment with tcz. in non-treated patients pre and post mean first and second evaluation, respectively. statistical rapid and severe covid- pneumonia with severe acute chest syndrome in a sickle cell patient successfully treated with tocilizumab first case of covid- in a patient with multiple myeloma successfully treated with tocilizumab cytokine release syndrome in severe covid- : interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention. jama. detection of sars-cov- in different types of clinical specimens genomic characterisation and in the eye of the storm: immune-mediated toxicities associated with car-t cell monocyte- derived il- and il- are differentially required for cytokine-release syndrome and neurotoxicity due to car t cells expression of acute-phase cytokines, surfactant proteins, and epithelial apoptosis in small airways of human acute respiratory distress syndrome acute respiratory distress syndrome mechanisms and pathologic significances in increase in serum interleukin- (il- ) and soluble il- receptor after administration of an anti-il- receptor antibody, tocilizumab, in patients with rheumatoid arthritis and castleman disease roche provides an update on the phase iii covacta trial of accesed september , . . sanofi-genzyme. sanofi provides update on kevzara® (sarilumab) phase trial in severe and critically ill covid- patients outside the pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid- tocilizumab treatment in covid- : a single center experience all categorical variables are expressed as number (%) and quantitative variables as median pao /fio : arterial oxygen tension -fraction of inspired oxygen ratio; ldh: lactate dehydrogenase; il- : interleukin- ; crp: c-reactive protein; pct: procalcitonin. significance was determined with the mann-whitney test. (c) the graph represents the predicted mean (dots) with % confidence intervals (bars) of pao /fio according to baseline il- levels and early or late tcz treatment. data were obtained with the command marginsplot of stata lactic dehidrogenase and c-reactive protein levels, lymphocyte blood count and need for imv, according to the multivariable analysis displayed in supplementary table (see methods for further information) survival curves of covid- patients grouped according to baseline il- levels and tcz treatment. statistical significance was established with log-rank test key: cord- -h iv crq authors: sumino, kaharu; tucker, jennifer; shahab, muhammad; jaffee, katy f.; visness, cynthia m.; gern, james e.; bloomberg, gordon r.; holtzman, michael j. title: antiviral ifn-γ responses of monocytes at birth predict respiratory tract illness in the first year of life date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: h iv crq background: viral respiratory tract infections are the leading cause of acute illness during infancy and are closely linked to chronic inflammatory airway diseases later in life. however, the determinants of susceptibility to acute respiratory tract infections still need to be defined. objective: we investigated whether the individual variation in antiviral response at birth determines the risk for acute respiratory tract illness in the first year of life. methods: we studied children who were enrolled in a birth cohort study of inner-city children with at least parent with allergy or asthma. we cultured cord blood monocytes and assessed ifng and ccl mrna production at hours after inoculation with respiratory syncytial virus. we also monitored the frequency of acute respiratory tract illness at -month intervals and analyzed nasal lavage samples for respiratory tract viruses at the time of illness during the first year. results: respiratory tract infection was reported for % of subjects, and respiratory tract viruses were recovered in % of symptomatic children. we observed a wide range of antiviral responses in cord blood monocytes across the population. furthermore, a decrease in production of ifng (but not ccl ) mrna in response to respiratory syncytial virus infection of monocytes was associated with a significant increase in the frequency of upper respiratory tract infections (r = − . , p < . ) and the prevalence of ear and sinus infections, pneumonias, and respiratory-related hospitalizations. conclusion: individual variations in the innate immune response to respiratory tract viruses are detectable even at birth, and these differences predict the susceptibility to acute respiratory tract illness during the first year of life. viral respiratory tract infections are a common cause of early childhood illness. most of these infections are short-lived and selflimited, but some can be severe enough to require hospitalization. indeed, viral respiratory tract infections are associated with % of all mortality in children less than years of age. in addition to the morbidity of the acute infection, viral respiratory tract infections with wheezing are strong indicators of subsequent asthma. , therefore predicting those infants at risk for respiratory tract infections is an important first step in preventing acute and chronic respiratory disease. previous studies have identified a variety of potential risk factors for viral lower respiratory tract infections during the first year of life. these factors include day care attendance, number of siblings, small lung size, exposure to tobacco smoke, low birth weight, and premature birth. [ ] [ ] [ ] [ ] infections caused by respiratory syncytial virus (rsv) are particularly implicated in acute illness and chronic lung disease in the first years of life. however, the majority of rsv infections in infants occur without any known risk factors. thus we still do not understand the precise mechanism for the wide variation in susceptibility to severe respiratory tract infections among children in these settings. one possible explanation for the range of susceptibility to viral respiratory tract infection in early childhood is that there are definable variations in the antiviral response, such as a congenital deficiency in the innate immune response that can be detected even at the time of birth. a central ingredient of the innate immune response to respiratory viruses is the system for interferon production and signaling. in that regard a decrease in ifn-g production from cord blood mononuclear cells (cbmcs) stimulated by pha or allergens has been associated with increased risk for acute respiratory tract illness during infancy. , perhaps more relevant to viral infection, the lack of a detectable ifn-g response to rsv in cbmcs was associated with decreased wheezing in the first year of life, but a detectable response was only found in a third of subjects, and therefore predictive power was limited. therefore in the present study we developed alternative methods to determine whether the innate immune response of virus-infected cbmcs could predict the later development of respiratory tract illness. we used rsv to activate cbmcs based on the well-established association of rsv infection with subsequent childhood asthma. , , however, to monitor the innate immune response to rsv, we determined the induction of the genes encoding ifn-g and the remarkably virus-responsive chemokine ccl based on sensitive and quantitative methods for mrna from we also monitored interferon signal transduction by tracking the level of signal transducer and activator of transcription (stat) activation in response to ifn-b stimulation. in both cases we used cultured/adherent cbmcs to select for monocytes versus the mixed cell population that included t cells in previous studies. we assessed whether each of these immune end points could predict the development of respiratory tract illness during the first year of life in a prospective birth cohort of children at high risk for asthma and allergic disease. the experimental matrix led to the unexpected finding of rsv-induced ifng gene expression in monocytes as a predictor of subsequent viral respiratory tract illness. we analyzed cord blood samples from newborns enrolled in the urban environment and childhood asthma (ureca) study. this group represents a subset of the children enrolled at the st louis site, which in turn was a subset of the total number of children enrolled at the baltimore, boston, and new york city sites between february and march , as described previously. [ ] [ ] [ ] subjects were required to have at least parent with allergic rhinitis, eczema, and/or asthma and to reside in an area with greater than % of the residents below the poverty level, as well as being born at weeks' gestation or later. at the st louis site, a small number of children without an allergic parent (n ) were also recruited for comparison. after enrollment, all subjects were monitored for any episodes of acute respiratory tract illness over the next year along with quarterly assessments of respiratory (and nonrespiratory) tract illness and wheezing by questionnaire. nasal lavage samples were obtained when a caregiver reported an acute respiratory tract illness and at the time of a -year follow-up visit. the washington university human research protection office approved the study protocol. cord blood samples were collected in the delivery room, and cbmcs were isolated by means of density gradient centrifugation with accuspin tubes (sigma, st louis, mo) within hours of collection, as described previously. , when sufficient amounts of sample were available (ie, in / subjects), the cells were resuspended in rpmi medium with % fbs, mmol/l l-glutamine, and mmol/l nonessential amino acids to a final concentration of per milliliter and plated in -well lab-tek chambers ( ml per well; nunc a/s, roskilde, denmark) for the real-time pcr assay and in -well lab-tek chambers ( ml per well) for the stat activation assay, as described below. nonadherent cells were removed after hours, and adherent cells were cultured for days with media changes on days , , and and removal of additional nonadherent cells. at the end of the cell-culture period, the adherent cells were greater than % positive for cd immunostaining as a marker of monocytic lineage and therefore designated as cord blood monocyte cultures. the approach avoided purification methods (eg, magnetic bead selection or fluorescence-activated cell sorting) that modify the cell membrane or cell-culture methods (eg, growth factor supplementation) that promote full differentiation and polarization and thereby aimed to obtain cells of the monocyte lineage that were representative of naive lung tissue monocytes and macrophages (the target for viral respiratory tract infection in vivo). on culture day , cord blood monocytes were infected with rsv (a strain) at a multiplicity of infection of . or an equivalent amount of uvinactivated respiratory syncytial virus (rsv-uv). cellular rna was isolated immediately and hours after inoculation with the rnaeasy mini kit (qiagen, valencia, calif) and transcribed to cdna by using the high-capacity cdna reverse transcription kit (applied biosystems, foster city, calif). single-target quantitative real-time pcr was used to monitor ifng and ccl mrna and rsv rna levels. for ifng and ccl mrna, primers were obtained from applied biosystems (hs _a and hs _a ). for rsv rna, primers f ( -tccctacggttgtgatcgataga- ) and r ( -tgatgggaagtagtagtgtaaagttggt- ) and probe t ( -aggtaatacagccaaatc- ) targeting the viral l gene were based on the sequence of rsv strain crd (genbank accession no. dq ). for glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna, primers f ( -cagccgagccacatccctcagacaccat- ) and r ( -ctttaccagagttaaaagcagccctggtgacca- ) and probe t ( -aggtcggagtcaaccgatttggtcgtattg- ) were used. plasmids encoding ccl and ifng (origene, rockville, md) and a portion of the rsv l gene (nt - ) and gapdh gene sequence (genbank accession no. nm_ ) were used to generate rna standards. rt-pcr was performed by using taqman real-time pcr master mix with ml of sample cdna in accordance with the manufacturer's protocol (applied biosystems). all data for gene copy number was normalized to gapdh level. in a subset of cord blood samples (n ) with an adequate number of cells, we also assessed interferon signal transduction by monitoring the level of stat phosphorylation in response to interferon stimulation. the corresponding cbmcs were processed as described above and serum starved on day of culture. on culture day , the cells were incubated with ifn-b ( u/ml) for minutes. cell lysate was harvested after treatment with cell lysis buffer (cell signaling, danvers, mass). the level of total stat was determined by using elisa (invitrogen, carlsbad, calif), and phosphorylated stat (tyr ) levels were determined by using a sandwich elisa (pathscan phospho-stat , cell signaling). nasal lavage samples were obtained during acute respiratory tract illnesses during the first year of life and at the -year follow-up visit. for illness samples, a respiratory symptom scorecard was completed as described previously. , when the score indicated a moderate-to-severe respiratory tract illness, the site staff obtained a nasal lavage sample within hours. all nasal lavage samples were processed for identification of respiratory tract viruses by using a pcr-based assay, as described previously. descriptive data were expressed as percentages, means sds, or medians with interquartile ranges for nonnormally distributed data. to test differences between specific groups, x or fisher exact tests were used to compare categorical variables, whereas unpaired t tests were used to compare continuous variables. appropriate log transformations were made to the data to yield an approximately normal distribution. for nonnormally distributed data, the wilcoxon rank sum tests (mann-whitney u tests) were used to compare groups. each measurement was standardized as the ratio over control to minimize variability in day-to-day experiments as follows: d ifng mrna response to rsv ifng mrna copies with rsv/ ifng mrna copies without rsv, d ccl mrna response to rsv ccl mrna copies with rsv infection/ccl mrna copies with no rsv infection, and rsv level was expressed as the rsv rna copy number with infection minus the value for no infection. spearman correlations were calculated to test for associations between variables. we examined the possibility of confounding by the following variables: sex, breast-feeding, maternal smoking during pregnancy and the first year of the child's life, an overall sum of bedroom allergen exposure (mus musculus, blattella germanica, and felis domesticus), and exposure to endotoxin (recombinant factor c assay) and ergosterol. we evaluated whether any of these variables were associated with the outcomes under analysis, and we found only that maternal smoking during the first year of life was related to the pneumonia outcome and the sum of bedroom allergen exposures was related to sinus infections. however, in logistic models controlling for these variables, the odds ratios changed by less than % when adjusted, and therefore we present unadjusted relationships here. all statistical tests were -tailed, and p values of less than . were considered statistically significant. statistical procedures were conducted with both sas . (sas institute, inc, cary, nc) and r . . software. we processed all cord blood samples that contained an adequate number of cells, representing of the total of children who were enrolled at the st louis site of the ureca cohort. among the newborns, % of the babies were african american, the mean age of the mother at the time of delivery was . years, and % of the infants had at least parent with asthma (table i) . subjects were reported to have an average of . upper respiratory tract infections (colds), . wheezing illnesses, and an all-cause hospitalization rate of % during the first year of life. the basic demographics and first-year outcomes for this group of children were not significantly different from those of the remaining group of children who were not part of the present analysis (see table e in this article's online repository at www.jacionline.org). we collected nasal lavage samples during symptomatic respiratory episodes (n ) and at routine -year follow-up visits (n ). respiratory tract viruses were detected in % of the symptomatic episodes and in % of the routine visits (table ii) . human rhinovirus was found with the highest frequency and rsv with the next highest frequency in subjects with symptomatic episodes, but both could also be detected in asymptomatic subjects. together, the findings indicate that the majority of respiratory tract illnesses during the first year of life are associated with detectable levels of respiratory tract viral pathogens, but asymptomatic infants also have a high rate of apparent viral carriage. the distribution of values for rsv-induced ifng and ccl mrna, rsv rna, and stat activation showed a broad range among the subject population, suggesting a degree of individual variation among subjects for each of these responses (table iii) . this finding further suggests that the differences in responses might translate to variable degrees of protection against viral infection. we next investigated whether individual variation in antiviral response was associated with the development of respiratory tract fig ) . there was no correlation between ifng response and available measures that might be associated with a subject's atopy (ie, number of parents with asthma, number of parents with hayfever, and presence of parental asthma at the time of initial screening). the relationship to respiratory tract infections was selective for the ifng response because we found no association between ccl response, rsv titer, or stat activation with the frequency of upper respiratory tract infections (r . , p . ; r . , p . ; and r . , p . , respectively). we found no association of ifng response, ccl response, rsv titer, or stat activation with the frequency of wheezing episodes in the first year of life (r . , p . ; r . , p . ; r . , p . ; and r . , p . , respectively). we also investigated whether there was an association of antiviral responses with the occurrence of infections at other respiratory and nonrespiratory sites (ie, sinus, ear, croup, and stomach), as well as any association with the number of reported hospitalizations for respiratory tract illness. we found that ifn-g responses to rsv in cord blood monocyte cultures were inversely related to the frequency of ear infections (p . ), sinus infections (p . ), pneumonias (p . ), and respiratory-related hospitalizations (p . , fig ) . we found no differences in ifn-g responses between those who did and did not have croup, ''stomach flu,'' or unexplained fevers (fig and data not shown) . together, these findings reinforce the association of a decreased ifn-g response to rsv with the development of increased viral respiratory tract infections in the first year of life. in this study we provide evidence that a decreased antiviral interferon response at the time of birth is selectively associated with an increase in acute respiratory tract infections in the first year of life among infants at high risk for asthma and allergic disease. in support of this relationship between antiviral response and respiratory tract infection, we show that ( ) rsv-driven induction of ifng mrna production in cord blood monocytes is variable among infants at birth; ( ) decreased levels of rsv-induced ifng mrna in cord blood monocytes are associated with a significant increase in the frequency of upper respiratory tract infections, as well as the prevalence of ear infections, sinus infections, pneumonias, and respiratory tract illnesses requiring hospitalization; ( ) levels of rsv-induced ccl mrna expression (another highly inducible antiviral system) and interferon-driven activation of stat (the downstream target of the interferon receptors) are not associated with this phenotype for subsequent illness; ( ) symptomatic respiratory tract illnesses were frequently associated with detectable levels of respiratory viral pathogens; and ( ) levels of rsv-induced ifng mrna were not linked to other types of infections, such as croup or stomach flu, which are caused by other types of pathogens. together, these findings provide for a close relationship between rsv-driven ifng mrna production and the development of viral respiratory tract illness and in turn suggest the possibility that a decrease in this type of response might lead to an increase in this type of illness. the present findings offer distinct insights from those obtained previously. for example, others found that pha-and allergenstimulated ifn-g production in cbmcs was inversely correlated with the frequency of viral respiratory tract infection in the first year of life. , however, these studies likely measured the responsiveness of t cells because nonadherent cells were not eliminated and t-cell mitogen and antigen were used for stimulation. moreover, the effect of viral infection itself was not assessed in these studies. other studies examined the capacity of pbmcs to produce ifn-g during rsv-induced illness in children, but in this case cells were activated with phorbol -myristate -acetate-ionomycin and cross-linking antibodies to t-cell costimulatory receptors, and the response was localized to cd t cells. similarly, others again made no attempt to purify cells and then stimulated cells with t-cell mitogen and did not separately quantify ifng mrna levels. the same approach was taken in previous reports of an association between decreased ifn-g production from cbmcs and an increased risk of allergic sensitization and recurrent wheezing. , , here again, this might reflect the focus on t-cell production of ifn-g and the proposed role of t h versus t h cytokines in the development of atopy and asthma. after these studies, we have come to better recognize the critical role of the innate immune system in controlling viral infection and postviral asthma. in particular, the interferon and monocyte-macrophage systems are required for protective immunity against respiratory tract viruses, such as rsv, [ ] [ ] [ ] and these same systems are capable of driving postviral asthma independent of the adaptive immune system, at least in experimental models. [ ] [ ] [ ] therefore the present approach was designed to directly assess the innate immune response to viral infection and was done so by using the relevant cell type (purified monocytes) and stimulus (rsv infection), as well as more specific and sensitive methods (real-time quantitative pcr) than applied previously. the upshot is the first evidence that the monocyte ifng gene also serves as a marker and might even participate in the susceptibility to infection during infancy. this unexpected finding provides a new lead for control of the antiviral response because the previous view was that ifng gene expression was silenced in the monocyte lineage and was only active in lymphoid cells. in that regard we note that ifn-g production is generally attributed to natural killer (nk) cells, nkt cells, and t cells, whereas monocytes and macrophages are solely a target of ifn-g action. indeed, studies of atopic disease in infancy often focus exclusively on t-cell production of ifn-g. this circumstance is also likely due to the lower levels of ifn-g produced by monocytes and macrophages under conditions used in previous studies. here we are able to measure ifn-g production by using a sensitive assay for the corresponding mrna. whether this level of interferon production has functional consequences is uncertain, but its utility as a biomarker for susceptibility to viral infection proved quite useful. in contrast, it appears that the monocytemacrophage lineage is critical for host defense against respiratory tract viruses (including rsv). in particular, lung macrophages are charged with clearance of infected cells without dying themselves, and this protection derives from an antiapoptotic survival function of the chemokine ccl . if this function is lost (eg, in mice that are ccl deficient), the host is more susceptible to viral respiratory tract infection. other work suggests that ccl is also needed to direct dendritic cell traffic in the face of viral infection. each of these observations are consistent with those linking ccl promoter gene polymorphisms to susceptibility to severe rsv-induced bronchiolitis. nonetheless, we did not find that rsv induction of ccl gene expression was significantly associated with viral respiratory tract infection rates in the first year of life. it is still possible, however, that ccl production at the level of the lung macrophage would be predictive of susceptibility to infection, especially given the heterogeneity of monocyte-macrophage populations in the circulation and the lung. our study was conducted in a population selected for high prevalence of atopic disease. however, we do not expect that ifng response or viral susceptibility is attributable to atopy. indeed, we found no association of the ifng response with the available measures associated with atopy in our subject group because full evaluation of atopic status in our subjects was not yet performed at year of age. similarly, others found no association between lower respiratory tract illness in the first year of life and the occurrence of parental atopy or subject eczema. these findings suggest that viral susceptibility can be independent of atopy, and certainly there is evidence that this can be the case in experimental models of viral respiratory tract infection. however, it will require a nonatopic cohort, follow-up of the present cohort, or both to formally test the relationship between virus-induced ifn-g production in monocytes, viral susceptibility, and atopic status in human subjects. the molecular mechanism for rsv induction of ifng gene expression still needs to be defined. thus the pathogen recognition receptor system is responsible for mediating viral induction of the various forms of ifn-a, ifn-b, and ifn-l, and rsv is remarkably effective in blocking the induction (and signaling) of these interferon species. , by contrast, the pathogen recognition receptor system of toll-like receptors and retinoic acid inducible gene i (rig-i)-like receptors (rlrs) does not appear to regulate ifng gene expression or signaling. instead, ifng gene expression is subject to a distinct type of positive and negative regulation at pretranscriptional, transcriptional, and posttranscriptional levels, at least in the case of lymphoid (nk, nkt, and t) cells. however, these regulatory mechanisms have not been studied in monocytes or in response to rsv in any cell type. we could not define this regulatory mechanism with such limited human samples, but our work should open this new field of research. rsv is the most common cause of serious respiratory tract illness during infancy, and severe rsv-induced bronchiolitis is linked to subsequent wheezing illness/asthma. furthermore, paramyxoviral infection is established as a high-fidelity experimental model of asthma. moreover, rsv and related paramyxoviruses are easily detected in monocytes and macrophages in the lung. hence we chose rsv for our study of newborns and their monocytes, unaware of course that the subsequent results would predict respiratory tract illness associated with rhinovirus. with such a severe limitation in cell sample size, we were unable to test multiple viruses, but an analysis of the response to other types of viruses (including rhinovirus) and viral strains (including other rsv types) would be a valuable goal in the future. in summary, we report that congenital variations in the innate immune response might predict the susceptibility to acute respiratory tract illness during the first year of life. our effort uncovered evidence that the ifng mrna monocyte response to virus rather than t-cell response to mitogen/allergen might be linked to the development of viral infections and eventually postviral asthma. we were able to define this relationship despite a relatively small sample size. sample availability for complex immunologic analysis served to limit the number of ureca participants that could be studied. however, the demographic characteristics of our cohort were no different from those of the overall group, suggesting that we studied a representative sample of subjects. in addition, our study included mostly african american children, and therefore the result might not be generalized to children with other racial backgrounds. interestingly, others have recently found that interferon-stimulated genes (eg, pyhin ) are also linked to the development of asthma, particularly in subjects of african descent. our findings are also consistent with observations of decreased ifn-g production in response to rsv in pbmcs from older children and adults with allergic asthma. , thus these early events in infancy might carry over to a similar deficit in antiviral defense in later life. together, the findings suggest that a full analysis of interferon production pathways might provide key insights into the susceptibility to viral respiratory tract infection and subsequent chronic obstructive lung diseases, such as asthma. world health organization. the world health report : make every mother and child count. geneva: world health organization wheezing rhinovirus illnesses in early life predict asthma development in high-risk children tucson children's respiratory study: to present day care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy prospective study of healthcare utilisation and respiratory morbidity due to rsv infection in prematurely born infants influence of attendance at day care on the common cold from birth through years of age respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis host epithelial-viral interactions as cause and cure for asthma cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life cord blood cytokines and acute lower respiratory illnesses in the first year of life bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life respiratory syncytial virus in early life and risk of wheeze and allergy by age years asthma and allergy patterns over years after severe rsv bronchiolitis in the first year of life virusinducible expression of a host chemokine gene relies on replication-linked mrna stabilization prenatal maternal stress and cord blood innate and adaptive cytokine responses in an inner-city cohort parental characteristics, somatic fetal growth, and season of birth influence innate and adaptive cord blood cytokine responses the urban environment and childhood asthma (ureca) birth cohort study: design, methods, and study population standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study decreased interferon-gamma response in respiratory syncytial virus compared to other respiratory viral infections in infants type and type cytokine imbalance in acute respiratory syncytial virus bronchiolitis association between antenatal cytokine production and the development of atopy and asthma at age years reduced interferon gamma production and soluble cd levels in early life predict recurrent wheezing by year of age the role of ifn in respiratory syncytial virus pathogenesis ccl -ccr interaction provides antiapoptotic signals for macrophage survival during viral infection airway epithelial versus immune cell stat function for innate defense against respiratory viral infection induction of high-affinity ige receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia persistent activation of an innate immune response translates respiratory viral infection into chronic inflammatory lung disease cutting edge: cd d neutrophils induce fcεri expression on lung dendritic cells in a mouse model of postviral asthma regulation of interferon-g during innate and adaptive immune responses high ifn-g production by cd t cells and early sensitization among infants at high risk of atopy controls for lung dendritic cell maturation and migration during respiratory viral infection rantes promoter gene polymorphisms and suceptibility to severe respiratory syncytial virus-induced bronchiolitis suppression of the induction of alpha, beta, and gamma interferons by the ns and ns proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory syncytial virus nonstructural proteins ns and ns mediate inhibition of stat expression and type i interferon responsiveness meta-analysis of genome-wide association studies of asthma in ethnically diverse north american populations atopic phenotype in children is associated with decreased virus-induced interferon-alpha release impaired virus-induced interferon-alpha release in adult asthmatic patients disclosure of potential conflict of interest: k. sumino has received research support from the national institutes of health (nih). j. e. gern is on the scientific advisory board for and owns stock options in v biosciences; has consulted for centocor, boeheringer ingelheim, glaxosmithkline, biota, medimmune, and theraclone; and has received research support from astrazeneca. g. r. bloomberg has received research support from the nih/national institute of allergy and infectious diseases. m. j. holtzman has consulted for and received research support from hoffman-la roche and forest laboratories. the rest of the authors declare that they have no relevant conflicts of interest. clinical implications: individual variations in the immune response to respiratory tract viruses are detectable at birth, and these differences predict the susceptibility to acute respiratory tract illness during first year of life. key: cord- - euaaspo authors: li, xiaochen; xu, shuyun; yu, muqing; wang, ke; tao, yu; zhou, ying; shi, jing; zhou, min; wu, bo; yang, zhenyu; zhang, cong; yue, junqing; zhang, zhiguo; renz, harald; liu, xiansheng; xie, jungang; xie, min; zhao, jianping title: risk factors for severity and mortality in adult covid- inpatients in wuhan date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: euaaspo abstract background in december , covid- outbreak occurred in wuhan. data on the clinical characteristics and outcomes of patients with severe covid- are limited. objective the severity on admission, complications, treatment, and outcomes of covid- patients were evaluated. methods patients with covid- admitted to tongji hospital from january , to february , were retrospectively enrolled and followed-up until march , . potential risk factors for severe covid- were analyzed by a multivariable binary logistic model. cox proportional hazard regression model was used for survival analysis in severe patients. results we identified ( . %) of patients as severe cases on admission. elder age, underlying hypertension, high cytokine levels (il- r, il- , il- , and tnf-a), and high ldh level were significantly associated with severe covid- on admission. the prevalence of asthma in covid- patients was . %, markedly lower than that in the adult population of wuhan. the estimated mortality was . % in nonsevere patients and . % in severe cases during the average days of follow-up period. survival analysis revealed that male, elder age, leukocytosis, high ldh level, cardiac injury, hyperglycemia, and high-dose corticosteroid use were associated with death in patients with severe covid- . conclusions patients with elder age, hypertension, and high ldh level need careful observation and early intervention to prevent the potential development of severe covid- . severe male patients with heart injury, hyperglycemia, and high-dose corticosteroid use may have high risk of death. the sars-cov- virus, as a betacoronavirus, shares % of two bat-derived sars-like coronaviruses and distances from sars-cov (around %) and middle east respiratory syndrome coronavirus (mers-cov, around %). sars and mers epidemics posed threats to global health due to high mortality rates of . % for sars-cov and . % for mers-cov globally. , epidemiological data released by the chinese center for disease control and prevention showed that , confirmed cases have been identified in wuhan and , in mainland china except wuhan as of march , . the mortality rate of covid- patients was . % in wuhan, which was close to that in the world ( . %) and much higher than that in mainland china except wuhan ( . %). this study aims to describe and compare the epidemiological, demographic, clinical, laboratory and radiological characteristics as well as the complications, treatment and outcomes of hospitalized patients with nonsevere and severe covid- . potential risk factors for severe covid- and factors associated with death in severe cases were analyzed to provide scientific data for relieve severe cases and reduce mortality. this study was an ambispective cohort study of consecutive hospitalized patients with covid- enrolled at sino-french new city branch of tongji hospital, huazhong university of science and technology in wuhan from january , to february , . the final date of follow-up was march , . sino-french new city branch of tongji hospital is one of the major nationally designated hospitals only providing medical care for adult covid- patients in wuhan. all cases with covid- enrolled in this study were diagnosed based on the who interim guidance and the diagnostic and treatment guideline for covid- issued by chinese national health committee (version ). detection of sars-cov- nucleic acids was shown in text in the online repository. this study was approved by institutional review board of tongji hospital, huazhong university of science and technology. written informed consent was waived in light of the urgent need to collect data. the epidemiological and demographic data were obtained by face-to-face or telephone interview. clinical symptoms, laboratory, and radiological findings on admission as well as the complications, treatment and outcomes during hospitalization were extracted from electronic medical records. serum cytokines (il- β, il- r, il- , il- , il- and tnf-α) were measured on admission. patient data were cross-checked for consistency before final data entry and then entered into a computerized database. the presence of underlying comorbidities was identified based on the international classification of diseases and injuries- diagnostic codes. the complications of covid- after admission were assessed and the definitions were shown in text in the online repository. cardiac injury was one of the complications, which was defined as a serum hypersensitive cardiac troponin i level higher than . pg/ml without acute coronary symptoms or abnormal electrocardiogram. the clinical outcomes were classified into discharge from hospital, in-hospitalization, and death. severe covid- was defined according to clinical practice guideline from infectious diseases society of america and american thoracic society for diagnosis and treatment of adults with community-acquired pneumonia. based on whether or not requiring ventilatory support on admission, severe cases upon admission were divided into two cohorts, severely ill and critically ill cases. the descriptive statistics are median and interquartile range (iqr) for continuous data. a total of patients with covid- were enrolled, of whom cases were included in the study. one case not meeting inclusion criteria was excluded due to inclusion criteria. almost half of the patients ( . %, of ) were identified as severe cases and . % ( of ) were nonsevere cases on admission. . % ( of ) of cases were positive for sars-cov- nucleic acid test pre-admission. comparison of findings between nonsevere and severe cases in the patients with positive viral nucleic acid test pre-admission showed essentially the similar differences to that in the total patients (see table e in the online repository). the epidemiological and demographic characteristics are shown in table . ( . %) of patients got the infection in hospital. ( . %) of patients were health-care workers and ( . %) patients were family members of health-care workers. nonsevere cases had a higher proportion of health-care workers and family members than severe cases (p< . ). the date of onset of the first reported case with covid- was december , . the median time from december , to the onset of covid- was days, ranging from days to days. the median age of study population was years (iqr - ), ranging from years to years, of whom ( . %) were aged years or older. the patients aged years or older in severe cases were almost twice as nonsevere cases of the same age ( . % vs. . %, p< . ). slightly more than half ( . %) of all patients were male and the proportion of males in severe cases is higher than in nonsevere cases ( . % vs. . %, p= . ). . % of patients with severe covid- were smokers (table ) asthma ( . %) were identified in total population. ( . %) of patients regularly took angiotensin-converting enzyme inhibitors or angiotensin ii receptor blockers; no significant difference was found between nonsevere and severe cases. sore throat ( . %), cough ( . %), chest pain ( . %), dyspnea ( . %), chest tightness ( . %), dizziness ( . %), confusion ( . %), headache ( . %), myalgia ( . %), vomiting ( . %), diarrhea ( . %), abdominal pain ( . %). patients were asymptomatic and diagnosed by ct screening. duration of fever pre-admission was significantly longer among severe cases compared with that in nonsevere cases (p= . ). severe cases experienced longer duration from onset to outpatient visit and longer duration from onset to hospitalization compared with nonsevere cases (p= . and p= . , respectively). ( . %) of patients were treated with corticosteroids delivered by oral or intravenous pre-admission. ( . %) of patients had received at least one of the following antiviral medications: umifenovir ( . %), oseltamivir ( . %), lopinavir/ritonavir ( . %), and ribavirin ( . %). ct scans for patients were evaluated pre-admission, and showed multilobar pulmonary infiltrates in patients ( table ). the median time from onset to pneumonia diagnosed by ct scan was days. on admission, oxygen saturation (spo ) less than . % on room air presented in . % of all patients, of whom ( %) were severe cases. . % of all patients experienced lymphopenia (< cells per mm ) and . % of all patients had thrombocytopenia (< , cells per mm ). compared with nonsevere cases, inflammation-related marker levels (hscrp, esr, and ferritin) were significantly higher in severe cases the levels of procalcitonin, globulin, lactate dehydrogenase (ldh), nt-prob-type natriuretic peptide (nt-probnp), d-dimer, alanine aminotransferase, aspartate aminotransferase, total bilirubin, conjugated bilirubin, blood urea nitrogen, and creatinine were elevated in . %, . %, . %, . %, . %, . %, . %, . %, . %, . %, and . % of all patients, respectively. serum cytokine levels of il- r, il- , il- , and tnf-α were significantly higher in severe patients than those in nonsevere patients (all of the severe cases, were classified as critically ill for requiring respiratory support. compared with severely ill cases, the time from december , to onset was shorter and the time from onset to outpatient visit was longer in critically ill cases (see table e in the online high procalcitonin, high nt-probnp, high ldh, high d-dimer, low albumin, and high creatinine) were observed in critically ill cases compared with severely ill cases (all p< . ). . % of critically ill patients received systemic corticosteroids pre-admission, the proportion of whom was significantly higher than that in severely ill cases ( . %, p< . ). compared with nonsevere cases, systemic corticosteroid use pre-admission was more common in severe cases with larger cumulative dose and longer duration (p= . ; p< . ; p< . ; respectively). stratification of patients by corticosteroid exposure is presented in table e in the online repository. severe patients treated with corticosteroids had higher ldh level compared with severe patients without corticosteroid use pre-admission (p< . ). nonsevere cases were more likely to receive antiviral drugs pre-admission, including umifenovir and oseltamivir (p< . and p= . ; respectively). in severe case subgroup, the patients receiving umifenovir were younger than those without umifenovir use (p< . ). a comparison of baseline demographic and clinical characteristics between patients with and without antiviral drug use revealed no marked difference in spo or laboratory findings in both nonsevere and severe case subgroups (see table e and e in the online repository). in the final logistic regression model, variables such as age or older (or . ; % ci . - . ), hypertension (or . ; % ci . - . ), ldh > u/l (or . ; % ci . - . ), and d-dimer > mg/l (or . ; % ci . - . ) were significantly associated with severe cases with covid- ( figure ) . in the follow-up period, the complications of covid- were assessed , including acute respiratory distress syndrome (ards) ( . %), cardiac injury ( . %), liver dysfunction ( . %), acute kidney injury ( . %), bacteremia ( . %), diffuse intravascular coagulation ( . %), and hyperglycemia ( . %) ( this study provided a comprehensive data on the epidemiological, demographic, clinical, laboratory, and radiological characteristics as well as the complications, treatment, and outcomes of hospitalized patients with nonsevere and severe covid- in wuhan. almost half of the patients in this study were identified as severe cases, which may differ from the results of the previous studies. the proportion of patients aged years or older were higher in our study than in nanshan zhong's study ( . % vs. . %, respectively). the time from december , to the onset of most patients was longer than days. during mid-january to early february, wuhan experienced the highest peak of covid- outbreak, with a family cluster and high prevalence of covid- in older adults. longer wait for access to medical care was observed in severe cases compared with that in nonsevere cases. more than half of the patients experienced at least two hospital visits, which may increase the risk of nosocomial transmission events. diagnosis and treatment may be delayed due to the long wait for access to medical care. severe covid- patients would likely develop ards and died of respiratory failure. although there are currently no effective antiviral drugs for sars-cov- , prompt identification and early respiratory support would relieve severe cases and reduce mortality. the severity of disease in patients with initial positive nucleic acid test was similar to that of all covid- patients. we thus propose that urgent timely diagnosis is crucial, and that early intervention should not be delayed based on nucleic acid test. proinflammatory cytokine levels (inf-γ, il- β, il- , il- , il- , and tnf-α) and were associated with the development of ards. - in our study, severe covid- patients had ( . %) in our study was markedly lower than that reported in the adult population of wuhan ( . %). - we thus speculate that th immune response in asthmatic patients may counter the inflammation process induced by sars-cov- infection. further studies are required to characterize the immune response and inflammation features of the majority of severe patients showed rapid progression and multiple organ dysfunction. the to onset in critically ill cases than that in severely ill cases may reflect a higher virulence of sars-cov- , or earlier onset of the risk factors for severity identified in this study included age, high ldh level, and high d-dimer level, consistent with those in previous reports. , however, different from the findings of previous studies , hypertension was the only comorbidity associated with the severity of covid- after adjustment for age, sex and smoking status. the distinct features of pneumonia and high severity in patients with covid- in this study may lead to this difference from previous reports. ace , a gateway to sars, was reported to be a protective factor against sars-cov-induced lung injury. , the association between ace expression and hypertension was confirmed in a previous study. this fact may partly explain the high prevalence of severe covid- in patients with hypertension. ldh has been recognized as a marker for severe prognosis in various diseases, including cancer and infection. the high ldh level in covid- in severe cases suggested that ldh may be associated with lung injury and tissue damage, warranting an investigation for the potential mechanism. this study evaluated pre-admission medications for patients with severe covid- . although the proportion of nonsevere cases in patients receiving oseltamivir was higher than that in patients without oseltamivir use, stratification analysis showed that there was no significant difference in case subgroup. therefore, oseltamivir use may just be an indicator of disease severity. the patients receiving umifenovir were younger than those without umifenovir use, indicating that younger patients may have easier access to drugs or prefer umifenovir. older age, leukocytosis and high ldh level were reported to be risk factors associated with in-hospital death in previous studies. , , the present study also revealed that hyperglycemia was related with increased mortality in covid- patients. the prevalence of hyperglycemia may be associated with underlying diabetes and corticosteroid therapy. however, the localization of ace expression in the pancreas in sars was reported to damage islets, resulting in hyperglycemia ; this finding suggested that hyperglycemia may also be an indicator of severe this study indicated that corticosteroid use was more common in severe cases than in nonsevere cases and that high-dose corticosteroid use was related to high risk of death in severe covid- patients. high-dose steroid use may be an indicator of disease severity rather than a predisposing factor. in a previous study, treatment with methylprednisolone was shown to be beneficial for covid- patients who developed ards. however, critically ill cases had more signs of infection and abnormal laboratory findings, including high leukocyte, high procalcitonin, high d-dimer, low albumin, and high creatinine levels. high-dose corticosteroid use should be used with cautious in critically ill patients to avoid aggravating complications. a recent study by bin cao et al showed that lopinavir/ritonavir treatment offered no significant benefit over the standard care for hospitalized adult covid- patients. cao's study also reported that lopinavir/ritonavir led to a shorter median time to clinical improvement than the standard care (hr . ; % ci . to . ) in a modified intention-to-treat analysis. compared with cao's study, the severity of patients was more serious and lopinavir/ritonavir treatment was associated with a lower risk of death in severe covid- patients in this study. however, our study was an observational study; thus, the benefit of lopinavir/ritonavir for severe covid- patients needs to be further confirmed. there were limitations to the current study. firstly, epidemiological data were collected respectively and recall bias might have occurred. secondly, missing data on some variables, such as detailed information of ct scan, may cause bias in the estimation and reduce the representativeness of the samples. thirdly, laboratory findings were measured upon admission and may indicate the severity of covid- . the causal relationship between abnormal laboratory findings and severity could not be determined. fourthly, this study was an observational study with limitations in terms of evaluating the efficacy of corticosteroids and antiviral drugs. finally, the absence of comparative data from covid- patients not admitted or from other critically ill patients were limitations of this study. in conclusion, covid- outbreak caused widespread concern and has threatened the global clinical characteristics of patients infected with sars-cov- in wuhan an increased prevalence of self-reported allergic rhinitis in major chinese cities from ace links amino acid malnutrition to microbial ecology and intestinal inflammation cholangiocytes may cause liver damage after -ncov infection binding of sars coronavirus to its receptor damages islets and causes acute diabetes risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease comorbidity and its impact on patients with covid- in china: a nationwide analysis where n is the total number of patients with available data. p values comparing nonsevere cases and severe cases are from χ² test, fisher's exact test, or mann-whitney u test cov- nucleic acid test was performed pre-admission table . complications and treatment during hospitalization and clinical outcomes of covid- data are expressed as median (iqr), n (%), or n/n (%), where n is the total number of patients with available data. p values comparing nonsevere cases and severe cases are from χ² test, fisher's exact test ards=acute respiratory distress syndrome key: cord- -j ayp tv authors: navel, valentin; chiambaretta, frédéric; dutheil, frédéric title: reply date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: j ayp tv nan reply q to the editor: we read with interest the relevant comment of gallo et al concerning our recent editorial. globally, the severe acute respiratory syndrome coronavirus (sars-cov- ) outbreak has upset the lifestyle of humans, increasing sedentary behaviors with quarantine (ie, staying at home, tele-working, screen activities), the fear of contagion (ie, social connections, mass transit, and compulsive disinfecting), and probably a city dweller migration to the periphery of crowed cities and surrounding countryside. as explained by the authors, this exceptional period seems to be salient to explore the complex interplay of interactions between exposome (ie, indoor and outdoor expositions), genetic heritage, lifestyle, and allergic diseases expression. interestingly, more than half the people stayed at home to prevent the spreading of sars-cov- during the several months of quarantine, involving a massive decrease in greenhouse gas emissions related to human activities. considering the irritative factor of air pollution for the respiratory tract, the eyes, and the skin, we could attempt a reduction in allergic exacerbations mediated by nonspecific inflammation. considering that filtering face piece respirators (ffps) reduced the penetration of fine particulates ranging from % (ffp ) to % (ffp ), their global use during the coronavirus disease pandemic could protect people against outdoor and indoor allergenic components. however, the authors rightly highlighted that people were particularly exposed to biological aeroallergens (ie, dust mites, domestic animals, or molds) and chemical components (ie, ammonia, solvent, tobacco, and cooking smokes) of home indoor environment during quarantine. similar to the concentration of diesel exhaust particles in a vehicle cabin exposed to heavy traffic, nonventilated houses could condense the toxicity of outdoor air pollution during several days. during the past decades, some countries have developed models of energy other than petroleum to promote more renewable energy sources, that is, wave power, offshore wing q power, photovoltaic, or wood. however, it is widely accepted that home exposure to wood smoke causes inflammation in the respiratory system. promoting warm and humid environment, indoor activities (ie, tele-working, cooking, and indoor sports) could improve the exposition of airborne fungal spores in damp houses, increasing the incidence of asthma and allergic diseases. outdoor sports practice being prohibited, some people could run or cycle in their home. moreover, evidence data showed that air pollutant exposure during exercise could decrease the pulmonary and vascular function in healthy individuals, increasing systemic and airways inflammation and amplifying the dose of pollutants during endurance physical activity. thus, the benefit of the decrease in air pollution could be attenuated by the massive exposure to indoor pollutants, aggravated by the toxicity of disinfectants such as bleach, rubbing alcohol, or quaternary ammonium-massively used to limit the spreading of sars-cov- . considering that the hygienic hypothesis highlighted the major role of microbiome to regulate the immune response to allergens, it would be very interesting to evaluate the impact of the extensive use of hygienic measures on the incidence of allergic diseases. because of the fear of contagion, people may avoid mass transit and increase their commuting by individual car, which may in turn promote air pollution-and the release of the highly allergenic fine particulate matter. we may therefore see a bimodal response: a decrease in air pollution during the confined period, followed by an increase in air pollution-concomitantly with the effort of worldwide governments to kickstart economic growth (despite ecological engagements). to conclude, it will be salient to analyze the aforementioned factors related to allergic diseases in forthcoming months, to better understand the complex relation between allergens, air pollution, immune response, and exacerbation of allergic diseases. global lockdown, pollution, and respiratory allergic disease: are we in or are we out? will environmental impacts of social distancing due to the sars-cov- pandemic decrease allergic disease? comparison of filtration efficiency and pressure drop in anti-yellow sand masks, quarantine masks, medical masks, general masks, and handkerchiefs wood stove pollution in the developed world: a case to raise awareness among pediatricians. curr problems pediatr adolesc health care indoor airborne fungal spores, house dampness and associations with environmental factors and respiratory health in children cardiorespiratory biomarker responses in healthy young adults to drastic air quality changes surrounding the asthma and the hygiene hypothesis: does cleanliness matter? key: cord- -sdbj ca authors: hosoki, koa; kimata, jason t.; chakraborty, abhijit; sur, sanjiv title: reply date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: sdbj ca nan reply q to the editor: we thank zwaveling et al for appreciating our review and for their insightful correspondence. they suggest that suppression of angiotensin-converting enzyme- (ace- ) by severe acute respiratory syndrome coronavirus (sars-cov- ) could impair the hydrolysis of des-arg -bradykinin and stimulate the bradykinin receptor type (bkb ) pathway to induce leakage of fluid into the lungs. in support of their hypothesis, they stated that loss of ace- in an animal model aggravated acid-induced pulmonary edema, and these effects are alleviated by administration of recombinant human ace- q . in addition, a report demonstrated that sars-cov infection downregulates the expression of ace- . however, other studies suggest that sars-cov- may upregulate the expression of ace- in patients with coronavirus disease (covid- ) or influenza pneumonia in alveolar epithelial cells, endothelial cells, and lymphocytes in perivascular tissue than in uninfected control autopsy lung. furthermore, single-cell rna sequencing analysis revealed that secretory cells in the upper airway epithelium have higher ace- expressions in covid- . thus, until the effect of sars-cov- on ace- levels or functionality is thoroughly addressed in peer-reviewed publications, it is difficult to precisely determine the contributory role of ace- in the bradykinin pathway during sars-cov- infection. zwaveling bradykinin receptor type (bkb ) could induce active fluid transfer through vascular pores (fig f - /c] , a). another explanation for extravascular fluid leakage into the lungs in covid- is secretion of proinflammatory cytokines such as tnf and il- during the cytokine storm (fig , a) . we favor a third hypothesis, where excessive and prolonged secretion of type i and type iii ifns in the airways contributes to loss of lung epithelial barrier function during covid- and other rna virus infections (fig , a) . , to test whether entry of sars-cov- through ace- is sufficient to induce type iii ifns without need for viral replication, we engineered a replication-deficient sars-cov- spike-hiv-luc pseudotype virus. infection of caco- cells, which naturally express ace- , with this engineered virus was sufficient to increase the mrna expression of ifn-l (fig , b and c) , indicating that virus replication is not required for upregulating its expression. because ifn-l contributes to loss of lung epithelial barrier function, we hypothesize that entry of sars-cov- via ace- can stimulate secretion of ifn-l and induce leakage of fluid into the lungs (fig , a) . zwaveling et al's hypothesis is most intriguing and certainly plausible. however, as acknowledged by the authors, there is no direct evidence showing increased levels of bradykinin or des-arg -bradykinin in the patients with covid- at this time. here, we provide evidence that entry of sars-cov- through ace- provides an adequate signal even without viral replication to stimulate ifn-l mrna expression, a cytokine that can cause damage to the epithelial barrier. further investigations are required to test the hypotheses outlined in figure , a, which may induce leakage of fluid into the lungs in covid- , and identify the most important pathway(s). koa hosoki, md, phd a * jason t. kimata, phd b * abhijit chakraborty, phd a sanjiv sur, md a pulmonary edema in covid- : explained by bradykinin? molecular mechanisms and epidemiology of covid- from an allergist's perspective angiotensin-converting enzyme protects from severe acute lung failure a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- covid- severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis cytokine prediction of mortality in covid patients type iii interferons disrupt the lung epithelial barrier upon viral recognition type i and iii interferons disrupt lung epithelial repair during recovery from viral infection key: cord- -kheek lx authors: carroll, kecia n.; gebretsadik, tebeb; minton, patricia; woodward, kimberly; liu, zhouwen; miller, e. kathryn; williams, john v.; dupont, william d.; hartert, tina v. title: influence of maternal asthma on the cause and severity of infant acute respiratory tract infections date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: kheek lx background: respiratory syncytial virus (rsv) and rhinovirus infections are the most common significant infant respiratory tract illnesses and are associated with increased but differential risks of childhood asthma. objective: we sought to determine whether maternal asthma is associated with higher odds of infant respiratory tract infection with rhinovirus versus rsv and increased infection severity. methods: mother-infant dyads were enrolled from - during an infant respiratory tract infection ( with rhinovirus and with rsv). mothers were classified into mutually exclusive groups (atopic asthma, nonatopic asthma, and no asthma). we determined viral cause using pcr and the severity of the infant’s respiratory tract infection using the bronchiolitis severity score. adjusted relative odds of maternal asthma with viral cause were calculated by using logistic regression. proportional odds models assessed the association of maternal asthma and infant infection severity. results: infants with a mother with atopic asthma compared with infants whose mothers did not have asthma were more likely to have rhinovirus versus rsv infection (adjusted odds ratio, . ; % ci, . - . ). similarly, among infants with rhinovirus, having a mother with atopic asthma was associated with increased infection severity (adjusted odds ratio, . ; % ci, . - . ). this relationship was not seen among infants with rsv. conclusions: clinically significant rhinovirus infection during infancy was more strongly associated with having a mother with atopic asthma than clinically significant rsv infection. having a mother with atopic asthma was associated with increased severity of infant rhinovirus but not rsv infections. infants with rhinovirus were more likely to have a familial atopic predisposition, which might partly explain the subsequent increased asthma risk. bronchiolitis, a lower respiratory tract infection (lrti) commonly caused by respiratory syncytial virus (rsv) and less commonly by human rhinovirus (hrv), affects an estimated % to % of children in the first year of life and is a leading cause of hospitalization during infancy. [ ] [ ] [ ] [ ] in addition to the acute morbidity seen with bronchiolitis, infants hospitalized with bronchiolitis and young children who experience virus-induced wheezing illnesses are at increased risk of recurrent wheezing and asthma in early childhood. [ ] [ ] [ ] [ ] [ ] the pathogenesis of the increased wheezing after viral bronchiolitis is not fully understood. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in efforts to learn whether children at risk of bronchiolitis are also at increased risk for asthma, studies have investigated whether a family history of asthma is associated with the severity or incidence of bronchiolitis during infancy, with some prior studies finding an association, whereas others did not. , , [ ] [ ] [ ] [ ] prospective birth cohorts in which all children have a familial predisposition to asthma, such as a cohort based in perth, australia, and the childhood origins of asthma (coast) cohort, have investigated the association of a viral cause of infections in early life and subsequent wheezing and asthma. , , in the coast cohort hrvinduced wheezing illnesses in early life were found to be stronger predictors of wheezing and asthma at age years than rsvinduced illnesses. , , however, it is not known whether infants with symptomatic hrv infections that lead to an unscheduled health care visit are more likely to have a familial predisposition to asthma than infants with symptomatic rsv infection. in this investigation that included mother-infant dyads enrolled in the tennessee children's respiratory initiative (tcri), we tested the hypothesis that a familial atopic predisposition was associated with viral cause and increased severity of viral acute respiratory tract infection (ari) during infancy. the tcri is a prospective cohort of term (> _ weeks), non-low-birth-weight (> _ g) infants and their mothers designed to investigate the association of characteristics of infant viral aris, such as severity and cause, and familial atopic predisposition on the development of early childhood asthma and atopy. this investigation included the mother-infant dyads in which the infant presented for an unscheduled clinic or emergency department visit or hospitalization and was determined to have a sole hrv-or rsv-induced ari. mother-infant dyads were enrolled at the time of an infant ari during viral respiratory seasons, september through may - . dyads were recruited in the inpatient, emergency department, and clinic settings at a single academic institution, and the children are currently being followed longitudinally through age years to ascertain asthma and atopy outcomes. each woman provided written informed consent for participation of herself and her infant. the vanderbilt university institutional review board approved the study. during study enrollment, research nurses administered an in-person structured questionnaire that included questions regarding demographics, the infant's home environment, the index infant's illness, previous medical history of the infant, and detailed family asthma and atopic disease history, including maternal responses to the international study of asthma and allergies in children questionnaire. at enrollment, research nurses obtained nasal and throat swabs from the infants for viral detection. through a structured medical chart review, information was abstracted regarding the infant's medical visit, including birth weight, room air pulse oximetry, requirement for supplemental oxygen, history of prior wheezing, and detailed medical information. final discharge diagnoses were obtained through chart review after discharge. self-reported maternal asthma status was defined as a positive response to the question ''have you ever had asthma?,'' which was asked as part of the international study of asthma and allergies in children questionnaire and/or to the question ''were you diagnosed with asthma as a child?'' , maternal atopy was determined based on skin prick test results or allergen-specific ige levels. preferentially, women underwent skin prick tests to saline, histamine, and aeroallergens: cat pelt, alternaria species, grass mix # , ragweed mix, oak mix, tricophyton species, mite mix, and cockroach mix (quintest extract tray; hollister-stier, spokane, wash). allergen-specific ige measurement (phadiatop; phadia, kalamazoo, mich) was performed on maternal blood samples for women who could not undergo skin prick tests or who had an inadequate skin test. multiallergen screens for specific ige (phadiatop) were measured with the immunocap (performed by the johns hopkins daci laboratory). a positive phadiatop result was defined as . ku a /l or greater by using the standards in place at the time the assays were performed. , among women for whom allergen sensitization status was determined based on skin prick test results or allergen-specific ige levels, we classified women by whether they reported a history of asthma into mutually exclusive categories: atopic asthma, nonatopic asthma, and no asthma. women with selfreported asthma and evidence of allergen sensitization (> _ positive skin prick test response or positive phadiatop result) were classified as having atopic asthma, women with self-reported asthma and without evidence of allergen sensitization were classified as having nonatopic asthma, and women who did not report self-reported asthma were in the no asthma group. nasal and throat swabs were obtained from infants at the time of enrollment, and the biospecimens were processed, placed in aliquots, and stored at c. the specimens were tested in batches for rsva and b, hrv, adenovirus, human metapneumovirus, coronaviruses, influenza a and b, and parainfluenza types , , and by using real-time rt-pcr with the cepheid smart cycler ii, as previously described. pcr results were used to identify infants with a sole rsv-or hrv-induced ari. infants included in this study had an hrv-or rsv-induced ari, either viral upper respiratory tract infections (urtis) or lrtis. children with a urti had a health care provider's diagnosis of a viral urti and/or symptoms, including fever, cough, congestion, hoarse cry, otitis media, and/or rhinorrhea without evidence of lower respiratory tract symptoms or respiratory distress. infants with a physician's diagnosis of bronchiolitis or wheezing, signs and symptoms consistent with bronchiolitis on chart review, or both were considered to have a viral lrti. , we determined the severity of the ari by using an ordinal bronchiolitis severity score with factors including respiratory rate, room air oxygen saturation, and the presence and extent of wheezing and flaring and retractions. scores range from to , with higher scores indicating more severe illness. , covariates other variables of interest obtained from the questionnaire administered at enrollment included self-reported maternal race/ethnicity, maternal education, secondhand smoke (shs) exposure, infant's insurance type (tennessee medicaid, private, or none), infant's birth weight (in grams), infant's sex, infant's age at enrollment (in weeks), and siblings. descriptions of demographics and characteristics of the infants with sole hrv or rsv infections are presented as frequencies and proportions for categorical variables and medians and interquartile ranges for continuous variables. univariate analyses were conducted to compare maternal asthma factors by the infant's hrv-or rsv-induced ari status by using the wilcoxon rank sum test for continuous variables or the pearson x test for categorical variables. in our analyses we first defined maternal asthma using self-reported asthma in the women. in addition, to examine the association of maternal asthma in combination with an objective measure of atopy, we repeated all analyses using a more detailed definition that classified women as having atopic asthma, nonatopic asthma, or no asthma by incorporating their skin prick test or allergen-specific ige findings. therefore we investigated whether ( ) having a mother with self-reported asthma and ( ) having a mother with atopic or nonatopic asthma was associated with an infant's ari with hrv or rsv. we assessed the association of measures of maternal asthma and virus type in the overall ari group (combined urti and lrti) and next among the lrti subgroup. we applied a logistic regression model with variable hrv( ) or rsv( ) as a binary outcome variable and maternal asthma as defined above, as our main factor along with covariates. because of our limited regression power determined by the hrv( ) group, we used propensity score adjustment to prevent overfitting because the propensity score analysis adjusts for many confounding factors simultaneously while preserving analytical power. variables in the propensity score model included a priori selected variables: maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. in our next set of analyses we examined whether having a mother with asthma (first defined by maternal self-report and then using the atopic asthma and nonatopic asthma classifications) was associated with increased severity of the infant's hrv-or rsv-induced ari. we conducted a separate analysis for infants with sole hrv-or rsv-induced ari and in the subgroups with lrtis. we used the proportional odds model to evaluate the association of maternal asthma with infant hrvor rsv severity using the bronchiolitis severity score. for infants with rsv, a priori selected variables in the multivariable j allergy clin immunol volume , number models included maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. analyses among hrv-induced lrtis were limited by small sample size for a full covariates model, and therefore we performed a propensity score-adjusted model that included infant's sex, age, and birth weight. we performed an interaction analysis to assess whether the association between maternal asthma and severity of the infant's ari was different depending on the infant's hrv or rsv status. the proportional odds model was used with a cross-product term of maternal history of asthma and virus positivity (rsv and hrv ) and adjustment for covariates. statistical analyses were performed with r version . . software. a total of infants with sole infection with either hrv or rsv were included in this study. table i highlights the demographics and characteristics of the cohort by infant ari cause, as determined by means of pcr: positive for hrv only (n ) or rsv only (n ). compared with infants with rsv, infants with hrv were more likely to be older ( vs weeks, p < . ), have medicaid insurance ( % vs %, p . ), have mothers who were african american ( % vs %, p < . ), have a urti versus an lrti ( % vs %, p < . ), and have a lower median bronchiolitis severity score ( vs . , p < . ). among infants with aris and the lrti subgroup, we determined the association of self-reported maternal asthma and infant hrv-or rsv-induced aris (both urtis and lrtis). infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . , table i ). in adjusted analyses, compared with infants whose mothers did not have asthma, infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced ari (adjusted odds ratio, . ; % ci, . - . ). in analyses limited to infants with lrtis, infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . ). in adjusted analyses infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced lrti (adjusted odds ratio, . ; % ci, . - . ). maternal allergen sensitization determined based on skin prick test results or allergen-specific ige levels was available for % of the mothers (n ) and was used to identify maternal atopic asthma. a larger percentage of infants with hrv-induced aris had a mother with atopic asthma than infants with rsv-induced aris ( % vs %, respectively), whereas the percentages of infants with a mother with nonatopic asthma were similar ( % vs %, respectively). having a mother with atopic asthma was associated with increased odds of an infant having hrv versus rsv infection when compared with having a mother without asthma (propensity score-adjusted odds ratio, . ; % ci, . - . ). having a mother with nonatopic asthma compared with no asthma was not associated with viral cause (adjusted odds ratio, . ; % ci, . - . ). a larger percentage of infants with hrv-induced lrtis had a mother with atopic asthma than infants with rsv-induced lrtis ( / [ %] vs / [ %]); however, the percentages of infants with a mother with nonatopic asthma were similar ( / [ %] vs / [ %]). in multivariable propensity score-adjusted analyses there was a statistically significant association with having a mother with atopic asthma compared with having a mother without asthma for infants with hrv-induced lrtis compared with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ). there was not a statistically significant difference between infants having a mother with nonatopic asthma compared with no asthma for infants with hrv-induced lrtis compared with those with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ) . in separate analyses for infants with either hrv or rsv infection, we examined whether the severity of the infant's respiratory tract infection was associated with having a mother with self-reported asthma. in infants with hrv, the association between self-reported maternal asthma and infant infection severity was not statistically significant in the ari group (both urtis and lrtis; adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). in infants with rsv, the association between self-reported maternal asthma and infant infection severity was not significant in the total ari group (adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). next, in separate analyses for infants with either hrv or rsv infections, we examined whether the severity of the infant's respiratory tract infection was associated with whether the infant's mother had atopic or nonatopic asthma. among infants with hrv-induced ari, having a mother with atopic asthma was associated with increased ari severity (fig ) , and in adjusted analyses there was a more than -fold increased relative odds of having more severe illness compared with infants whose mothers did not have asthma (adjusted odds ratio, . ; % ci, . - . ; table ii ). the interaction analysis investigating a differential effect of maternal atopic asthma by whether the infant had hrvor rsvon infection severity was also statistically significant (p . ). this relationship was not seen when limited to the subgroup of infants with hrv-induced lrtis; however, the number of infants with hrv-induced lrtis was very small (n ) and could be adjusted through propensity scores for only the infant's age, sex, and birth weight (table ii) . there was not an association between maternal atopic asthma and infection severity in infants with rsv-induced aris or the rsv-induced lrti subgroup (table ii) . rsv and hrv are the most common viruses associated with infant aris, and rsv-and hrv-induced lrtis are a leading cause of respiratory morbidity and hospitalizations in the first year of life. - viral lrtis during infancy and early childhood are also well established to be associated with an increased risk of asthma later in childhood. [ ] [ ] [ ] , because of the known differential risk of early childhood asthma after rsv-and hrv-induced infant infections, we were interested in studying whether a familial predisposition to asthma and allergies was associated with the viral cause of the infant's ari and the severity of the ari. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] several small studies have not found an association between familial predisposition and bronchiolitis; however, in our prior large, population-based cohort investigation of infants, we found that having a mother with asthma was associated with increased risk and severity of bronchiolitis during infancy. in the current investigation we sought to expand on previous findings by addressing the research questions of whether infants with hrvinduced aris or lrti subgroups are more likely to have a familial predisposition to asthma than those with rsv-induced infections and whether a familial predisposition to asthma is associated with more severe infant hrv-or rsv-induced aris. in our previous work we found that although, as a group, infants with a history of bronchiolitis had an increased risk of early childhood asthma, infants who had bronchiolitis during hrvpredominant months had a % increased risk of early childhood asthma compared with infants who had bronchiolitis during rsvpredominant months. furthermore, in the coast birth cohort, in which all children have a familial predisposition to asthma, , investigators found that hrv-induced wheezing illnesses in the first years of life were associated with a . -fold relative odds of asthma at years compared with a . -fold increase among children with rsv. we were able to assess whether maternal asthma and atopy were associated with the viral cause and the virus-specific severity of the infant's infection leading to an unscheduled health care visit because this cohort consisted of infants with and without a familial predisposition to asthma, and we used objective measures of maternal atopy and molecular techniques to determine the viral cause of the infant's respiratory tract infection. we found that infants with a mother with atopic asthma had an increased relative odds of having hrv-induced aris than rsv-induced aris compared with infants whose mothers did not have asthma. it is notable that although the prevalence of self-reported maternal asthma is higher in children with hrv ( %), the % prevalence of maternal asthma among infants with rsv-induced aris is higher than the asthma prevalence in the adult population in the united states. these study findings suggest that for infants with hrv-induced aris in particular, a familial predisposition to asthma might partly explain the increased risk of asthma seen later in life and/or that a familial predisposition to asthma predisposes infants to clinically significant hrvinduced aris during infancy, a viral infection that, unlike rsv, can often be isolated from young children without clinically obvious respiratory symptoms. we also found that infants with hrv who had a mother with atopic asthma had more than a -fold increased relative odds of having more severe illness than infants whose mothers did not have asthma. among those with rsv-induced aris, there was no relationship between maternal asthma and atopy and ari severity. the relationship between maternal atopic asthma and the infant's hrv infection severity is intriguing given the described altered host response to hrv, as has been found in vitro and among subjects with asthma. , [ ] [ ] [ ] [ ] [ ] these data support the notion of differential susceptibility to hrv among patients with atopic asthma. continued follow-up of these children until the age of years will further delineate whether it is the subset of infants with hrv-induced aris and a familial atopic predisposition who will have asthma and allergic diseases later in childhood or have more severe asthma or recurrent exacerbations. ultimately, this might help us to understand whether there is an altered host response or increased susceptibility to hrv among patients with atopic asthma. there are several limitations of this work. this study included a convenience sample of mother-infant dyads in which all infants presented for an unscheduled health care visit and not a cohort followed from birth. however, the study included participants recruited during viral seasons over years, which should serve to strengthen the generalizability of the findings. a single episode of rsv-or rhinovirus-induced ari was captured. it is likely that children had additional viral infections during infancy. the primary focus of this study was the association of a maternal atopic predisposition and the infant's ari severity, and therefore we were not able to investigate the outcome of bronchiolitis incidence as in our larger, population-based retrospective cohort of mother-infant dyads. in addition, maternal asthma was determined based on self-report and not based on objective criteria, such as airway reversibility testing. however, we used a validated instrument and self-reported asthma in young adults in whom there is little overlap with other diseases, and thus there is high specificity. furthermore, this study included objective measures of atopy. lastly, we cannot completely rule out the possible influence of unknown or unmeasured potential confounding factors, as with all observational studies. in summary, infant hrv infections requiring clinical care were more strongly associated with having a mother with atopic asthma than infant rsv infections. in addition, infants with hrv-induced aris who had a mother with atopic asthma had more than a -fold increased relative odds of having a more severe ari compared with that seen in infants with hrv whose mothers did not have asthma. this relationship was not seen in infants with rsv infections. these findings suggest that there is likely an underlying genetic basis for the risk of and response to respiratory tract infections during infancy and that the mechanisms underlying the increased asthma risk after hrv-and rsv-induced bronchiolitis might be different. future longitudinal investigations successful at preventing or modifying the host response to infant viral infections will provide insight into the relationship of infant viral infections and early childhood asthma, as will investigations that assess the atopic host and nonatopic host response to select respiratory pathogens. key messages d infants whose mothers had atopic asthma had increased relative odds of having a rhinovirus-induced ari than rsv infection compared with the odds in infants whose mothers did not have asthma. d in infants with rhinovirus, having a mother with atopic asthma was associated with -fold increased relative odds of more severe illness, a relationship not seen in infants with rsv. d for infants with rhinovirus-induced aris, a familial atopic predisposition might partly explain the subsequent increased risk of asthma and differential susceptibility to rhinovirus among asthmatic patients. bronchiolitis-associated hospitalizations among us children risk factors for respiratory syncytial virus-associated lower respiratory illnesses in the first year of life the increasing burden and risk factors for bronchiolitis-related medical visits in infants enrolled in a state healthcare insurance plan earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age the severity-dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma rhinovirus illnesses during infancy predict subsequent childhood wheezing the impact of respiratory viral infection on wheezing illnesses and asthma exacerbations atopy does not predispose to rsv bronchiolitis or postbronchiolitic wheezing wheezing, asthma, and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy asthma and wheezing in the first six years of life. the group health medical associates respiratory syncytial virus in early life and risk of wheeze and allergy by age years viral infections, atopy, and asthma: is there a causal relationship? the childhood origins of asthma (coast) study childhood infections, the developing immune system, and the origins of asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease respiratory status and allergy nine to years after acute bronchiolitis family history of atopy and clinical course of rsv infection in ambulatory and hospitalized infants maternal asthma and maternal smoking are associated with increased risk of bronchiolitis during infancy host and viral factors associated with severity of human rhinovirusassociated infant respiratory tract illness wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the tennessee children's respiratory initiative: objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases relationship of maternal vitamin d level with maternal and infant respiratory disease international study of asthma and allergies in childhood (isaac): rationale and methods in vitro assays for the diagnosis of ige-mediated disorders in vitro diagnosis of atopic allergy in children. a comparison between total ige, conventional rast and a new multi rast (phadiatop) phadiatop compared to skin-prick test as a tool for diagnosing atopy in epidemiological studies in schoolchildren dexamethasone and salbutamol in the treatment of acute wheezing in infants prednisolone plus albuterol versus albuterol alone in mild to moderate bronchiolitis the central role of the propensity score in observational studies for causal effects r: a language and environment for statistical computing respiratory syncytial virus infection and recurrent wheeze/asthma in children under five years: an epidemiological survey community study of role of viral infections in exacerbations of asthma in - year old children the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the september epidemic of asthma exacerbations in children: a search for etiology rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing allergic sensitization is associated with rhinovirus-, but not other virus-, induced wheezing in children season of infant bronchiolitis and estimates of subsequent risk and burden of early childhood asthma asthma prevalence, health care use, and mortality: united states asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma rhinoviruses in the pathogenesis of asthma: the bronchial epithelium as a major disease target rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo validation of questionnaire and bronchial hyperresponsiveness against respiratory physician assessment in the diagnosis of asthma we conducted an analysis of mother-infant dyads enrolled in the tcri to investigate the association of a familial atopic predisposition with the viral key: cord- -nemh wdo authors: sims, jonathan t.; krishnan, venkatesh; chang, ching-yun; engle, sarah m.; casalini, giacomo; rodgers, george h.; bivi, nicoletta; nickoloff, brian j.; konrad, robert j.; de bono, stephanie; higgs, richard e.; benschop, robert j.; ottaviani, silvia; cardoso, anabela; nirula, ajay; corbellino, mario; stebbing, justin title: characterization of the cytokine storm reflects hyperinflammatory endothelial dysfunction in covid- date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: nemh wdo background physicians treating covid- patients increasingly believe that the hyperinflammatory acute stage of covid- results in a cytokine storm. the circulating biomarkers seen across the spectrum of covid- have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. objective to identify and characterize the host inflammatory response to sars-cov- infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. methods blood samples from adult patients hospitalized with covid- were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of markers. results results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within covid- patients, and strong biomarker association with patient response as measured by ordinal scale. as patients progress, we observe statistically significant dysregulation of ifnγ, il- ra, il- , il- , il- , mcp- , - , - , cxcl , cxcl , cxcl , enrage and parp- . furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. conclusion these wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. they also provide insights into mechanisms of sars-cov- pathogenesis and the host response. background: physicians treating covid- patients increasingly believe that the hyperinflammatory acute stage of covid- results in a cytokine storm. the circulating biomarkers seen across the spectrum of covid- have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. objective: to identify and characterize the host inflammatory response to sars-cov- infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. methods: blood samples from adult patients hospitalized with covid- were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age-and sex- matched healthy controls to provide insights into differential regulation of markers. results: results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within covid- patients, and strong biomarker association with patient response as measured by ordinal scale. as patients progress, we observe statistically significant dysregulation of ifnγ, il- ra, il- , il- , il- , mcp- , - , - , cxcl , cxcl , cxcl , enrage and parp- . furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine the covid- pandemic created an overwhelming need to define host-derived molecular mediators of disease severity evident in hospitalized patients. one approach to dissect protective and pathological immune responses to covid- infection is to measure plasma biomarkers correlated to various stages of covid- and determine which pro-inflammatory mediators are modulated in response to therapies improving patient outcomes( ). we utilized multiplex methodology from olink proteomics to assess analytes, spanning a broad range of cytokines and chemokines involved in inflammation (inf i) and cardiovascular- linked processes (cvd ii), and, based on the inflammatory responses described in sars-cov- -infected lung epithelium, we also measured il- levels using a highly sensitive and specific assay as previously described( , , ). analysis of the differential protein regulation patterns within severity classes of covid- cases compared to hc revealed strong dysregulation of ifnγ, il- , il- , mcp- , cxcl , cxcl , cxcl , enrage, parp- , and il- ra (fig. a , b-bottom, c). these changes were significant and ranged from increases of -fold for ifnγ, -fold for il- , -fold for mcp- , to -fold for cxcl , to more than fold for mcp- and - , our analysis is consistent with a more prominent increase in ifnγ and il- levels in covid- patients as seen in the moderate/severe and critical patients who are more likely to suffer from adult respiratory distress syndrome( ). while interferon signaling is certainly implicated with the j o u r n a l p r e -p r o o f dramatic increase in ifnγ, additional insights into type i interferon family members was out of scope of this manuscript due to the lack of reliable ultrasensitive assays for these analytes. furthermore, we observed ptx , gal , and cd a upregulation in all patients regardless of severity (fig. a clinical inflammatory markers, such as ldh, crp, and ferritin, but we specifically observed that mcp- , ctsl , and ptx were more meaningfully correlated to changes in the ordinal scale than these widely used readouts (supplementary table ). we observed several analytes inversely correlated to the same magnitude (cor<- . ) with both ldh, crp, and ferritin and the ordinal scale: mmp , gif, gdf- , boc, scf, and cd (fig. a, b- in addition to these identified immunological changes, there is a growing consensus on the changes in parp- were most pronounced in the severe, and critical patients and this marker have been implicated in cardiovascular disease( ). it is also well known that parp- activity correlates with viral infection and, conceivably, this marker reflects the added tissue viral burden in such patients; however, in this limited set, parp- levels did not correlate with change in the ordinal scale or viral load (fig. b) . furthermore, it has been reported that gdf- (bmp- ) plays a protective role against inflammation-mediated damage on endothelial and cardiac tissues. likewise, we observed downregulation of gdf- in the plasma of covid- patients (fig. b) . in gdf- -/mice subjected to transverse aortic constriction, loss of gdf- activity promoted cardiac fibrosis and impairs heart function( ). therefore, reductions in gdf- potentially may result in endothelial and cardiac damage in an inflammatory condition, and therapeutic restoration of factors such as gdf- , such as observed with baricitinib-treated patients, can help stabilize cardiac function in these at-risk patients (fig. d) . changes in cytokines and other biomarkers in a series of patients treated with baricitinib offers insights in the pleiotropic nature of this jak /jak inhibitor. the biomarkers that were most robustly decreased longitudinally in the baricitinib-treated cohort compared to baseline include il- , mcp- , il- , cxcl , and ifnγ ( fig. -middle; fig. the authors would like to thank healthcare workers across the world for their response to covid- . we thank jochen schmitz for scientific input and support. we thank cynthia abbott and diane stothard for manuscript preparation support. moderate/severe (middle), and critical (right) patients compared to hc. the y-axis is the -log of the p-value with higher numbers reflecting greater significance and the x-axis represents the log fold change, wherein color designation of blue or red represents decreased or increased presence of these markers relative to hc, respectively. covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal world health organization mechanism of baricitinib supports artificial intelligence-predicted testing in covid- patients clinical relevance of serum il- levels in psoriasis and atopic dermatitis using a sensitive and specific novel immunoassay journal of machine learning research homogenous -plex pea immunoassay exhibiting high sensitivity, specificity, and excellent scalability the landscape of lung bronchoalveolar immune cells in covid- revealed by single-cell rna sequencing exuberant elevation of ip- mcp- and il- ra during sars-cov- infection is associated with disease severity and fatal outcome covid- : consider cytokine storm syndromes and immunosuppression immune cell promotion of metastasis fms-like tyrosine kinase receptor ligand (flt l)-dependent development and function of conventional and plasmacytoid dendritic cells. proceedings of the national academy of sciences ccr and cxcr agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type ii a systems approach to inflammation identifies therapeutic targets in sars- cov- infection remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial comparison of an ordinal endpoint to time-to-event, longitudinal, and binary endpoints for use in evaluating treatments for severe influenza requiring hospitalization anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia thromboembolic risk and anticoagulant therapy in covid- patients: emerging evidence and call for action soluble cd ligand, interleukin (il)- , and hemostatic parameters in metabolic syndrome patients with and without overt ischemic heart disease mapping of loci for plasma protein biomarkers in cardiovascular disease role of poly(adp-ribose) polymerase (parp- ) in cardiovascular diseases: the therapeutic potential of parp inhibitors protein reduces cardiac fibrosis and improves cardiac function in heart failure ptx in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation circulating levels of the long pentraxin ptx correlate with severity of infection in critically ill patients a single-cell atlas of the peripheral immune response in patients with severe covid- key: cord- -qhl fm authors: gelardi, matteo; cassano, michele; ciprandi, giorgio title: the clinical relevance of the clinical cytological grading in patients with chronic rhinosinusitis with nasal polyps date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: qhl fm nan the clinical relevance of the clinical cytological grading in patients with chronic rhinosinusitis with nasal polyps to the editor: smell dysfunction is a common symptom and has a diagnostic value in the workup of patients with chronic rhinosinusitis with nasal polyps (crswnp) as recently outlined by mullol et al. notably, loss of smell has been proposed as one of the main criteria for an indication of and response to the new biological treatments. of note, it has been very recently reported that a significant part ( %- %) of the patients with coronavirus disease (covid- ) experienced the loss of smell; anosmia can be the first presenting symptom and so the european rhinologic society advised that patients with sudden-onset loss of smell should be considered to be covid- positive (www.europeanrhino logicsociety.org). crswnp is characterized by a type inflammation and is frequently associated with some comorbidity, including asthma, aspirin sensitivity, and allergy. type inflammation leads to eosinophils and mast cells recruitment and activation in upper airways. therefore, to document the presence of inflammatory cells in the nose by nasal cytology is a simple and fruitful way to assess the severity of disease and evaluate the response to treatments. nasal cytology is a wellstandardized method that may be applied to all nasal inflammatory disorders. therefore, nasal cytology may be performed as a useful step in the workup of patients with crswnp. as a consequence, a clinical cytological grading (ccg), including the documentation of possible asthma, allergy, or aspirin sensitivity comorbidity, and the presence of eosinophils and/or mast cells in the nose, has been proposed as a precision medicine-based approach in the management of patients with crswnp. in particular, ccg grades were defined on the basis of the score: low ( - ), medium ( - ), and high (> _ ). a recent study provided evidence that a ccg value of more than was a reliable (area under the curve, . , and adjusted odds ratio, . ) cutoff to identify patients with crswnp with olfactory dysfunction. smell loss is a clinical marker of crswnp severity and is associated with type inflammation: both conditions indicate a biological approach. the crswnp overall prevalence has been approximately estimated to be % to % of the general population and approximately % of these patients also have asthma. on the basis of these epidemiological considerations, a large group of italian outpatients with crswnp ( ; males, mean age, . years) was recruited in a real-world study to define the distribution of the ccg grades. the preliminary results showed that ( . %) outpatients had low-grade ccg, ( . %) medium, and ( . %) high (fig ) . high-grade ccg was frequently characterized by mixed cytological phenotype and severe progress. patients with crswnp deserve adequate management and optimal identification of the besttailored therapy; in this regard, ccg could be a fruitful tool. in particular, considering the estimated prevalence of crswnp and the current findings, it could be reasonably expected that at least million europeans have crswnp with high-grade ccg. therefore, intercepting the most severe patients and launching an early and tailored treatment, including the new biologics, could positively modify the natural history and improve the quality of life. the sense of smell in chronic rhinosinusitis epos : european position paper on rhinosinusitis and nasal polyps euforea consensus on biologics for crswnp with or without asthma role of nasal cytology standardization of the nasal cytology in the work-up of allergic rhinitis non-surgical management of chronic rhinosinusitis with nasal polyps based on clinical cytological grading: a precision medicine-based approach olfactory dysfunction in patients with chronic rhinosinusitis with nasal polyps is associated with clinical-cytological grading severity key: cord- -e q mck authors: holgate, stephen t. title: genetic and environmental interaction in allergy and asthma()() date: - - journal: j allergy clin immunol doi: . /s - ( ) - sha: doc_id: cord_uid: e q mck asthma is an inflammatory disorder of the airways involving coordinate up-regulation of t(h) -type cytokines encoded in a cluster on chromosome q( - ) on t cells and inflammatory cells. there is also a requirement for local airway susceptibility factors that, together with t(h) polarization, results in hyperresponsiveness, variable airflow obstruction, and, over time, remodeling of the airway wall. asthma has strong genetic and environmental components that interact both in the induction and subsequent expression of the disease phenotypes. multiple genes are involved and probably interact. whole genome screens are beginning to identify gene-rich regions of special relevance to asthma and atopy, although a novel disease-related gene has yet to be discovered from these. by contrast, there are a plethora of candidate genes whose function in relation to disease pathophysiologic mechanisms and response to treatment are known. two examples are polymorphisms involving il- receptors and the enzymes controlling cysteinyl leukotriene production. abnormal signaling between the epithelium, which is in contact with the environment, and the underlying (myo)fibroblasts and dendritic cells indicating reactivation of the epithelial mesenchymal trophic unit, which is involved in fetal lung development and branching, provide a basis for asthma that encapsulates both t(h) polarization and airway wall remodeling. (j allergy clin immunol ; : - .) asthma is a complex disorder involving a combination of genetic and environmental interactions that culminate in a specific type of inflammation involving mast cells, eosinophils, and macrophages and polarization of t cell-mediated immunity toward enhanced production of cytokines encoded in a cluster on the long arm of chromosome . these include il- , il- , il- , il- , and gm-csf, which orchestrate the isotype switching of b lymphocytes to produce ige, maintain the persistence of t-helper (t h ) lymphocytes with their enhanced capacity to secrete cytokines of the il- gene cluster and recruit and maintain the survival of mast cells, basophils, and eosinophils. in contrast to the many animal models of t h polar-ization in the lung, which tend to be self-limiting, asthma is a chronic disorder characterized by exacerbations and remissions and an underlying bronchial hyperresponsiveness (bhr) of the airways to a wide variety of environmental factors. in established disease a major part of the bhr can be separated from the inflammatory response and most likely represents structural changes to the airways with deposition of matrix proteins throughout the airway wall and "remodeling" of the formed elements including epithelial goblet cell metaplasia, smooth muscle hypertrophy, and an increase in microvessels and nerves. whether these features occur as a consequence of or in parallel with the inflammatory response is not known, but increasingly in asthma it is becoming appreciated that many of these structural elements are themselves altered to produce cytokines, growth factors, and mediators that may contribute to the sustenance of the inflammatory response. although for therapeutic reasons it has become convenient to consider asthma as a single disease entity, this clearly is not the case, with many variants occurring. from a clinical standpoint, a minimal subdivision includes atopic asthma, cough variant asthma, brittle asthma, intrinsic asthma, aspirin-intolerant asthma (aia), and occupational non-ige-dependent asthma. when disease severity and responsiveness to treatment are added to this categorization, the number of variations becomes very large. if these manifestations are controlled by specific genes and environmental interactions, then the true complexity of this disease becomes appreciated. atopy, the propensity to generate ige against common environmental allergens, is the strongest single risk factor that has so far been identified for asthma, increasing the risk in those affected by -to -fold. although atopy and asthma interact, they are not interchangeable. thus, although a high proportion of children and young adults are atopic, even in countries with a high prevalence of atopy (eg, united kingdom, australia, and new zealand) full-blown chronic asthma will develop in only in atopic patients. moreover, in adults, especially at the severe end of the asthma spectrum, atopy diminishes as a risk factor. the phenotype of other chronic inflammatory diseases, such as crohn's disease or psoriasis, presents little diagnostic uncertainty but, in the case of asthma, there is a lack of diagnostic precision. this has led to the use of intermediate phenotypes reflecting asthma and atopy, such as bhr, serum total and allergen-specific ige, skin prick test (spt) positivity, and circulating eosinophil counts. although these provide quantitative measures, bhr, for example, can be assessed in many different ways (eg, histamine or methacholine provocation, exercise or cold air challenge, sulfur dioxide challenge, peak expiratory flow variability, etc), each measure describing different airway characteristics. in addition, there are numerous ways in which an individual can be designated atopic. the danger in using intermediate phenotypes is the assumption that their genetic basis will be the same as that of the disease state. several large epidemiologic studies have established that the presence of asthma corresponds to high serum levels of total ige. , however, when analyzed on a family basis, a tendency to be a high ige producer proved to be only one factor related to the inheritance of asthma susceptibility and, in itself, had a limited ability to predict asthma inheritance. an alternative approach has been to pool multiple measures of asthma (eg, respiratory questionnaire, spt, specific and total ige) to generate summary scores using, for example, principal component regression analysis. this method can also be used to construct a quantitative trait in the form of an asthma score in which the influence of atopy is removed (fig ) . although it has long been known that asthma and related atopic diseases cluster in families, the genetic basis for this has eluded definition. in families a striking association has been shown between asthma in parent and offspring, hay fever in parent and offspring, and eczema in parent and offspring, suggesting that, in addition to genetic factors determining allergen sensitization, there are also important genetic factors that determine the end-organ in which this was expressed, a conclusion drawn from further large epidemiologic studies. , one method that can be used to great effect in complex disorders to determine the relative contribution of genes and the environment is to study the concordance of a trait in monozygotic (mz) and dizygotic (dz) twins, in which the former have identical genotypes and the latter share on average only half their genes. thus a disease that has a strong genetic component will show a higher rate of concordance in mz than in dz twins. in a large survey of twin pairs, concordance rates for asthma, eczema, and hay fever were all substantially higher for mz than for dz twins. this observation has also been shown for bhr, spt responses, and serum total and specific ige. , in a study deliberately designed to disaggregate genetic from environmental effects, both allergic disease and partial phenotypes were compared in mz and dz twins reared together and apart. whether together or separated, mz twins showed a greater concordance than dz twins, indicative of a strong genetic contribution. this has been further strengthened by the findings in a recent , danish twin pairs with use of additive and genetic and nonshared environmental modeling that % of asthma susceptibility was genetic and a substantial part of the variation liability of asthma was the result of environmental factors. segregation analysis is a method for estimating the pattern of inheritance of a disease by observing how it is distributed through family pedigrees. several different modes of inheritance have been proposed for elevated serum total ige, including autosomal recessive, autosomal dominant, and polygenic inheritance. [ ] [ ] [ ] applica- tion of more sophisticated segregation models to asthma and its partial phenotypes have shown that it is unlikely that any single gene predominates, although for physician-diagnosed asthma in us hispanics a major autosomal codominant gene has been proposed. in contrast to single-gene disorders, which are rare, subject to severe mutations and deletions, and exert a large phenotypic effect often independent of environmental factors, complex genetic traits such as asthma and related allergic disorders are common and result from mild mutations in multiple genes each of which has a small effect on the phenotype and requires subtle genegene (epistasis) or gene-environmental interactions for optimal expression. two fundamental approaches are being used to discover susceptibility genes in asthma and atopy: linkage analysis with functional cloning and association analysis for mutations of "candidate" genes thought to be involved in disease pathogenesis. linkage analysis uses family data to follow the transmission of genetic information between or across generations to enable genes to be identified by their position on the genetic map; no prior knowledge of disease pathophysiologic features is required. linkage studies require families usually enriched with the disease, which must be accurately phenotyped. depending on the approach, families can be nuclear families (ie, children and their parents), extended pedigrees, or inbred populations. linkage analysis requires the saturation of the entire human genome at regular intervals with microsatellite markers comprising variable nucleotide tandem repeats whose precise position on the genetic map are known. whole-genome screens use to dinucleotide, trinucleotide, or tetranucleotide markers spaced to cm across the genome. the proximity of a marker to a disease-related gene is estimated by measuring the number of recombination events between them; the closer loci are, the less chance they will be separated at meiosis (fig ) . the significance of linkage is measured by the "lod score" (log of the odds ratio of the likelihood of a hypothesis of linkage to the likelihood of a hypothesis of no linkage). for example, for a lod score of , there is a % chance of a false-positive linkage. a lod score of . is suggestive of linkage, . significant linkage, and . highly significant linkage. this type of parametric linkage analysis requires an a priori knowledge of the mode of inheritance of the disease or its partial phenotype, which, for asthma and allied allergic disorders, is not known. under such circumstances nonparametric linkage analysis or allele sharing methods are more reliable but less powerful. affected sib-pair analysis is most commonly used, which involves studying affected relatives in a pedigree to see how often a particular copy of a chromosomal region is shared identical by descent (ie, inherited from a common ancestor within a pedigree). for example, siblings can share or copies of any locus. if the disease or partial phenotype is linked to a certain marker, then the affected siblings will inherit identical alleles of the marker more frequently than expected by chance alone. when assessed by a chi-square test a lod score of > . is considered significant. association studies are case control studies based on a comparison of unrelated affected and unaffected individuals from a population. although association can be performed for any random dna polymorphisms, they are most meaningful when applied to functionally significant variations in genes having a clear biologic relationship to the trait. positive association may occur under circumstances: ( ) the allele is contributing to the phenotype, ( ) population (racial or ethnic) admixture, and ( ) when alleles at loci are close together (linkage disequilibrium). an alternative type of case control is the transmission disequilibrium test, which avoids the confounding effects of an incorrectly matched control population and uses a trio design of parents and affected sibling. a number of whole-genome scans for asthma have now been completed (table i) . [ ] [ ] [ ] [ ] [ ] although there is an emerging consensus for some chromosomal regions, in different populations there exist many where linkage has not been reproduced. although this may represent true heterogeneity on the basis of racial differences, it is of concern that all the whole genome screens have been conducted on relatively small numbers of families ( - ), whereas it is predicted on statistical grounds that in excess of sibling pairs is needed to provide absolute confidence for linkage to a specific marker. one possible way around this is to pool data from different studies either by meta-analysis or through establishing a voluntary network in which separate groups pool their results in a single analysis. even when a chromosomal region has been narrowed down to - cm, the task of identifying which gene (or genes) within this stretch of dna is contributing to the disease phenotype is daunting. this requires the construction of physical maps of the region with use of overlapping genomic dna clones and techniques such as exon trapping and complementary dna selection to identify genes in a given section of dna. with the availability of integrated genetic and physical maps it is possible to obtain an inventory of genes mapping to the specified genetic interval and scan this for genes of known function (positional-candidate approach). once a specific gene and its mutations have been identified, their expression can be assessed in diseased tissue by a combination of pcr and in situ hybridization and functional studies undertaken by overexpressing and underexpressing the gene in appropriate human cell lines and transgenic mice. at the time of writing, no candidate genes for asthma have yet been reported that have emerged from whole genome screens. a large number of mutations have been described for candidate genes that influence functions relevant to known disease mechanisms in asthma and allied disorders. in all cases, altering the level of expression or function of a specific protein accounts for only a very small component of the disease phenotype. table ii displays candidate genes that have found widest acceptance in different populations. in many cases it is highly likely that several mutations involving a number of components of a particular metabolic or physiologic pathway are needed to fully manifest the effect of a candidate gene. for example, for il- a polymorphism at - involving a c→t substitution in the promoter region on chromosome q results in increased transcription of il- and therefore increased responsiveness to il- (eg, by enhanced ige production). clearly, there may be other functionally active polymorphisms influencing il- secretion. moreover, at least common polymorphisms have also been described in the coding region of the il- receptor gene on chromosome p . , of which lead to amino acid changes of the gene product (fig ) . ) decrease stat activation, whereas others appear to have no effect on il- signalling. the role of glutamine (gln) arginine (arg) is not clear. , , when or more il- r polymorphisms occur together (eg, ser prol and gln arg, stat phosphorylation is reduced and irs / phosphorylation is increased. if the level of il- secretion is also increased (eg, in the - promoter polymorphism), then any genetic effects mediated through the il- r or il- receptor are likely to be magnified. another example of gene-gene interaction may occur in the leukotriene pathway. the cysteinyl leukotrienes ltc , ltd , and lte are critical mediators of airway narrowing, microvascular leakage, mucus secretion, and eosinophilia in bronchial asthma. , the terminal enzyme for cysteinyl leukotriene (cyst-lt) synthesis is ltc synthase encoded on chromosome q . we have shown that in patients with aspirin-intolerant asthma (aia) the expression of this enzyme in mast cells and eosinophils is increased -fold in parallel with enhanced cyst-lt production. this might explain why such patients find particular benefit from being treated with cyst-lt receptor antagonists (ltras) such as montelukast. an a→c polymorphism at the - position of the ltc synthase promoter has been shown to be strongly associated with aia (odds ratio . ). this base substitution creates an extra activator protein- transcription factor binding site, leading to increased enzyme transcription. we have also shown that the a/a or a/c allele is also found more commonly in patients with severe asthma and is accompanied by a greater ability to produce ltc on ex vivo activation of the peripheral blood eosinophils and a tendency toward enhanced responsiveness to the antileukotriene drug zafirlukast. -lipoxygenase ( -lo) is the first committed enzyme in the biosynthesis of leukotrienes. a series of naturally occurring mutations have been discovered within the glucocorticoid-rich transcription factor binding region in the promoter of the -lo gene. these involve deletion of , deletion of , or addition of zinc finger (sp /egr ) binding sites. when transfected, the mutant alleles result in reduced sp /egr binding, and reporter gene transcription is reduced by % and % compared with the wild-type sequences. in patients with asthma, only those possessing mutant -lo alleles were relatively resistant to treatment with the -lo inhibitor abt- ; the mean fev improved by ~ % compared with ~ % for the wild-type and heterozygotes. thus, if there were mutations both of the cysteinyl lt synthase and -lo in favor of greater ltc production, then a subtype of "leukotriene-dependent" asthma can be envisaged. with the recent cloning and expression of the cyst-lt receptor, variation in expression, ligand binding, or transduction signaling may further the appearance of leukotriene dependence. such mechanisms may help explain the responder-nonresponder phenomenon that is emerging in clinical studies of asthma involving ltras. , although asthma and related allergic disorders are closely linked to atopy, there are other important gene-environmental interactions that are of central importance in the clinical expression of disease. in the case of asthma, exposure to oxidant air pollutants (eg, ozone, no x , particulates, tobacco smoke) has been linked to worsening disease. respiratory virus infections, especially those caused by rhinoviruses and coronaviruses, account for the majority of asthma exacerbations in children and adults. , because both these environmental factors operate by increasing the activation of proinflammatory transcription factors in epithelial and inflammatory cells (eg, nuclear factor-κb), genetic factors regulating this cascade are likely to be of considerable importance in determining susceptibility to exacerbations of continuing disease. the complex cellular events that are linked to altered stress responses at the mucosal surface on exposure to pollutants or respiratory viruses are only just being revealed, but key among them is the ability of the epithelium to protect itself from such insults by antioxidant pathways. , these include glutathione peroxidase, glutathione synthase, and xanthine oxidase. in susceptible mice genetic linkage has shown that ozone-induced lung inflammation is directed by genes encoded on chromosome , including the strong candidate tnf-α, a pleiotropic cytokine generated during oxidant-induced cell injury. there are numerous studies linking asthma with a reduced antioxidant status. , because antioxidants may also be provided in the diet (eg, vitamins a, e, and c), asthmatic subjects defective in endogenous antioxidant-generating capacity may be especially susceptible to dietary deficiencies. [ ] [ ] [ ] a similar case can be made for another dietary link to asthma. both in animals and in in vitro studies in humans omega- polyunsaturated fatty acid (pufa)-enriched diets has been shown to reduce allergen-induced inflammatory responses and cyst-lt generation, respectively. , on this basis, it has been suggested that supplementation of the diet with omega- fatty acids in the form of fish oil might protect asthmatic patients by reducing the capacity to generate cyst-lts. unfortunately, clinical trials with diet supplementation have been disappointing both with allergen provocation and in clinical asthma. [ ] [ ] [ ] however, broughton et al have recently shown that only those patients who have high urine secretions of lte responded favorably to omega- fatty pufa dietary supplementation, whereas in those who were low excretors the asthma deteriorated or was unchanged rather than improved. because urinary lte excretion is a measure of the activity of the -lo pathway, , it is possible that responders and nonresponders to this dietary intervention could be determined by prior genotyping. it has been suggested that reduced exposure to bacteria or their products during early infancy is a key factor in programming the immune response toward an allergic phenotype. thus children brought up in livestock farming communities have a substantially lower risk for development of allergies. it is suggested that exposure to bacteria or their products (eg, endotoxin) polarizes the immune response toward a protective t h phenotype by enhanced il- (or il- ) production by professional antigen-presenting cells such as dendritic cells, thereby providing a negative signal for t h polarization by enhanced ifn-γ production. the lower prevalence of allergic disease in formerly communist countries and the subsequent increase that has been observed since reunification has also been cited as supporting the "hygiene hypothesis" of allergic disease, as has the increased urban-to-rural gradient of allergic sensitization in african communities. however, what is notable about the changes in prevalence being observed in the former eastern bloc countries and in africa is that allergic sensitization has increased but asthma and bhr has not. this adds further evidence to the view that asthma and allergy are not equivalent or even linearly related and that local organ-derived factors are important. the recent description of a polymorphism of cd , the endotoxin receptor that is linked to the development of atopy in children, is of great interest and will encourage a search for other candidate gene polymorphisms linked to susceptibility to early life infection, including ifn-γ, natural resistance-associated macrophage protein- , natural resistance-associated macrophage protein- , and the mannose-binding protein. although genetic and environmental factors that operate to direct the immune response toward the t h phenotype are fundamental to understanding the origin and pathogenesis of allergic diseases, genetic and the environmental factors that direct this response to selected organs are of key importance. eczema is a strong predictor of asthma persistence and, in chronic severe disease, there is evidence for gastrointestinal epithelial permeability and inflammation. therefore the epithelium may play a particularly important role in directing the inflammatory and remodeling responses in chronic allergic disease. subepithelial deposition of interstitial (repair) collagens, impaired epithelial proliferative responses to damage, enhanced inflammatory and cytokine growth factor secretion, and evidence for altered subepithelial myofibroblast function in asthma suggest a fundamental abnormality in the epithelial-mesenchymal trophic unit that is involved in fetal lung development. , abnormal signaling between the epithelium and myofibroblast and to dendritic cells provides a basis for asthma that encapsulates both t h polarization and airway wall remodeling. whether this hypothesis can be sustained will require research at the gene by environment interface and its application to disease as it occurs in humans. cytokine networks in the pathogenesis of bronchial asthma: implications for therapy the inflammation-repair cycle in asthma: the pivotal role of the airway epithelium airways remodelling: the chronicity of asthma, quaeritur focus viii cytokine production by cell cultures from bronchial subepithelial myofibroblasts difficult asthma reversing the trend: reducing the prevalence of asthma proportion of asthma attributable to sensitisation to aeroallergens association of asthma with serum ige levels and skin-test reactivity to allergens relation between airway responsiveness and serum ige in children with asthma and in apparently normal children the relationship between parental and children's serum ige and asthma genetic analysis of atopy and asthma as quantitative traits and ordered polychotomies linkage of asthma to markers on chromosome in a sample of families using quantitative phenotype scores the familial incidence of allergic disease genetic risk for asthma, allergic rhinitis and atopic dermatitis allergy in twin pairs total and specific ige (rast) in atopic twins bronchial reactivity pattern in non asthmatic parents of asthmatics atopic disease and immunoglobulin e in twins reared apart and together genetic and environmental influence on asthma: a population-based study of , danish twin pairs a genetic study of immunoglobulin e genetics of total serum ige levels: a regressive model approach to segregation analysis detection of a recessive major gene for high ige levels acting independently of specific response to allergens analytic options for asthma genetics evidence for mendelian inheritance of serum ige levels in hispanic and non-hispanic white families genetic dissection of complex traits: guidelines for interpreting and reporting linkage results genetic dissection of complex traits transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (iddm) a genome-wide search for quantitative trait loci underlying asthma collaborative study on the genetics of asthma. a genome-wide search for asthma susceptibility loci in ethnically diverse populations genetic influences of chromosome q -q and q on specific ige responsiveness to common inhaled allergens among african american families: collaborative study on the genetics of asthma genome-wide search for asthma susceptibility loci in a founder population a genome-wide search for linkage to asthma: german asthma genetics group report on the working group on phenotype approaches workshop on genetics of asthma: methodological approaches promoter polymorphisms in the chromosome gene cluster in asthma and atopy common polymorphisms in the coding part of the il- -receptor gene ileu val variant of il- a upregulates ige synthesis and associates with atopic asthma the polymorphisms s p and q r in the interleukin- receptor * gene are associated ith atopy and influence signal transduction effect of allergy associated mutation in human il- ra (q r) on human il- induced signal transduction the association of atopy with a gain of function mutation in the * subunit of the interleukin receptor leukotriene antagonists and synthesis inhibitors: new directions in asthma therapy inflammatory mechanisms in asthma over expression of leukotriene c synthase in bronchial biopsies from patients with aspirin-intolerant asthma drug therapy: treatment of asthma with drugs modifying the leukotriene pathway leukotriene c synthase promoter polymorphism and risk of aspirin-induced asthma variant ltc synthase gene enhance in vitro ltc synthesis and clinical response to zafirlukast, international symposium: aspirin intolerance and related syndromes: a multidisciplinary approach naturally occurring mutations in the human -lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription egr- and sp interact functionally with the -lipoxygenase promoter and its naturally occurring mutants pharmacogenetic association between alox promoter genotype and the response to anti-asthma treatment characterisation of the human cysteinyl leukotriene cys lt receptor oral montelukast, inhaled beclomethasone and placebo for chronic asthma: a randomised control trial panel studies for investigating the acute health effects of air pollution community study of role of viral infections in exacerbations of asthma in school children in the community respiratory viruses and exacerbations of asthma in adults is nk-kb the sensor of oxidative stress? toxicological mechanisms underlying oxidant pollutant induced airway injury linkage analysis of susceptibility to ozone-induced lugn inflammation in inbred mice dietary antioxidant vitamin intake and lung function in the general population a prospective study of diet and adult-onset asthma effect of fresh fruit consumption on lung function and wheeze in children n- polyunsaturated fatty acids and cytokine production in health and disease effect of dietary intake of omega- and omega- fatty acids on severity of asthma in children effect of dietary fish oil supplementation on the antigen-induced late phase response in the skin dietary fish oil effects on seasonal hay fever and asthma in pollen sensitive subjects reduced asthma symptoms with n- fatty acid ingestion are related to -series leukotriene production microbial stimulation as an aetiologic factor in atopic disease prevalence of hay fever and allergic sensitisation in farmer's children and their peers living in the same rural community: scarpol team development of t-cell memory agonist inhalant allergens: risks for the future the epidemiology of childhood asthma the rising trends in asthma and allergic disease polymorphism in the -flanking region of the cd gene is associated with circulating soluble cd levels and with total serum immunoglobulin e the bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma the attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit key: cord- -nrrl kjc authors: rivas, magali noval; porritt, rebecca a.; cheng, mary hongying; bahar, ivet; arditi, moshe title: covid- associated multisystem inflammatory syndrome in children (mis-c): a novel disease that mimics toxic shock syndrome. the superantigen hypothesis date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: nrrl kjc nan as of mid-september, the novel severe acute respiratory syndrome coronavirus (sars-cov- ) has infected more than million people, resulting in approximately one million deaths worldwide, including over , deaths in the usa alone. fever, dry cough, breathing difficulties and gastrointestinal (gi) symptoms are typical features of coronavirus disease- . although % of infected people develop a mild disease, approximately % progress to severe covid- , which is associated with lung damage and breathing difficulties, and may lead to respiratory failure and death. exacerbation of the covid- immune response manifested by extensive cytokines release, called cytokine storm, may lead to multisystem inflammatory syndrome that is fatal in % of cases . children can also be infected with sars-cov- (< %), however the majority of confirmed pediatric cases have a less severe outcome and milder in late april, , reports from europe described the emergence of a new febrile pediatric entity that involved persistent fever, systemic hyperinflammation, multiorgan involvement with prominent and severe gi symptoms, and cardiogenic shock and hypotension, requiring pediatric icu care in the majority of cases. some who developed this syndrome, referred to as covid- -associated multisystem inflammatory which are all very rare in kd . in addition, the overall clinical picture of mis-c is similar in many respects to the late, severe covid- phase in adults, which is characterized by a cytokine storm, hyperinflammation, and multiorgan damage, and often includes severe myocarditis and acute kidney injury, and laboratory and clinical features of tss . a causal link between sars-cov- infection and mis-c has not yet been clearly established, however, many mis-c patients were reportedly exposed to someone known or suspected to have covid- . although only around a third of mis-c patients are positive for sars-cov- by pcr, a large majority are pcr-negative but positive serologically for sars-cov- antibodies and/or have a history of mild covid- infection or exposure several weeks before presentation. such timing suggests that mis-c is a post-infectious disease or immune or autoimmune disease. moreover, the virus may still be present in the gi tract of these patients, as they demonstrate very severe gi symptoms. through structure-based computational modeling, we discovered that the sars- cov- s gene encodes a high-affinity sag-like sequence motif near the s /s cleavage adult covid- patients . the pathologic mechanisms leading to these symptoms remain unknown. interestingly, sag-induced tss has been associated with long-term neuropsychologic deficits in adults, including cognitive decline , and we identified a homology between the sag motif of sars-cov- and neurotoxin-like sequences which are able to bind the tcr . notably, sars-cov- spike contain other neurotoxin-like motifs as well, including in particular the segment t -y which has been recently observed to be a highly cross-reactive epitope that triggers cd + t cell response . it will be interesting to determine whether these neurotoxin-like sequences in the sars- cov- s protein contribute to the neurological manifestations observed in children with why only a small fraction of sars-cov- infected children develop mis-c remains unclear. it is possible that a poor initial antibody response to the virus in a subset of children fails to produce neutralizing antibodies, leading to immune enhancement following sars-cov- re-exposure. alternatively, some human leukocyte antigen (hla) types may be more permissive, and respond more robustly to certain viral antigenic structures . indeed, among the reported cases from london , % of mis-c patients were of afro-caribbean descent, which suggests a possible genetic component for mis-c susceptibility. finally, our findings suggest that immunomodulatory therapeutic approaches used for tss, such as ivig and steroids, may also be effective for mis-c. indeed, most mis-c patients respond well to intravenous immunoglobulin (ivig; gm/kg) and aspirin, with or without steroids . given the structural similarities between seb and the sars- j o u r n a l p r e -p r o o f cov- s protein sag motif , it is possible that antibodies within ivig that neutralize seb cross-react with sars-cov- s, which may in part explain the beneficial response of mis-c cases to ivig. in addition, in the mouse model of tss, lethal seb superantigen challenge can be prevented by short peptide mimetics of the sag motif . therefore, it would be important to investigate the therapeutic potential of peptide mimetics of sars-cov- spike sag-like region in covid- -induced hyperinflammatory syndromes in future studies. further elucidation of the parameters affecting the interaction between sars-cov- s glycoprotein and immune cells will be necessary to design effective preventive and therapeutic interventions. we gratefully acknowledge support from nih awards p gm (to ib) and r ai (to ma). table the trinity of covid- : immunity, inflammation and intervention clinical characteristics of children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov- multisystem inflammatory syndrome related to covid- in previously healthy children and adolescents in new york city novel paediatric presentation of covid- with ards and cytokine storm syndrome without respiratory symptoms superantigenic character of an insert unique to sars-cov- spike supported by skewed tcr repertoire in patients with hyperinflammation binding of superantigen toxins into the cd homodimer interface is essential for induction of cytokine genes that mediate lethal shock the immunology of multisystem inflammatory syndrome in children with covid- engagement of mhc class ii molecules by staphylococcal superantigens activates src-type protein tyrosine kinases neurological associations of covid- persistent neuropsychological sequelae of toxic shock syndrome key: cord- - l k q authors: ijspeert, hanna; driessen, gertjan j.; moorhouse, michael j.; hartwig, nico g.; wolska-kusnierz, beata; kalwak, krzysztof; pituch-noworolska, anna; kondratenko, irina; van montfrans, joris m.; mejstrikova, ester; lankester, arjan c.; langerak, anton w.; van gent, dik c.; stubbs, andrew p.; van dongen, jacques j.m.; van der burg, mirjam title: similar recombination-activating gene (rag) mutations result in similar immunobiological effects but in different clinical phenotypes date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: l k q background: v(d)j recombination takes place during lymphocyte development to generate a large repertoire of t- and b-cell receptors. mutations in recombination-activating gene (rag ) and rag result in loss or reduction of v(d)j recombination. it is known that different mutations in rag genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes. objective: we sought to study the immunologic mechanisms causing the clinical spectrum of rag deficiency. methods: we included patients with similar rag mutations (c. delt or c. _ delaa) resulting in n-terminal truncated rag protein with residual recombination activity but presenting with different clinical phenotypes. we studied precursor b-cell development, immunoglobulin and t-cell receptor repertoire formation, receptor editing, and b- and t-cell numbers. results: clinically, patients were divided into main categories: t(−)b(−) severe combined immunodeficiency, omenn syndrome, and combined immunodeficiency. all patients showed a block in the precursor b-cell development, low b- and t-cell numbers, normal immunoglobulin gene use, limited b- and t-cell repertoires, and slightly impaired receptor editing. conclusion: this study demonstrates that similar rag mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. we postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients. defects in v(d)j recombination result in a block in b-and t-cell differentiation because formation of immunoglobulin and t-cell receptors (trs) is perturbed. this results in a combined immunodeficiency (cid) of b and t cells. v(d)j recombination is initiated by the recombination-activating gene (rag) and rag proteins by creating double-stranded breaks in the immunoglobulin and tr loci. subsequently, these breaks are processed and repaired by proteins involved in nonhomologous end joining. thus far, genetic defects have been identified in the rag , rag , artemis, ligase iv (lig ), xlf (cernunnos), and dna-pkcs genes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the immunologic phenotypes and clinical presentations of these mutations are different, depending on the type of genetic defect (ie, null mutations or hypomorphic mutations with residual v[d]j recombination activity). especially for the rag genes, many different mutations have been described that give rise to residual activity of the mutated rag protein. different rag mutations can result in a broad spectrum of clinical phenotypes, including severe combined immunodeficiency (scid), rag deficiency (ragd) with skin inflammation and ab t-cell expansion (classical omenn syndrome [os]), ragd with skin inflammation but without t-cell expansion (incomplete os), ragd with maternofetal transfusion, ragd with gd t-cell expansion, late-onset scid, ragd with granulomas, and ragd with cd cytopenia and thymus hypoplasia. , this broad spectrum of clinical phenotypes impedes timely recognition of ragd and might thus delay treatment (hematopoietic stem cell transplantation). in this study we selected patients with ragd with similar n-terminal truncating rag mutations to study the effect of a similar mutation on the clinical phenotype. these patients could be divided into main clinical phenotypes (ie, scid, os, and cid, which includes the other phenotypes). we studied whether key immunologic parameters (eg, precursor b-cell development, b-and t-cell numbers, and b-and t-cell repertoire) might explain the differences in clinical phenotypes. peripheral blood (pb), bone marrow (bm), and clinical data were obtained according to the guidelines of the medical ethics committee of the erasmus mc rotterdam. flow cytometric analysis was performed, as previously described. , , rag analysis and in vitro v(d)j recombination assay the rag and rag genes were amplified by means of pcr and sequenced, as previously described. the level of recombination activity of the rag expression constructs was determined by using the recombination plasmid pdvg , as described previously. , a taqman-based realtime quantitative (rq)-pcr was used to measure rag and rag transcription levels in bm mononuclear cells, as described previously. t-cell receptor b analysis t-cell receptor b (trb) gene rearrangements were studied, as described previously. vk-ck junctions were amplified in a multiplex pcr by using primers specific for vk - families (vki: -gtaggagacagagtcaccatcact- , vkii: -tggagagccggcctcca-tctc- , vkiii: -gggaaagagccacc ctctcctg- , and vkiv: -ggcgagagggcc-accatcaac- ) and a ck primer ( -actttggcctctctggata- ). pcr products were cloned in the pgem-teasy vector (promega, madison, wis) and prepared for sequencing on the abi prism xl fluorescent sequencer (applied biosystems, foster city, calif). obtained sequences were analyzed with the imgt database (http://imgt. cines.fr/) to assign the vkand jk genes. , the productive and unique sequences were used to determine the frequency of the vk and jk genes. the vh-jh junctions were amplified from post-ficoll pbmcs in a multiplex pcr by using the vh - fr and jh consensus biomed- primers. the primers were adapted for sequencing by adding the forward a or reverse b adaptor, the ''tcag'' key, and the multiplex identifier adaptor. pcr products were purified by using gel extraction (qiagen, valencia, calif) and agencourt ampure xp beads (beckman coulter, fullerton, calif). subsequently, the pcr concentration was measured with the quant-it picogreen dsdna assay (invitrogen, carlsbad, calif). the purified pcr products were sequenced on the gs junior instrument according to the manufacturer's recommendations by using the gs junior titanium empcr kit (lib-a), sequencing kit, and picotiterplate kit ( life sciences; roche, branford, conn). by using the clc genomic workbench software, the samples were separated based on their multiplex identifier sequence and trimmed, and reads with a quality score of less than . and less than bp were discarded. the reads were uploaded to imgt highv-quest software. subsequently, these output files were uploaded to the custom galaxy platform. [ ] [ ] [ ] further processing was done in the r programming language to generate the tabular and graphic outputs. the complementary determining region (cdr ) amino acid patterns were visualized with weblogo (http://weblogo.berkeley.edu/). , statistics differences in absolute numbers of lymphocyte subsets were analyzed by using the -tailed t test for independent samples (p < . was considered significant) in graphpad prism software (graphpad software, la jolla, calif). over the past years, we identified one of the mutations resulting in n-terminal truncating rag mutations in patients (tables i and ii ). these c. delt (hereafter abbreviated as delt) and c. _ delaa (hereafter abbreviated as delaa) mutations have been described before in several patients. , [ ] [ ] [ ] [ ] [ ] they were found to be hypomorphic , because translation can be reinitiated from the alternative start site methionine (m ) or m , resulting in an n-terminal truncated rag protein with the same (comparable) residual rag activity (< % compared with wild type; fig , a) . sixteen patients were homozygous for the delaa or delt mutation, and patients were compound heterozygous (table i) . three rag mutations found on the second allele were also analyzed in the in vitro recombination assay, showing no residual rag activity (fig , a) . in addition, we determined the presence of polymorphisms in the rag gene because these might influence the recombination activity of rag . the only polymorphism found was p.arg his, which was shown not to affect recombination activity. n-terminal truncating rag mutations result in a spectrum of clinical phenotypes although all patients had similar rag mutations, resulting in the same n-terminal truncation of the rag protein, the clinical phenotypes varied substantially. the patients could be divided into main clinical phenotypes: ''classical'' t b scid (n ), os (n ), and cid (n , tables i and ii). the patients with ''classical'' scid were defined as having low b-and t-cell numbers and age at diagnosis before the first year of life. the patients with os all had generalized and pronounced erythroderma. the patients with cid were given a diagnosis after the first year of life and had greater than % gd t cells or normal levels of t cells (p and p ). despite the same n-terminal truncation of rag in the patients, the range of clinical phenotypes strongly suggests that factors other than residual rag activity contribute to the clinical phenotype. ragd results in a block in the precursor b-cell differentiation in bm at the b-cell stages during which v(d)j recombination of the immunoglobulin genes takes place. the relative distribution of pro-b, pre-bi, pre-bii, and immature b cells was assessed in bm from of patients to investigate precursor b-cell differentiation. in healthy children pro-b and pre-bi cells constitute % to % of the precursor b cells (fig , b) . all patients with ''classical'' scid and os, except p , showed a complete block before the pre-bii-cell stage (fig , b) , whereas most of the patients with cid had a leaky block with greater than % pre-bii and immature b cells (fig , b) . rag and rag transcription levels were determined in the bm mononuclear cells to exclude that differences in rag transcription levels caused these difference in precursor b-cell composition. it is known that rag and rag transcription levels are correlated, and that rag and rag levels in bm mononuclear cells depend on the number of cells expressing rag (pre-bi and pre-bii cells). in all studied patients, the rag transcription level was correlated to rag (fig , c) , indicating that the differences in severity of the precursor b-cell block were not caused by differences in expression of rag . b-cell numbers in pb were undetectable or very low in most patients, except p , who had normal levels (table ii) . correlating the percentage of pre-bii and immature cells in bm with the number of peripheral b cells showed that only patients with greater than % pre-bii and immature b cells in bm (p , p , p , p , and p ) had detectable b cells in pb. collectively, these data indicate that most patients with cid have a milder block in the precursor b-cell composition and that only patients with a leaky block have detectable b cells in the pb. in those patients with detectable peripheral b cells, we studied the igh v(d)j recombination repertoire. igh gene rearrangements were amplified from mononuclear cells derived from pb, bm, or both and subsequently sequenced by using nextgeneration sequencing in healthy control subjects (pb and bm) and patients with cid (p , p , and p ). the frequency of unique sequences in igh genes was significantly lower in patients with ragd than in control subjects (table iii) , which is a reflection of the low numbers of b cells present in pb. despite the low recombination activity, ighv, ighd, and ighj gene use was not restricted (fig and see fig e in this article's online repository at www.jacionline.org). forty-eight of the ighv genes used in control subjects were identified in the patients with ragd because were the ighd genes and all ighj genes. ighv, ighd, and ighj gene uses were similar to those seen in control subjects, although some genes were used with different frequencies (fig and see fig e ) . most strikingly, jh use was lower whereas jh use was higher compared with that seen in control subjects. the patients with ragd had a significantly lower frequency ( . % to . % vs . % to . % in control subjects) of unproductive rearrangements (table iii) , as reported previously. unproductive rearrangements were defined as out-of-frame rearrangements or rearrangements with a stop codon. therefore even though the patients with ragd had reduced v(d)j recombination, leading to a limited tr and immunoglobulin repertoire, the igh gene use was similar to that seen in control subjects without preferential use of the proximal or distal genes. os is characterized by autoimmune-like clinical features, including severe erythroderma, hepatosplenomegaly, and lymphadenopathy. , the immune dysregulation in patients with os might be caused by the severe abnormalities of thymic architecture and impaired expression of autoimmune regulator and tissue-specific antigens. , in addition, hypomorphic rag mouse models have shown a disturbance in b-cell tolerance. , in addition, patients with os and also patient with ''classical'' scid and patients with cid had autoimmunity, and all displayed idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, or both (table i) . unfortunately, the thymic architecture and autoimmune regulator and tissue-specific antigen expression could not be studied in our patients, but we were able to evaluate parameters in the igh sequences that are associated with autoimmunity. these are characterized by long cdr s, and the frequency of ighv - , which is known to encode intrinsically self-reactive cold agglutinin antibodies that recognize carbohydrate antigens on erythrocytes. , the distribution of the cdr length of the unique junctions in bm and pb was similar to that seen in control subjects (fig , a) , except patient , who seemed to have increased numbers of junctions with a cdr of and amino acids. these junctions with a cdr length of amino acids displayed high similarity (fig , b) . no sequence similarity was found when all -amino-acid cdr s were compared (fig , b) , but . % of these junctions used ighv - , and all these junctions had a highly similar cdr sequence (fig , b) , which suggests that they might recognize a common antigenic determinant. the frequency of long cdr s (> _ amino acids) was significantly lower in p and p (p < . ) but not in p (fig , c) . the frequency of ighv - use was significantly higher in p (p < . ) and p (p < . ; fig , d) . from the patients we analyzed, p had autoimmunity, which was reflected by the high frequency of ighv - use. in addition to selection against long cdr s, b-cell tolerance is also generated by receptor editing of self-reactive b cells. these self-reactive b cells are induced to express the rag proteins and edit their receptor light chains through available upstream vk and downstream jk genes to change the affinity of their receptors. therefore the vk-jk junctions were amplified from patients with os and patients with cid. the igkv gene use was not significantly different from that seen in control subjects (fig , e) , but less igkj genes were used in the patients with ragd (fig , f) . therefore receptor editing seems partly affected, as deduced from the very low igkj use. the hallmark of classical os is an expansion of autologous t cells with an hla-dr cd ro phenotype and an oligoclonal ab t-cell repertoire. consistent with this, most of the patients with os had normal or increased cd t-cell numbers; in addition, patients with cid had normal numbers (p and p ), whereas all other patients had low absolute numbers of cd t cells (table ii) . remarkably, many patients had high percentages (> %) of gd t cells, including patients with ''classical'' scid, patient with os, and patients with cid (table ii ). in addition, we determined the t-cell proliferation by determining the drec-cja t-cell receptor excision circle (trec) content per ng of dna in patients with ''classical'' scid, patients with os, and patients with cid. in patients trecs were not detectable, and in the other patients (p , p , p , and p ), the number of trecs/ ng of dna was less than compared with trecs/ ng of dna in control subjects (n ; age, months to years; data not shown), meaning that the t cells that were present in these patients showed extensive proliferation. furthermore, the t-cell repertoire was determined by testing the trb gene rearrangements in patients with ''classical'' scid, patients with os, and patients with cid. in all patients the trb repertoire was clearly restricted (fig ) . taken together, the t cells that were present in the patients with ragd showed extensive proliferation and had a restricted tr repertoire. many different rag mutations have been reported to the rag mutation database. although most are null mutations, several have been described to result in residual recombinase activity. , , , , previously, it was hypothesized that null mutations in rag would result in ''classical'' t b scid and that partial reduction of rag activity would result in os or an intermediate late-onset scid or os phenotype. over the last few years, the spectrum of reported clinical phenotypes of ragd has broadened and now also includes ragd with gd t-cell expansion, ragd with skin inflammation but without t-cell expansion (incomplete os), ragd with granulomas, ragd with maternofetal transfusion, and ragd with cd cytopenia and thymus hypoplasia. , a few case reports have shown that the same rag mutation can result in a different clinical phenotype. , , , this study is the first to report an in-depth immunobiological evaluation of patients with ragd with similar rag mutations, resulting in the same n-terminal truncation of the rag protein. these similar mutations result in different clinical phenotypes, which indicates that a specific mutation does not predict a patient's clinical phenotype. because all patients had similar mutations, the residual rag protein activity was expected to be comparable among all patients. the n-terminally truncated rag protein is produced through translation starting from an alternative start site (m or m ), and hence the amount of protein is dependent on how efficiently these start sites are used. because the rag transcription level correlated with that of rag , we assume that all patients had similar expression of the mutant rag protein (fig , c) . we cannot exclude that epigenetics and modifier genes accounted for small differences in rag protein expression. although a previous attempt to identify such modifier genes in human subjects was not successful, studies in mouse models could shed more light on the contribution of epigenetics and modifier genes. in our cohort v(d)j recombination was not completely abolished but was strongly reduced because of the low residual activity of the rag protein. reduced v(d)j recombination was characterized by normal ighv, ighd, and ighj gene use, without preferential use of proximal or distal genes. however, as shown previously, the frequency of unproductive sequences was significantly lower than in healthy control subjects, . e and f, the percentage of igkv and igkj genes was determined in control subjects, patients with os, and patients with cid. igkv use was normal (fig , e) , but hardly any igkj gene was used (fig , f) . indicating that the b cells in the patients with ragd did not correct unproductive rearrangements by means of recombination of the second igh allele. as a consequence of the reduced v(d)j recombination, fewer b and t cells with a functional receptor can be produced. the proliferation of the lymphocytes is increased to compensate for low circulating b-and t-cell numbers. this idea is corroborated by the low numbers of trecs in patients with ragd. the increased proliferation of t cells might result in normal or increased t-cell counts, especially in the patients with os; however, the corresponding tr repertoire in all the patients with ragd remains restricted. most patients with ragd showed clinical signs of immune dysregulation, such as erythroderma, lymphadenopathy, hepatosplenomegaly, idiopathic thrombocytopenic purpura, and autoimmune hemolytic anemia. b cells have been shown to contribute to the immune dysregulation seen in rag mouse models. , sera from these mice contained high-affinity anti-double-stranded dna and tissue-specific autoantibodies, and b cells displayed impaired receptor editing. in addition, these mice had increased serum b cell-activating factor levels, which might rescue autoreactive b-cell clones. this increase in serum b cell-activating factor levels was also seen in patients with rag-, artemis-, and x-linked scid. similar to observations in mice, most patients with ragd did not use the igkj gene, whereas igkv gene use was normal. this suggests that receptor editing in this group of patients with ragd was slightly impaired, which can either be a result of reduced recombination activity caused by the rag mutation or by low b-cell numbers leading to reduced selection against autoreactive b cells. the igh repertoire was investigated for long cdr s and increased ighv - use, which are associated with autoreactive antibodies. , from the patients with ragd we analyzed, only p had autoimmunity, which was reflected by an increased vh - gene use. the patients divided into the main clinical ragd groups hardly differed in their immunobiological parameters, and consequently, we could not find any specific pattern that could explain the different clinical phenotypes. on the basis of our results and earlier reported data, we propose an explanatory model for the development of different clinical phenotypes in patients with ragd with similar mutations (fig ) . if ragd results in reduced v(d)j recombination, low b-and t-cell numbers are produced with some (compensatory) clonal expansion. this expansion might increase the b-and t-cell numbers to even normal levels but does not change the limited repertoire. in such limited repertoire the selection against autoreactive cells is impaired. provided the deficient immune system is not activated, patients with ragd are asymptomatic. however, when the immune system will be activated by potentially a wide range of different (auto)antigens, the type of antigen and activated effector lymphocyte will have important consequences for the clinical phenotype. in addition, the impaired negative and positive selection of thymic lymphocytes and reduced number of regulatory t cells might result in autoimmunity when patients are exposed to autoantigens. this phenomenon can occur at any early stage, even in utero, as shown by the fact that patients with os can have severe erythroderma already at birth, which is unlikely to be triggered by infections. additionally, directly after birth, the skin and gastrointestinal tract become colonized by commensal bacteria, which can trigger the chronic diarrhea seen in most patients with ragd. key steps in the development of a certain clinical phenotype will be the b-and t-cell repertoire, the type of (auto)antigen exposure, the specificity of the antigen receptors and timing, the cell type involved in the immune activation, and the potential influence of genetic variations in modifier genes. variability in any of these factors might eventually lead to different clinical phenotypes, despite a similar genetic defect. in conclusion, this study clearly shows that the type of rag mutation and the level of residual rag recombinase activity are not the only determinants predicting the clinical phenotype, as previously assumed. the clinical outcome of an individual patient with ragd depends on a complex interplay between the (limited) immune receptor repertoire, (auto)antigen exposure, the specificity of antigen receptors, and the timing and cell type involved in immune activation. therefore the clinical outcome of patients with ragd with similar mutations is extremely difficult to predict. we thank b. h. barendregt and i. pico-knijnenburg for technical assistance, s. de bruin-versteeg for making the figures, d. zessen for help with the repertoire analysis, and professor a. j. cant for discussion and advice. clinical implications: ragd can result in a broad spectrum of clinical presentations, but the level of residual rag activity is not always predictive for the clinical outcome. v(d)j recombination rag mutations in human b cell-negative scid artemis, a novel dna double-strand break repair/v(d)j recombination protein, is mutated in human severe combined immune deficiency xlf interacts with the xrcc -dna ligase iv complex to promote dna nonhomologous end-joining cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly an overview of three new disorders associated with genetic instability: lig syndrome, rs-scid and atr-seckel syndrome a new type of radiosensitive t-b-nk severe combined immunodeficiency caused by a lig mutation more than just scid-the phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (rag) and idiopathic cd t lymphopenia without autoimmunity or granulomatous disease in the slipstream of rag mutations the immunophenotypic and immunogenotypic b-cell differentiation arrest in bone marrow of rag-deficient scid patients corresponds to residual recombination activities of mutated rag proteins radiosensitive scid patients with artemis gene mutations show a complete b-cell differentiation arrest at the pre-b-cell receptor checkpoint in bone marrow n-terminal truncated human rag proteins can direct t-cell receptor but not immunoglobulin gene rearrangements fusion gene transcripts and ig/tcr gene rearrangements are complementary but infrequent targets for pcr-based detection of minimal residual disease in acute myeloid leukemia design and standardization of pcr primers and protocols for detection of clonal immunoglobulin and t-cell receptor gene recombinations in suspect lymphoproliferations: report of the biomed- concerted action bmh -ct - imgt databases, web resources and tools for immunoglobulin and t cell receptor sequence analysis in an attempt to compensate for low numbers, b and t cells start to proliferate, but the repertoire remains limited and imbalanced, so that selection and immune regulation are impaired. most likely the type of antigenic stimulation together with the incomplete and imbalanced repertoire that has been imgt, the international immunogenetics database imgtÒ tools for the nucleotide analysis of immunoglobulin (ig) and t cell receptor (tr) v-(d)-j repertoires, polymorphisms, and ig mutations: imgt/v-quest and imgt/highv-quest for ngs galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences galaxy: a web-based genome analysis tool for experimentalists galaxy: a platform for interactive large-scale genome analysis r: a language and environment for statistical computing. vienna: r foundation for statistical computing weblogo: a sequence logo generator sequence logos: a new way to display consensus sequences identical mutations in rag or rag genes leading to defective v(d)j recombinase activity can cause either t-b-severe combined immune deficiency or omenn syndrome a novel immunodeficiency associated with hypomorphic rag mutations and cmv infection n-terminal rag frameshift mutations in omenn's syndrome: internal methionine usage leads to partial v(d)j recombination activity and reveals a fundamental role in vivo for the n-terminal domains v(d)j recombination defects in lymphocytes due to rag mutations: severe immunodeficiency with a spectrum of clinical presentations from severe combined immunodeficiency to omenn syndrome after hematopoietic stem cell transplantation in a rag deficient family ig gene rearrangement steps are initiated in early human precursor b cell subsets and correlate with specific transcription factor expression lack of nonfunctional b-cell receptor rearrangements in a patient with normal b cell numbers despite partial rag deficiency and atypical scid/omenn syndrome combined immunodeficiency and reticuloendotheliosis with eosinophilia familial reticuloendotheliosis with eosinophilia aire deficiency in thymus of patients with omenn syndrome early defects in human t-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of omenn syndrome homeostatic expansion of autoreactive immunoglobulin-secreting cells in the rag mouse model of omenn syndrome expansion of immunoglobulin-secreting cells and defects in b cell tolerance in rag-dependent immunodeficiency nucleotide sequence analysis of the v regions of two igm cold agglutinins. evidence that the vh - gene segment is responsible for the major crossreactive idiotype variable region gene analysis of pathologic human autoantibodies to the related i and i red blood cell antigens restricted heterogeneity of t lymphocytes in combined immunodeficiency with hypereosinophilia (omenn's syndrome) human t cell leukemias with continuous v(d)j recombinase activity for tcr-delta gene deletion rag mutation database partial v(d) j recombination activity leads to omenn syndrome a novel homozygous mutation in recombination activating gene in relatives with different clinical phenotypes: omenn syndrome and hyper-igm syndrome recombinase-activating gene immunodeficiency: different immunological phenotypes in three siblings gvhd-associated cytokine polymorphisms do not associate with omenn syndrome rather than t-b-scid in patients with defects in rag genes atypical vh-d-jh rearrangements in newborn autoimmune mrl mice predominant autoantibody production by early human b cell precursors key: cord- -gg xespb authors: navel, valentin; chiambaretta, frédéric; dutheil, frédéric title: will environmental impacts of social distancing due to the sars-cov- pandemic decrease allergic disease? date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: gg xespb nan the burden of climatic change and air pollution represents a massive challenge for humanity, affecting the development of allergic diseases and upsetting the exposome. air pollution, a causative factor of climate change, contributes to nine million deaths by years, and more than million people have an allergic disease around the world. in urban area of industrial countries, the ocular surface, the respiratory tracts and the skin are daily exposed to intense burden of particulate matter (pm), ozone (o ), carbon dioxide (co ), nitrogen dioxide (no ) and sulfur dioxide (so ) produced by diesel combustion and industrial fumes. air pollution is a causative factor for both various symptoms such as bronchospasm, rhinorrhea, eye redness, and irritation, as well as various allergic diseases such as asthma, chronic rhinitis, nasal polyposis, atopic dermatitis, seasonal or perennial allergic conjunctivitis, and vernal or atopic keratoconjunctivitis. increasing asthma exacerbations and the global mortality. putative etiologic relation between atmospheric changes and allergic diseases is mediated by inflammation and oxidative stress in tissues exposed enhancing immunologic responses to allergens, carrying aeroallergens into the airways, and inducing epithelial permeability. interestingly, nasa's aura satellite described a no decrease by % across china from january - , (before the quarantine) to february - (during the quarantine). similar decrease was measured in most megalopolis of developing countries around the world where containment was set for limiting the spreading of the sars-cov- ( figure ). following containment for limiting the spreading of the sars-cov- pandemic (satellite images from european spatial agency (esa)). emerging concepts and challenges in implementing the exposome paradigm in allergic diseases and asthma: a practall document global, regional, and national comparative risk assessment of behavioural, environmental and occupational, and metabolic risks or clusters of risks, - : a systematic analysis for the global burden of disease study obesity may enhance the adverse effects of no exposure in urban schools on asthma symptoms in children climate change and the impact on respiratory and allergic disease change research program effects on asthma and respiratory allergy of climate change and air pollution airborne nitrogen dioxide plummets over china nitrogen dioxide concentrations over france key: cord- -p mxc authors: van singer, mathias; brahier, thomas; ngai, michelle; wright, julie; weckman, andrea m.; erice, clara; meuwly, jean-yves; hugli, olivier; kain, kevin c.; boillat-blanco, noémie title: covid- risk stratification algorithms based on strem- and il- in emergency department date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: p mxc background the covid- pandemic has led to surges of patients presenting to emergency departments (ed) and potentially overwhelming health systems. objective this study aimed to assess the predictive accuracy of host biomarkers at clinical presentation to the ed for adverse outcome. methods prospective observational study of pcr-confirmed covid- patients in the ed of a swiss hospital. concentrations of inflammatory and endothelial dysfunction biomarkers were determined at clinical presentation. we evaluated the accuracy of clinical signs and these biomarkers in predicting -day intubation/mortality, and oxygen requirement by calculating the area under the receiver operating characteristic curve (auroc) and by classification and regression tree analysis. results of covid- patients included, were outpatients or hospitalized without oxygen requirement, hospitalized with oxygen requirement and intubated/died. we found that soluble triggering receptor expressed on myeloid cells (strem- ) had the best prognostic accuracy for -day intubation/mortality (auroc . ; % ci . - . ) and interleukin- (il- ) measured at presentation to the ed had the best accuracy for -day oxygen requirement (auroc . ; % ci . - . ) .an algorithm based on respiratory rate and strem- predicted -day intubation/mortality with % sensitivity and . nlr. an il- -based algorithm had % sensitivity and . negative likelihood ratio (nlr) for -day oxygen requirement. conclusion strem- and il- concentrations in covid- in the ed have good predictive accuracy for intubation/mortality and oxygen requirement. strem- - and il- -based algorithms are highly sensitive to identify patients with adverse outcome and could serve as early triage tools. strem- and il- concentrations in covid- in the ed have good predictive accuracy for intubation/mortality and oxygen requirement. strem- -and il- -based algorithms are highly sensitive to identify patients with adverse outcome and could serve as early triage the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic has led to surges of patients that can overwhelm health systems ( ) ( ) ( ) ( ) . medical resource constraints and scarcity are a new reality including lack of hospital beds, oxygen concentrators, and ventilators in both high-and low-income countries ( , ) . there is an urgent need to have guidance allowing the rational allocation of scarce medical equipment and resources. in this context, it is critical to have triage tools in place permitting the early recognition of patients at risk of adverse outcomes, and allow for optimal resource allocation ( - ). current prediction models to support clinical decision making for coronavirus disease (covid- ) patients were developed based on demographics, clinical signs and symptoms, imaging techniques, biomarkers or a combination of these variables, however most are poorly validated and at risk of bias ( ). cytokine dysregulation during covid- is suspected to contribute to disease severity and multiple clinical trials are underway to test the efficacy of immuno-modulatory therapies ( - ). a recent study identified different inflammatory cytokine profiles according to the stage of the disease, suggesting their usefulness for risk stratification in covid- patients ( ). interleukin (il- ), interleukin (il- ) and c-reactive protein (crp) have been proposed for monitoring and prognosis purpose in the context of covid- ( - ). triggering receptor inflammatory response ( , ) . in support of this contention, several studies have reported an association between markers of a pro-coagulable state (e.g. d-dimers and fibrinogen) and patients outcome ( ) ( ) ( ) . in a french study, level of angiopoietin- (ang- ) as marker of endothelial activation predicted with a medium accuracy the icu admission for covid- ( ). this prospective observational cohort study of covid- patients was conducted in the ed of lausanne university hospital (chuv), a tertiary care center in switzerland. we prospectively screened all patients (age ≥ years) upon arrival at the ed with symptoms of an acute lower respiratory tract infection (cough, sputum, dyspnea or chest pain for < days) between february th and april rd ( ). patients were included in this study if covid- was confirmed by real time polymerase chain reaction (rt-pcr) for sars-cov- from a nasopharyngeal swab. patients demographics, comorbidities, symptoms, vital signs and laboratory results performed during routine care were recorded in the ed using a standardized electronic case report form in redcap (research electronic data capture). easy-to-measure bedside clinical scores to identify patients at risk of poor outcome were calculated at inclusion: (i) quick sequential a plasma sample was collected in the ed at enrollment and stored at - °c without freeze- thaw until analysis. plasma concentrations of endothelial and immune mediators (ang- , il- , il- , and strem- ) were analyzed head-to-head on a multi-analyte ella tm platform with custom-developed reagents from protein simple (california, usa) as described ( ). c- reactive protein (crp) was quantified by enzyme-linked immunosorbent assays (r&d duoset, minneapolis, mn). statistical analyses differences between the three groups were evaluated by one-way anova, kruskal-wallis or chi-squared, as appropriate. a bilateral p value < . was considered indicative of statistical significance. the primary outcome was -day intubation/mortality. secondary outcomes were -day intubation/mortality, -and -day oxygen requirement (all patients hospitalized with oxygen requirement, including those who were intubated or died, as they all received oxygen). these outcomes are important for pragmatic clinical decision-making in the ed. they are objective and therefore more reproducible in other settings than clinician decision to admit a patient to hospital or to the intensive care unit. we hypothesized first, that patients intubated the dependent variable was assessed via the box-tidwell procedure and by inspecting the partial residuals. non-linear variables were transformed for multivariate logistic regression. the predictive validity of a combinatorial model adding top biomarkers to vital signs or clinical severity scores was measured using logistic regression, and the predicted probabilities were used to generate auroc. the combinatorial models were compared using the delong all included patients signed an informed consent form. table shows patient demographics, clinical characteristics and laboratory results. while sex distribution, prevalence of co-morbidities and duration of symptoms were similar between the three severity groups, patients in the intubation/mortality group were significantly older than table ). the need for -day oxygen supplementation was best predicted by il- (auroc . , % ci . - . ) and il- (auroc . , % ci . - . ) ( figure b ). strem- and crp showed an acceptable performance, which was not statistically lower than il- . ang- performed significantly worse than il- , il- and strem- (p< . ). table ). the combination of respiratory rate with strem- performed significantly better than the respiratory rate alone (p = . ), but not than strem- alone. we found similar results when combining clinical scores and strem- (table ) . after having evaluated the magnitude of the association between clinical signs, scores, biomarkers and adverse outcome, we performed a crt analysis to find optimal variables and cut-points suitable to generate simple algorithms. the resulting classification tree represents visual decision making. since it is also important to identify patients who require immediate medical attention, we also assessed the performance of this algorithm to predict -day intubation/death. the sensitivity was maintained at %, the specificity at % and the negative and positive likelihood ratio were . and . respectively. since we could use this triage tool to identify patients requiring oxygen, we tested this algorithm to predict -and -day oxygen requirement. while it was highly specific ( %), its sensitivity was low ( %) in this setting. the crt analysis performed with all clinical signs, severity scores and biomarkers to predict since we could also use this triage tool to identify patients at high risk of poor outcomes, we tested this algorithm to predict -and -day intubation/death. it had an excellent sensitivity ( %) but a poor specificity ( %) for -day intubation/death and showed a similar performance for -day intubation/death (sensitivity at % and specificity at %). sensitivity. however, the performance of this algorithm to identify patients with a lower level of severity (those requiring oxygen) was not optimal with a low sensitivity. our data do not support the use of a respiratory rate/strem- triage algorithm for oxygen requirement. nevertheless, our data support strem- and il- as potential candidates for poct, which could be used as a triage tool at presentation to predict disease severity in covid- . in this study both algorithms were very sensitive with a low negative likelihood ratio assuring safe management of patients. ultimately biomarkers-based algorithms such as these, could enhance clinical decision making and resource allocation; however, this will require further prospective trials to confirm their risk-stratification performance in actual practice. acknowledgements we thank all the patients who accepted to participate and make this study possible. we thank professor carron, head of the emergency department, who supported the study. we thank all healthcare workers of the emergency department, internal medicine ward, infectious disease nonparametric roc curves were generated and auroc were plotted to illustrate the ability of these markers to discriminate between patient groups. each auroc was compared to other using delong method. aurocs for the outcome of each biomarker are presented to the right of its respective forest plot, with % cis in parentheses. * angiopoietin- performed significantly worse than strem- , il- and il- (p< . ) to predict -day oxygen requirement. no other comparison reached a statistically significant difference (p< . ). for all algorithms, the cost of misclassifying a patient who was intubated or died was designated as times the cost of misclassifying a patient that survived without intubation. aurocs were calculated from the predictive probabilities of logistic regression models to -day mortality/intubation. auroc of clinical parameters alone and combined with strem- or il- are presented. differences in aurocs were assessed using the delong method. * p value < . comparing the clinical parameter auroc versus the combined clinical parameter with strem- or il- auroc. covid- : how doctors and healthcare systems are tackling coronavirus worldwide critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response covid- in europe: the italian lesson -the lancet lessons learned from the coronavirus health crisis in madrid, spain: how covid- has changed our lives in the last weeks fair allocation of scarce medical resources in the time of covid- clinical ethics recommendations for the allocation of intensive care treatments in exceptional critical care impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand imperial college london elevated levels of il- and crp predict the need for mechanical ventilation in covid- the role of interleukin- in monitoring severe case of coronavirus disease clinical and pathological investigation of patients with severe covid- metalloproteinases shed trem- ectodomain from lipopolysaccharide-stimulated human monocytes. the journal of immunology cutting edge: inflammatory responses can be triggered by trem- , a novel receptor expressed on neutrophils and monocytes. the journal of immunology pathological inflammation in patients with covid- : a key role for monocytes and macrophages heart rate, bpm; median (iqr) kruskal-wallis or chi-squared, as appropriate. # bmi > kg/m . * heart failure, coronary disease. † stroke, dementia, parkinson. ‡ stade iii-v according to ckd classification. ® autoimmune or chronic inflammatory disease. missing values: obesity , duration of symptoms , fever , cough , vital signs , blood count , chest radiograph key: cord- - d r authors: renz, harald title: autophagy: nobel prize and allergy and asthma research date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: d r nan the nobel prize in physiology or medicine was awarded to yoshinori ohsumi, professor at the tokyo institute of technology, for ''recycling.'' recycling on the cellular level is termed autophagy and is a fundamental process for degrading and recycling cellular components. it is now well regarded that this process plays an important role not only in physiologic cellular homeostasis but also in a variety of diseases. the story starts about years ago with the discovery of the lysosome. lysosomes are specialized organelles containing enzymes that digest proteins, carbohydrates, and lipids. for the discovery of this process, christian de duve, a belgian scientist, was awarded the nobel prize in physiology or medicine in . the next important step in this journey was the observation that under certain conditions large cellular components and sometimes whole organelles can be found inside lysosomes. a new type of vesicle was discovered and named autophagosomes, describing a process of ''self-eating'' to understand the mechanism of how such large cargos get into the lysosome. a few years later, researchers discovered an additional system to degrade proteins, although not as large ''fragments'' but rather one by one (fig ) . this system was termed the proteasome, and aaron ciechanover, avram hershko, and irwin rose were awarded the nobel prize in chemistry in for the discovery of ubiquitin-mediated protein degradation (table i) . ohsumi and colleagues now discovered many steps and the molecular tools involved in this complex process of recycling. [ ] [ ] [ ] [ ] he recognized that external cellular stress, such as heat, radiation, starvation, and infection, turn on the cellular recycling machinery to promote survival of the cells. today, more than , proteins have been catalogued, among them about dozen proteins involved in autophagy and regulators involved in recycling of survival-eminent materials. ohsumi and colleagues conducted a series of brilliant experiments in the early s using the bakers' yeast saccharomyces cerevisiae to identify the essential genes for this process. through random mutation, he knocked out one after the other of these relevant genes and elucidated the underlying mechanism for autophagy. through this process, he discovered genes, originally termed autophagy-related genes (ats) atg to atg and today renamed atg genes. today, a disturbed process of autophagy has been linked to many diseases, including parkinson disease, type ii diabetes, alzheimer disease, certain cancers, and many infections. furthermore, this process also regulates our own appetite. the process starts in certain neurons in the hypothalamus, where, under starvation conditions, mediators are produced under the control of autophagy. these mediators then trigger our appetite. recent data indicate that autophagy plays an important role in the inflammatory pathways in allergy and asthma. in this issue of the journal radonjic-hoesli et al investigate a mechanism that leads to release of cytotoxic granules from eosinophils. cytolysis is one of these mechanisms and leads to the release of intragranules, so-termed clusters of eosinophil granules. the investigators used state-of-the-art technologies, including time-lapse fluorescence microscopy, electron microscopy, and immunohistochemistry, to prove that cytoplasmic membrane disintegration and intragranule release depend on a signaling pathway, including receptor-interacting protein kinase and mixed lineage kinase-like protein. importantly, if they activate autophagy in eosinophils, this counterregulates eosinophil cytolysis. therefore the authors not only demonstrate in a very elegant fashion a new pathway for eosinophil cytolysis, they were also able to link cytolysis with counterregulating autophagy and open new pathways for at least experimental intervention. autophagy has also been previously linked to the pathogenesis of asthma. although little work has been carried out in this regard thus far, there are convincing data available linking autophagy in airway epithelium, and autophagy might be essential for bronchial epithelial mucus secretion, as has been shown in an allergic asthma model. interestingly, autophagy, rather than endoplasmic reticulum stress, contributed to il- -induced eotaxin- peptide secretion also from human airway epithelial cells. [ ] [ ] [ ] a link between the epithelial-mesenchymal transition unit and autophagy has been shown in other models of allergic asthma. , a further link between autophagy and asthma has been established for human rhinovirus, respiratory syncytial virus, coronavirus, adenovirus, and influenza a virus through the role of autophagy in the defense against respiratory tract viruses, which represent a major exacerbation factor for asthma. in this regard autophagy impairs viral replication, and autophagy is enhanced in the airways of asthmatic patients. , finally, a single nucleotide polymorphism has been discovered in the atg gene, which is one of the genes regulating autophagy. this polymorphism was associated with asthma and low fev levels. atg transcripts are enhanced in nasal epithelial cells in patients with acute asthma exacerbations. this could further strengthen the functional link between autophagy and asthma (exacerbation). in summary, the study of autophagy opens a new field in allergy and asthma research. autophagy is a key process involved in immune responses, inflammation, and antiviral immunity. further studies are warranted to establish this link and also, more importantly, to explore the possibilities of therapeutic intervention in this regard. we are looking forward to reading more articles about autophagy, allergy, and inflammation in the journal. isolation and characterization of autophagy-defective mutants of saccharomyces cerevisiae autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction a protein conjugation system essential for autophagy a ubiquitin-like system mediates protein lipidation eosinophil cytolysis requires the ripk -mlkl signaling pathway and is counter-regulated by autophagy autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target il activates autophagy to regulate secretion in airway epithelial cells the complex roles of endoplasmic reticulum stress and autophagy in modulating eotaxin- production and secretion from human airway epithelial cells anti-nerve growth factor antibody reduces airway hyperresponsiveness in a mouse model of asthma by downregulating the level of autophagy in lungs astragalin inhibits autophagy-associated airway epithelial fibrosis autophagy is involved in influenza a virus replication matrix protein of influenza a virus blocks autophagosome fusion with lysosomes genetic and histologic evidence for autophagy in asthma pathogenesis the first autophagy gene, atg ohsumi key: cord- -qq qg authors: siniorakis, eftychios; arvanitakis, spyridon; sfakianaki, titika; katsianis, antonios; sinaniotis, athanasios; papagiannopoulou, veneta title: covid- and upper respiratory tract: collecting swab specimens from patients inhaling corticosteroids date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: qq qg nan correspondence covid- and upper respiratory tract: collecting swab specimens from patients inhaling corticosteroids q to the editor: jian et al in a recent article refer to the immune reaction of the upper respiratory tract (urt) in patients infected by severe acute respiratory syndrome coronavirus (sars-cov)- (coronavirus disease [covid- ] pandemic). the authors wonder about the consequences of inhaled corticosteroids (icss) on immune reaction. we share the queries of jian et al, from a practical point of view. since late february , when the covid- outbreak exploded in greece, emergency departments (eds) of major hospitals were enrolled in triaging patients with suspicious clinical presentation. medical staff was called to perform nasopharyngeal and oropharyngeal swabbing, irrespective of specialization, and forwarding the samples for a pcr. training was offered through educational videos. the authors of this letter are going to maintain such a triage during the summer months when a wave of tourists from countries with different prevalence of covid- is going to visit greece. pitfalls in performing nasopharyngeal or oropharyngeal swabbing are not negligible. learning from the experience of previous medical personnel engaged in ed, we focus on an intriguing problem we are going to cope with. given the pneumonologic character of the hospital and the seasonal allergies, % to % of patients admitted to the ed are expected to suffer exacerbations of chronic obstructive pulmonary disease, asthma, rhinitis, and rhinosinusitis. most of them are under chronic or seasonal therapies with icss, oral or intranasal. with the urt sprayed by icss, we are wondering about the diagnostic accuracy of specimens collected. asking patients to blow their nose, or postponing the procedure is not feasible, as far as we ignore the washout period needed for an ics, especially in chronic administration. icss are likely to modify the replication of commensal and pathogenic virome in the nasopharynx, oropharynx, and saliva. medical literature regarding the effects of icss on pathogenic virome of the urt is scarce. we try to extrapolate data from previous coronavirus epidemics, namely, sars-cov, mers . this is likely for inhaled cortisone, prednisolone, dexamethasone, and fluticasone. contrarily, mometasone inhibits the replication of sars-cov and hcov- e. similarly, budesonide degrades the virulence of hcov- e. recently, the ics ciclesonide was found to suppress the replication of sars-cov- , by directly attacking the nsp viral endoribonuclease, demonstrating an antiviral function. therapies with icss modulate the innate defensive mechanisms of the urt, interfering with cytokines, while at the same time perturbate the abundance and replication of commensal and pathogenic viruses. , depending on the chemical composition of icss, the impact on the virome of the urt is unpredictable. with this in mind, swabbing the urt of coronavirus-infected patients under an ics regimen renders the quality of the viral load collected questionable. taking alternative specimens from pleural effusions, stools, or even blood is of dubious diagnostic value. with all this scepticism in our minds, we are preparing ourselves to cope with the intricacies of triaging in a covid- era, performing with responsibility the swabbing techniques, ensuring specimens of high diagnostic accuracy. perspective: covid- , implications of nasal diseases and consequences for their management how to obtain a nasopharyngeal swab specimen determinants of the nasal microbiome: pilot study of effects of intranasal medication use long-term impact of inhaled corticosteroid use in asthma and chronic obstructive pulmonary disease (copd): review of mechanisms that underlie risks the inhaled corticosteroid ciclesonide blocks coronavirus rna replication by targeting viral nsp inhibitory effects of glucopyrronium, formoterol, and budesonide in coronavirus hcov- e replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells identification of antiviral drug candidates against sars-cov- from fda-approved drugs key: cord- -rx ajiw authors: jian, li; yi, wei; zhang, nan; wen, weiping; krysko, olga; song, woo-jung; bachert, claus title: perspective: covid- , implications of nasal diseases and consequences for their management date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: rx ajiw nan the severe acute respiratory syndrome-coronavirus- (sars-cov- ) has caused a global health emergency. with increasing numbers of infected people and deaths worldwide reported daily since the beginning of the year, we have to urgently focus on a new pandemic caused by the sars-cov- , a betacoronavirus related to sars-cov. we have to urgently learn more about this virus, its ways of transmission to spread infection so fast all over the world, the pathomechanisms involved in human infection and intracellular entry, the consecutive spreading within the body, and finally the factors that determine the difference between a mild or even asymptomatic infection in one and a deadly disease in another patient. obviously, the age and health condition of the infected person including hypertension, diabetes, and cardiovascular disease play an important role in the clinical course of a coronavirus disease. urgently, we need to find adequate treatments, and there might be more than adequate masks and social distancing on the one hand and vaccination on the other hand. sars-cov- , which causes coronavirus disease (covid- ), is highly contagious. it is of utmost importance to understand how the virus succeeds in infecting an individual person and entering first an outer surface cell and starts replicating, because this might offer therapeutic approaches in the near future. the major entrance door to the patient obviously is the nose and nasopharynx. in a study of infected patients, sars-cov- viral load was detected higher in the nose than in the throat. also, we have painfully learned from the fact that many health care professionals, specifically also ear-nose-and-throat (ent) specialists, died from covid- . surgery in the nose of an infected patient has been shown to not only put the surgeon at risk, but everyone in the operation room, specifically when drills and endoscopes are used and aerosols are formed during the surgery. furthermore, it was quite apparent early on that one of the early symptoms of covid- is a loss of smell and consecutively of taste, clearly differentiating the sars-cov- infection from a normal common cold or a flu. although most reports are still anecdotal, anosmia/hyposmia was reported in up to % of the patients, even among those without typical symptoms such as cough, fever, or dyspnea (https://www.entuk.org/categories/covid- ). this led to the statements by the american and british ent societies, proposing a particular attention to recent-onset olfactory dysfunction as an early possible sign of covid- . it has been confirmed later that olfactory epithelial support cells and stem cells express both of these genes discussed below, as do cells in the nasal respiratory epithelium. what do we know about upper airway conditions such as chronic rhinosinusitis (crs) with or without polyps, or allergic rhinitis; would these diseases increase the risk of infection? these are diseases affecting % to % of the population in the united states and europe, and most often type immune reactions associated with an anyway weak immune defense and reduced ifn production upon infection. we have learned, also from the related coronavirus sars-cov, causing sars outbreak in , that sars-cov- binds to the angiotensin-converting enzyme (ace ) exposed at the airway surface and uses it as receptor (fig ) . to invade the cell, there is need for another player, the transmembrane protease tmprss , a serine protease expressed in airway epithelial cells, mucosal glands, and inflammatory cells such as macrophages, that activates the viral s protein and enables human airway cell entry. how is this receptor and the protease regulated in the nasal cavity, and how does that change with age, sex, or disease status, and would that possibly explain some of the questions posed above? of patients hospitalized for sars-cov- infection from wuhan and zhuhai hospitals, only patient reported crs, allergic rhinitis, and chronic pharyngitis as preexisting diseases (li jian, personal communication, ). during infection, only patients reported pharyngeal dryness and pain, and nasal obstruction as symptoms, whereas reported loss of smell, in accordance with recent reports. , differences in interviewing the patients may account for different prevalences of loss of smell. in the data sets from healthy individuals generated by the human cell atlas consortium, when assessing the rna expression of the coronavirus receptor (ace ) as well as the viral s protein priming protease tmprss , nasal epithelial cells displayed the highest ace pcr measurements of all cells analyzed. , however, it needs to be mentioned that the protein expression was not demonstrated. furthermore, the corona virus receptor ace may be an ifn-stimulated gene. this could imply that any viral infection could facilitate sars-cov- infection by releasing ifns and upregulating ace . however, type immune conditions such as crs without nasal polyps (crswnp) with a pronounced type immune reaction and a deficit in ifns might rather downregulate ace expression. in fact, we observed a reduced expression of ace in nasal polyp disease versus control tissue based on rt-pcr in nasal samples (o. krysko, n. zhang, and c. bachert, unpublished data, ). in a rat model of allergic asthma, the expression of ace in lungs was decreased compared with negative control. in summary, these findings might suggest that type inflammatory condition in the airways could have a protective effect against covid- infection or its severity. however, further studies are warranted to clarify causal relationships. meanwhile, it also needs to be clarified whether ace expression in the nasal tissues was influenced by intranasal corticosteroid (incs) treatment often used in these patients. this leads us to the question whether treatment in patients with allergic rhinitis, normally incs, or in severe patients with crswnp, nowadays including biologics to suppress type immune reactions, should be continued in case of a sars-cov- infection. the global initiative for asthma statement (https://ginasthma. org/recommendations-for-inhaled-asthma-controller-medications/) advised that patients with asthma should not stop their prescribed inhaled corticosteroid controller medication (or prescribed oral corticosteroids), because stopping inhaled corticosteroid may lead to the potentially dangerous worsening of asthma, and avoiding oral corticosteroids during severe asthma attacks may have serious consequences. the administration of oral corticosteroids for covid- lung injury is not advised by the world health organization (https://www.who.int/publications-detail/clinical-management-of-severe-acute-respiratory-infec tion-when-novel-coronavirus-(ncov)-infection-is-suspected); however, the world health organization does not discourage the use of corticosteroids when indicated independently from covid- . referring to the global initiative for asthma statement, the allergic rhinitis and its impact on asthma-european academy of allergology and clinical immunology statement authored by bousquet et al based on a delphi process advised to continue incs treatment for allergic rhinitis, because there is no current evidence that incs would increase infectivity or symptoms of sars-cov- infection. however, discontinuation of corticosteroid therapy could induce more coughing or more sneezing because of the loss of control of inflammation, and therefore may increase the risk of infection of so far healthy subjects. sneezing may spread the virus over several meters, distancing of m clearly is not enough, and face masks are advised in this situation. sars-cov- may also infect patients with severe asthma and crswnp, who might be under treatment with a type biologic drug such as dupilumab, omalizumab, or mepolizumab. again, no data are available for any on the type biologics today, but balancing the risk of losing disease control and the lack of evidence or expectation of increased infectivity or mortality, there already are recommendations to continue the treatment with biologics and even start new treatments (eg, from the german allergy society [https://dgaki.de/]). in fact, in a situation in which surgery of the sinuses is not advised to prevent infection of the operation theater staff, a biologic may in fact offer a possibility to severe patients with nasal polyps. we (ulrike f€ orster, charite berlin) actually just report on a patient on dupilumab for recurrent severe crswnp, who developed a sars-cov- infection without any additional difficulties. the smell, which was restored after dupilumab, disappeared as the only symptom under the cov infection, but returned fast thereafter. no other symptoms or changes have been observed. the emergence of covid- will bring a major change to our practice. however, we begin to recognize that diseases of the upper airways or their management by corticosteroids and biologics do not seem to increase the risk of infection nor the risk for severe covid- . a narrow follow-up certainly is advised. because health care workers at ent departments are prone to the viral exposure, a consensus is needed very urgently on protective measures for the professionals. in research perspective, because the airway passage of nose and nasopharynx is the main entry for respiratory viruses including the sars-cov , the expression and its regulation of the ace receptor and the tmprss protease are key topics for research and targets for interventions. however, given the potential of new respiratory viral outbreaks in the future, further attention should be given to how to modulate protective roles of upper airway mucosa against different viral infections. clinical characteristics of patients infected with sars-cov- in wuhan, china sars-cov- viral load in upper respiratory specimens of infected patients letter: precautions for endoscopic transnasal skull base surgery during the covid- pandemic non-neural expression of sars-cov- entry genes in the olfactory epithelium suggests mechanisms underlying anosmia in covid- patients sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor cleavage of the sars coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro sars-cov- entry genes are most highly expressed in nasal goblet and ciliated cells within human airways sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues activation of angiotensin-converting enzyme (ace ) attenuates allergic airway inflammation in rat asthma model intranasal corticosteroids in allergic rhinitis in covid- infected patients: an aria-eaaci statement key: cord- -f orw ro authors: li, hailan; liu, huaping title: regarding “ruxolitinib in treatment of severe coronavirus disease (covid- ): a multicenter, single-blind, randomized controlled trial” date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: f orw ro nan regarding ''ruxolitinib in q treatment of severe coronavirus disease (covid- ): a multicenter, singleblind, randomized controlled trial'' q to the editor: we read with great interest the article titled ''ruxolitinib in treatment of severe coronavirus disease (covid- ): a multicenter, single-blind, randomized controlled trial'' by cao et al. the study, which finally included patients, evaluated the efficacy and safety of ruxolitinib for severe covid- cases. they finally found that ruxolitinib recipients had a significant chest computed tomography (ct) improvement, a faster recovery from lymphopenia, and favorable side-effect profile. however, we think it is appropriate to comment on the methods used in this study. first, the duration from illness onset to randomization, days for the control group and days for the ruxolitinib group, was not appropriate because according to literature reports, - the course (from illness onset to discharge) of severe patients was about to days. therefore, the patients may have passed the dangerous period at the th day and be in the end stage of the disease, which corresponds to the improvement period. second, in terms of the extent of ct abnormalities, covid- showed a marked increase from the subclinical period through the first and second weeks after illness onset, then decreased gradually in the third week. [ ] [ ] [ ] therefore, it is difficult to say whether the absorption of patients after the th day was the process of disease outcome or the effect of ruxolitinib. finally, using the data of ct improvement at day in table iii , we found that the p value between the control group and the treatment group was not . but . . nevertheless, these small issues in no way detract from the outstanding work of cao et al, which sheds light on the treatment of covid- . ruxolitinib in treatment of severe coronavirus disease (covid- ): a multicenter, singleblind, randomized controlled trial risk factors associated with disease severity and length of hospital stay in covid- patients clinical characteristics and outcomes of hospitalised patients with covid- treated in hubei (epicentre) and outside hubei (non-epicentre): a nationwide analysis of china a trial of lopinavir-ritonavir in adults hospitalized with severe covid- temporal changes of ct findings in patients with covid- pneumonia: a longitudinal study imaging features and evolution on ct in covid- pneumonia patients in wuhan, china radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study key: cord- -t r l r authors: toivonen, laura; camargo, carlos a.; gern, james e.; bochkov, yury a.; mansbach, jonathan m.; piedra, pedro a.; hasegawa, kohei title: association between rhinovirus species and nasopharyngeal microbiota in infants with severe bronchiolitis date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: t r l r among infants with severe bronchiolitis, rhinovirus species related to distinct nasopharyngeal microbiota. infants with rhinovirus-a were more likely to have haemophilus-dominant microbiota profile, while those with rhinovirus-c were more likely to have moraxella-dominant profile. to the editor: bronchiolitis is the leading cause of hospitalization in us infants. rhinovirus (rv) is the second most common cause of severe bronchiolitis (ie, bronchiolitis requiring hospitalization) following respiratory syncytial virus (rsv). rvs are rna viruses consisting of more than genotypes that are classified into species (rv-a, rv-b, and rv-c). rv-a and rv-c are more frequently found than rv-b in children with acute respiratory infections (aris) and wheezing illnesses. , emerging evidence suggests a complex interplay between viral infection, airway microbes, and host immune response in the pathobiology of ari. studies have shown that rv infection in children is associated with increased detection of pathogenic bacteria in the airways. , furthermore, detection of rv together with specific airway pathogens (eg, moraxella catarrhalis) is associated with increased ari and asthma symptoms. recently, rv-a and rv-c were reported to differentially associate with detection of pathogenic bacteria in school-age children. however, no study has investigated the relationships between rhinovirus species and airway microbiota in infants, let alone infants with bronchiolitis. to address the knowledge gap, we examined the association between rhinovirus species and the nasopharyngeal airway microbiota determined by s rrna gene sequencing in infants with severe bronchiolitis. this was a post hoc analysis of data from the th multicenter airway research collaboration (marc- ) cohort study-a multicenter prospective cohort study of infants hospitalized for bronchiolitis. the details of the study design, setting, virus and microbiota measurements, and analysis are described in this article's online repository at www.jacionline.org. briefly, infants (age < year) hospitalized for bronchiolitis were enrolled in sites across us states (see table e in this article's online in pbmc-engrafted mice (n - per group) without or with injection of neutrophils and subsequent intranasal challenge with birch pollen extract (bpe) or pbs. i.n., intranasal; i.p., intraperitoneal. # p < . of bpechallenged mice with/without neutrophils; §p < . of bpe/pbschallenged mice. **p < . and ***p < . of bpe/pbs-challenged mice with activated neutrophils, -way anova. repository at www.jacionline.org). bronchiolitis was defined according to the american academy of pediatrics guidelines. the institutional review boards at participating sites approved the study. informed consent was obtained from the infants' parent or legal guardian. nasopharyngeal samples were collected within hours of hospitalization and stored at c locally. these samples were processed and tested for respiratory pathogens by real-time pcr and for microbiota using s rrna gene sequencing at baylor college of medicine (houston, tex). singleplex real-time pcr was used to detect rv, and positive a b fig . between-virus difference in nasopharyngeal microbiota in infants hospitalized for bronchiolitis. a, between the virus categories, the proportion of nasopharyngeal microbiota profiles differed. for example, compared with infants with rsv-only bronchiolitis, those with rv-a infection were more likely to have a haemophilus-dominant, mixed, or moraxella-dominant profile than a streptococcus-dominant profile. infants with rv-c infection were more likely to have a moraxella-dominant profile. p values were derived from adjusted multinomial logistic regression model. corresponding relative rate ratios are presented in table i . *p < . . b, between the virus categories, the distribution of relative abundance of most common genera in the nasopharyngeal microbiota differed. data are presented using violin plots, which are boxplots with a rotated kernel density plot on each side. p values adjusted for multiple comparisons are presented in table e . specimens were further genotyped by using molecular typing assay at the university of wisconsin (madison, wis). by using partitioning around medoids unsupervised clustering with the use of weighted unifrac distance, distinct nasopharyngeal microbiota profiles were derived as previously described. in the current analysis, we grouped infants into mutually exclusive virus categories: solo rsv (reference), rv-a, rv-b, and rv-c. we tested the association between these virus categories and nasopharyngeal microbiota profiles by constructing multinomial logistic regression model adjusting for covariates. data were analyzed using r version . . . of enrolled infants, were in of the prespecified virus categories ( rsv-only, rv-a, rv-b, and rv-c) and had high-quality microbiota data; they comprised the analytic sample. overall, the median age was . months (interquartile range, . - . ), % were male, and % infants received intensive care therapy. compared with infants with rsv-only, those with rv-a or rv-c were older and more likely to have previous breathing problems (p < . ; see table e in this article's online repository at www.jacionline.org). across the virus categories, there was a significant difference in the likelihood of nasopharyngeal microbiota profiles (p < . ; see table e in this article's online repository at www.jacionline.org). for example, while infants with rsv had the highest likelihood of streptococcus-dominant profile (the reference virus and microbiota profile), those with rv-a had the highest likelihood of haemophilus-dominant profile (fig , a) , corresponding to an adjusted relative rate ratio of . ( % ci, . - . ; p < . ; table i ). in contrast, infants with rv-c were more likely to have moraxella-dominant profile than streptococcus-dominant profile (adjusted relative rate ratio, . ; % ci, . - . ; p . ) . similarly, at the genus-level (fig , b ; see table e in this article's online repository at www.jacionline.org), compared with infants with rsv-only, those with any rv species had lower relative abundance of streptococcus (p . ) and those with rv-a had a higher abundance of haemophilus (p . ). earlier studies reported that rv-c infection is associated with higher risks of subsequent ari in young children and that enrichment of moraxella abundance in the upper airways is related to higher frequency of aris. furthermore, a recent analysis from rhinogen study ( children [aged - years] with or without asthma, using quantitative pcr for bacteria) reported that rv-a and rv-c are differentially associated with increased quantity of h influenzae, m catarrhalis, and s pneumoniae. our observations-for example, the association between rv-c and higher likelihood of moraxella-dominant microbiota-corroborate these earlier findings, and extend them by applying s rrna gene sequencing to the airway samples of a large multicenter prospective cohort of infants with severe bronchiolitis. the underlying mechanisms of the virus-microbiota relationships are beyond the scope of our data. the observed associations may be causal-that is, specific respiratory virus species (eg, rv-c) alters the airway microbiota. alternatively, unique microbiota profiles in conjunction with airway immune response might have contributed to susceptibility to specific virus infection. these potential mechanisms are not mutually exclusive. despite this complexity, the identification of the association between specific virus species and airway microbiota in infants with bronchiolitis is important given their relation to subsequent respiratory health in children. our study has potential limitations. first, the study design precluded us from examining the relation between the temporal pattern of airway microbiota and respiratory health in children. to address this question, the cohort is currently being followed longitudinally for years with serial examinations of microbiota. second, the current study did not have healthy controls. however, the study aim was to determine the association of virus species with microbiota among infants with bronchiolitis. finally, although the study cohort comprised a racially/ethnically diverse us sample of infants, we must generalize the inferences cautiously beyond infants with severe bronchiolitis. regardless, our data are highly relevant for , us children hospitalized with bronchiolitis each year. in summary, on the basis of this multicenter prospective cohort study of infants with severe bronchiolitis, we observed that compared with infants with rsv-only infection, infants with rv-a or rv-c infection had distinct nasopharyngeal microbiota profiles-for example, those with rv-c infection had a higher likelihood of moraxella-dominant microbiota profile, whereas those with rv-a infection had a higher likelihood of haemophilus-dominant profile. although causal inferences remain premature, our data should advance research into delineating the complex interrelations between respiratory viruses, airway microbiome, and respiratory outcomes in children. we thank the marc- study hospitals and research personnel for their ongoing dedication to bronchiolitis and asthma research (table e ) to the editor: eosinophilic gastrointestinal disorders (egids) are a set of conditions characterized by eosinophil-rich inflammation in various segments of the gastrointestinal tract in the absence of known causes, such as infection and systemic diseases. most attention on egid has focused on eosinophilic esophagitis (eoe). this increased focus on eoe is, in part, related to the agreed-upon diagnostic criteria for eoe. these consensus criteria are more straightforward than the limited, nonconsensus criteria of other egids because the esophagus, unlike the rest of the gastrointestinal mucosa, is devoid of eosinophils under healthy normal conditions. among egids, there is relatively little information concerning eosinophilic colitis (ec), currently defined as a clinicopathologic disorder that primarily affects the colon with eosinophil-rich inflammation in the absence of known causes of eosinophilia. [ ] [ ] [ ] [ ] even without agreed-upon diagnostic criteria for ec, previous studies have estimated its prevalence in adult and pediatric populations to be . to . per , individuals on the basis of the international classification of diseases (icd) or systematized nomenclature of medicine (snomed) coding. , herein, we aimed to ( ) identify cases of colonic eosinophilia in a tertiary pediatric medical center; ( ) determine associated diseases; and ( ) estimate the prevalence of cases considered to be ec in the hospital and geographic populations. retrospective analyses were performed using independent approaches at cincinnati children's hospital medical center (cchmc) by searching ( ) the electronic medical record (emr) (epic, verona, wis) and ( ) cchmc's pathology database. in both approaches, both local and referral patients were included. in the emr approach, a computer script was generated to identify all records since with icd- / codes for ec ( . ; k . ), whereas in the pathology database search, specimens (since ) were queried for the word ''eosinophil'' in biopsy descriptions. these samples were then limited to colonic biopsies by searching for specimens with the following terms: ''colon,'' ''sigmoid,'' ''rectum/sigmoid,'' ''sigmoid/rectum,'' ''rectum,'' ''rectal,'' or ''recto.'' cases identified by either approach were narrowed down to include patients who met criteria for colonic eosinophilia. colonic eosinophilia was defined as having an eosinophil count greater than the upper limit of normal in at least colonic site (> eosinophils/hpf in the ascending colon, > eosinophils/hpf in the transverse and descending colon, > eosinophils/hpf in the rectosigmoid colon). , a chart review of patients who met the criteria for colonic eosinophilia was performed to identify patients with ec or associated diseases. patients identified from the pathology database were cross-checked with the emr results to prevent ''double counting'' of individuals identified by both searches. patients were considered to have ''definite'' ec if they had ( ) an eosinophil count greater than the upper limit of normal in at least colonic site (thresholds as mentioned above); ( ) documentation of colonic symptoms; ( ) no underlying conditions known to be definitely associated with colonic eosinophilia; and ( ) a clinical diagnosis of ec by a gastroenterologist at cchmc as determined by manual chart review. this was a post hoc analysis of data from the th multicenter airway research collaboration (marc- ) e -a multicenter prospective cohort study of infants hospitalized with bronchiolitis (ie, severe bronchiolitis). marc- is coordinated by the emergency medicine network (emnet), a collaboration of participating hospitals. e site investigators enrolled infants (age < year) hospitalized with bronchiolitis at sites across us states (table e ) using a standardized protocol during consecutive bronchiolitis seasons (from november through april ) during the period to . bronchiolitis was defined by the american academy of pediatrics guidelines as an acute respiratory illness with some combination of rhinitis, cough, tachypnea, wheezing, crackles, and retractions e and was diagnosed by an attending physician. we excluded infants who were transferred to a participating hospital more than hours after the original hospitalization, those for whom consent was provided more than hours after hospitalization, or those with known heart-lung disease, immunodeficiency, immunosuppression, or gestational age of less than weeks. all patients were treated at the discretion of the treating physicians. the institutional review board at each of the participating hospitals approved the study. written informed consent was obtained from the parent or guardian. clinical data (patients' demographic characteristics and family, environmental, and medical history, and details of the acute illness) were collected via structured interview and chart reviews. e all data were reviewed at the emnet coordinating center (boston, mass), and site investigators were queried about missing data and discrepancies identified by manual data checks. in addition to the clinical data, blood and nasopharyngeal airway samples were collected within hours of hospitalization and stored at c using standardized protocols. e nasopharyngeal samples were analyzed for respiratory pathogens using real-time pcr assays e as well as for microbiota using s rrna gene sequencing at baylor college of medicine (houston, tex). singleplex real-time pcr was used to detect rv at baylor college of medicine. of rv-positive specimens, their species and genotypes were identified by using molecular typing assay that targets a variable fragment in untranslated region of the viral genome flanked by highly conserved motifs at the university of wisconsin (madison, wis). e s rrna gene sequencing of nasopharyngeal airway microbiota s rrna gene sequencing methods were adapted from the methods developed for the national institutes of health human microbiome project. e ,e all samples were processed with a low-biomass extraction protocol to avoid sample loss and degradation and to maximize yield. bacterial genomic dna was extracted using mo bio powersoil dna isolation kit (mo bio laboratories; carlsbad, calif). the s rdna v region was amplified by pcr and sequenced in the miseq platform (illumina; san diego, calif) using the bp paired-end protocol yielding pair-end reads that overlap almost completely. the primers used for amplification contain adapters for miseq sequencing and single-end barcodes allowing pooling and direct sequencing of pcr products. e ,e sequencing read pairs were demultiplexed on the basis of unique molecular barcodes, and reads were merged using usearch v . . , e allowing mismatches and a minimum overlap of bases. rarefaction curves of bacterial operational taxonomic units (otus) were constructed using sequence data for each sample to ensure coverage of the bacterial diversity present. samples with suboptimal amounts of sequencing reads were resequenced to ensure that most bacterial taxa were encompassed in our analyses. s rrna gene sequences were clustered into otus at a similarity cutoff value of % using the uparse algorithm. e otus were determined by mapping the centroids to the silva database e containing only the s v region to determine taxonomies. a custom script constructed a rarefied otu table (rarefaction was performed at only sequence depth) from the output files generated in the previous steps for downstream analyses of alpha-diversity (eg, shannon index) and beta-diversity (eg, weighted unifrac). e ,e the processes involving microbial dna extraction, s rrna gene amplification, and amplicon sequencing included a set of controls that enabled us to evaluate the potential introduction of contamination or off-target amplification. nontemplate controls (extraction chemistries) were included in the microbial dna extraction process and the resulting material was subsequently used for pcr amplification. in addition, at the step of amplification, another set of nontemplate controls (pcr-mix) was included to evaluate the potential introduction of contamination at this step. similarly, a positive control composed of known and previously characterized microbial dna was included at this step to evaluate the efficiency of the amplification process. before samples (unknowns) were pooled together, sequencing controls were evaluated and the rejection criteria were the presence of amplicons in any of the nontemplate controls or the absence of amplicons in the positive control. in the present study, no amplicons were observed in the nontemplate controls and a negligible amount of raw reads was recovered after sequencing. s rrna gene sequencing of the nasopharyngeal airway samples from the enrolled infants (n ) obtained , , high-quality merged sequences, of which , , ( . %) were mapped to the database. of infant samples, ( . %) had sufficient sequence depth (rarefaction cutoff, reads per sample). the primary outcome was nasopharyngeal microbiota profile. as previously described, e by using the partitioning around medoids method e with weighted unifrac distance, an unsupervised clustering method, we derived distinct microbiota profiles: ( ) haemophilus-dominant profile, ( ) moraxella-dominant profile, ( ) mixed profile, and ( ) streptococcus-dominant profile. the number of clusters was determined using the average silhouette score. e to examine the association of respiratory viruses with nasopharyngeal microbiota profiles, we first grouped infants into mutually exclusive virus categories: rsv-only (reference), rv-a, rv-b, and rv-c. infections with multiple rv species were excluded. we compared the patient characteristics and clinical presentation between the virus categories, by using chi-square test and wilcoxon-mann-whitney test, as appropriate. we also compared the relative abundances of most abundant genera between the virus categories by using -way anova, adjusting for multiple comparisons with the use of the benjamini-hochberg false-discovery rate method. e we then tested the association of these virus categories with nasopharyngeal microbiota profiles by constructing a multinomial logistic regression model adjusting for potential confounders (ie, age, sex, race/ethnicity, gestational age, siblings in the household, breast-feeding, history of breathing problems, and lifetime history of systemic antibiotic use). these potential confounders were chosen on the basis of clinical plausibility and a priori knowledge. e ,e -e we reported all p values as -tailed, with p < . considered statistically significant. the data were analyzed with the use of r version . . (r foundation, vienna, austria) with the phyloseq package for the microbiota analysis. iqr, interquartile range. data are n (%) of infants unless otherwise indicated. *of these, had coinfection with a non-rsv/non-rv, which did not have statistically significant association with the microbiota profiles (p . ). a novel role for neutrophils in ige-mediated allergy: evidence for antigen-presentation in late-phase reactions human t-cell clonal anergy is induced by antigen presentation in the absence of b costimulation membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation the t lymphocyte glycoprotein cd binds the cell surface ligand lfa- interaction of cd with its ligand, lfa- , in human t cell proliferation cd /cd is the primary costimulatory pathway in human cd cd t cells blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model cd -mediated regulatory t-cell activation inhibits the development of disease in a humanized mouse model of allergic airway disease infectious pathogens and bronchiolitis outcomes rhinoviruses and their receptors: implications for allergic disease human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human rhinovirus species c infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions acquisition and transmission of streptococcus pneumoniae are facilitated during rhinovirus infection in families with children detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations association of rhinovirus species with common cold and asthma symptoms and bacterial pathogens association of nasopharyngeal microbiota profiles with bronchiolitis severity in infants hospitalised for bronchiolitis maturation of the infant respiratory microbiota, environmental drivers and health consequences: a prospective cohort study association of nasopharyngeal microbiota profiles with bronchiolitis severity in infants hospitalised for bronchiolitis clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis prospective multicenter study of children with bronchiolitis requiring mechanical ventilation improved molecular typing assay for rhinovirus species a, b, and c human microbiome project consortium. a framework for human microbiome research human microbiome project consortium. structure, function and diversity of the healthy human microbiome qiime allows analysis of high-throughput community sequencing data ultra-high-throughput microbial community analysis on the illumina hiseq and miseq platforms search and clustering orders of magnitude faster than blast uparse: highly accurate otu sequences from microbial amplicon reads the silva ribosomal rna gene database project: improved data processing and web-based tools unifrac: a new phylogenetic method for comparing microbial communities unifrac: an effective distance metric for microbial community comparison linking long-term dietary patterns with gut microbial enterotypes silhouettes: a graphical aid to the interpretation and validation of cluster analysis controlling the false discovery rate: a practical and powerful approach to multiple testing household siblings and nasal and fecal microbiota in infants maturation of the infant respiratory microbiota, environmental drivers and health consequences: a prospective cohort study the infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development key: cord- -eiphxwmn authors: trouillet-assant, sophie; viel, sebastien; gaymard, alexandre; pons, sylvie; richard, jean-christophe; perret, magali; villard, marine; brengel-pesce, karen; lina, bruno; mezidi, mehdi; bitker, laurent; belot, alexandre title: type i ifn immunoprofiling in covid- patients date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: eiphxwmn covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous ifn-α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. sophie trouillet-assant , * , phd, sebastien viel , , , * , pharmd, phd, alexandre gaymard merazga for their excellent work. we thank fabien subtil for his helpful advice for statistical analysis. we also thank the life (lyon immunopathology federation) community for fruitful discussion. capsule summary: covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. to the editor, severe acute respiratory syndrome coronavirus (sars-cov- ) infection (covid- ) is characterized by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper respiratory tract illness that can evolve into respiratory failure or rapidly progressing severe viral pneumonia with acute respiratory distress syndrome (ards). disease severity depends on viral strain and host risk factors have been identified such as age and male gender. in addition, an excessive immune response has been identified in patients showing a cytokine storm associated with ards . various immunosuppressive drugs, including il- blockers or jak-stat signaling inhibitors have been suggested for the treatment of sars-cov- infection whereas additional clinical trials are evaluating the use of recombinant interferon to foster host antiviral response. (clinicaltrials nct , nct ). type i interferons (ifn-i) are major components of the innate immune system and represent critical antiviral molecules . to date, ifn-i response has not been evaluated in covid- patients and its contribution to the viral control and inflammation is unknown. in this study, we assessed the kinetics of plasma ifn-i in covid- patients with a spectrum of severity degree. this study was approved by an ethical committee for biomedical research (comité de protection des personnes hcl). (supplemental material and method of this article online repository). firstly, we explored three patients issued from the first covid cluster diagnosed in france (les contamines, haute savoie, france) in february . we took advantage of the new digital elisa technology single-molecule arrays (simoa) and analyzed the kinetics of plasma inflammatory cytokines. interleukin (il)- , c-reactive protein (crp) and interferon γ-induced protein (ip- ) were elevated in the two symptomatic patients (pt , ) (supplementary figure in the online repository). strikingly, no ifn-α was detectable in these two patients. in contrast, il- , crp and ip- elevation of plasmatic ifn-α was observed. viral loads were low with no obvious quantitative difference between all three patients. we further explored a larger cohort of critically ill covid patients from one of the intensive care unit (icu) at hospices civils de lyon (lyon, france). of note, all the patients were treated with standard of care and none received antiviral or immunotherapies. considering the first days of infection, more than half of critically ill patients required invasive mechanical ventilation ( / ). we observed that patients demonstrated a peak in ifn-α at day - of symptoms onset corresponding to the viral replication phase, that decreased overtime to low but still detectable ifn-α the timing of interferon exposition may be critical to control the virus and avoid immunopathogenesis. channappavanar et al. have shown that delayed ifn-i expression can be detrimental in mice in the context of sars-cov- infection . our data suggests that screening patients for ifn production is instrumental to select those who could benefit from early intervention with ifn. following day , il- remains increased while ifn-α tapered. this kinetics highlight that cytokine inhibitors could be helpful at the second phase of the disease following ifn-i decrease. viral characteristic or individual genetic susceptibility should be explored to understand the defect of ifn- α production in some covid patients. some ifn-α positive patients also experienced fatal outcome highlighting the multifactorial causes of disease severity. we acknowledge limitations of this study, related to the small number of included patients and the technical limitation for the measurement of ifn-β and ifn-λ, in this proof of concept study. here, we provide new argues for an early intervention with recombinant ifn-α and we also highlight the window of opportunity for immunosuppressors at the second phase of the disease, delay between symptom onset and icu admission (days) [ - ] [ - ] . bacterial co-infection during icu stay (n (%)) ( %) ( %) diabetes (n (%)) ( %) ( %) chronic obstructive pulmonary disease (n (%)) ( %) ( %) cardiovascular disease (n (%)) ( %) ( %) hypertension (n (%)) ( %) ( %) cancer (n (%)) ( %) ( %) active smokers (n (%)) ( %) ( %) mortality at d after symptom onset(n(%)) ( %) ( %) . crp -c-reactive protein, icu -intensive care unit, bmi -body mass index table -clinical characteristics of covid- patients in intensive care unit p-value are calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones. a. plasma ifn-α concentrations (fg/ml) were determined by single molecule array (simoa) b.c.d. il- , crp and ip- concentrations were measured using a multiplexed assay with the ella platform. e. viral load is represented as cycle threshold of ip rt-qpcr using assay designed by pasteur institut in paris. ifn-interferon ; il- -interleukin ; crp -c-reactive protein ; ip- -interferon γ-induced protein a. ifn score is a transcriptionnal signature defined by interferon-stimulated gene (isg) quantified using nanostring technology and obtained from paxgene tubes in covid- patients. b-d. normal values for healthy volunteers was indicated by grey area. vertical bar indicates median delay between symptom onset and icu admission. concentrations of ifn-γ were quantified in only / patients because of lack of material. clinical features of patients infected with novel coronavirus in wuhan, china covid- : consider cytokine storm syndromes and immunosuppression type i interferons (α/β) in immunity and autoimmunity / and directive / /ec) and the french data protection law (law n° - on / / and décret n° - on / / ), we obtained consent from each patient or his next of kin usa) on plasma samples of covid- patients. the assay was based on a -step protocol using an hd- analyzer (quanterix). il- , crp and interferon γ-induced protein (ip- ) concentrations were measured using a multiplexed assay with the ella platform (protein simple© ca, usa), according to manufacturer's instructions. plasma il a/b and il- (type iii interferon) have been quantified by elisa (pbl laboratories rna integrity was then evaluated by agilent rna microarray (agilent technologies© data standardization was obtained using the geometric mean of internal control and housekeeping genes count number. interferon score was calculated as previously described . virus quantification load viral load was quantified from nasopharyngeal swabs or endotracheal aspirates. rna extraction was performed by the automated nuclisens® easymag® (biomérieux, marcy l'etoile, france) using manufacturer's instructions. a μl reaction contained μl of rna p-value were calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones comparison of rt-qpcr and nanostring in the measurement of blood interferon response for the diagnosis of type i interferonopathies walzer international center of research in infectiology, lyon university, inserm u , cnrs umr , ens, ucbl, lyon, france we explored the first three sars-cov- positive patients diagnosed in france (les contamines, france) in february . patient : a high risk contact (a -year-old man) initially negative for sars-cov- developed fever and cough with respiratory crackles at auscultation on the fifth day of hospital isolation. a bilateral interstitial syndrome at the ct-scan with bilateral ground-glass opacification predominant on the left. sars-cov was detected from endotracheal aspirates (eta), all nasopharyngeal swabs were always negative. the daily follow-up revealed a short-lasting excretion with only two successive eta for these three patients, no other respiratory pathogens were detected. these patients did not need oxygenation, nor antibiotics, steroids or antiviral agents. plasma samples and paxgene® tubes were collected from covid- patients hospitalized in the university hospital of lyon (hospices civils de lyon), france. diagnosis of covid- was confirmed in all patients by rt-pcr.all critically ill patients, admitted to icu, were included in the mir-covid study. this study was registered to the french national data protection agency under the number - and was approved by an ethical committee for biomedical research (comité de protection des personnes hcl) under the number n° - . in agreement with the general data protection regulation (regulation key: cord- -ygtbuy k authors: nan title: corrigenda date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: ygtbuy k nan corticosteroids on covid- outcomes. this is highlighted by preliminary findings in a recent study demonstrating the efficacy of dexamethasone in reducing mortality in critically ill patients with covid- , but showing no benefit in those not requiring respiratory support. furthermore, our study demonstrated a ''dose-response,'' with greater degrees of respiratory allergy being associated with larger reductions in angiotensin-converting enzyme gene expression in the nasal epithelium, and the impact of suppressing type inflammation in these individuals is not currently known. thus, we agree that strategies targeting the upper airway for the prevention and/or treatment of covid- are of significant interest. studies with nasal/inhaled corticosteroids as postexposure prophylaxis for very early stages of the disease (prehospitalization) may be worth considering. furthermore, various additional approaches are available for consideration, including topical therapy with intranasal antivirals, immune stimulants, and/or vaccines. covid- : start with the nose sars-cov- reverse genetics reveals a variable infection gradient in the respiratory tract detection of rhinovirus rna in lower airway cells during experimentally induced infection effect of dexamethasone in hospitalized patients with covid- : preliminary report association of respiratory allergy, asthma, and expression of the sars-cov- receptor ace ), it has been brought to the editors' attention that the authors' names were incorrect as printed. the names were shown as gregori silvia, phd, and aiuti alessandro, md, phd. the first and last names of both authors were inadvertently reversed and should be silvia gregori key: cord- - zs m authors: shilts, meghan h.; rosas-salazar, christian; turi, kedir n.; rajan, devi; rajagopala, seesandra v.; patterson, megan f.; gebretsadik, tebeb; anderson, larry j.; peebles, r. stokes; hartert, tina v.; das, suman r. title: nasopharyngeal haemophilus and local immune response during infant respiratory syncytial virus infection date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: zs m nan page libraries were then sequenced on an illumina miseq platform with x base pair reads. we subsequently processed the s rrna paired sequences using the dada pipeline, grouped sequences into amplicon sequence variants (asvs), and assigned taxonomy using the silva reference database. we used the relative abundance of haemophilus and haemophilus asvs (expressed as simple proportions) for statistical analyses. in addition, we used the basic local alignment search tool (blast) to search the asv sequences against a reference database and available whole metagenomic sequencing (wms) from a selected number of nasal washes (n= ) to identify haemophilus species. the luminex xmap data was processed using a method that uses median fluorescence intensities (mfis) of individual beads instead of the usual standard curve-based data-processing method to increase the sensitivity and accuracy of high-throughput immunoassays. to examine the unadjusted and adjusted associations of the relative abundance of haemophilus and haemophilus asvs with rsv viral load and the local immune mediator mfis, we used spearman correlation and multivariable linear regression, respectively. our primary models included the infant's age at the time of the rsv ari, sex, and race and ethnicity as covariates. we also created secondary models replacing race and ethnicity with maternal asthma. the statistical analyses were conducted in r. we used the benjamini and hochberg method to control for multiple comparisons when appropriate. one parent of each infant provided informed consent for their participation. the institutional review board of vanderbilt university medical center approved this study. further details are also available in the online repository. one hundred and five (~ %) of the infants with rsv-only aris enrolled in inspire had ≥ nasal wash with s rrna sequencing and immune mediator data available -obtained as page part of separate projects , -and were thus included in our study. the median (interquartile range [iqr] ) age at the rsv ari in these infants was . ( . - . ) months. the majority of these infants were males, white non-hispanic, and born via vaginal delivery ( table e in the online repository). in comparison to those included in the study, those not included were more likely to be born via vaginal delivery and to have federal or state insurance ( table e in using unsupervised methods, we identified clusters of infants based on their nasopharyngeal immune response patterns (figure ) . cluster # was mostly comprised of infants with higher local immune mediator levels when compared to cluster # . cluster # also mainly included infants with a higher relative abundance of haemophilus in their nasopharynx (figure ) in unadjusted analyses, the nasopharyngeal relative abundance of haemophilus was positively correlated with the levels of (~ %) of the local immune mediators after controlling for multiple comparisons (figure a and table e in the online repository). these associations page remained significant for (~ %) of the local immune mediators after adjusting for potential confounders in our primary and secondary models ( figure b and table e in the online repository). in general, the nasopharyngeal relative abundance of haemophilus was associated with higher levels of local immune mediators with anti-viral and/or pro-inflammatory functions, as well as those important in the chemotaxis, differentiation, proliferation, and survival of numerous immune cells (e.g., tnf-α, ifn-α, il- β, il- , il- , il- , il- , and il- a). in contrast, it was not associated with the levels of the main type (e.g., ifn-γ) or type (e.g., il- a) local immune mediators. in regard to type immune responses, there was a positive association with il- , but not with il- or il- , which may be related to the temporal expression of these cytokines. the nasopharyngeal relative abundance of haemophilus was also associated with il- (figure ) several members of the haemophilus genus are commonly found in the nasopharynx of healthy infants, although usually at low relative abundances. the aggregated evidence suggests that early-life rsv aris can lead to a rapid increase in the nasopharyngeal relative abundance of haemophilus throughout infancy (a critical period of lung and immune development) and that this could in turn impact the pathophysiology of rsv disease. - in our study, we show that the relative abundance of haemophilus (most likely h. influenzae) in the nasopharynx during an rsv ari in infancy is positively associated with multiple local immune mediators, particularly those with anti-viral and pro-inflammatory functions, which could be a mechanism through which these viral-bacterial interactions influence short-and long-term rsv-related outcomes. one prior study found that a nasopharyngeal microbial profile dominated by haemophilus was associated with delayed viral clearance during rsv ari in infancy; although exactly how the combination of anti-viral and pro-inflammatory immune responses impact viral clearance during rsv ari in infancy is unknown. to our knowledge, only one other study has examined the effect of nasopharyngeal haemophilus on the local immune response during an rsv ari in infancy. that study was smaller, used elisa-based immunoassays, and only included of the local immune mediators we examined, finding similar results for il- . likewise, in vitro studies found similar results for il- and il- , which also support our findings. , our study has multiple strengths, including the population-based design of the parent study, the close surveillance during the infants' first winter viral season to capture their initial rsv ari, the use of next-generation sequencing and high-throughput immune assays, and the use of statistical analysis adjusting for potential confounders. we also acknowledge several limitations. page first, we decided to focus on haemophilus due to the multiple prior studies assessing the whole nasopharyngeal microbiome that have identified this taxa as a key determinant of rsv-related outcomes - but it is certainly possible that haemophilus could just be a marker for other bacterial pathogens or that other taxa could also have important or stronger immune-modulatory effects. second, our sample size was small, and we may not have been powered to detect certain associations. the number of nasal washes with wms data was also limited, so the wms analyses should be regarded as exploratory as it is possible that we did not capture all relevant haemophilus species. third, it is possible that some of the associations found were driven by a small number of observations, as the nasopharyngeal relative abundance of haemophilus was overall low in most infants. however, several of the statistical tests we used are robust to the effect of outliers. furthermore, the effect of a particular species on a microbial ecosystem is not directly related to its abundance. thus, haemophilus could be a crucial taxa impacting the rsv- we subsequently processed the s rrna sequences using the dada pipeline by following its standard operating procedure (available at: https://benjjneb.github.io/dada /tutorial.html, last accessed november , ). to this end, sequences were grouped into amplicon sequence variants (asvs) and taxonomy was assigned using the silva reference database. sequences were subsequently processed through the r package decontam to remove any suspected contaminants that were found in the negative control samples. the local immune mediator mfis were generalized log transformed prior to all statistical analyses. † the unadjusted analyses were conducted using spearman correlation. the spearman correlation coefficients (ρ) and their corresponding p-values are shown. the p-values after controlling for multiple comparisons using the benjamini and hochberg method (i.e., q-values) are also shown. the shaded cells indicate nonsignificant p-and q-values (i.e., ≥ . ). ‡ the adjusted analyses were conducted using multivariable linear regression. the β coefficients and their corresponding % lower and upper cis are shown. model included the infant's age at the time of the rsv ari, sex, and race and ethnicity as covariates. model included the infant's age at the time of the rsv ari, sex, and maternal asthma as covariates. the β coefficient can be interpreted as the increase in that local immune mediator's generalized-log-transformed mfi per % increase in the nasopharyngeal relative abundance of haemophilus during an rsv ari after controlling for the model covariates. only the estimates for local immune mediators with q-values < . in unadjusted analyses and that remained significant in adjusted analyses are shown. definition of abbreviations: ari = acute respiratory infection, ci = confidence interval, mfi = median fluorescence intensity, rsv = respiratory syncytial virus. objectives, design and enrollment results from the infant susceptibility to pulmonary infections and asthma following rsv exposure study (inspire) evidence of a causal role of winter virus infection during infancy in early childhood asthma the burden of respiratory syncytial virus infection in young children research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy associated with increased childhood asthma risk following respiratory syncytial virus infection in infancy respiratory infection nasal immune-response patterns and their association with subsequent childhood recurrent wheeze application of taqman low-density arrays for simultaneous detection of multiple respiratory pathogens real- time reverse transcription-polymerase chain reaction assay for sars-associated coronavirus minimally invasive sampling method identifies differences in taxonomic richness of nasal microbiomes in young infants associated with mode of delivery bei resources: supporting antiviral research high-resolution sample inference from illumina amplicon data silva: a comprehensive online resource for quality checked and aligned ribosomal rna sequence data compatible with arb simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data blast: at the core of a powerful and diverse set of sequence analysis tools fastq screen: a tool for multi-genome mapping and quality control trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform initial sequencing and analysis of the human genome accurate read-based metagenome characterization using a hierarchical suite of unique signatures key: cord- - eqdlef authors: schaller, matthew; hogaboam, cory m.; lukacs, nicholas; kunkel, steven l. title: respiratory viral infections drive chemokine expression and exacerbate the asthmatic response date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: eqdlef a number of investigations have linked respiratory vial infections and the intensity and subsequent exacerbation of asthma through host response mechanisms. for example, it is likely that the immune-inflammatory response to respiratory syncytial virus can cause a predisposition toward an intense inflammatory reaction associated with asthma, and adenovirus might cause exacerbation of the immune response associated with chronic obstructive pulmonary disease. in each of these situations, the host's immune response plays a critical mechanistic role through the production of certain cytokines and chemokines. specific aspects of these augmented immune responses are determined by the biology of the virus, the genetic variability of the host, and the cytokine-chemokine phenotype of the involved tissue. for instance, the type /type cytokine ratio in the airways during infection with rhinovirus determines how long the viral infection endures. by this same theory, it has been demonstrated that chemokine levels produced during respiratory syncytial virus infection determine host responses to later immune stimuli in the lung, with the potential to augment the asthmatic response. further research in this area will clarify cytokines, chemokines, or cell targets, which will provide the basis for next-generation therapies. increasing clinical evidence supports the concept that certain respiratory viral infections play an important mechanistic role in the initiation of acute lung pathology, as well as set the foundation for longer-term chronic effects that were initiated during the original virus-host interaction. a number of clinical epidemiology studies have identified that the exposure to respiratory syncytial virus (rsv) during early childhood can provide the underpinnings for the development of chronic asthma in later childhood. furthermore, respiratory viruses, such as rsv and rhinovirus, appear to be intimately linked to exacerbations in the physiologic and immunologic intensity of an asthmatic response in many individuals. these exacerbations are associated with increased airway hyperresponsiveness and a significant influx of leukocytes into the lungs. the intensity of the inflammatory response has been directly correlated to the expression of chemokines by virally infected pulmonary structural cells, resident immune cells, and infiltrating leukocytes. in the clinical arena investigations have incriminated a number of chemokines, including cxcl (il- ), ccl (macrophage inflammatory protein a), and ccl (rantes), as major mediators released during respiratory viral infection, and the level of these chemokines correlates with the severity of disease. these studies collectively indicate an intimate connection between chemokine expression and respiratory viral infections. they support the growing notion that exposure to infectious agents in early life profoundly influences subsequent immune events that might facilitate the development of severe chronic obstructive airway disease. it is becoming clear that not all respiratory viruses induce the same lung pathology; instead, they are responsible for disparate immune responses. clinical studies examining patients infected with respiratory viruses have found that rsv, rhinovirus, and adenovirus cause airway obstruction and in some cases cause decreased forced expiratory volume readings. interestingly, this correlation is not present in patients infected with the influenza virus. additionally, studies in mouse models have shown that although rsv infection causes the production of (t h ) cytokines, as measured on the basis of mrna, this is not the case for influenza. one experimental mouse model examining the role between influenza and allergy has determined that influenza infection can decrease the allergic response, although the mechanism is not clear. these studies are supported by clinical investigations that demonstrate that a relatively low number of influenza cases, versus rsv or rhinovirus infections, actually cause exacerbation of asthma and acute bronchiolitis in adults. [ ] [ ] [ ] some of the differences in the end physiologic response to viral infection likely come from the differences in the profile of inflammatory mediators that are produced in response to each virus. one particular difference in the host response to infection with rsv or rhinovirus is the chemokine profile that each infection elicits. although there are often cellular similarities in the response to infection, there are clear differences in the chemokines that are present, the levels of those chemokines expressed, and the presence of the predominant chemokine-generating cell types. chemokines, or chemotactic cytokines, were first described for their ability to attract leukocytes to sites of inflammation, a phenomenon known as chemotaxis. however, data now support a varied and eclectic role for these cytokines during immune-inflammatory processes. chemokines are produced by a variety of cells, including stromal cells, epithelial cells, and all immune cells. these molecules are known to have functions other than chemotaxis, including regulation of inflammation, cellular proliferation, mucus production, tumorigenesis, and angiogenesis. in addition, various chemokines have been found to be important in the initiation and maintenance of the host's response to pathogens. several studies have examined the differences in the host response to respiratory viral infection in human subjects. although none of these studies have come up with a single cytokine or chemokine that can account for the differences in physiologic and immunologic responses that occur on infection, several interesting findings have been published. although many studies have looked at the production of ccl and ccl in vitro and in vivo, there is no difference in the level of these ligands found in the airways of patients infected with influenza or rsv. , however, one report demonstrated that in patients infected with either rsv or influenza, higher levels of ccl protein levels in the bronchoalveolar lavage (bal) fluid correlated with hypoxic bronchiolitis, which was not the case for ccl . another study found that patients infected with rsv have higher serum levels of ccl , soluble intercellular adhesion molecule, il- , il- , and ige than patients infected with influenza. this latter study suggests the importance of examining additional compartments for cytokine and chemokine levels and not only the lung when studying respiratory viral infections. cxcl (il- ) is the most studied chemokine and appears to correlate with the severity of respiratory disease. some differences have been found in cxcl production in patients infected with respiratory viruses. a comparative study examining the effects of respiratory viruses on cxcl production found that although levels of cxcl protein in the nasal lavage fluid (nlf) of influenza virusand rhinovirus-infected patients correlated with a higher symptom score, this was not the case for rsv-infected patients. interestingly, in this same study the rsv-infected group had the greatest incidence of wheezing, an indicative correlate for the development of asthma. another group found that influenza infection was associated with higher cxcl levels in nasal washes than rsv. additionally, the peak production of cxcl is different when comparing infections. in experimental rsv infection of human subjects, there is an initial spike of cxcl protein levels in nlf on day , with a more significant peak production occurring between days and of infection. however, during influenza infection, cxcl protein in nlf peaks between days and and is back to baseline by day . rhinovirus infection mimics the cxcl secretion pattern seen in influenza infection, with the peak production occurring between days and and dropping off by day . these data clearly demonstrate that the biology between the viruses is different, which might account for some of the differences in inflammation and pathophysiology of the different pathogens. differences have also been found in the levels of some nonchemotactic cytokines when comparing respiratory viruses, specifically influenza and rsv. one study found that il- levels were higher in the nlf of rsv-infected patients than in uninfected control subjects, which was not the case for influenza-infected patients. an additional difference between the groups was that although levels of il- were increased in asthmatic subjects infected with rsv, this was not the case for influenza-infected asthmatic patients. experimental infection of human subjects with viruses has yielded additional valuable data on the duration of infection and peak of chemokine and cytokine production. although a direct comparative study has not been conducted in experimental infection, it is clear that rsv infection lasts longer than influenza or rhinovirus infection. rsv is detected in nasal washes between days and , whereas influenza and rhinovirus titers peak at day and disappear by day . , , in contrast, adenovirus might be able to establish a latent infection that is correlated with chronic obstructive pulmonary disease (copd). thus the phenotypic cytokine and chemokine production profiles and disease severity are likely affected by the ability of the virus to propagate. infecting cells in vitro and measuring chemokine production has been beneficial in understanding which chemokines are relevant during viral infection. although several chemokines, such as ccl , ccl , ccl , and cxcl , are consistently found to be produced in cell culture systems upon viral infection, microarray data from rsv-infected epithelial cells suggests that other chemokines might also play a role in the response to viral infection. interestingly, there seems to be some disparity in the chemokines produced during a given viral infection. for example, pbmcs infected with influenza do not produce cxcl , [ ] [ ] [ ] [ ] and epithelial cells infected with adenovirus do not produce ccl (summarized in fig ) . , , chemokines and their receptors might also play a more direct role in the outcome of viral infection than cell recruitment or activation. for example, in vitro ccl production has been shown to inhibit binding of rsv to cells. additionally, the rsv g protein was shown to mimic the ligand for cx cr and might inhibit t cells from migrating to the lung during rsv infection. although the host response to viral infection differs depending on the infectious agent, perhaps the most interesting and clinically relevant differences occur well after the infection has taken place. for example, many have speculated that rsv infection can result in childhood asthma, and research has been published to suggest this hypothesis might be true. other research suggests that adenovirus infection might predispose children to chronic obstructive brochitis. another report implicated that exacerbation of copd might be the result of latent adenoviral infection. it is clear that some viruses can have a lasting effect on the structure and function of the lung as shown in fig . interestingly, no research studies to date draw a correlation between influenza infection and the later development of respiratory disorders. one study did find that the percentage of influenza-infected children affected by bronchiolitis, which is thought be a marker for wheezing later in life, was only %. the most powerful research suggesting that rsv infection predisposes children to later asthma followed the same cohort of children for years. one group of these children was hospitalized as infants for rsvinduced bronchiolitis, whereas the control group did not have an rsv infection during infancy. the studies show that children infected with rsv have increased wheezing and allergies when compared with control subjects. , analyzing the cytokine secretion of t cells from these individuals in response to a panel of aeroallergens revealed that t cells from children hospitalized for rsv in their infancy secreted more il- in response to aeroallergens than control subjects. another publication demonstrated that although rsv infection at a young age leads to a significant increase in wheeze up to age years, the incidence of wheeze in rsv-infected individuals decreases by age years. two other studies found that although there was no correlation between rsv infection in infancy and clinically diagnosed asthma later in life, children infected with rsv had impaired lung function when compared with control subjects. , these investigators argue that rsv does not cause skewing of the immune system, but rather those children affected by rsv infection have preexisting lung abnormalities. these same abnormalities cause decreased lung function later in life. in accordance with this research, another study followed a cohort of children admitted to a hospital for acute bronchiolitis, the cause of which was not determined. nine years after admission, children in the index group had a higher incidence of asthma when compared with control subjects. further research will clarify whether viral infection is necessary for predisposition to asthma or whether it merely uncovers abnormalities in individuals who are already predisposed to have asthmatic responses. murine studies have also yielded conflicting results in regard to the ability of viral infection to enhance subsequent allergic responses. although some research suggests that rsv infection occurring during the allergen sensitization phase prolongs airway hyperreactivity and increases inflammation and mucus production in the lungs, other reports suggest that rsv given before allergen sensitization reduces airway hyperreactivity, eosinophilia, and il- production. another study with repeated rsv infections before and during allergen challenge demonstrated a decrease in mucus-producing cells and alveolitis, although infection did not alter lymphocytic infiltration into the lungs. this research suggests that the timing of rsv infection is critical in determining how the immune system responds to subsequent stimuli. if applied clinically, these data could determine which rsv-infected patients receive treatment. several studies in which mice were infected with rsv and then sensitized to allergen suggest ccl might be important in predisposing virally infected mice to more severe allergies. in these studies rsv infection was initiated days before allergen sensitization. in control mice previous rsv infection increased airway hyperreactivity and the level of chemokines in the lungs. blocking il- during rsv infection reduced the levels of chemokines, as well as airway hyperreactivity, in the lungs of mice that were subsequently sensitized and challenged with allergen. when ccl was blocked during the course of rsv infection, subsequent allergen sensitization and challenge was reduced to the phenotype of allergen-sensitized uninfected mice. this was correlated to a reduced level of leukotriene production. additionally, the absence of ccr in this model caused a reduction in airway hyperreactivity and mucus, which was accompanied by a reduced amount of il- in the lungs, as well as reduced numbers of t cells and eosinophils. these studies implicate that ccl and other ccr ligands might be important in setting up the immune system in the lung to respond inappropriately to allergens after rsv infection. fig shows some of the chemokines induced during rsv infection and the cell types on which the receptors for these chemokines are present. many viruses have been implicated in the exacerbation of allergic asthma, including influenza, rhinovirus, rsv, adenovirus, and coronaviruses. experimental infection of human subjects with rhinovirus demonstrated that exacerbation was associated with increased airway hyperreactivity in response to allergen. interestingly, rsv appears to cause the most severe exacerbations, whereas rhinovirus appears to be responsible for the majority of exacerbations. , for this reason, the bulk of human studies have examined the role of rhinovirus in exacerbation of allergic responses. the exacerbation of allergic asthma by viruses seems to be correlated with both ccl and cxcl . there is a definite link between rhinovirus infection in exacerbation of asthma and increased cxcl levels. one report found that cxcl levels correlated with severity of symptoms in asthmatic subjects who had rhinovirus infection, which mimics what is seen in subjects infected with rhinovirus alone. not surprisingly, there was also a correlation between cxcl levels and neutrophils found in the airway. this report found no differences in airway hyperreactivity of asthmatic subjects on rhinovirus infection, which is in contrast to other published studies, but did find an increased sensitivity to histamine challenge. another investigation examining the different cell types recruited to the respiratory tract during the common cold found a difference in the number of mast cells present in allergic patients versus those seen in nonallergic patients on viral infection. this result could account for the increased sensitivity to histamine that was observed by van benten et al. this report also found that the number of ccl -and ccl -producing cells increased on viral infection in both allergic and nonallergic individuals. the investigators also observed an increase in almost every type of inflammatory cell, including eosinophils, t cells, macrophages, and neutrophils, regardless of allergen sensitization. other research has demonstrated a correlation between ifn-g levels and symptom scores in rhinovirus-infected asthmatic subjects. those patients with a higher ratio of ifn-g/il- mrna in the airways had less severe symptom scores. interestingly, these subjects also had no detectable virus days after infection, whereas those patients with a lower ifn-g/il- mrna ratio had worse symptoms and detectable virus at days after infection. the implications of this latter study are that the virus itself might not cause the exacerbation of asthma, but rather the individual's host response to the virus could be responsible. in this case perhaps higher levels of il- or lower levels of ifn-g are responsible for delayed viral clearance. the mechanism could be due to recruitment of inappropriate cells or unnecessary regulation of cells already recruited. although these results suggest that there are few differences in the types of inflammatory processes that occur in allergic individuals infected with a respiratory virus when compared with nonallergic individuals, it is likely that exacerbation of allergic asthma by viral infection still exists. one criterion that might cause the exacerbation of asthma appears to be the presence of allergen at the time of viral infection. if both viral infection and allergen exposure do not occur at the same time, then the increase in inflammation caused by the virus has a limited ability to exacerbate an allergic response. confirmation of this hypothesis stems from a report demonstrating that there was a higher risk of hospital admission for exacerbation of asthma in patients who were both infected with a respiratory virus and exposed to allergen at the same time. another study in which allergic individuals were infected week after allergen exposure reported that subjects infected with rhinovirus had no differences in airway hyperreactivity when compared with infected nonallergic individuals. this provides further evidence that viral infection and allergen exposure need to be concurrent for exacerbation to occur. however, concomitant exposure to allergen and virus is likely not the only cause of asthma exacerbation because nonallergic asthmatic subjects can also experience periods of increased wheezing. a number of studies have been done in murine models to examine the role of viral infection in allergen-sensitized mice. these models sensitize mice to an allergen and then infect the mice with virus before allergen challenge. although several models have proved that influenza and rsv can increase inflammation in the lungs of allergic mice on infection and augment the allergic response, other studies have actually proved the opposite. for example, one study shows that influenza infection actually inhibits the recruitment of t h cells to the bal fluid of allergic mice. a report comparing the effects of several viruses on animals previously sensitized to allergen showed that although both influenza and rsv induced similar patterns of inflammation, influenza downregulated the expression of type cytokines in the lung. it is possible that some of the changes in inflammation that take place on respiratory viral infection of allergic mice are due to differences in chemokine expression. for instance, rsv infection increased levels of ccl , ccl , and ccl in the lungs of allergic mice (schaller and lukacs, unpublished data). the increase in chemokines could cause an increase in t cells to the lungs and bal fluid of allergen-sensitized mice. this has been shown in both an influenza and an rsv model of exacerbated asthma (schaller and lukacs, unpublished data). the research using the influenza model demonstrated that allergen-specific cells can be recruited to the lymph node on viral infection and secrete il- . this suggests a prominent role for t cells during the virally exacerbated response. further evidence supporting the hypothesis that increased t-cell recruitment might be responsible for asthma exacerbation comes from research using an influenza virus expressing the mhc i and mhc ii epitopes of the ovalbumin (ova) peptide. these studies have shown that ova-specific t cells of both the cd and cd subsets can be recruited to the lung and bal fluid nonspecifically, even in the absence of an influenza infection. , viral infection with wild-type influenza increased recruitment of these t cells to the lung and lymph node; however, only the influenza virus expressing ova peptide was able to induce activation and expansion of the ova-specific t-cell population. these studies provide insight into the mechanism of asthma exacerbation. although respiratory viral infection causes increased recruitment of t cells to the lung and lymph node, only some of these t cells are specific for viral antigen. in addition, some of the recruited t cells might be specific for inhaled allergens. if allergen exposure occurs at the time of viral infection, the increase in allergen-specific t cells to the lung would cause an increased allergic response. in addition to an ongoing viral infection, this could cause increased airway hyperreactivity and increased pathology in the respiratory tract. the recruitment of allergen-responsive t cells to the lung and draining lymph nodes has been linked to ccr in a murine model of rsv-induced exacerbation of allergic asthma (schaller and lukacs, unpublished data). studies initiated in our laboratory have demonstrated that rsv infection increases the numbers of both cd and cd t cells in the lungs and lymph nodes of allergic mice. this was correlated with increased inflammatory cytokine levels and increases in the chemokines ccl and ccl . these studies also provide evidence that ccr might be in part responsible for exacerbation because ccr / mice do not have an exacerbated phenotype. ccr / mice exhibited a reduction in recruitment of allergen-specific cd t cells to the lymph node. thus it is possible that ccr is part of the chemokine receptor profile of allergen-specific and virus-specific t cells, and the production of ccr ligands during viral infection could cause the recruitment of t cells to the site of infection and enhance allergic responses. the role of ccr in t cell-mediated allergen exacerbation correlates well with data from multiple laboratories indicating that ccl is one of the most highly expressed chemokines during virally induced disease. , , a subset of memory cd t cells can also express the cxcr receptor and chemotax in response to cxcl . these t cells are activated, expressing high levels of perforin and granzyme, and are more cytotoxic than the larger population of memory cd t cells. interestingly, in patients infected with influenza, a high percentage of cxcr cd t cells are specific for the virus. conclusions taken together, a variety of investigations suggest an important role for chemokines during respiratory viral infection. in cases of viral infection alone, the differences in production of chemokines, such as ccl , ccl , ccl , and cxcl , on infection might cause differences in the host response. these chemokines might also be important in setting up later immune responses in the lung. the way the host responds to infection with different viruses might cause the association of various viral infections with chronic diseases. for example, it is possible that the host response to rsv can cause a predisposition toward asthma and that adenovirus causes exacerbation of copd, thereby leading to do the production of certain cytokines and chemokines. although some of this response is determined by the biology of the virus, genetic variability of the host also plays a role. for instance, the t h /t h cytokine ratio in the airways during infection with rhinovirus determines how long the viral infection endures. by this same theory, it is possible that the amount of ccl produced during rsv infection determines host responses to later immune stimuli in the lung and that the amount of cxcl produced during adenoviral infection correlates with the degree of exacerbation of a patient with copd. further research in this area might clarify what cytokines, chemokines, or cell types need to be targeted to prevent viral infection from influencing other immune responses. although certain viruses might set up later immune responses in the respiratory tract, it is likely that all respiratory viruses are able to exacerbate asthmatic responses in the lung. this is because the production of excess chemokines in the lung will not only recruit virus-specific t cells but also allergen-specific t cells. these allergen-specific cells will augment any allergic response that is already ongoing in the lung. although a previous study has shown that ccr is responsible for this, there are likely other chemokine receptors that are also shared between allergenspecific and virus-specific cells. with the identification of the chemokine receptors responsible for virally induced respiratory diseases, treatments might become more likely as therapies develop that could target cells that express these receptors. although it is clear that many different cell types are responsible for clearing viral infection, perhaps targeting ones that are responsible for much of the pathology could eliminate many of the side effects of viral infection that result in skewing of later immune responses. for example, by targeting ccr cd t cells, but not cxcr cd t cells, viral clearance would not be delayed, and the pathology of viral infection could be reduced. a better understanding of how the host responds to different respiratory viral infections will also contribute significantly to our understanding of immunology in general. for example, how virus biology affects cell signaling is key in understanding how the host later responds to an infection. because there are clear differences in which chemokines are produced on viral infection, this might be a good beginning in our understanding of how different viral infections cause disparate responses in the host. relationships among specific viral pathogens, virus-induced interleukin- , and respiratory symptoms in infancy respiratory syncytial virus, pneumonia virus of mice, and influenza a virus differently affect respiratory allergy in mice influenza a virus infection inhibits the efficient recruitment of th cells into the airways and the development of airway eosinophilia respiratory syncytial virus infection in elderly and high-risk adults the relationship between respiratory syncytial virus infections and the development of wheezing and asthma in children respiratory viruses and exacerbations of asthma in adults macrophage inflammatory protein- alpha and rantes are present in nasal secretions during ongoing upper respiratory tract infection a comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants interleukin- , interleukin- , interleukin- , and interferon-gamma levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza a virus infection chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense rhinovirus stimulation of interleukin- in vivo and in vitro: role of nf-kappab effect of induced interferon in experimental rhinovirus infections in volunteers latent adenoviral infection in the pathogenesis of chronic airways obstruction expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cdna microarrays selective induction of monocyte and not neutrophil-attracting chemokines after influenza a virus infection differential mononuclear leukocyte attracting chemokine production after stimulation with active and inactivated influenza a virus chemokine receptor expression and chemotactic responsiveness of human monocytes after influenza a virus infection influenza a and sendai viruses induce differential chemokine gene expression and transcription factor activation in human macrophages cell-specific expression of rantes, mcp- , and mip- alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus expression of cytokine and chemokine genes by human middle ear epithelial cells induced by influenza a virus and streptococcus pneumoniae opacity variants rhinovirus infection up-regulates eotaxin and eotaxin- expression in bronchial epithelial cells cytokines contribute to airway dysfunction in antigen-challenged guinea pigs: inhibition of airway hyperreactivity, pulmonary eosinophil accumulation, and tumor necrosis factor generation by pretreatment with an interleukin- receptor antagonist the release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid rsv infection of human airway epithelial cells causes production of the beta-chemokine rantes expression of rantes by normal airway epithelial cells after influenza virus a infection proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells adenovirus vector-induced inflammation: capsid-dependent induction of the c-c chemokine rantes requires nf-kappa b rhinovirus replication causes rantes production in primary bronchial epithelial cells human metapneumovirus elicits weak ifn-g memory responses compared with respiratory syncytial virus defense against influenza a virus infection: essential role of the chemokine system airway epithelial cell-induced activation of monocytes and eosinophils in respiratory syncytial viral infection influenza virus a stimulates expression of eotaxin by nasal epithelial cells respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line influenza virus a infection induces interleukin- gene expression in human airway epithelial cells type-specific induction of interleukin- by adenovirus cytokine (tumor necrosis factor, il- , and il- ) production by respiratory syncytial virus-infected human alveolar macrophages il- release from human neutrophils by the respiratory syncytial virus is independent of viral replication rhinoviruses induce interleukin- mrna and protein production in human monocytes inhibition of respiratory syncytial virus infection with the cc chemokine rantes (ccl ) cx c chemokine mimicry by respiratory syncytial virus g glycoprotein respiratory syncytial virus g protein and g protein cx c motif adversely affect cx cr t cell responses persistent adenoviral infection and chronic obstructive bronchitis in children: is there a link? persistent adenoviral infection and chronic airway obstruction in children influenza-a infection in children severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age enhanced il- responses in children with a history of respiratory syncytial virus bronchiolitis in infancy respiratory syncytial virus in early life and risk of wheeze and allergy by age years respiratory morbidity years after rsv infection in infancy wheezing, asthma, and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy respiratory status and allergy nine to years after acute bronchiolitis respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges timing of infection and prior immunization with respiratory syncytial virus (rsv) in rsv-enhanced allergic inflammation respiratory syncytial virus predisposes mice to augmented allergic airway responses via il- -mediated mechanisms respiratory syncytial virus-induced ccl /rantes contributes to exacerbation of allergic airway inflammation respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon ccr -associated immune responses rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood community study of role of viral infections in exacerbations of asthma in - year old children effect of experimental rhinovirus colds on airway hyperresponsiveness to histamine and interleukin- in nasal lavage in asthmatic subjects in vivo prolonged nasal eosinophilia in allergic patients after common cold relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection synergism between allergens and viruses and risk of hospital admission with asthma: case-control study are rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure? allergic vs nonallergic asthma: what makes the difference? allergic airway inflammation is exacerbated during acute influenza infection and correlates with increased allergen presentation and recruitment of allergen-specific t-helper type cells influence of respiratory syncytial virus infection on cytokine and inflammatory responses in allergic mice recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness antigen-specific and non-specific cd ( ) t cell recruitment and proliferation during influenza infection the role of antigen in the localization of naive, acutely activated, and memory cd ( ) t cells to the lung during influenza pneumonia chemokine concentrations in nasal washings of infants with rhinovirus illnesses cutting edge: expression of chemokine receptor cxcr on human effector cd t cells il- responsiveness defines a subset of cd t cells poised to kill key: cord- - oaobtlx authors: lee, pui y.; platt, craig d.; weeks, sabrina; grace, rachael f.; maher, george; gauthier, kasey; devana, sridevi; vitali, sally; randolph, adrienne g.; mcdonald, douglas r.; geha, raif s.; chou, janet title: immune dysregulation and multisystem inflammatory syndrome in children (mis-c) in individuals with haploinsufficiency of socs date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: oaobtlx abstract objectives to identify the mechanisms underlying the development of infection-driven autoimmune cytopenias. background we studied two unrelated patients with immune thrombocytopenia and autoimmune hemolytic anemia in the setting of acute infections. one patient developed multisystem inflammatory syndrome in children (mis-c) in the setting of a severe acute respiratory syndrome coronavirus (sars-cov- ) infection. methods whole exome sequencing was performed on both patients and the impact of the identified variants were validated by functional assays using the patients’ peripheral blood mononuclear cells (pbmcs). results each patient was found to have a unique heterozygous truncation variant in socs . suppressor of cytokine signaling (socs ) is an essential negative regulator of type i and type ii interferon (ifn) signaling. the patients’ pbmcs showed increased levels of stat phosphorylation and a transcriptional signature characterized by increased expression of type i and type ii interferon stimulated genes and pro-apoptotic genes. the enhanced ifn signature exhibited by the patients’ unstimulated pbmcs parallels the hyperinflammatory state associated with mis-c, suggesting the contributions of socs in regulating the inflammatory response characteristic of mis-c. discussion heterozygous loss-of-function socs mutations are associated with enhanced interferon signaling and increased immune cell activation, thereby predisposing to infection-associated autoimmune cytopenias. suppressor of cytokine signaling (socs ) is an essential negative regulator of type i and type ii interferon signaling. of the socs family members, socs binds with highest affinity to the substrate binding pocket of janus kinases (jak) and jak , thereby inhibiting phosphorylation of signal transducer and activator of transcription (stat) and stat . complete deficiency of socs in mice causes perinatal lethality due to type i and type ii interferon (ifn)-driven inflammatory disease that can be reduced by neutralizing antibodies to either type i or type ii ifns or genetic deletion of their respective receptors. , furthermore, socs -haploinsufficient mice exhibit features of systemic lupus erythematosus, indicating the importance of bi-allelic socs expression for self-tolerance. , in a recent study detailing outcomes of whole-genome sequencing for patients with primary immunodeficiency, socs haploinsufficiency was briefly described in two individuals with recurrent bacterial infections and severe multi-systemic autoimmunity. however, the immunologic sequelae of socs haploinsufficiency in humans remain incompletely understood. individuals with evans syndrome (es) present with immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), and/or immune neutropenia arising from either primary or secondary causes. the relative risk of es is significantly higher in children with monogenic disorders of immunity, while es secondary to malignancy is the most common association in adults. additionally, viral and bacterial infections have been identified as triggers of es. in this report, we present two unrelated children with different heterozygous loss-of-function variants in socs , aberrant ifn signaling, and es. one patient developed a sars-cov- infection, precipitating multisystem inflammatory syndrome of children (mis-c), which responded well to treatment with intravenous immunoglobulins (ivig) and corticosteroids. patient presented at five months of age with fever, otitis media, oral ulcers, and developed itp ( x platelets/µl, normal - x platelets/µl; fig. a ). at two years of age, in addition to itp and immune neutropenia, he developed a warm antibody aiha with a hemoglobin of . g/dl (normal . - . g/dl). he was initially treated with corticosteroids and subsequently transitioned to mycophenolate mofetil due to recurrence of aiha with corticosteroid tapering. in addition to immune cytopenias, he had mild cd + t cell lymphopenia and a predominance of naïve igd + cd -b cells ( has not been previously reported in the genome aggregation database (gnomad). the mutation arose de novo as it was absent in both parents. patient was diagnosed with itp at years of age when he presented with diffuse petechiae and a platelet count of x cells/µl (normal - x cells/µl; fig. c ). over the course of several years, he developed a weakly positive direct antiglobulin test indicative of erythrocyte autoantibodies without evidence of hemolysis. he subsequently developed neutropenia and lymphopenia (fig. c) . his thrombocytopenia improved with the thrombopoietin receptor agonist eltrombopag and mycophenolate mofetil, but he had persistent neutropenia and lymphopenia with reduced numbers of cd + and cd + t cells and a predominance of naïve igd + cd + b cells ( d ). this variant is also absent from the gnomad database. in march , at the age of years, patient presented with shock-like physiology after having two days of fever, recurrent emesis, diarrhea, and dehydration. upon presentation, he was tachycardic and hypoxic with an oxygen saturation of %. chest radiography showed no evidence of acute pneumonia. laboratory evaluation revealed metabolic acidosis with a bicarbonate level of mmol/l (normal - mmol/l) and increased lactate level of mmol/l (normal . - mmol/l). he had a warm igg autoimmune hemolytic anemia, cd + and cd + leukopenia, and thrombocytopenia ( table ) . he had ongoing hemolysis, as evidenced by his hemoglobin of . g/dl, an elevated lactate dehydrogenase level of , units/l (normal - units/l), undetectable haptoglobin, and indirect hyperbilirubinemia (indirect bilirubin . mg/dl; normal - . mg/dl). his c-reactive protein was elevated at . mg/l (normal < . mg/l), as was his procalcitonin ( . mg/dl, normal < . mg/ml). his prothrombin time was elevated at . sec (normal - . sec). nasopharyngeal swab was positive for sars-cov- by rt-pcr. even after receiving methylprednisolone ( mg/kg every hrs) for one day, he had a markedly elevated soluble il- receptor level ( , pg/ml) that was increased from his prior level of , pg/ml (normal - pg/ml), reflecting widespread t cell activation. although his aiha was initially thought to be the most prominent feature of his sars-cov- infection, his history of fever, involvement of the gastrointestinal, hematologic, and respiratory systems, and elevated inflammatory markers were consistent with the subsequently described we assessed expression of isgs in the patients' unstimulated pbmcs. notably, patient had been treated with prednisone and mycophenolic acid for over two months at the time of this monocytes from both patients compared to healthy controls (fig. c) . in contrast, the expression of cd , which is not regulated by interferon signaling, was similar between patients and controls (fig. c) . consistent with these findings, qpcr of unstimulated pbmcs from both patients showed increased expression of two additional isgs, ifi and isg , compared to six healthy controls (fig. d) . the specificity of these observations was supported by both patients' fcgr a fcgr b fcgr c fi s a s a s lgals bp gca gbp pygl ankrd ifi ifit ifit ifit ifi isg epsti ifit rsad ifi ly e cxcl cecr ankrd fcgr a s a s a gbp gca s a lgals bp pygl pglyrp cxcl ly e epsti ifit ifi ifit ifi l ifit ifi ifi isg ifit chronic obstructive pulmonary disease rsv, respiratory syncytial virus mgus, monoclonal gammapathy of unknown significance evans syndrome in a patient with covid- simultaneous onset of covid- and autoimmune haemolytic anaemia cold agglutinin autoimmune haemolytic anaemia associated with novel coronavirus (covid- ) covid- infection associated with autoimmune hemolytic anemia autoimmune haemolytic anaemia associated with covid- infection j o u r n a l p r e -p r o o f lopez et al. zagorski et al. capes et al. lazarian et al. age ( lopez et al. zagorski et al. capes et al. lazarian et al. age ( key: cord- -veavzt d authors: ueland, thor; heggelund, lars; lind, andreas; holten, aleksander r.; tonby, kristian; michelsen, annika e.; jenum, synne; jørgensen, marthe j.; barratt-due, andreas; skeie, linda g.; nordøy, ingvild; aanensen fraz, mai sasaki; quist-paulsen e, else; pischke, søren e.; johal, simreen k.; hesstvedt, liv; bogen, mette; fevang, børre; halvorsen, bente; müller, fredrik; bekken, gry kloumann; mollnes, tom e.; dudman, susanne; aukrust, pål; dyrhol-riise, anne m.; holter, jan c. title: elevated plasma stim- levels in severe covid- patients date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: veavzt d background the pathogenesis of covid- is still incompletely understood, but seems to involve immune activation and immune dysregulation. objective we examined parameters of activation of different leukocyte subsets in covid- infected patients in relation to disease severity. methods we analyzed plasma levels of myeloperoxidase (mpo, neutrophil activation), soluble (s) cd and soluble t cell immunoglobulin mucin domain- (stim- ) (markers of t cell activation and exhaustion) and scd and scd (markers of monocyte/macrophage activation) in covid- infected patients at hospital admission and two additional times during the first days in relation to the need for icu treatment. results our major findings were: (i) severe clinical outcome (icu) was associated with high plasma levels stim- and mpo suggesting activated and potentially exhausted t cells and activated neutrophils, respectively. (ii) in contrast, scd and scd showed no association with need for icu treatment. (iii) scd , stim- and mpo were inversely correlated with the degree of respiratory failure as assessed by p/f ratio and positively correlated with the cardiac marker n-terminal pro-b-type natriuretic peptide. conclusion our findings suggest that neutrophil activation and in particular activated t cells may play an important role in the pathogenesis of covid- infection, suggesting that t cell targeted treatment options and downregulation of neutrophil activation could be of importance in this disorder. patients with severe disease, as compared to those with mild to moderate disease [ ] [ ] [ ] . there are also reports of lymphopenia, and in particular t cell lymphopenia, in those with severe disease , . however, so far the role of the different leukocyte subsets in the pathogenesis of covid- infection has not been fully elucidated. in the present study we examined plasma parameters of activation of different j o u r n a l p r e -p r o o f informed consents were obtained from all patients or next-of-kin if patients were incapacitated of giving consent. the study was approved by the south-eastern norway regional health authority (reference number: ). outcome was defined as the need for treatment at the icu during hospitalization. indication for admission to icu was respiratory failure that required mechanical ventilation support or non-invasive ventilation support that could not be given at the hospital ward. peripheral blood was collected with ml vacutainer ® (bd biosciences, san diego, ca) with edta as anti-coagulant. samples were immediately stored on ice, processed within minutes and plasma was isolated by centrifugation at g for minutes at °c to obtain platelet-poor plasma. plasma were immediately stored at - c in several aliquots until analysis. samples were thawed only once. plasma levels of scd , scd , scd , stim- and mpo were measured in duplicate by cardiac markers and hscrp were measured at department of medical biochemistry at the two centers: ouh: ntprobnp and hscrp were analyzed on cobas (roche diagnostics, basel, switzerland). drammen hospital: hscrp was analyzed on alinity/architect ci (abbott, abbott park, il) and ntprobnp on a cobas e (roche). reference values for nt-probnp: women: < years (y) ≥ ng/l; - y ≥ ng/l; ≥ y ≥ ng/l, men: < y ≥ ng/l; - y ≥ ng/l; ≥ y ≥ ng/l. patient characteristics were compared using student's t-test or chi-square for continuous and categorical variables, respectively (table and (table ) . correlation analysis between selected markers was also performed at individual time-points (pearson, figure ). to limit multiple comparisons, post-hoc testing was only performed on variables where outcome or outcome*time interaction was significant using multivariate regression at each time-point with egfr and age as covariate ( figure ). in addition, we multiplied the pvalues with the number of leukocyte markers assessed, i.e. with five in the overall univariate and partial correlation analysis ( figure and table ) and with three in the post-hoc and individual correlation analysis (figure and ). p-values are two-sided and considered significant when < . . spss release . . . was used for statistical analysis. the sponsor of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and takes final responsibility for the decision to submit for publication. table . of the covid- patients, were admitted to icu (death n= , due to respiratory failure, due to multi-organ failure related to . the icu patients were characterized by low p/f ratio and lymphocyte counts and high nt-probnp and hscrp levels. no significant differences in age, time from symptoms to admission, major comorbidities or egfr were detected in relation to icu ( table ) . the temporal course of leukocyte markers in relation to icu admission (icu n= ; non-icu n= ) is shown in figure . patients admitted to icu were characterized by high levels of the t cell activation marker scd and stim- in à priori analysis, and for stim- also in posthoc testing. moreover, whereas scd displayed stable levels between samples obtained at - and - days, stim- levels increased until - days in the icu group. finally, scd and stim- levels were markedly higher than control levels at all time-points. the neutrophil marker mpo was significantly higher in the icu group compared with the non-icu group at all time points (figure ). mpo levels in the non-icu patients were comparable to control levels. however, in contrast to the t cell markers, mpo in the icu patients decreased during follow-up. in contrast to the t cell markers and mpo, plasma levels of scd and scd , reflecting activation of monocytes/macrophages, were similar in icu and non-icu patients ( figure ). for the above analysis of outcomes, age and egfr were included as covariates when comparing time-points, but excluding these covariates gave similar results. table shows correlations between leukocyte activation markers and p/f ratio as a marker of respiratory function and nt-probnp as a marker of cardiac involvement during the course of the study. scd , stim- and mpo were negatively correlated with the p/f ratio and positively correlated with nt-probnp. notably, these correlations were consistent with a similar pattern at all time-points, in particular for stim- ( figure whereas mpo levels were positively correlated with neutrophil counts (table ) , none of the t cell markers correlated with lymphocyte counts, of which t cells is the dominating cell subset, suggesting that the increased levels of scd and stim- do not merely reflect altered numbers of t cells. all markers were correlated with kidney function, but importantly, the associations of scd , stim- and mpo with icu admission was seen also after adjustment for egfr. finally, the two markers of t cell activation, scd and stim- , were strongly correlated (r= . , p< . ). with severe clinical outcome, characterized by high levels of inflammatory cytokines and low lymphocyte counts [ ] [ ] [ ] . several studies demonstrate that lymphopenia, and in particular low number of t cells, is an important feature of covid- infection , , , , . we show that despite signs of t cell depletion, severe covid- infection as reflected by icu admission is also characterized by raised levels of stim- suggesting activated and potentially exhausted t cells. there are a few reports of elevated stim- levels in infectious disorders such as hiv , hepatitis b and c , malaria falciparum and pulmonary tuberculosis . this is, however, to the best of our knowledge, the first report on stim- finding may suggest that excessive t cell and neutrophil activation also could be involved. increasing evidence indicate that covid- infected patients are at higher risk of developing cardiac involvement or cv related death , and accordingly, a majority of icu patients had elevated nt-probnp levels in our study. the spectrum of cardiac involvement in covid- infection is ranging from type and type myocardial infarction to myocarditis, and in more general terms, t cells are known to participate in these conditions , the outbreak of coronavirus disease (covid- )-an emerging global health threat hospital surge capacity in a tertiary emergency referral centre during the covid- outbreak in italy a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov- spike glycoprotein the pivotal link between ace deficiency and sars-cov- infection clinical characteristics of coronavirus disease in china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study complex immune dysregulation in covid- patients with severe respiratory failure clinical features of patients infected with novel coronavirus in wuhan, china down-regulated gene expression spectrum and immune responses changed during the disease progression in covid- patients retrospective analysis of laboratory testing in patients with severe-or critical-type novel coronavirus pneumonia acute myocardial injury is common in patients with covid- and impairs their prognosis soluble plasma programmed death (pd- ) and tim- in primary hiv infection tim- as a marker of exhaustion in cd (+) t cells of active chronic hepatitis b patients soluble t-cell immunoglobulin mucin domain- is associated with hepatitis c virus coinfection and low-grade inflammation during chronic human immunodeficiency virus infection soluble markers of neutrophil, t-cell and monocyte activation are associated with disease severity and parasitemia in falciparum malaria elevated expression of tim- on cd t cells correlates with disease severity of pulmonary tuberculosis elevated serum levels of checkpoint molecules in patients with adult still's disease soluble plasma programmed death (pd- ) and tim- in primary hiv infection reduction and functional exhaustion of t cells in patients with coronavirus disease (covid- ) t-cell exhaustion in chronic infections: reversing the state of exhaustion and reinvigorating optimal protective immune responses t cells and viral persistence: lessons from diverse infections neutrophil extracellular traps in covid- cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) role of t-cells in myocardial infarction restricted usage of t cell receptor v alpha-v beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy patients (n= ) women, n (%) ( ) ( ) age, years ± ± time from symptoms, days - . ± . caucasian, n (%) ( ) ( ) current smoker, n (%) ( ) ( ) p/f ratio (kpa) - ± need for oxygen therapy, n (%) - ( ) comorbidities cardiovascular, n (%) ( ) ( ) pulmonary, n (%) ( ) ( ) renal, n (%) ( ) ( )liver, n (%)obesity, n (%) ( ) ( ) diabetes, n (%) ( ) ( ) rheumatic, n (%) ( ) ( ) j o u r n a l p r e -p r o o f key: cord- -u aa authors: kainulainen, leena; vuorinen, tytti; rantakokko-jalava, kaisu; Österback, riikka; ruuskanen, olli title: recurrent and persistent respiratory tract viral infections in patients with primary hypogammaglobulinemia date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: u aa background: the occurrence of respiratory tract viral infections in patients with primary hypogammaglobulinemia has not been studied. objective: we conducted a prospective -month follow-up study of respiratory tract infections in adult patients with primary hypogammaglobulinemia. methods: nasal swab samples and induced sputum samples were taken at the onset of acute respiratory tract infection and every months thereafter. samples were tested for bacteria and viruses. pcr tests were performed for respiratory tract viruses. in case the results for rhinovirus were positive, follow-up nasal swab samples were taken every weeks until rhinoviral pcr results became negative. patients completed symptom diaries, which were collected every month. the spouses of the patients served as healthy control subjects. results: during the -month period, the patients had episodes of acute respiratory tract infections, and the spouses had acute episodes (p < . ). respiratory tract viruses were found in sputum in % of the infections. rhinovirus was the most common virus. in more than half of our patients, rhinoviral pcr results stayed positive for more than months. the most long-acting persistence with the same rhinovirus was months. conclusions: despite adequate immunoglobulin replacement therapy, patients with primary hypogammaglobulinemia have increased susceptibility to respiratory tract viral infections. rhinoviral infections are frequent and prolonged. patients with primary hypogammaglobulinemia typically experience recurrent bacterial infections. it is generally considered that patients with hypogammaglobulinemia are not more prone to viral infections than immunocompetent subjects. enteroviruses, however, are an exception. systemic enterovirus infections (echoviruses, coxsackieviruses, and vaccine-related polioviruses) have caused severe morbidity and high mortality rates in patients with hypogammaglobulinemia. , fatal enterovirus-induced meningoenchephalitis has been described in case reports in patients with primary x-linked agammaglobulinemia (xla). however, chronic enterovirus-induced meningitis has become less common in recent years since the use of higher immunoglobulin doses. the occurrence of other picornaviruses, such as rhinoviral infections in patients with primary hypogammaglobulinemia, is not known. thus far, respiratory tract viral infections have not been studied in patients with primary hypogammaglobulinemia. using modern diagnostic techniques, we wanted to study the occurrence of respiratory tract infections, especially viral infections, in patients with primary hypogammaglobulinemia who were receiving regular immunoglobulin replacement therapy. twelve adult patients with primary hypogammaglobulinemia participated in the study (table i) . two patients had xla, and had common variable immunodeficiency (cvid). their ages ranged from to years (median, . years). cvid was characterized by decreased serum immunoglobulin levels (> sds less than the age-adjusted mean), defective in vitro antibody formation, and exclusion of other known causes of humoral immune defects. the diagnosis of xlawas based on early-onset, very low serum immunoglobulin concentrations; male sex; and a lack of circulating mature b lymphocytes in the peripheral blood and mutation analysis of the bruton tyrosine kinase (btk) gene. all patients were receiving regular immunoglobulin replacement therapy (dose, - mg/kg/mo). the trough serum igg concentration was greater than g/l in all patients; patients had serum igg levels of . g/l or greater. no patients were receiving prophylactic antibiotic treatment at the beginning of the study. all patients were nonsmokers. eleven spouses of the patients served as healthy control subjects. in families there were school-aged children. one of the patients was a widow and lived alone. respiratory tract infections. the study was carried out at turku university hospital, finland. informed consent was obtained from the patients and healthy control subjects, and the study was approved by the joint commission on ethics of the turku university hospital and the university of turku. the primary outcome variable was an acute episode of respiratory tract infection (the common cold) defined on the basis of new symptoms (ie, excessive sneezing, sore throat, nasal discharge, nasal obstruction, and cough). the secondary outcome variable was viral cause of this acute respiratory episode. we searched for viruses: adenovirus; coronaviruses oc , e, hku , and nl ; enteroviruses; human bocavirus; human metapneumovirus; influenza a and b viruses, parainfluenza type , , and viruses; rhinovirus and respiratory syncytial virus (rsv). at the first visit and follow-up visits every months, patients were examined (l.k.). four nasal swabs were taken, and the patients provided induced sputum samples. at the onset of respiratory tract infection, the patients were asked to contact the investigator (l.k.) if they presented with new symptoms of respiratory tract infection. the patient was examined (l.k.) within days. three nasal swabs were taken, and the patients provided an induced sputum sample. enteroviral and rhinoviral pcr, as well as viral and bacterial cultivation, were performed from the swab and sputum specimens. if rhinoviral pcr results were positive in the nasal swab samples, sputum samples, or both, new nasal swabs were taken every weeks until rhinoviral rna results became negative. the research nurse taught the patient to take nasal swabs at home, and swabs were sent in vials by post to the laboratory. if the bacterial culture of the sputum sample was positive, the patient received antibiotic treatment. a new induced sputum sample was taken after the antibiotic treatment. antibiotic treatment was also given in some patients with bacterial culture-negative acute infections. samples from healthy control subjects. if the spouse of the patient had acute symptoms of respiratory tract infection, she or he took nasal swabs at home according to the instructions of the research nurse and sent the vials by post. in cases in which the rhinoviral rna results were positive, new nasal swabs were taken every weeks until they became negative. symptom diary. patients and spouses completed a common symptom diary, recording fever, nasal discharge, cough, sputum amount and color, absenteeism from work, and antibiotic treatments. the diaries were returned monthly and checked by the research nurse. if questions arose, the research nurse called the patient or the spouse. nasal swab. nasal swabs were obtained from the nostrils at a depth of to cm by using a sterile cotton swab. the nasal swab for respiratory tract viral pcr was inserted into a dry vial. the nasal swab for viral culture was inserted into a vial containing . ml of viral transport medium ( % tryptose phosphate broth, . % bsa, and antibiotics in pbs). the nasal swab for the bacterial culture was inserted into a culture medium vial. induced sputum sample. sputum production was induced by means of inhalation of % hypertonic saline solution according to the method of zar et al. the quality of the sputum sample was assessed by means of light microscopy. the samples were classified in categories, of which classes to (ie, those with > leukocytes and < squamous cells per field) were further processed. the samples were cultured on standard media, and potential respiratory tract pathogens were identified and tested for antimicrobial susceptibility. viral analysis. nasal swab samples were analyzed immediately for viral cultivation and pcr analysis for enteroviruses and rhinovirus. sputum samples for viral cultivation were stored at c until analysis. viral cultivation from nasal swabs and induced sputum specimens was performed according to standard roller tube methods in llc-mk , hela ohio, and a cells and in human foreskin fibroblasts. nasal swabs were eluted by vortexing with ml of pbs. nucleic acids were extracted from -ml aliquots of the specimens with the qiaamp minelute virus spin kit (qiagen, hilden, germany), according to the manufacturer's instructions, and from -ml induced sputum specimens with the nuclisens easymag automated extractor (biomerieux, boxtel, the netherlands) with an elution volume of up to ml. extracts from nasal swabs and sputum specimens were analyzed for enteroviruses, rhinovirus, and rsv according to standard pcr protocols used in the virus diagnostic laboratory of the department of virology, turku university. extracts from the sputum specimens were also analyzed for adenovirus and human bocavirus by using pcr assays in accordance with standard protocols of the department of virology, turku university, and for human metapneumovirus by using the amplitect quantification kit for metapneumovirus genomes (ame biosciences, toroed, norway). a multiplex pcr assay (the seeplex respiratory virus detection assay; seegene, inc, seoul, korea) was used for detection of respiratory tract viruses (adenovirus, influenza a and b viruses, coronaviruses oc /hku and e/nl , rhinovirus, human metapneumovirus, rsv a and b, and parainfluenza type , , and viruses). rhinovirus-positive cdnas were amplified for sequence analysis, as described earlier. the primers from the noncoding region generate -bp-long amplicons, which were sequenced in the dna sequence service laboratory of the turku center for biotechnology. the partial sequences were aligned with clustalw software. viruses with greater than % sequence homology were considered the same type. the phylogenetic tree was computed by using the jukes-cantor algorithm and the neighbor-joining method. phylogenetic analyses were conducted by using the mega program and the bootstrap consensus tree inferred from replicates. statistical analysis were performed with spss for windows software (version . ; spss, inc, chicago, ill). a paired-samples t test was conducted to compare respiratory tract infection episodes in patients and spouses and to compare respiratory tract viral infections and rhinoviral infections, respectively. (table ii ). in patients the rhinoviral pcr results were positive for or more weeks in nasal swabs taken every weeks. in a patient with xla rhinovirus, pcr results stayed positive for months. the same rhinoviral type, with more than % partial nucleotide sequence homology, was found for months (fig ) . in a patient with cvid, the same rhinoviral type persisted for weeks (score of % similarity). the majority of rhinoviral infections occurred during the fall months (fig ) . adenovirus was found in the sputum samples of patients times. in patient adenovirus was found times during months; (table ii) . the mean number of respiratory tract infections during the -month period per patient was (range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and the mean number of received antibiotic treatments was (range, - ). the most common clinical signs and symptoms of acute respiratory tract infections were cough and rhinitis and increased sputum volume in patients with chronic cough. fever (> . c) was present in of episodes. there were no differences in the symptoms in patients with viral infection or viral-bacterial coinfection (data not shown). the mean duration of symptoms was days. two patients required hospitalization because of pneumonia. results for rhinovirus and adenovirus were positive in sputum before pneumonia in patient. bacteria were not found in the blood or sputum of the patients with pneumonia. six of the spouses had acute respiratory tract infections during the -month period. five had no infections. rhinovirus was found in only ( %) of episodes, and results were negative in a control sample weeks later. parainfluenza viruses were detected in episodes. in family both the patient and the spouse had parainfluenza type viral infections at the same time. there was a significant difference in the number of respiratory tract infection episodes in patients and spouses: patients had . episodes more than the spouses ( % ci, . - . ; p < . ). patients had also significantly more viral infections than the spouses (mean difference, . ; % ci, . - . ; p . ) and more rhinoviral infections (mean difference, . ; % ci, . - . ; p . ), respectively. our prospective -month study has new observations. first, despite adequate immunoglobulin replacement therapy, most patients with primary hypogammaglobulinemia had increased susceptibility to respiratory tract viral infections. second, many patients specifically had recurrent and persistent rhinoviral infections. in this study patients with hypogammaglobulinemia had a median of episodes of respiratory tract infection in a year, which was significantly more than seen in their spouses. it is well established that otherwise healthy adults have annually to episodes of respiratory tract infection. half of the respiratory episodes in our patients were associated with respiratory tract viruses, showing the increased susceptibility to respiratory tract viral infections. there are no previous studies focused on respiratory tract viruses in acute or chronic respiratory tract infections in patients with hypogammaglobulinemia. we have earlier searched for respiratory tract viruses in bronchoalveolar lavage fluid and maxillary sinus lavage samples at the time when patients with hypogammaglobulinemia were free from acute infection. , an interesting observation in this study was that patients with primary hypogammaglobulinemia had recurrent and persistent rhinoviral infections. rhinovirus was the most common pathogen in respiratory tract infections, and results for it were positive in one third of the infections. rhinovirus was found both as a sole pathogen and also together with bacteria. rhinoviruses have been shown to induce proinflammatory response in the lower airways. [ ] [ ] [ ] chronic inflammation might predispose patients to a progression of pulmonary changes, even if the immunoglobulin replacement therapy is adequate. previously, chronic rhinoviral infection with the same virus in the lower respiratory tract has been described in immunocompromised lung transplant recipients. in immunocompetent adults rhinoviral clearance is usually rapid, on average to weeks. , in immunocompetent children rhinoviral shedding has been shown to persist for up to weeks, but prolonged shedding for months has not been documented in immunocompetent subjects. consequently, continuous viral rna detection for months suggests active replication and persistent infection. in more than half of our patients, rhinoviral pcr results were positive for more than months. frequent rhinoviral infections raise the question of the possible role of the absence of secretory iga in the defense against rhinoviruses. our patients also had adenoviral, metapneumoviral, and coronaviral infections. it was of interest that patients had a positive adenoviral pcr result in sputum samples, and in patients it was found more than once. four of the patients with recurrent adenovirus had prominent bronchiectasis. there are several studies showing that persistent or latent adenovirus might contribute to chronic respiratory diseases. , furthermore, in episodes adenovirus was found concomitantly with other viruses or bacteria. in the present study enteroviruses were found on occasions in the respiratory tract, although the initial presumption was that the patients would have recurrent enteroviral infections because it is known that patients with primary immunodeficiency are unusually susceptible to enteroviral infections. chronic enteroviral encephalitis is frequently a fatal complication in patients with agammaglobulinemia. , in a review of cases of chronic enteroviral infections in patients with primary immunodeficiency, enteroviruses were usually isolated from the cerebrospinal fluid, and the major signs and symptoms were neurological. echoviruses were the most common enteroviruses. in only patients were enteroviruses documented in the respiratory tract. a more recent report of patients with xla also showed that enteroviruses were the most common causes in central nervous system infections. several vaccine-and non-vaccine-associated poliomyelitis case reports have been published, and patients with hypogammaglobulinemia have been reported to chronically excrete poliovirus. [ ] [ ] [ ] patients with primary hypogammaglobulinemia appear to have a more severe clinical course of certain viral infections, such as hepatitis c virus, herpes simplex virus, and varicella zoster virus, than immunocompetent subjects. [ ] [ ] [ ] wheat et al studied the prevalence of human herpesvirus infection in patients with cvid and found that patients with granulomatous or lymphocytic interstitial lung disease had a markedly higher prevalence of human herpesvirus than patients without granulomatous/lymphocytic interstitial lung disease. these data further suggest that antiviral competence is not intact in a group of patients with primary hypogammaglobulinemia. acute respiratory tract infections were common even if patients had a trough serum igg concentration of more than . g/l. repeated antibiotic treatments were considered necessary in many patients. this is in agreement with the observations of a recent follow-up study in italy that showed that patients with primary hypogammaglobulinemia tend to have recurrent bacterial bronchitis despite replacement therapy. mixed viral-bacterial coinfection was detected in one fourth of the patients at the onset of acute infection. viral-bacterial infection might induce more severe inflammation in the lower respiratory tract than sole viral or bacterial infection. it has recently been shown that rhinovirus induces impairment of the antibacterial host defense and thereby predisposes the patient to coinfections with bacteria and probably with other viruses and furthermore to development of pulmonary changes. the mechanisms of increased susceptibility to respiratory tract viral infections in hypogammaglobulinemic patients are unclear. in patients with xla, btk is defective. btk is a cytoplasmic nonreceptor tyrosine kinase and a member of the tec family. btk is important in toll-like receptor (tlr) and tlr signalling. both tlr and tlr are important in the activation of host defense against viral and bacterial infections. defective triggering of tlrs leads to impaired production of proinflammatory cytokines, such as tnf-a and il- . tlr recognizes single-stranded rna. tlr recognizes unmethylated cpg motifs that exist in both viral and bacterial dna. patients with xla have impaired il- , which might explain their susceptibility to viral infections. patients with cvid also presented with prolonged persistence of rhinovirus. impaired il- and il- production, as well as tlr activation defects, have been demonstrated in patients with cvid. this might partly explain the persistence of rhinovirus. our study has some limitations that should be taken into account in interpreting the results. the number of patients was small. the spouses did not visit the study clinic. nasal swabs were done based on self-assessment, and their quality might not be comparable with those taken by the research nurse. in addition, we used cotton swabs, and flocked swabs might have provided a better yield. rhinoviral shedding in healthy spouses could not be properly studied because only spouse had rhinoviral infection. furthermore, association of rhinoviral pcr positivity with clinical illness is not yet fully understood. in conclusion, our study shows that in addition to bacterial lower respiratory tract infections, patients with primary hypogammaglobulinemia are prone to recurrent respiratory tract viral infections. especially rhinoviral infections were frequent and prolonged. recurrent and persistent respiratory tract viral infections, often in association with bacteria, most probably predispose patients to chronic pulmonary changes. adequate immunoglobulin replacement therapy is not effective in preventing respiratory tract viral infection, and other kinds of treatments are needed. unfortunately, the availability of antivirals is still very limited. however, our observations suggest that the possibility of respiratory viruses should be investigated by means of pcr more often in patients with hypogammaglobulinemia because viral infection often explains the respiratory signs and symptoms of the patient. infections in patients with common variable immunodeficiency failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man encephalomyelitis in primary hypogammaglobulinemia enteroviral infections in primary immunodeficiency (pid): a survey of morbidity and mortality international union of immunological societies. primary immunodeficiency diseases rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infection sputum induction as a diagnostic tool for community-acquired pneumonia in infants and young children from a high hiv prevalence area epidemiology of viral respiratory infections bacteria and viruses in maxillary sinuses of patients with primary hypogammaglobulinemia viruses and bacteria in bronchial samples from patients with primary hypogammaglobulinemia rhinovirus infects the lower airways relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection the presence of rhinovirus in lower airways of patients with bronchial asthma pulmonary abnormalities in patients with primary hypogammaglobulinemia chronic rhinoviral infection in lung transplant recipients enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increase viral load in the respiratory tract picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season latent adenoviral infection in the pathogenesis of chronic airway obstruction amplification of inflammation in emphysema and its association with latent adenoviral infection persistent and fatal central-nervous-system echo virus infections in patients with agammaglobulinemia chronic enteroviral meningoenchephalitis in agammaglobulinemic patients x-linked agammaglobulinemia: report on a united states registry of patients antigenic analysis of polioviruses isolated from a child with agammaglobulinemia and paralytic poliomyelitis after sabin vaccine administration paralytic poliomyelitis in a child with agammaglobulinemia failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man x-linked agammaglobulinemia: an analysis of patients long-term outcome of chronic hepatitis c virus infection in primary hypogammaglobulinaemia common variable immunodeficiency: clinical and immunological features of patients possible role of human herpes virus in the lymphoproliferative disorders in common variable immunodeficiency longterm follow-up and outcome of a large cohort of patients with common variable immunodeficiency incidence and characteristics of viral community-acquired pneumonia in adults rhinovirus exposure impairs immune responses to bacterial products in alveolar macrophages signalling by toll-like receptors and requires bruton's tyrosine kinase tlr activation is defective in common variable immunodeficiency clinical implications: respiratory tract viruses should be investigated by means of pcr more often in patients with primary hypogammaglobulinemia. bacterial and viral coinfections might predispose patients to chronic pulmonary changes. speirs ultimately gave up on championing the theoretical aspects of his interpretation, which had become contested and controversial. nonetheless, the unique images he had recorded through radioisotope labeling and electron and inventive phase microscopy remain a noteworthy contribution to the archival collections of functional cytology and histopathology. . speirs rs. cells involved in hypersensitivity and immunity. blood ; : - .electron micrograph of eosinophil in close contact with vacuolated mononuclear cell. the membranes appear to be broken in places, with cytoplasm flowing from one cell into the other. a nearby eosinophil granule is undergoing change s (provided by dr r. s. speirs).electron micrograph of rosette of eosinophils attached to macrophage (provided by dr r. s. speirs). key: cord- -uo oab authors: jartti, tuomas; waris, matti; niesters, hubert g.m.; allander, tobias; ruuskanen, olli title: respiratory viruses and acute asthma in children date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: uo oab nan to the editor: we read with great interest the article by khetsuriani et al on the prevalence of respiratory tract viruses in children with asthma. the results of pcr analysis of combined nasopharyngeal and throat swabs for different viruses were positive in % of patients with asthma exacerbation in the -year study. rhinovirus was the most common virus detected. we disagree with the low virus detection rate reported by khetsuriani et al because many studies in wheezing children have shown virus detection rates of close to %. we have also recently reported the viral cause of acute wheezing (specific clinical diagnoses were not reported) in hospitalized children. , of these children, had acute asthma that fulfilled the criteria of the national heart, lung, and blood institute. the median age of the asthmatic children was . years (range, months to years), and % of them were boys. thirtyseven ( %) children had symptoms of acute respiratory tract infection (ie, runny nose, fever, or both). virus culture, virus antigen detection, pcr techniques (for viruses), and serologic testing were used. a potential causative agent was identified in ( %) patients (table i) . among them, the results of pcr were positive for or more viruses in %, virus culture in %, virus antigen detection in %, and virus serology in %. rhinovirus, enteroviruses, parainfluenza viruses, and human bocavirus were the most common viruses. all patients with human bocavirus had low virus genome copy numbers (< copies/ml), and in no case was bocavirus detected in serum, which might suggest long-term virus carriage rather than symptomatic infection. no patients with positive results for influenza a virus, metapneumovirus, or coronaviruses were found, as also reported by khetsuriani et al. interestingly, or more viruses were detected in % of the children compared with % in the study by khetsuriani et al. the differences between our findings and those of khetsuriani et al might be due to many differences in these studies. we enrolled patients over a -year period, the children in our study were younger, and all were hospitalized. furthermore, nasopharyngeal aspirates were obtained and tested for viruses by using both molecular and traditional diagnostic techniques. finally, the sensitivities of methods for some viruses might be different. our findings suggest that nearly all exacerbations of asthma in children necessitating hospitalization are associated with viral infection. in nearly half of the cases, there is evidence for other coexisting viruses. the gradual disappearance of rhinovirus rna within to weeks after acute wheezing suggests that the occurrence of rhinovirus rna is associated with an acute symptomatic infection, and therefore our study also supports the view that rhinovirus infection is closely associated with exacerbation of asthma in children. prevalence of viral respiratory tract infections in children with asthma respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children human bocavirus and acute wheezing in children guidelines for the diagnosis and management of asthma-update on selected topics persistence of rhinovirus and enterovirus rna after acute respiratory illness in children available online april key: cord- - bagohw authors: navel, valentin; chiambaretta, frédéric; dutheil, frédéric title: reply date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: bagohw nan parts of china with continental rainy-cold climates. these provinces are particularly industrialized with considerable pollution in cities, ensuring populations are heavily exposed to particulate matter (pm), ozone (o ), carbon dioxide (co ), nitrogen dioxide (no ), and sulfur dioxide (so ) produced by diesel combustion. similar to italy and china, the north-eastern population of france is more affected by covid- than the population of the atlantic coast and mediterranean regions. , similar to the italian peninsula, france could be isolated into macro-area zones on both sides of the river loire, highlighting a clear north-west/ south-east line coinciding with the french demarcation line during world war ii (fig [f - /c] ). the populations living next to coasts with daily exposure to oceanic or mediterranean climates seem to be less at risk of covid- . however, the putative link between covid- and geographic zones could be associated with genetic variations in the population and explained by the importance of human exchanges around continental frontiers areas (eg, north-eastern part of france, benelux countries, and urban area of ruhr). similar to the environmental exposures affecting the incidence and decompensation of allergic diseases, covid- could be affected by internal and external exposome. in crowded areas and industrialized territories exposed to hot warm season, the high levels of o seem to be associated with variations in epigenetic modulation. globally, emerging data may identify air pollution as a modulator to dna methylation (dnam) disturbing the inflammation process, allergic diseases development, and exacerbation risk. as such, no and pm exposures during pregnancy could significantly deregulate dnam of antioxidants or anti-inflammatory genes related to the oxidative stress pathway in utero. , interestingly, lower dnam of il- and ifn-g genes was identified in adults exposed to pm, black carbon, and o , involving an increase in specific immune system. the change in dnam was often observed at specific locations within the promoter region, deregulating the expression of genetic heritage. considering that sars-cov- infection involves a proinflammatory cytokine storm as il- and il- b, a putative hypothesis could explain that populations exposed to chronic air pollution are associated with a different covid- incidence in line with chronic epigenetic deregulation. affecting the immune system and the inflammatory pathways, dnam related to air pollution could explain the disparities in covid- in geographic zones in which genetically predisposed populations were living in climate favoring sars-cov- distribution. the bimodal sars-cov- outbreak in italy as an effect of environmental and allergic causes will environmental impacts of social distancing due to the sars-cov- pandemic decrease allergic disease? minist ere des solidarit es et de la sant e. epidemiologic situation of sars-cov- in france a cross-sectional comparison of epidemiological and clinical features of patients with coronavirus disease (covid- ) in wuhan and outside wuhan, china climate change and outdoor aeroallergens related to allergy and asthma: taking the exposome into account air pollution and dna methylation: effects of exposure in humans air pollution during pregnancy and placental adaptation in the levels of global dna methylation maternal residential proximity to major roadways, birth weight, and placental dna methylation air pollution and gene-specific methylation in the normative aging study: association, effect modification, and mediation analysis key: cord- -rwkfev j authors: kaplan, allen p.; ghebrehiwet, berhane title: pathways for bradykinin formation and interrelationship with complement as a cause of edematous lung in covid- patients date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: rwkfev j nan include dry cough, dyspnea, tachypnea, a feeling of drowning, pulmonary edema, unilateral or bilateral pneumonia, mottling and ground glass opacifies on ct scan, and progression to the acute respiratory distress syndrome (ards) requiring ventilatory support( ). hypoxemia is particularly prominent throughout and a hyaline membrane of dead cells can be observed at autopsy. once infection takes hold, a cascade of inflammatory events is initiated including the release of cytokines such as i- , il- , ip- , mcp- , tnfα( ) (and many more) which has been referred to as a "cytokine storm". in addition, the prominent edema seen throughout the lung and the association of ace inhibition with severe angioedema has focused attention on another innate inflammatory cascade; namely, the overproduction of bradykinin( ) which is the focus of this editorial. there are two general pathways for the production of bradykinin, the first being the release of cellular tissue kallikrein which cleaves low molecular weight kininogen (lk) to release lys- bradykinin (fig. ) . tissue kallikrein is secreted as an active enzyme (i.e. processed intracellularly) and is a particularly prominent product of the lung, pancreas, kidney, salivary glands, and the prostate. there are homologous gene products, three of which can produce bradykinin (klk , , and ), klk being the most prominent. j o u r n a l p r e -p r o o f and high-molecular weight kininogen (hk)( ). prekallikrein circulates primarily as a bimolecular complex with hk (about - % bound) as does coagulation factor xi ( % is bound). they compete for a single overlapping binding site but there is sufficient hk present to bind both. both factor xii and prekallikrein possess minute levels of proteolytic activity relative to their respective active enzymes which may be the initial spark needed for activation to proceed. all three proteins are also bound to bimolecular sites on the surface of endothelial cells ( figure ). factor xii binds primarily to u-par-cytokeratin (ck- ) while hk binds to gc qr-cytokeratin with pk attached to the hk (fig. ). once activation proceeds, factor xii is converted to two forms of the activated enzyme, factor xiia ( kd) and factor xiif ( . - kd; β fxiia). both can convert prekallikrein to kallikrein and kallikrein digests hk to release bradykinin (arg-pro-pro-gly-phe-ser-pro-phe-arg). factor xii activation proceeds by a relatively slow autoactivation process to produce a small amount of factor xiia and a very rapid positive feedback in which the initial kallikrein formed activates all remaining factor xii in seconds to yield factor xiia and then factor xiif. tissue kallikrein does not activate factor xii. the larger kd factor xiia is the clotting factor that converts factor xi to factor xia to continue the intrinsic coagulation pathway (fig. ). factor xiif, lacks a surface binding site, loses - % of cov- . bradykinin causes vasodilation and increases vascular permeability by interacting with constitutively expressed b- receptors on small venules. the same is true of lys-bradykinin produced by tissue kallikrein (fig. ) although the lys is rapidly removed by aminopeptidase p. bradykinin is degraded primarily by ace, a dipeptidase which removes the c-terminal phe-arg, which inactivates it, followed by removal of ser-pro. an alternative process requires carboxypeptidase activity (carboxypeptidase n in plasma and carboxypeptidase m on pulmonary vascular endothelial cells) to first remove the c-terminal arg from either bradykinin (plasma cascade) or lys-bradykinin (tissue kallikrein product) (fig. ) . this leaves des-arg bradykinin (arg-pro-pro-gly-phe-ser-pro-phe) which is minimally reactive with b- . however this peptide binds to the b- receptor which also mediates vasodilation and vascular permeability. the b- receptor is not normally present but is induced by il- or tnfα (produced by febrile viral illnesses such as covid- ) as well as gc qr. it's ligands are des-arg bradykinin( ) as well as des-arg lys-bradykinin ( fig. ) . there are many observations and theories regarding a prominent role for bradykinin and perhaps des-arg bradykinin in the pathogenesis of the pulmonary dysfunction of covid- which is linked in part to changes in the renin-angiotensin system (ras). studies of gene expression in bronchoalveolar lavage specimens of covid- patients( ), when compared to normal control specimens, reveal upregulation of multiple components that lead to bradykinin production and expression for c -inh was decreased -fold which would render the plasma bradykinin cascade labile and overreactive as we see in c -inh deficiency (types i and ii hae) in which enzymes not adequately inhibited by c -inh include both forms of activated factor xii, plasma kallikrein, and c r. by contrast, gene expression for ace was decreased -fold so that bradykinin would not be inactivated normally. while viral binding to ace- limits its enzymatic activity( ) so that des-arg bradykinin is not degraded (ace- removes c-terminal phe) and lowered ace levels also limit des-arg bradykinin degradation (it removes c-terminal ser-pro-phe acting then as a tripeptidase rather than a dipeptidase). with the markedly augmented bradykinin receptor production, a "bradykinin storm" can result. our own preliminary observations (unpublished) reveal upregulation and secretion of gc qr by infected cells which creates the cell surface platform for activation of the bradykinin cascade and secreted gc qr also upregulates the b- receptor( ). the renin-angiotensin system( ) can also be contributory in that decreased ace limits formation of the vasoconstrictor angiotensin ii from angiotensin i. as angiotensin i accumulates, ace- removes c-terminal phe to produce angiotensin - . this moiety stimulates angiotensin- receptors to cause vasodilation and can do so synergistically with bradykinin( ). if significant amounts of angiotensin ii were produced, ace- can then convert it to another vasodilator, angiotensin - active through the mas receptor( ). here, the balance of decreased ace- via viral binding and internalization( ) and increased ace- , as seen when covid- bal fluids are examined( ), needs to be quantified at the protein level (cell surface and interstitial fluid) rather than the dna level to determine the net enzymatic effect. there are numerous reports of a possible therapeutic role for antagonists of cytokines such as il- (anakinra) or il- (tocilizumab) to treat covid- ( , , ) . we suggest use of lanadelumab to block plasma kallikrein ( ) can release hsp- and prolylcarboxypeptidase. both convert pk to plasma kallikrein if pk is a pneumonia outbreak associated with a new coronavirus of probable bat origin kallikrein-kinin blockade in patients with covid- to prevent acute respiratory distress syndrome the plasma bradykinin-forming pathways and its interrelationships with complement a mechanistic model and therapeutic interventions for covid- involving a ras-mediated bradykinin storm soluble gc qr is an autocrine signal that induces b r expression on endothelial cells sars-cov- as a factor to disbalance the renin-angiotensin system: a suspect in the case of exacerbated il- production covid- as an acute inflammatory disease structure-function studies using deletion mutants identify domains of gc qr/p as potential therapeutic targets for vascular permeability and inflammation key: cord- -tna e dw authors: kimura, hiroki; francisco, dave; conway, michelle; martinez, fernando d.; vercelli, donata; polverino, francesca; billheimer, dean; kraft, monica title: type inflammation modulates ace and tmprss in airway epithelial cells date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: tna e dw abstract background sars-cov- has dramatically changed our world, country, communities and families. there is controversy regarding risk factors for severe covid- disease. it has been suggested that asthma and allergy are not highly represented as co-morbid conditions associated with covid- . objective to extend our work in interleukin (il)- biology to determine if airway epithelial cell expression of two key mediators critical for sars-cov- infection, angiotensin-converting enzyme (ace ) and transmembrane protease, serine (tmprss ) are modulated by il- . methods we determined effects of il- treatment on ace and tmprss expression ex vivo in primary airway epithelial cells from participants with and without type asthma obtained by bronchoscopy. we also examined expression of ace and tmprss in two datasets containing gene expression data from nasal and airway epithelial cells from children and adults with asthma and allergic rhinitis. results il- significantly reduced ace and increased tmprss expression ex vivo in airway epithelial cells. in two independent datasets, ace expression was significantly reduced and tmprss was significantly increased in the nasal and airway epithelial cells in type asthma and allergic rhinitis. ace expression was significantly negatively associated with type cytokines while tmprss expression was significantly positively associated with type cytokines. conclusion il- modulates ace and tmprss expression in airway epithelial cells in asthma. this deserves further study with regard to any effects asthma and atopy may render in the setting of covid- infection. since its recognition in december , the outbreak of covid- caused by sars- cov- infection has generated strong concern among individuals presenting with underlying medical conditions. individuals with chronic lung diseases or asthma are at higher risk for developing severe complications from covid- ( ). interestingly, among patients with allergy and asthma, there is controversy as to whether these represent comorbid conditions that increase risk for covid- . one study that reported cases of covid- in wuhan, china, indicated no self-reported cases of asthma, allergic rhinitis, atopic dermatitis, and food allergy among infected patients ( ). moreover, a report by dong and colleagues suggested a low prevalence of asthma and allergy in pediatric cases, and also found a low prevalence of rhinitis and atopic infection globally and reported a low prevalence of co-existing respiratory disease overall in patients with acute covid- infection ( ). however, a recent report from seattle, washington describing cases of acute lung injury associated with covid- described of patients who had asthma and had been treated with oral corticosteroids within one week of presenting due to an asthma exacerbation ( ). a recent publication from covid- -associated hospitalization surveillance network (covid-net) describing patients admitted to hospitals in the united states from march - , reported that % carried the diagnosis of asthma ( ). however, information regarding severity, medication requirements and co-morbid conditions of these patients are not currently known. therefore, the controversy exists, as it is well known that viral infections exacerbate asthma ( ). for effective host cell entry, sars-cov- relies on two critical proteins, angiotensin-converting enzyme (ace ) ( ) and transmembrane serine, protease (tmprss ) ( ). here, we demonstrate that interleukin (il)- , a cytokine associated with type (t ) asthma, suppresses ace receptor expression and significantly increases tmprss expression in airway epithelial cells from participants with t asthma and atopy. based on these findings, we hypothesized that t cytokines modulate ace and tmprss expression in the airway epithelial cell in asthma and atopy. participants were recruited from the population in tucson, arizona and the surrounding areas. informed consent was obtained from each participant ( - years of age). asthmatic participants met gina criteria for mild and moderate asthma including the presence of reversibility of airflow obstruction or airways responsiveness with a provocative concentration of methacholine resulting in a % fall in fev (pc fev ) of ≤ mg/ml or < mg/ml if they were taking inhaled corticosteroids ( ). the presence of atopy was determined using skin testing; peripheral eosinophils and fractional exhaled nitric oxide (feno) was measured. healthy participants had no evidence of airflow obstruction, and no history of pulmonary disease; the presence of atopy was not an exclusion. exclusion criteria included an exacerbation of asthma within four weeks of study requiring antibiotics and/or corticosteroids, greater than - pack year history of tobacco use or any cigarette use in the last year and any other significant medical conditions. participants underwent bronchoscopy with endobronchial-protected brushing as previously described ( ). the brushing of the proximal airways to obtain bronchial epithelial cells was performed under direct visualization using a separate protected cytologic brush for each pass, for a total of eight passes. participants were discharged when their forced expiratory volume in one second (fev ) achieved % of their pre- bronchoscopy, post-albuterol value. freshly isolated airway bronchial epithelial cells from endobronchial brushings were cultured with pneumacult-ex plus (stemcell technologies, vancouver, bc, canada). after reaching confluence, cells were trypsinized and seeded onto collagen- coated polyester transwell ® insert membranes of -mm diameter (corning, lowell, ma, usa), at a concentration of × /well. the cells were then cultured at air-liquid interface (ali) using pneumacult-ali (stemcell technologies) and allowed to differentiate for weeks as previously described ( ). cells were stimulated with ng/ml il- (peprotech, rocky hill, nj, usa) for hours. epithelial cells were collected for rt-pcr analysis of ace and tmprss (see pcr methods below). conditions were performed in triplicate with appropriate unexposed controls. total mrna was extracted according to rneasy plus mini kit instructions (qiagen) and cdna was synthesized using µg of rna (applied biosystems, foster city, ca, usa). real-time pcr was performed on a cfx touch™ (bio-rad, hercules, ca, usa) using taqman™ probes (applied biosystems) specific for ppia, ace , and tmprss . the data were presented per the -∆∆ct once analysis was complete. we used linear mixed effect models to evaluate il- treatment and asthma status on ∆ct values. the mixed model accommodates the nesting structure of participants within asthma group, and treatment replicates for each participant. for analysis of the public databases, differences among the groups were analyzed using wilcoxon rank sum test or steel's multiple comparison test, as appropriate. the trend among the groups was analyzed using the jonckheere-terpstra test. in categorizing t low and t high groups in gse and gse datasets respectively, unsupervised hierarchical clustering including values of three t signature genes, calcium-activated chloride channel regulator (clca ), periostin (postn), and serpin peptidase inhibitor, clade b, member (serpinb ) using the euclidean metric with complete linkage was performed, as previously described ( ). correlations between nonparametric data were undertaken using spearman's rank correlation. statistical significance was defined as p< . . all analyses were performed using r version . . . software (the r foundation; http://www.r-project.org/) or graphpad results participant demographics for the ex vivo study are shown in table . the asthmatic participants were mild to moderate in severity based upon medication requirements with one patient requiring controller medication. asthmatic participants exhibited good to fair asthma control. all asthma and non-asthma participants had a history of atopy based upon their clinical history, presence of allergic rhinitis, and elevated feno. total and allergen specific ige were not available for these participants. il- reduces ace and increased tmprss expression in airway epithelial cells from both the asthma and non-asthma atopic groups (fig ) . as it been suggested that ciclesonide may possess anti-viral properties ( ), we excluded the one asthmatic participant who used inhaled corticosteroids and repeated the analysis. we did not find significant differences in ace and tmprss expression (data not shown). in study gse , nasal epithelial cells of participants with asthma and/or allergic rhinitis demonstrate lower ace expression compared to healthy participants (fig a) . assessment of ace expression by severity of symptoms (healthy, rhinitis without asthma, rhinitis with controlled asthma, and rhinitis with uncontrolled asthma) shows that participants with rhinitis and uncontrolled asthma (ua) demonstrate the lowest nasal epithelial cell ace expression (the jonckheere-terpstra trend test, fig b ). in addition, participants who are defined as t high based upon three gene signatures (clca , postn, serpinb ) ( ) also demonstrate lower nasal epithelial ace expression compared to the t low and healthy control groups (fig c) . we next examined the association of nasal epithelial cell ace expression with t cytokines. il- , but not il- and il- significantly negatively correlates with ace expression (fig a-c) . t -driven genes clca , postn and serpinb show an inverse correlation with ace expression (fig e ) . in dataset gse containing airway epithelial cell mrna, ace expression is lower (albeit not significantly) in asthmatic participants compared to control participants (p= . ) (fig a) . when asthmatic participants are grouped into t high and t low based upon the three gene signature expressions ( ), the t high participants demonstrate lower expression of ace compared to healthy participants while t low participants demonstrate similar expression as compared to healthy participants (fig b) . and il- in asthmatic participants (fig a-c) . furthermore, two t cytokine-induced genes (i.e., clca and serpinb , but not postn) significantly negatively correlate with ace expression when asthma and healthy groups are combined (see supplement, fig e ) . in dataset gse , participants with asthma and/or allergic rhinitis demonstrate increased nasal epithelial cell expression of tmprss compared to healthy participants whether combined (fig a) or evaluated as separate groups (fig b) . moreover, the t high participants demonstrate significantly higher expression of tmprss while the expression by t low participants is similar to healthy participants ( fig c) . there was a significant correlation between nasal epithelial tmprss gene expression with clca , postn and serpinb , but not with il- , il- or il- (fig e ) . while gene expression of tmprss is not different between healthy and asthmatic participants (fig a) , tmprss expression is higher (albeit not significantly) in t high asthmatic compared to healthy participants (p= . ). there is no difference in tmprss expression between healthy and t low asthma (p= . ) (fig b) . in addition, a significant correlation between airway epithelial tmprss and il- expression is present when both groups are combined as well as for asthma only (fig e ). this relationship is not demonstrated for il- and il- (fig e ) . airway epithelial cell tmprss expression significantly positively correlates with expression of clca and serpinb , but not postn when asthma and control groups are combined (fig e ) . to our knowledge, this is the first report to show that il- decreases ace and increases tmprss expression in t high asthma and in allergic rhinitis without asthma. these ex vivo observations are supported by interrogation of two public databases which include children and adults and in which we show that ace expression is reduced and tmprss expression is increased in t asthma and rhinitis. in addition, ace and tmprss expression correlate with t cytokines, although in opposite ways: ace inversely correlates with t or t driven genes, while tmprss expression positively correlates with t or t -driven genes. these analyses are exploratory and provide a direction for future investigation. the role of ace in asthma is just beginning to be elucidated. the renin- angiotensin system (ras) is a critical component in regulating multiple tissue and organ functions, such as those of the cardiovascular system, kidney, lung, and liver, specifically by maintaining homeostasis of blood pressure, electrolyte balance, and inflammatory responses ( , ). renin, a protease produced predominantly in the kidneys, cleaves angiotensinogen to generate angiotensin i (ang i). subsequently, angiotensin-converting enzyme (ace) cleaves ang i to produce ang ii. ang ii is hydrolyzed by various angiotensinases, such as ace . ace is a terminal carboxypeptidase and a type i transmembrane glycoprotein and a potent negative regulator of the ras. the imbalance in enzymatic activity of ace/ace has been suggested to involve in pathogenesis in several diseases, including lung diseases ( ). in asthma, ace may have a role due to its anti-inflammatory nature and ability to inactivate ang ii and activate ang - , the two counteractive systems strongly related to asthma. in an ovalbumin-challenged mouse model of t asthma, ang - modulated ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes, and whether inhaled or nasal corticosteroids are protective against sars-cov- infection merits comment, as it has been suggested that ciclesonide may possess anti- viral properties due to its ability to block viral replication ( ). in our ex vivo study, one of our asthmatic participants used inhaled corticosteroids. we excluded this participant and repeated the analysis. we did not find significant differences in ace and tmprss expression (data not shown). participants whose nasal airway epithelial cell data were included in dataset gse did not receive treatment with nasal corticosteroids for at least one month prior to enrollment. similarly, participants whose airway epithelial cells are included in dataset gse did not take inhaled or oral corticosteroids for one month prior to enrollment. thus, the association of ace and tmprss gene expression with t cytokines, atopy and t high asthma are not dependent upon the effect of corticosteroids in this analysis. this issue should be addressed in future investigation. our study has several strengths: using airway epithelial cells from well- phenotyped atopic, non-asthma and asthmatic participants from our own laboratory, we have shown that il- : ) suppresses ace gene expression and ) increases tmprss expression. we have observed similar results from two distinct datasets in children and adults with rhinitis and asthma. this approach adds robustness to the observation that il- modulates the receptor system for covid- . recently, jackson et al. has reported that underlying allergy and allergen exposure reduces the expression of ace ( ) . our data extends their findings by demonstrating ) contrasting changes in tmprss in response to il- stimulation ex vivo ) the effect of underlying allergy was t -specific, using unsupervised hierarchical clustering including t -induced genes. limitations of our study included: ) our sample size for the ex vivo studies is small ) data from publicly available datasets do not include detailed clinical information. in summary, we provide evidence that t inflammation suppresses expression of ace and increases expression of tmprss in nasal and airway epithelial cells in asthma and atopy. these observations may provide a foundation to elucidate the relative role of these two mediators in cell entry and how t cytokines modulate susceptibility to covid- . online repository values are presented as mean ± sd. clinical characteristics of patients infected with sars-cov- in wuhan eleven faces of coronavirus disease novel coronavirus ( -ncov) outbreak: a new challenge covid- in critically ill patients in the seattle region -case series hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states epidemiology of virus- induced asthma exacerbations: with special reference to the role of human rhinovirus angiotensin- converting enzyme is a functional receptor for the sars coronavirus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor analysis of relative gene expression data using real- time quantitative pcr and the (-delta delta c(t)) method. methods distinct epithelial gene expression phenotypes in childhood respiratory allergy national heart, lung, and blood institute and the national asthma education and prevention program genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids t-helper type -driven inflammation defines major subphenotypes of asthma. am j respir crit care med accumulation of intraepithelial mast cells with a unique protease phenotype in t(h) - high asthma the inhaled corticosteroid ciclesonide blocks coronavirus rna replication by targeting viral nsp expression of renin-angiotensin system components in the heart, kidneys, and lungs of rats with experimental heart failure distinct epithelial gene expression phenotypes in childhood respiratory allergy t-helper type -driven inflammation defines major subphenotypes of asthma. am j respir crit care med the authors would like to acknowledge pascal barbry, ph.d. and prescott woodruff, m.d. for use of their data in databases gse and gse , respectively. the authors would also like to acknowledge ronald schunk, r.t. for his assistance with participant recruiting and bronchoscopy and bianca sierra for her assistance with ex vivo experiments. of il- , il- , and il- with ace in the airway epithelial cells from asthmatic participants and when asthma and healthy groups are combined. r s : spearman's rank correlation coefficient. key: cord- -l adr authors: maggi, enrico; canonica, giorgio walter; moretta, lorenzo title: covid- : unanswered questions on immune response and pathogenesis date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: l adr abstract the novel coronavirus disease (covid- ) has rapidly increased in pandemic scale since it first appeared in wuhan, china, in december . in these troubled days the scientific community is asking rapid replies to prevent and combat the emergency. it is generally accepted that only achieving a better understanding of the interactions between the virus and host immune response and of the pathogenesis of infection is crucial to identify valid therapeutic tools to control virus entry, replication and spread as well as to impair its lethal effects. based on the recent research progress of sars-cov- and the results on previous coronaviruses, in this contribution we underscore some of the main unsolved problems, mostly focusing on pathogenetic aspects and host immunity to the virus. on this basis, we also touch important aspects regarding the immune response in asymptomatic subjects, the immune-evasion of sars-cov- in severe patients and differences in disease severity by age and gender. the recent spread of the sars-cov- pandemia revealed an unprecedented threat for humanity. as for other novel pathogens we are virtually disarmed against sars-cov- , while an efficient protection will be possible only after development of an effective vaccine. in these days, while the virus is rapidly spreading through the world, many urgent questions arise on how to combat its deleterious effects in infected patients. this implies to be able to answer many questions on the virus itself, on the pathogenesis of infection, on the host immune response and to identify therapeutic tools to control virus entry into the cells, its replication and spread as well as its lethal effects. here, we underscore some of the main unsolved problems, mostly focusing on pathogenetic aspects and host immune response to virus. on this basis, we will also touch important aspects regarding asymptomatic subjects and differences in severity of the disease by age and gender. ) which, when triggered by the virus, leads to higher ifn and lower il- production. - . hospitalized case series indicate that this group of diseases, in particular asthma, does not represent a risk factor for sars-cov- morbidity and mortality. , the few suitable data, however, do not allow to establish whether these disorders may constitute a protective factor for the infection or the related mechanisms. among them we could hypothesize that: i. the atopic status itself associated to the impairment of some regulatory mechanism may favor anti-viral immunity, ii. the higher proportion of activated eosinophils in allergy may play some protective role, while these cells are usually strongly reduced in -impaired circulating nk cells and t cell subsets in mild and severe patients. -relative higher frequency of cd + than cd + t cells in recovered patients. -highly activated cd + and cd + t cells with predominant type or type profiles in severe patients. -high type cytokines present in sera of patients with severe diseases. -strong memory t cell responses correlating with high nabs serum levels. -t cells specific for structural proteins (s, e, m, n epitopes) and memory cd + t cells detectable over yrs from infection. -early onset of cd + t cells correlating with disease severity. -predominance of memory cd + t cells with th or th profiles in survived patients. -higher t cell response in survived patients than in fatal cases. -time of onset, phenotype, repertoire, functional profile and amplitude of t cell response still unknown. -reduction of circulating nk cells and t cell subsets in relation to severity of disease. -few data on the recruitment of nk cells and t cell subsets and their functions (scrnaseq) in balf of patients with pneumonia. humoral immune responses sars-cov -seroconversion few days after the disease onset and specific igg detectable in most patients by days. -long lasting specific igg and nabs reported yrs after infection. -nabs specific for s, n, m epitopes, including rbd domain. -delayed or weak abs responses associated with severe outcome. -seroconversion within - weeks from disease onset still detectable until months after infection. -delayed or weak abs responses associated with severe outcome. -igm abs detectable - days after disease onset and serocoversion developed in most patients recovered. -unfrequent ab specificity for rbd domain of s protein. entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor cell clinical characteristics of patients infected withsars-cov- in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study inhibitory effects of glycopyrronium, formoterol, and budesonide on coronavirus hcov- e replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells asthma and expression of the sars-cov- receptor, ace innate immune evasion by human respiratory rna viruses sars and mers: recent insights into emerging coronaviruses natural killer cells and dendritic cells: rendez vous in abused tissues diversity, mechanisms and significance of macrophage plasticity • key: cord- -xh cw ao authors: papadopoulos, nikolaos g.; megremis, spyridon; kitsioulis, nikolaos a.; vangelatou, olympia; west, peter; xepapadaki, paraskevi title: promising approaches for the treatment and prevention of viral respiratory illnesses date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: xh cw ao viral respiratory tract infections are the most common human ailments, leading to enormous health and economic burden. hundreds of viral species and subtypes have been associated with these conditions, with influenza viruses, respiratory syncytial virus, and rhinoviruses being the most frequent and with the highest burden. when considering prevention or treatment of viral respiratory tract infections, potential targets include the causative pathogens themselves but also the immune response, disease transmission, or even just the symptoms. strategies targeting all these aspects are developing concurrently, and several novel and promising approaches are emerging. in this perspective we overview the entire range of options and highlight some of the most promising approaches, including new antiviral agents, symptomatic or immunomodulatory drugs, the re-emergence of natural remedies, and vaccines and public health policies toward prevention. wide-scale prevention through immunization appears to be within reach for respiratory syncytial virus and promising for influenza virus, whereas additional effort is needed in regard to rhinovirus, as well as other respiratory tract viruses. discuss this article on the jaci journal club blog: www.jacionline.blogspot.com. the respiratory system is one of the main portals of entry for human pathogens. although precise calculations are challenging because of methodology and inherent variability, the number of potentially infectious viruses we breathe every day can be in the range of many thousands. thus it is not surprising that viral respiratory tract infections (vrti) are the most common human diseases, leading to enormous health and economic burden. a wide variety of conditions fall within the spectrum of vrtis. many of these are by themselves major public health concerns: influenza, acute bronchiolitis, viral pneumonia, and common colds. together with their downstream effects (ie, acute exacerbations of asthma and chronic obstructive pulmonary disease [copd] ), all result in vast amounts of morbidity, mortality, and health care costs, including primary care visits, hospitalizations, and deaths but also inappropriate use of antibiotics, loss of productivity, and effects on quality of life. [ ] [ ] [ ] respiratory tract viruses have been isolated and characterized during the last century, starting from influenza virus (ifv) in the s and followed by respiratory syncytial virus (rsv), coronaviruses, adenoviruses, and rhinoviruses in the to s; nevertheless, ''new'' viruses or subtypes, such as human metapneumovirus or rhinovirus c, are still being identified. , even though several of these viruses are typically associated with a clinicopathologic entity (eg, ifv with influenza, rsv with bronchiolitis, and rhinovirus with the common cold), there is also extensive overlap, and it is often difficult to identify the etiologic agent based on clinical grounds alone. consequently, when considering prevention and treatment of vrti, potential targets include specific pathogens, the immune response, disease transmission, or just symptoms. here we provide an overview of the options and highlight some of the most promising approaches in vrti treatment, including symptomatic medication, immunomodulatory drugs, antiviral agents, and natural products, as well as in vrti prevention, ranging from vaccines to immunostimulators and public health policies. this is a vast field, and thus we emphasize advances that might be relevant in tackling the virus-induced aspects of allergic disease, such as asthma exacerbations. most mild viral respiratory illnesses are managed symptomatically with over-the-counter medications, such as nasal decongestants, antipyretics/analgesics, antitussives, or expectorants, on which no major improvements are foreseen. although generally well tolerated for short-term relief, some agents can have adverse effects, especially in young children. therefore the us food and drug administration has issued a warning against the use of over-the-counter cough and cold products in children younger than years of age. furthermore, the use of decongestants should be minimized, especially in children, whereas codeine has been restricted in children by the european medical agency since . selective cox inhibitors, such as celecoxib and mesalazine, have been widely used in clinics for their antipyretic, analgesic, and antiinflammatory properties in patients with airway diseases, whereas their combination with neuraminidase inhibitors (nais) has significantly improved the survival of ifv-infected mice. recent studies have revealed a new genus of specialized proresolving lipid mediators (spms), including lipoxins, resolvins, protectins, and maresins, enhancing anti-inflammatory, antiviral, and proresolving mechanisms. medications interfering with prostanoid and lipoxygenase biosynthesis and signaling, thus affecting resolution and spm switching, such as aspirin and nonsteroidal anti-inflammatory drugs, have been suggested as potential agents modulating antiviral immunity, , whereas several spm resolution agonists are in clinical development programs. symptomatic relief can also be sought in severe cases. noninvasive ventilation can reduce respiratory distress in patients with acute viral bronchiolitis. very recently, new devices delivering totally conditioned gas ( c at % relative humidity) through a very high-flow nasal cannula (up to l/min) have been indicated for bronchiolitis mainly as rescue therapy to reduce the need for admission to the intensive care unit. immune and antiviral pathway modulators although vrtis are most often short-lived events, impaired antiviral clearance and/or activation of inflammatory pathways lead to important downstream complications, such as exacerbations of asthma or copd. the immune and antiviral mechanisms leading from infection to exacerbation have been scrutinized, and medications targeting these pathways are being evaluated as promising candidates to reduce disease burden. impaired interferon production has been observed in patients with various obstructive respiratory diseases, potentially contributing to enhanced susceptibility to and/or severity of virus-induced acute airway exacerbations. although inhaled ifn-b supplementation has not shown a clear effect in preventing virus-induced symptom worsening in patients with mild asthma, subanalysis in patients with severe asthma showed a protective effect. interestingly, in an experimental model exogenous administration of ifn-l induced a strong and more prolonged antiviral state than ifn-b. moreover, experimental studies in an allergic asthma model showed that ifn-l supplementation enhanced t h immunity by inducing ifn-g and suppressing t h and t h responses through modulation of lung cd c dendritic cell function. , novel antibody-based drugs with antirhinovirus and immunomodulatory effects act through ifn-b induction and suppression of t h responses in experimental models. the prototype synthetic toll-like receptor (tlr) antagonist eritoran (e ) and anti-tlr igg therapy have been shown to block ifv lethality in mice by suppressing lung pathology, clinical symptoms, and viral titers. , other innate immune receptors, such as tlr , also have potential for host-targeted therapeutic approaches. interestingly, omalizumab, an anti-ige mab, prevents asthma exacerbations either by decreasing the duration and shedding of rhinovirus infection or by blocking the synergistic effect of rhinovirus infection on allergy. , because high-affinity ige receptor (fcεri) cross-linking on plasmacytoid dendritic cells reduces ifn-a responses after viral infections, it is plausible that omalizumab enhances virus-induced ifn-a production in asthmatic patients, thus limiting virus spreading and infection severity. ''severe cytokine storm,'' an entity associated with markedly higher levels of proinflammatory cytokines, has been associated with severe influenza infections; immunomodulatory agents have been proposed as potential therapeutic strategies. peroxisome proliferator-activated receptor g agonists (eg, rosiglitazone and pioglitazone) are critical regulators of inflammation and have been promising in improving the clinical outcome of severe influenza infections. their development slowed down from to because of possible cardiovascular side effects; however, in , the us food and drug administration lifted restrictions based on new safety data. moreover, sphingosine- -phosphate receptor agonists , which are located mainly on pulmonary endothelial cells, exhibit cytokine storm-blunting activity by suppressing both innate cellular and cytokine/chemokine responses, particularly when combined with antiviral agents. there is increasing interest in the use of macrolides to treat or prevent virus-induced asthma exacerbations, although microbial resistance remains a major hurdle, and therefore they are not currently indicated. early in vivo evidence suggested that azithromycin has anti-inflammatory and antiviral effects through induction of interferon-stimulated gene mrna expression and reduced viral replication and release in patients with asthma and chronic obstructive lung disease. , in a randomized clinical trial including wheezing preschool-aged children, early azithromycin administration significantly reduced the likelihood of a severe lower respiratory tract infection. novel macrolides (mycobacterium avium complex ) with anti-inflammatory, antibacterial, and, more importantly, interferon-augmenting activity in airway epithelium have been identified. finally, in vitro models have demonstrated that a -antitrypsin exerts anti-inflammatory effects in airway epithelial cells from rhinovirus-infected patients with copd, potentially through inhibition on caspase- activity, suggesting a -antitrypsin as a potential anti-inflammatory agent. antivirals vrtis are usually characterized by an acute and self-limiting course, which means that the peak of viral replication usually precedes or parallels the appearance of clinical symptoms. as a result, the time window from verification and/or typing of the pathogen, allowing a specific therapeutic intervention, is extremely narrow. additional challenges need to be overcome, such as the structural variation of viral proteins, multiple genotypes, and high mutation rates. accordingly, only a very limited number of specific antiviral drugs are currently licensed, and promising approaches mostly aim to control severe complications, reduce disease burden, or transmission. antiviral strategies aim to block particular stages of the viral lytic cycle, including attachment and entry to the host cell, replication, transcription, and translation (fig ) . in principle, preventing a viral pathogen from entering the host cell represents the ideal antiviral strategy because the virus is not allowed to ''hack'' the host: ifv nais have been successfully used to competitively bind the sialic acid-binding pocket of neuroaminidase and are good examples of this approach. oseltamivir and zanamivir have been used as anti-flu therapies, whereas laninamivir and peramivir show antiviral activity against wild-type but also against oseltamivir-resistant and nai-resistant strains, respectively. , the nonenveloped rhinoviruses use viral capsid structures to bind their receptors (intercellular adhesion molecule [icam- ], low-density lipoprotein receptor, and cadherin-related family member ). even though more than % of rhinovirus strains use icam- for cell entry, an icam- competitor, tremacamra, did not make it into the clinic despite initially promising results, and no anti-icam- drugs are currently available. another strategy is to prevent capsid uncoating and further assembly of new virions. this strategy has been successfully used against ifv and severe acute respiratory syndrome (sars)coronavirus, which use a class i fusion mechanism. das (fludase, nexbio, inc, san diego, calif) is a fusion construct that cleaves the sialic acid receptors on host cells, and its antiviral spectrum includes ifv and parainfluenza viruses (pivs). nonenveloped viruses, such as rhinovirus, release their genomes through a conformational shift of the capsid proteins accompanied by an expansion of the viral shell along with the opening of symmetry-related channels (pores) from which the genome is released (virus uncoating). , various capsid-binding compounds against rhinoviruses have been tested (r and win series) without ultimate success. pleconaril, bta (vapendavir), and pocapavir (v- ) are still under clinical evaluation. of note, a major drawback of capsid binders is the rapid emergence of resistance. several fusion inhibitors are being developed for the treatment of rsv and have been reviewed elsewhere. , because of their limited coding capacity, viruses rely on the production of polyproteins that need to be cleaved into functional subunits by viral proteases. the enterovirus polyprotein is cleaved by a family of cysteine proteases, which are highly conserved among different subtypes but lack homology with human proteases. unfortunately, after failed attempts with ruprintrivir (ag ) and ag , which showed antiviral activity in vitro but not in vivo, no similar agents are being pursued currently. the use of hiv protease inhibitors, such as lopinavir and ritonavir, in patients with sars has not been associated with any proved benefit, although retrospective studies reported that severe outcomes (acute respiratory distress syndrome or death) occurred less often in those receiving a combination of lopinavir/ritonavir and ribavirin with corticosteroids. polymerase inhibitors (nucleoside/nucleotide analogs) act by leading to termination of the polynucleotide chain elongation. ribavirin has been used for the treatment of severe rsv-related disease in high-risk infants and in combination with protease inhibitors in patients with sars, but its use has been limited because of cost and unconfirmed efficacy toward severe outcomes. als- is a promising orally bioavailable prodrug of the novel rsv replication inhibitor als- (a cytidine nucleoside analogue), which inhibits rsv replication. other promising polymerase inhibitors include amiloride (competitive inhibitor of coxsackie virus b rna polymerase) and gpc-n (multiple genera in picornaviridae) but are still in the early stages. favipiravir (t- ) is an antiviral drug that selectively inhibits the rna-dependent rna polymerase of ifv, as well as several other viruses. umifenovir has been shown to inhibit various human respiratory rna viruses, including several strains of ifv-a and ifv-b, rsv, piv , and rhinovirus b . it also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases. finally, a most promising but also challenging antiviral approach is through use of antisense oligonucleotides. antisense oligonucleotides are single-stranded deoxyribonucleotide oligomers with a sequence complementary to a target mrna transcript. thus viral genomic rna or viral mrna can be targeted directly. antisense technology and rna interference have been experimentally explored in targeting measles virus, sars-coronavirus, coxsackievirus, enteroviruses and rhinoviruses, piv, human metapneumovirus, ifv, and rsv genomes. , the rna inhibition-based therapeutic that is furthest advanced in clinical development at this time is against rsv. aln-rsv is an unmodified, naked, small interfering rna designed to inhibit the replication of rsv by interrupting the synthesis of the viral n protein. the sequence of the target is well conserved throughout naturally occurring rsv a and b genotypes. in all, new antivirals are being explored continuously, particularly for life-threatening viruses, such as ifv (influenza) and rsv. rhinoviruses, even though simple in terms of genome organization and protein coding, have proved extremely difficult to target, mostly because of their high diversity and immuneevading strategies but also to some extent to the underestimation of rhinovirus infection clinical consequences. within the past few years, scientific communities all over the world have shown renewed interest in the search for novel immune-stimulating or antiviral agents of plant origin for either treatment or prevention, often using ethnopharmacologic approaches. natural compounds are widely recognized as privileged structures trimmed by evolutionary processes to interact with macromolecular targets. plants use a diverse set of biochemical pathways to generate several secondary metabolites representing ecosystem adaptations to help plants to survive various environmental stresses and protect them from infections and infestations. the antiviral potential of plant extracts or compounds varies among viruses. natural compounds occupy an equally large and complex chemical space as synthetic compounds. in the case of antiviral agents, % of entities registered in the last approximately years ( - ) can be classified as natural product botanicals, synthetic but natural product mimics, natural product pharmacophores, or a combination of the latter . oseltamivir, a success story in ifv drug synthesis, has its roots in nature: the abundant plant constituents quinic acid and shikimic acid are used as its starting materials. a screening strategy was applied to investigate crude extracts from plant species on their inhibiting potential toward nais of clostridium perfringens. moreover, bioactive compounds from cleistocalyx operculatus buds were discovered by using an anti-ifv screening approach. the chinese academy of medical sciences tested more than , plants. among them, a pronounced neuroaminidase-inhibiting effect was observed for the herb extract of elsholtzia rugulos. some extracts from agrimonia pilosa, echinacea purpurea, and prunus mume or the multicomponent mixtures polyphenol fractions from punica granatum and secoiridoid glucosides from ligustrum lucidum have shown a significant reduction of virus-induced cytopathic effects and in general antiviral or anti-influenza activity. a % to % risk reduction of common cold incidence with the use of echinacea species supplements has been shown. moreover, a recent meta-analysis demonstrated benefit on long-term ( - months) prevention with echinacea species on recurrent respiratory tract infections (rtis). another promising compound is bno , a fixed combination of herbal substances that significantly reduced symptoms and led to faster recovery in patients with acute viral rhinosinusitis. reported antiviral effects from natural products, regardless of whether obtained from clinical trials or empiric knowledge, can only give clues for further research. however, it appears that we are entering a new golden age of natural product drug discovery. prevention of viral respiratory illness is attempted by either avoiding exposure or strengthening immune defenses, either nonspecifically with immunostimulators or specifically with vaccines. often, but not always, interventions are targeted toward high-risk groups for a particular infection (eg, rsv in infants and the elderly and ifv in patients with asthma). a variety of compounds (of microbial, herbal, or synthetic origin) have been used and are still being developed as nonspecific immunostimulatory agents to enhance or modulate the immune response against respiratory pathogens in a preventive or sometimes also therapeutic context. the effectiveness of these agents is usually moderate, and therefore they are only used as secondary supportive measures. as such, however, their potential should not be underestimated. among several agents based on bacterial components (om- bv, lw , pmbl, d , and ru ), om- bv, a lyophilisate of water-soluble fractions of bacteria commonly detected in patients with rtis, has been extensively studied, and a role in the prevention of both acute and recurrent rtis has been shown. , mechanistic studies have confirmed pleiotropic immunomodulating effects on both innate and adaptive immunity. , pidotimod, a synthetic dipeptide molecule, induces a variety of immunomodulatory effects , and has shown some efficacy in preventing rtis, although this was not always confirmed. , probiotic supplementation has been shown to reduce the incidence, duration, and severity of upper respiratory tract infections through immune modulation and in particular rhinovirus infection through altering nasal innate inflammatory responses. vitamin d ( -hydroxyvitamin d) has a modulatory role in host defense, inflammation, immunity, and epithelial repair after respiratory tract infections. a recent meta-analysis has confirmed that vitamin d supplementation reduces the overall risk of acute respiratory tract infections. data from in vitro rhinovirus-infected human primary bronchial epithelial cells showed that exogenous vitamin d can reduce rhinovirus replication through increasing interferon and cathelicidin gene expression. a significant amount of research is still dedicated to the efficacy of vitamin d supplementation, although not without controversy. hopefully, specific indications will be consolidated soon. despite widespread use and a multitude of studies, the role of vitamins c or zinc supplements is still inconclusive in relation to their action against the common cold. interestingly, meditation and exercise might significantly contribute to the reduction of rti burden, suggesting that the immunostimulatory capacity of nonpharmacologic measures should also be considered. the high transmission rate and epidemic nature of respiratory tract viruses indicate that effective public health measures to reduce transmission can have a substantial role in the overall prevention of these infections. a plethora of studies and metaanalyses delineated the important contribution of health policies in reducing transmission of epidemic respiratory tract viruses. in an elegant randomized control trial, an automated web-based intervention that maximized handwashing intention was associated with fewer episodes of influenza-like illness, shorter duration of symptoms, and fewer antibiotic prescriptions in the intervention group. although similar results regarding handwashing have been confirmed in a cochrane meta-analysis, hand hygiene interventions in educational settings were not as unequivocally effective. , low adherence to hand hygiene recommendations was correlated with higher incidence of ifv infection among health care workers during the pandemic. the use of face masks has been shown to be highly effective in the interruption of respiratory viral spread. this has been demonstrated further in a cluster randomized trial in which a reduced odds ratio of influenza infection secondary attack was observed in the intervention group. face masks are now regularly worn in some communities, especially in asia, but much less so in western societies. taken together, it seems that public health measures might provide a valuable ally in decreasing the burden of respiratory tract infections in the community. both vaccines and mabs (passive immunization) are relevant interventions. vaccines for ifv, rhinovirus, and rsv were initially developed as long ago as the s to s, although with mixed success, mostly because of rapid virus evolution. improved understanding of vaccine immunology and technologic developments place us now closer than ever to developing highly effective vaccines against the major respiratory tract viruses. mab therapies to viral infections, such as ebv (rituximab) or rsv (palivizumab), provide passive immunization and are licensed, whereas similar agents targeting influenza and other viruses are in preclinical development. neutralizing antibodies can bind and inactivate viruses, inhibit viral cell entry (blocking receptor binding or conformational changes), prevent the release of virions from the cell, or modulate immune effector functions. , engineering and production strategies to produce antibody fragments, higher-affinity binding, and longer half-life are contributing to a lower overall cost for therapy, although vaccines are still considered preferable in most cases. it is notable that effective neutralizing mab epitopes can also inform the rational design of vaccines. different types of vaccines to respiratory viruses exist, and these are shown in fig . traditionally, either live attenuated or inactivated viruses are used. more recently, subunit vaccines made of detergent-disrupted whole viruses or purified viral proteins are also common. furthermore, promising approaches use microparticle/nanoparticle material and recombinant technologies to produce broadly immunogenic, often self-adjuvanting, reproducible, and safe vaccine responses. these delivery systems include synthetic polymers, virosomes, virus-like particles (vlps), liposomes, lipid nanoparticles, proteins, emulsions, and immune-stimulating complexes. currently, naturally occurring particles are favored because of safety concerns, even though synthetic polymers, such as poly lactic-co-glycolic acid, are in use, and gold nanoparticles have shown promising results. self-assembling protein nanoparticles, such as ferritin cages and vaults, have also shown promising preclinical data. , layer-by-layer peptide-fabricated vaccine containing alternately charged poly-l-glutamic acid and poly-l-lysine layers with rsv peptides added have been efficacious in animals. a virosomal adjuvanted vaccine composed of reconstituted ifv envelope, effectively removing the core proteins and rna, has been available for years with excellent tolerability and efficacy. several vlp vaccines based on hepatitis b virus surface antigen have been approved for viral infections, such as human papilloma virus and other microbes (eg, malaria ), although an ifv candidate has not progressed. nevertheless these and other vlps offer promise because of their valency, similar immune presentation to pathogens, and antigenic preservation. adjuvants form a vital part of many vaccines; however, only aluminum hydroxide and oil in water emulsions are currently approved. a number of novel adjuvants, such as microcrystalline tyrosine, matrix m, pathogen-associated molecular patterns, and chitosan, are in development. [ ] [ ] [ ] [ ] dna and rna vaccines induce an immune response to the nucleic acid-encoded antigen. impressive results have been reported in animals for a single low-dose intradermal, nonreplicating dna vaccine for rsv; however, whether this will translate effectively to human subjects is not yet known. to enhance immunogenicity, rna vaccines have been encapsulated in nanoparticles, achieving sterilizing immunity for zika virus in mice, as well as being incorporated into virus-based self-replicating constructs known as replicons. , active ifv vaccination already forms the core of the global strategy against severe seasonal and pandemic influenza. trivalent and more recent quadrivalent vaccines are largely efficacious in healthy adults provided an adequate match between circulating and vaccine strains. higher-dose ( mg) and mf -adjuvanted vaccines are available for elderly patients. , similarly, pandemic vaccines can offer greater cross-clade protection because of the presence of improved (as or mf ) adjuvants. the current frontier of ifv vaccine development is ''universal'' vaccines (table i) . ideally, these would protect not only from circulating and pandemic strains but also from novel epitopes that might evolve in the future. many such vaccines are currently in preclinical and early clinical stages. heterosubtypic cross-reactive antibodies to ifv-a against the hemagglutinin (ha) stalk have been isolated from immune subjects, leading to mabs now in phase . , similar multilineage ha-stalk antibodies to ifv-b have also been reported. other conserved proteins have also been targeted, and an anti-m e antibody is in development. therefore passive immunization or postinfection treatment might soon become another tool to combat ifv. , , ha-stalk and chimeric head/stalk-based vaccines have also shown encouraging preclinical results. , [ ] [ ] [ ] a further vaccine strategy based on conserved epitopes in proteins, such as m , np, and pb , involves induction of cd and cd t-cell immunity, leading to development of a promising mva viral vector vaccine. other vaccines use multiepitope peptides to induce ifv-specific t-cell responses, reducing viral shedding in human subjects. , self-replicating rna nanoparticles also encoding multiple proteins and hepatitis b virus-based vlps expressing m e and ha epitopes also appear promising. there are currently no licensed vaccines and only mab (palivizumab) approved for the prevention of rsv infection. however, there are numerous candidates in clinical trials, as recently reviewed. suptavumab, an anti-f mab, has reached phase iii trials in preterm infants. medi offers -fold greater potency than palivizumab and has extended half-life in primates, suggesting a once per season dosing. candidate vaccines are based on live attenuated strains, subunit, vector, and nanoparticle technologies with a range of adjuvants. chimeric and combination vaccines using expression vectors in vlps show much promise. recent preclinical results exhibit effective neutralization of rsv. [ ] [ ] [ ] [ ] [ ] the most advanced of these is the novavax f-protein vlp nanoparticle vaccine with aluminum hydroxide adjuvant, which is in phase iii for maternal vaccination. , transplacental transmission of neutralizing antibodies has been demonstrated in preclinical studies, although this has not conferred significant protection from rsv. recombinant dna vaccines are also promising because of their apparent ability to induce a balanced t h /t h response, with a broad igg/iga profile mimicking live rsv challenge. intranasal and oral vaccine formulations are now in the early stages of clinical studies. fig . vaccine types. a, live attenuated vaccines are grown in culture to make them less virulent but can have the problem of reversion. b, inactivated vaccines are treated with uv or formaldehyde to crosslink proteins and make them nonviable. c, proteins can be purified, extracted, or dissolved by using detergents. d, naked nucleic acids are also used as vaccines. e, nanoparticle vaccines encompass natural and synthetic materials. membranes can be used to make liposomes to contain and deliver an antigen to a target cell. f, viruses can have nucleic acid and core protein removed to form virosomes. g, viral proteins, such as ha stalks or antigens, can be engineered onto immunogenic core proteins (eg, ferritin or vaults). this example is ha on ferritin adapted from pbd codes bve and c s. h, viruses, such as the vaccinia virus ankara, with coat proteins and genetic material removed can be engineered to express other antigens, such as influenza m ion channel protein. i, vlps can be engineered to express antigens and naturally glycosylated proteins and have adjuvants incorporated into the coat. pamp, pathogen-associated molecular pattern. j, synthetic nanoparticles made from polymers (polystyrene or poly lactic-co-glycolic acid), gold, or carbon nanotubes can have peptides adsorbed, admixed, or encapsulated. ag, antigen. initial vaccination attempts and more recent preclinical experiments show that inactivated rhinovirus vaccines are type specific and not cross-neutralizing. however, although in animals rhinovirus antibody responses might be weakly cross-neutralizing, data from human subjects suggest that responses are mainly misdirected to internal epitopes. understanding the full extent of rhinovirus diversity would probably be required to develop a panspecies vaccine. multiple strategies are being developed to reduce the burden of viral respiratory illnesses. it is likely that many of these strategies will find a relevant indication: antiviral strategies will most probably make sense in severe life-threatening situations or when a window of opportunity is clearly present, such as in specific virus seasons and susceptible populations. ideally, prevention at a wide scale through immunization will be able to reduce the overall burden of respiratory infections with a huge effect. this appears to be within reach for rsv and ifv, whereas additional effort is needed toward rhinovirus. in the meantime, symptomatic and immunostimulatory measures provide relief, and they hold promise in relation to postviral reactive airway disease. public health measures should be expanded because they can be critical in reducing the effect and contain potential epidemics. drivers of airborne human-to-human pathogen transmission global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics estimates of hospitalization attributable to influenza and rsv in the us during - , by age and risk status asthma research in europe: a transformative agenda for innovation and competitiveness influenza: exposing the true killer identification of new respiratory viruses in the new millennium a polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques over-the-counter medications: update on cough and cold preparations limits and danger of ephedrine and pseudoephedrine as nasal decongestants comprehensive evidence-based review on european antitussives cyclooxygenase : its regulation, role and impact in airway inflammation delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus the resolution code of acute inflammation: novel pro-resolving lipid mediators in resolution targeted prostaglandin e inhibition enhances antiviral immunity through induction of type i interferon and apoptosis in macrophages role of the lipoxygenase pathway in rsv-induced alternatively activated macrophages leading to resolution of lung pathology non-invasive ventilation improves respiratory distress in children with acute viral bronchiolitis: a systematic review high-flow warm humidified oxygen versus standard low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase , randomised controlled trial pathogenesis of viral infection in exacerbations of airway disease viruses and bacteria in acute asthma exacerbations-a ga( ) len-dare systematic review novel strategies for targeting innate immune responses to influenza the effect of inhaled ifn-beta on worsening of asthma symptoms caused by viral infections. a randomized trial efficacy of ifn-lambda to protect human airway epithelial cells against human rhinovirus b infection il- a (ifn-lambda ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease host dna released by netosis promotes rhinovirus-induced type- allergic asthma exacerbation efficacy of novel antibody-based drugs against rhinovirus infection: in vitro and in vivo results the tlr antagonist eritoran protects mice from lethal influenza infection the toll-like receptor antagonist eritoran protects mice from lethal filovirus challenge preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations omalizumab is associated with reduced acute severity of rhinovirus-triggered asthma exacerbation interferon at the crossroads of allergy and viral infections the cytokine storm of severe influenza and development of immunomodulatory therapy immunomodulatory therapy for severe influenza fda drug safety communication: fda eliminates the risk evaluation and mitigation strategy (rems) for rosiglitazone-containing diabetes medicines dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection azithromycin induces anti-viral responses in bronchial epithelial cells azithromycin induces anti-viral effects in cultured bronchial epithelial cells from copd patients early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial identification of novel macrolides with antibacterial, anti-inflammatory and type i and iii ifn-augmenting activity in airway epithelium the anti-inflammatory effect of alpha- antitrypsin in rhinovirus-infected human airway epithelial cells current progress in antiviral strategies influenza neuraminidase inhibitors: synthetic approaches, derivatives and biological activity japanese surveillance systems and treatment for influenza detection and management of antiviral resistance for influenza viruses viral entry pathways: the example of common cold viruses efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection: a randomized clinical trial viral membrane fusion drugs to cure avian influenza infection-multiple ways to prevent cell death viral uncoating is directional: exit of the genomic rna in a common cold virus starts with the poly-(a) tail at the '-end picornavirus rna is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes selective inhibitors of picornavirus replication replication and inhibitors of enteroviruses and parechoviruses challenges and opportunities in developing respiratory syncytial virus therapeutics advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses als- for respiratory syncytial virus infection favipiravir (t- ), a novel viral rna polymerase inhibitor arbidol as a broad-spectrum antiviral: an update the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses gene knockdown in human rhinovirus b using '-ome-modified sirnas results in the reactivation of the interferon response traditional use of medicinal agents longevity extension by phytochemicals a review on antiviral activity of the himalayan medicinal plants traditionally used to treat bronchitis and related symptoms influenza neuraminidase: a druggable target for natural products neuraminidase inhibitors from reynoutria elliptica structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities c-methylated flavonoids from cleistocalyx operculatus and their inhibitory effects on novel influenza a (h n ) neuraminidase anti-viral properties and mode of action of standardized echinacea purpurea extract against highly pathogenic avian influenza virus (h n , h n ) and swine-origin h n (s-oiv) in vitro inhibition of human influenza a virus infection by fruit-juice concentrate of japanese plum (prunus mume sieb. et zucc) pomegranate (punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir in vitro evaluation of secoiridoid glucosides from the fruits of ligustrum lucidum as antiviral agents echinacea for preventing and treating the common cold echinacea reduces the risk of recurrent respiratory tract infections and complications: a meta-analysis of randomized controlled trials herbal drug bno is safe and effective in the treatment of acute viral rhinosinusitis impact of a mixed bacterial lysate (om- bv) on the immunogenicity, safety and tolerability of inactivated influenza vaccine in children with recurrent respiratory tract infection clinical and immunological benefits of om- bacterial lysate in patients with allergic rhinitis, asthma, and copd and recurrent respiratory infections om- is an immunomodulator of interferon-beta production and inflammasome activity bacterial lysate increases the percentage of natural killer t cells in peripheral blood and alleviates asthma in children pidotimod: the state of art metabolomic profile of children with recurrent respiratory infections pidotimod for the prevention of acute respiratory infections in healthy children entering into daycare: a double blind randomized placebo-controlled study efficacy and safety of pidotimod in the prevention of recurrent respiratory infections in children: a multicentre study probiotics for preventing acute upper respiratory tract infections effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection-a randomised controlled trial vitamin d modulation of innate immune responses to respiratory viral infections vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data vitamin d increases the antiviral activity of bronchial epithelial cells in vitro prevention and treatment of the common cold: making sense of the evidence meditation or exercise for preventing acute respiratory infection: a randomized controlled trial an internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (primit): a primary care randomised trial physical interventions to interrupt or reduce the spread of respiratory viruses effectiveness of hand hygiene interventions in reducing illness absence among children in educational settings: a systematic review and meta-analysis can a school-based hand hygiene program reduce asthma exacerbations among elementary school children? risk factors for influenza among health care workers during pandemic the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial monoclonal antibodies for prophylactic and therapeutic use against viral infections the growth and potential of human antiviral monoclonal antibody therapeutics converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play antibody recognition of a highly conserved influenza virus epitope harnessing nanoparticles for immunomodulation and vaccines assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles current advances in research and clinical applications of plga-based nanotechnology consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies layer-by-layer nanoparticle vaccines carrying the g protein cx c motif protect against rsv infection and disease eleven years of inflexal v-a virosomal adjuvanted influenza vaccine human papillomavirus and hpv vaccines: a review as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase , individually randomised, controlled trial m e-based universal influenza a vaccine virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development comparison of a novel microcrystalline tyrosine adjuvant with aluminium hydroxide for enhancing vaccination against seasonal influenza matrix m h n vaccine induces cross-h clade humoral immune responses in a randomized clinical trial and provides protection from highly pathogenic influenza challenge in ferrets safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults chitosan-a versatile semi-synthetic polymer in biomedical applications genetic immunization is a simple method for eliciting an immune response development of an intradermal dna vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus modified mrna vaccines protect against zika virus infection dna and rna-based vaccines: principles, progress and prospects replicon rna viral vectors as vaccines transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response efficacy and safety of high-dose influenza vaccine in elderly adults: a systematic review and meta-analysis effectiveness of mf -adjuvanted seasonal influenza vaccine in the elderly: a systematic review and meta-analysis a systematic review and meta-analysis of cross-reactivity of antibodies induced by oil-in-water emulsion adjuvanted influenza h n virus monovalent vaccines preclinical pharmacokinetics of mhaa a, a human monoclonal antibody to influenza a virus, and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza a efficacy and safety of treatment with an anti-m e monoclonal antibody in experimental human influenza safety and upper respiratory pharmacokinetics of the hemagglutinin stalk-binding antibody vis support treatment and prophylaxis based on population modeling of seasonal influenza a outbreaks priming by a novel universal influenza vaccine (multimeric- )-a gateway for improving immune response in the elderly population a highly potent extended half-life antibody as a potential rsv vaccine surrogate for all infants safety and immunogenicity of a sf insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine immunogenicity and safety of a respiratory syncytial virus fusion protein (rsv f) nanoparticle vaccine in older adults evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial caparr os-wanderley w. synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans a t cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np m in adults aged over years a phase iia study to assess the safety and efficacy of a new influenza candidate vaccine mva-np m in healthy adults-flu clinical study report evaluating the immunogenicity and safety of a biondvax-developed universal influenza vaccine (multimeric- ) either as a standalone vaccine or as a primer to h n influenza vaccine back to the future: immunization with m- prior to trivalent influenza vaccine in / enhanced protective immune responses against / epidemic strain safety and immunogenicity of multimeric- -a novel universal influenza vaccine implication of respiratory syncytial virus (rsv) f transgene sequence heterogeneity observed in phase evaluation of medi- , a live attenuated parainfluenza type vectored rsv vaccine phase-i study medi- , of a live, attenuated intranasal vaccine against respiratory syncytial virus and parainfluenza- virus in seropositive children a randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (f) nanoparticle vaccine in healthy women of childbearing age efficacy of motavizumab for the prevention of respiratory syncytial virus disease in healthy native american infants: a phase randomised double-blind placebo-controlled trial motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial trivalency of a nanobody specific for the human respiratory syncytial virus fusion glycoprotein drastically enhances virus neutralization and impacts escape mutant selection efficacy, safety, and pharmacokinetics of a new % liquid intravenous immunoglobulin containing high titer neutralizing antibody to rsv and other respiratory viruses in subjects with primary immunodeficiency disease treatment with novel rsv ig ri- controls viral replication and reduces pulmonary damage in immunocompromised sigmodon hispidus structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses broadly cross-reactive antibodies dominate the human b cell response against pandemic h n influenza virus infection highly conserved protective epitopes on influenza b viruses human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza a viruses tackling influenza with broadly neutralizing antibodies influenza virus vaccine based on the conserved hemagglutinin stalk domain chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies chimeric hemagglutinin constructs induce broad protection against influenza b virus challenge in the mouse model epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza a virus influenza vaccine research funded by the european commission fp -health- -innovation- project antibodies to watch in protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized f protein of respiratory syncytial virus immunogenicity of rsv f dna vaccine in balb/c mice rsv fusion (f) protein dna vaccine provides partial protection against viral infection chimeric virus-like particles containing a conserved region of the g protein in combination with a single peptide of the m protein confer protection against respiratory syncytial virus infection baculovirus-expressed virus-like particle vaccine in combination with dna encoding the fusion protein confers protection against respiratory syncytial virus co-immunization with virus-like particle and dna vaccines induces protection against respiratory syncytial virus infection and bronchiolitis respiratory syncytial virus: infection, detection, and new options for prevention and treatment immunogenicity and efficacy of codon optimized dna vaccines encoding the f-protein of respiratory syncytial virus mucosal vaccines against respiratory syncytial virus prevention of colds by vaccination against a rhinovirus: a report by the scientific committee on common cold vaccines challenges in developing a cross-serotype rhinovirus vaccine misdirected antibody responses against an n-terminal epitope on human rhinovirus vp as explanation for recurrent rv infections key: cord- -v lll xy authors: zhu, zhaozhong; hasegawa, kohei; camargo, carlos a.; liang, liming title: investigating asthma heterogeneity through shared and distinct genetics: insights from genome-wide cross-trait analysis date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: v lll xy abstract: asthma is a heterogeneous respiratory disease reflecting distinct pathobiological mechanisms. these mechanisms are based, at least partly, on different genetic factors shared by many other conditions, such as allergic diseases and obesity. investigating the shared genetic effects enables better understanding the mechanisms of phenotypic correlations and is less subject to confounding by environmental factors. the increasing availability of large-scale genome-wide association study (gwas) for asthma has enabled researchers to examine the genetic contributions to the epidemiological associations between asthma subtypes, and those between coexisting diseases/traits and asthma. studies have found not only shared but also distinct genetic components between asthma subtypes, indicating that the heterogeneity is related to distinct genetics. this review summarizes a recently compiled analytical approach—genome-wide cross-trait analysis—to determine shared and distinct genetic architecture. the genome-wide cross-trait analysis features in several analytical aspects: genetic correlation, cross-trait meta-analysis, mendelian randomization, polygenic risk score and functional analysis. in this article, we discuss in detail the scientific goals that can be achieved by these analyses, their advantages and limitations. we also make recommendations for future directions: ) ethnicity-specific asthma gwass, and ) application of cross-trait methods to multi-omics data to dissect the heritability found in gwas. finally, these analytical approaches are also applicable to complex and heterogeneous traits beyond asthma. traditionally, examining the phenotypic correlation or coexistence of other factors is a useful way to investigate the heterogeneity of asthma. however, this approach may have residual confounding and provide insufficient biological insight as to which underlying mechanism(s) drive the association. a major advantage going from phenotypic correlations to genetic correlations is to improve understanding of the mechanism(s)-shared genetic components can be identified at different levels, from whole genome to individual variants, providing insights into the reasons why asthma and coexistent diseases or traits are correlated. furthermore, genetic correlations are less subject to confounding by environmental factors for several reasons. after adequately controlling for population ancestry, genetic correlation would occur only if the germline genetic variant is causal or in linkage disequilibrium (ld) with the causal variant of both traits. a purely environmental confounding factor (e.g., air pollution) would not lead to genetic correlation because it is not associated with any genetic variant (figure a and b) . in contrast, if an environmental factor is an intermediary step between the genetic variant and the trait, then it is in the causal pathway, and it is not considered a confounder-i.e., it does not create a false genetic correlation between the two traits (figure c) . population stratification is arguably the only confounding factor in gwas but it can be effectively controlled using principal components from genome-wide genetic markers. once the genetic effect on diseases and traits are robustly established, the genetic correlation between diseases and traits can be reliably estimated and replicated. [ ] [ ] [ ] [ ] in the following sections, we will discuss a range of detailed analyses that compile a comprehensive investigation between asthma and other coexistent diseases or traits. applicable to asthma and many other diseases/traits. the design has been successfully applied to the uk biobank and gwas consortia datasets, and determined the shared genetic architecture between asthma and allergic diseases, obesity, and mental health disorders, which were reproducible in other studies. - a genome-wide cross-trait analysis features several analyses: genetic correlation, cross-trait meta-analysis, mendelian randomization, polygenic risk score, and gwas functional analysis. each component is discussed in more detail in subsequent sections and depicted in figure . a glossary of the cross-trait gwas terminology may be found in table . a summary of genome-wide cross-trait analysis methods may be found in suggests obesity-to-asthma effects, reduction of body mass index (bmi) in patients with obesity might counteract the genetic effect, thereby potentially preventing the development of asthma. therefore, distinguishing horizontal pleiotropy from vertical pleiotropy where both contribute to genetic correlations are important and can be challenging. we discuss methods for these analyses in the sections below on cross-trait meta-analysis and mendelian randomization. the sensitivity analyses showed that the rg estimate from ldsc is unbiased to overlapping and asthma, the use of asset identified loci that are jointly associated with adhd and asthma, one locus with anxiety disorder and asthma, loci with major depressive disorder and asthma. of note, the human leukocyte antigen (hla) region (chr : - mb) was found to be shared in the cross-trait meta-analysis of allergic disease and asthma as well as that between major depressive disorder and asthma. the hla region was commonly reported to have definition cross-trait meta-analysis a meta-analysis that tests the null hypothesis that none of the traits being examined is associated with the genetic variant. one genetic variant is tested at a time. expression quantitative trait loci (eqtls) genetic variants that are associated with the gene expression levels. assuming all genetic variants have some effect on a trait and their effect size follow a gaussian distribution (called the infinitesimal model), the genetic correlation between two traits (a and b) measures the pearson's correlation between the genetic variant effect on traits a and b. genome-wide association study (gwas) an analytical method that tests the association between each genetic variant and a specific phenotype (a disease status or a quantitative trait). one genetic variant is tested at a time. human leukocyte antigen (hla)/ major histocompatibility complex (mhc) region a genomic region of approximately . mb genome sequence located on the chromosome p , which is mainly known for its pervasive pleiotropic effect and immune-related function. the extended mhc region is at - mb on chromosome . horizontal pleiotropy a genetic variant or gene having independent effects on multiple traits, which do not have causal effect on each other. variables that are associated with the modifiable exposure or risk factor of interest and affect the outcome only through the exposure or risk factor. mendelian randomization an analytic approach that examines the causality of an observed association of a modifiable exposure or risk factor with an outcome of interest using one or more genetic instrumental variables. polygenic risk score a score based on a set of disease/trait-associated genetic variants, commonly defined as weighted sum of their genotypes. weights are chosen by their association effect on the disease/trait, directly from gwas or further modified based on suitable statistical model incorporating all genetic variants on the genome. vertical pleiotropy (genetic causality) a genetic variant or gene having an effect on a trait, which has causal effect on another trait. advantages disadvantages examples of application in asthma or complex traits/pmid gobal initiative for asthma. the global asthma report asthma: epidemiology, etiology and risk factors comorbidities of asthma during childhood: possibly important, yet poorly studied asthma symptoms and bronchial reactivity in school children sensitized to food allergens in infancy association between atopic sensitization and asthma and bronchial hyperresponsiveness in swedish adults: pets, and not mites, are the most important allergens gender differences in asthma development and remission during transition through puberty: the tracking adolescents' individual lives survey (trails) study early age at menarche, lung function, and adult asthma differences between adult and childhood asthma shared genetic and experimental links between obesity-related traits and asthma subtypes in uk biobank effects of bmi, fat mass, and lean mass on asthma in childhood: a mendelian randomization study association between attention deficit hyperactivity disorder and asthma: a systematic review and meta-analysis and a swedish population-based study mental disorders among adults with asthma: results from the world mental health survey stress and inflammation in exacerbations of asthma are psychiatric disorders associated with worse asthma control and quality of life in asthma patients asthma: a syndrome composed of heterogeneous diseases genetic architectures of childhood-and adult-onset asthma are partly distinct shared and distinct genetic risk factors for childhood-onset and adult-onset asthma: genome-wide and transcriptome-wide studies causal associations between risk factors and common diseases inferred from gwas summary data detection of widespread horizontal pleiotropy in causal relationships inferred from mendelian randomization between complex traits and diseases distinguishing genetic correlation from causation across diseases and complex traits mendelian randomization accounting for correlated and uncorrelated pleiotropic effects using genome-wide summary statistics allergy, asthma, and the risk of breast and prostate cancer: a mendelian randomization study polygenic risk and the development and course of asthma: an analysis of data from a four-decade longitudinal study genome-wide polygenic scores for common diseases identify individuals with risk equivalent to monogenic mutations what are polygenic scores and why are they important modeling linkage disequilibrium increases accuracy of polygenic risk scores association of asthma and its genetic predisposition with the risk of severe covid- joint analysis of psychiatric disorders increases accuracy of risk prediction for schizophrenia, bipolar disorder, and major depressive disorder efficient cross-trait penalized regression increases prediction accuracy in large cohorts using secondary phenotypes clinical use of current polygenic risk scores may exacerbate health disparities an integrated encyclopedia of dna elements in the human genome integrative analysis of reference human epigenomes the genotype-tissue expression (gtex) project immunological genome project c. the immunological genome project: networks of gene expression in immune cells statistical testing of shared genetic control for potentially related traits heritability enrichment of specifically expressed genes identifies disease-relevant tissues and cell types ten years of pathway analysis: current approaches and outstanding challenges two-step epigenetic mendelian randomization: a strategy for establishing the causal role of epigenetic processes in pathways to disease the prevalence of food allergy: a meta-analysis racial and ethnic disparities in current asthma and emergency department visits: findings from the national health interview survey association study in african- admixed populations across the americas recapitulates asthma risk loci in non-african populations finding the missing heritability of complex diseases epigenetic mechanisms in asthma mediation analysis: a practitioner's guide causal structure learning key: cord- -jkik lb authors: paolo, gisondi; stefano, piaserico; luigi, naldi; paolo, dapavo; andrea, conti; piergiorgio, malagoli; valerio, marzano angelo; federico, bardazzi; massimo, gasperini; simone, cazzaniga; antonio, costanzo; sacchelli, lidia; pezzolo, elena; messina, francesco; lasagni, claudia; bigi, laura; cattaneo, angelo; carrera, carlo giovanni; arancio, luisa; ribero, simone; rozzo, giulia; damiani, giovanni; facheris, paola title: incidence rates of hospitalization and death from covid- in patients with psoriasis receiving biological treatment: a northern italy experience date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: jkik lb introduction whether biologic therapies enhance the risk of covid- or affect the disease outcome in patients with chronic plaque psoriasis remains to be ascertained. objective investigating the incidence of hospitalization and death for covid- in a large sample of patients with plaque psoriasis receiving biologic therapies compared with the general population. materials and methods this is a retrospective multicenter cohort study including patients with chronic plaque psoriasis (n= , ) being treated with biologic therapy and regularly followed up at the divisions of dermatology of several main hospitals in the northern italian cities of verona, padua, vicenza, modena, bologna, piacenza, turin, and milan. incidence rates (ir) of hospitalization and death per , person-months with exact mid-p % confidence intervals (ci) and standardized incidence ratios (sir) were estimated in the psoriasis patients and compared with the general population in the same geographic areas. results the ir of hospitalization for covid- was . ( % ci: . - . ) per , person-months in psoriasis patients and . ( % ci: . - . ) in the general population; the ir of death from covid- was . ( % ci: . - . ) and . ( % ci: . - . ) in psoriasis patients and the general population, respectively. the sir of hospitalization and death in psoriasis patients compared with the general population was . ( % ci: . - . ; p= . ) and . ( % ci: . - . ; p= . ) respectively. conclusions our data did not show any adverse impact of biologics on covid- outcome in psoriasis patients. we would not advise biologic discontinuation in patients on treatment since more than months and not infected with sars-cov- to prevent hospitalization and death from covid- . istat, istituto nazionale di statistica; sd, standard deviation; ir, incidence rate; ci, confidence intervals; sir, standardized incidence ratios; ibd, inflammatory bowel disease; psa, psoriatic arthritis; j o u r n a l p r e -p r o o f introduction italy has been deeply affected by the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic particularly in the northern regions ( ) . there is substantial concern among physicians regarding an increased risk of covid- in patients who are being treated with biologic therapies ( ) . however, whether biologics enhance this risk and/or if the disease course is worsened by the immunosuppressive/immunomodulating treatment remains to be determined. it is debated whether biologics for psoriasis should be interrupted for preventing severe complications of sars-cov- infection such as interstitial pneumonia. notably, sars-cov- infection seems to be most fatal when it triggers a cytokine storm, including tumor necrosis factor (tnf)-α, interleukin (il)- and il- ( ) ( ) . therefore, biologics are being investigated as treatments for covid- ( ) . in this study, we evaluated the incidence of hospitalization and death for covid- in a large sample of patients with plaque psoriasis receiving biologic therapies compared with the general population. j o u r n a l p r e -p r o o f the characteristics of the studied population are reported in the (table) . there were no further deaths for other causes during the study period. we had , out of , (i.e. . %) patients affected by psa. four out of hospitalized psoriasis patients had psa, whereas none of the dead ones had psa. all the hospitalized patients fully recovered from the viral infection and then re-started biologic therapy because of psoriasis relapse after a period of time ranging from to weeks from the hospital discharge. they are currently on biologic. the major finding of our study is that, although psoriatic patients treated by biologics are burdened by higher rates of metabolic and cardiovascular comorbidities, there was no evidence for an increased risk of hospitalization or death from covid- in those patients compared with the general population. accordingly, some preliminary data on tnf--α inhibitors and il / inhibitors in inflammatory bowel disease (ibd) patients j o u r n a l p r e -p r o o f showed that these therapies do not worsen the clinical course of covid- compared with sulfasalazine/mesalamine or no treatment ( ) . on the opposite, biologics appeared to be associated with a better outcome, even though there were insufficient data to make definite statements ( ) . some systemic complications caused by sars-cov- infection appear to be associated with excessive inflammatory and cytokine responses ( ). therefore, treatments that reduce the host inflammatory response, including agents blocking tnf-α, il- or il- pathways, in combination with therapies that have direct anti-viral activity, have been proposed, and are currently under investigation for the treatment of covid- ( ) ( ) ( ) . we acknowledge the limitations of our study, including the absence of serological or molecular investigations for the diagnosis of sars-cov- infection in asymptomatic patients with psoriasis. the objective of our study was not to investigate the prevalence of the sars-cov- infection, but to report the occurrence of hospitalization and death, as indicators of severe outcomes related to covid- . despite a cohort of , psoriatic patients receiving biologic treatment, we collected relatively few covid- cases, with wide cis. the low rates in patients treated with biologics are reassuring, especially considering that these patients had a high prevalence of comorbidities that are usually associated with a worse covid- course. despite a great effort has been made in retrieving covid- patients, we acknowledge that there is still a possibility that we have missed important cases. however, based on a simulation analysis on our sample size, we estimated that around missed deaths would be required to observe a sir= and at least deaths for a sir> with p-value< . . on the other hand, we would have missed at least hospitalized patients to observe a sir> . with p-value< . . therefore, it is unlikely that we have missed a number of patients needed to completely change our results. there weren't new patients starting biological treatment from february th to may st in our divisions of dermatology. this is because this time period was overlapping with the lockdown imposed by the italian government. during that period, public health measures required citizen to stay at home and shield. the clinical dermatological activity was significantly reduced and mostly dedicated to teleconsultation for those patients already on treatment ( ) . the access to the hospitals was limited only to symptomatic patients with fever, suspected for sars-cov- infection. the major strengths of our study are the cohort study design, the focus on italian regions most affected by the sars-cov- pandemic, and the completeness of the database. we acknowledge that patients on biologic drugs may have self-isolated more efficiently, thus limiting their own infectious risk. we can rule out that there have been deaths at home that we are not aware of and/or that patients have gone to hospitals outside their catchment area. our findings are consistent with those of another study which reported that patients with psoriasis on biologics were not at an increased risk of intensive care unit admission or death; conversely, the study found that patients were at a higher risk for testing positive for sars-cov- , to be self-quarantined at home or hospitalized ( ) . in conclusion, the results of our study show that the continuation of biologic therapies during the pandemic does not influence the development of severe complications of the sars-cov- infection. a prophylactic treatment discontinuation in an attempt to prevent a negative outcome of covid- may not be required ( ) . larger studies with longer follow-up periods are needed to confirm these findings. for descriptive purposes data were presented as means with standard deviations (sd) or numbers with percentages for continuous and categorical variables respectively. incidence rates (ir) were calculated as the number of observed cases per , person-months j o u r n a l p r e -p r o o f with exact mid-p % confidence intervals (ci). for comparison purpose, standardized incidence ratios (sir) were calculated by using indirectly standardized rates from the number of observed events in the study population and the number of expected events derived by applying, to the target population, age, gender and regional specific rates of the general adult population of the same geographical areas during the same period of time. sir were reported along with their exact mid-p % ci and p-values. a stratified sir analysis by age and class of biologic was also performed for covid- hospitalization. differences in the distribution of demographics and comorbidities between groups were assessed by using pearson's Χ test and two-sample t-test for nominal and continuous variables respectively. all tests were considered significant at p-value < . . analyses were carried out with r software v. . . (r foundation for statistical computing, vienna, austria). the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak should biologics for psoriasis be interrupted in the era of covid- ? elevated plasma level of selective cytokines in covid- patients reflect viral load and lung injury associations between immune-suppressive and stimulating drugs and novel covid- -a systematic review of current evidence. ecancermedicalscience trials of anti-tumour necrosis factor therapy for covid- are urgently needed secure-ibd database public data update. covidibd.org. last accessed on outcomes of covid- in patients with ibd in italy: an ig-ibd study should we stimulate or suppress immune responses in covid- ? cytokine and anti-cytokine interventions a randomized, open-label, controlled trial for the efficacy and safety of adalimumab injection in the treatment of patients with severe novel coronavirus pneumonia (covid- a randomized, blinded, controlled, multicenter clinical trial to evaluate the efficacy and safety of ixekizumab combined with conventional antiviral drugs in patients with novel coronavirus pneumonia dermatologists and sars-cov- : the impact of the pandemic on daily practice biologics increase the risk of sars-cov- infection and hospitalization, but not icu admission and death: reallife data from a large cohort during red-zone declaration guidance on the use of biologic agents during covid- outbreak american academy of dermatology (last accessed key: cord- -vik rgnq authors: ohkubo, kimihiro; baraniuk, james n.; hohman, robert; merida, marco; hersh, louis b.; kaliner, michael a. title: aminopeptidase activity in human nasal mucosa()()()() date: - - journal: j allergy clin immunol doi: . /s - ( ) - sha: doc_id: cord_uid: vik rgnq background: aminopeptidases activate bradykinin and degrade many inflammatory peptides. objective: the objective of this study was to identify the types of aminopeptidase activities in human nasal mucosa. methods: human nasal mucosa was homogenized (n = ), and cytoplasmic (s ) and membrane-rich (p ) fractions were obtained. several aminopeptidase (ap) activities were defined by ( ) substrate specificity with leucine-enkephalin (leu-ap) and alanine-nitroanilide (ala-ap), ( ) inhibitor studies with puromycin and bestatin, ( ) enzyme activity histochemistry (zymography), ( ) immunohistochemistry, and ( ) gel electrophoresis. human volunteers had methacholine, histamine, and allergen nasal provocations to determine the mechanisms controlling nasal aminopeptidase secretion in vivo. results: p was the largest reservoir of puromycin-resistant aminopeptidase activity ( pmol leu-enk/min/mg protein). s contained pmol leu-enk/min/mg activity, with % representing puromycin-resistant activity and % puromycin-sensitive aminopeptidase (ps-ap). ala-ap was detected in both p and s fractions and was localized by zymography to epithelial and gland cells. anti–rat brain–soluble ps-ap igg detected immunoreactive material in epithelium, glands, and endothelium. in nasal provocation studies, leu-ap correlated with glandular exocytosis but not vascular leak. conclusions: the predominant aminopeptidase in human nasal epithelial and submucosal gland cells was membrane-bound puromycin-resistant aminopeptidase. a novel soluble puromycin-resistant aminopeptidase and lower amounts of soluble ps-ap were also detected. (j allergy clin immunol ; : - .) a wide range of peptidolytic activities has been described including aminopeptidases that digest peptides from their n-terminus, carboxypeptidases, and neutral endopeptidase (nep) and angiotensin-converting enzyme (ace) that cleave internal peptide bonds. in airway mucosa, aminopeptidases may be critical for the generation of bradykinin but may also reduce the duration of action of bradykinin, neuropeptides released from nociceptive neurons (eg, substance p), and other proinflammatory peptides. [ ] [ ] [ ] [ ] aminopeptidase activities have frequently been identified with the use of leucine-enkephalin (enkephalindegrading aminopeptidase, leu-ap) as a substrate. , the major enkephalin-degrading aminopeptidase activities were initially described on the basis of their arylamidase activity but have subsequently been referred to as either aminopeptidase m (apm, also known as aminopeptidase n, cd ) or the puromycin-sensitive aminopeptidase (ps-ap). [ ] [ ] [ ] [ ] apm is an integral membrane protein that is relatively resistant to inhibition by puromycin with coefficients of inhibition (ki) of µmol/l to mmol/l. , ps-ap is predominantly a cytosolic enzyme but can be found associated with membranes. it is more sensitive to inhibition by puromycin (ki approximately µmol/l). , both apm and ps-ap are inhibited by bestatin. the cdnas for rat kidney apm and human psap have been cloned and their respective amino acid sequences deduced. to begin defining the physiology of aminopeptidases in airways, we have used multiple assays to characterize aminopeptidase activity in soluble and membrane fractions of human nasal mucosa. substrate specificity was assessed with the use of leucine-enkephalin (leu-ap activity) and alanine-nitroanilide (ala-ap activity). inhibition of leu-ap by puromycin defined ps-ap and rela-tively resistant activities. similar studies were performed with bestatin. ala-ap activities were localized on cryostat tissue sections by enzyme activity histochemistry (zymography), whereas ps-ap-immunoreactive material was identified by immunohistochemistry. this study complements previous analyses of neutral endopeptidase (neprilysin, nep, cd , calla, gp ), ace, and carboxypeptidase n (cpn) that also modulate neurogenic and other peptide-mediated inflammatory events. human inferior nasal turbinates were obtained at the time of surgery from patients with nasal airway obstruction as previously described. [ ] [ ] [ ] tissue was dissected from the conchal bones within minutes of excision and immediately prepared for future use. six mucosal specimens were used for enzyme assays and were frozen in -methylbutane on dry ice for seconds and then stored at °c. [ ] [ ] [ ] frozen turbinates were weighed (average wet weight ± mg, range to mg), finely dissected with razor blades, suspended in volumes (volume/wet tissue weight) of . mol/l sucrose in mmol/l tris hcl (ph . ), and homogenized with a polytron homogenizer (std tissumizer, tekmar, cincinnati, ohio) at °c at maximum speed for seconds twice. the homogenates were centrifuged at g for minutes at °c and supernatants were collected. the pellets were suspended again in the same buffer and recentrifuged at the same rate and the pellet discarded. both supernatants were combined and designated s . s was centrifuged at , g for minutes at °c and the resulting supernatant was collected (s ). the , g pellet (membrane fraction) was washed and resuspended in mmol/l tris hcl (ph . ) and was designated as p . nasal extract samples ( µl) were incubated in µl of mmol/l tris hcl buffer (ph . ) with µl of [ h]leuenkephalin ( . ci/mmol, nendupont, wilmington, del; . nmol/l final concentration) as a tracer and leucine-enkephalin ( µmol/l final concentration; peninsula laboratories, belmont, calif) as substrate. , [ ] [ ] [ ] phosphoramidon ( µmol/l final concentration; peninsula) and captopril ( µmol/l final concentration; provided as a gift from squibb, princeton, nj) were added to inhibit nep and ace activities, respectively. incubations were stopped after minutes by addition of µl of n hcl. the metabolite [ h]tyrosine was separated from nonmetabolized enkephalin by chromatography on a porapack q - mesh polystyrene bead column (waters, milford, mass). blanks were obtained from parallel incubations performed in the presence of mmol/l puromycin (calbiochem, la jolla, calif) or µmol/l bestatin (calbiochem). porcine aminopeptidase m (sigma chemical co, st louis, mo) was used as a standard for confirming assay conditions and inhibitor activities. enkephalin-degrading aminopeptidase activity was expressed as picomoles of leuenkephalin metabolized per minute per milligram of protein for extracts, or as picomoles of leu-enkephalin metabolized per minute per milliliter for nasal lavage fluid samples. puromycin and bestatin were added to the leu-ap in concentrations of - to - mol/l and the ki defined as the concentration reducing leu-ap activity by %. alanine aminopeptidase (ala-ap) assay samples ( µl) were added to cuvettes containing . ml of mmol/l tris hcl buffer (ph . ), µmol/l phosphoramidon, and µmol/l captopril, mixed thoroughly, and incubated for minute. l-alanine- nitroanilide ( µl, ala-na; sigma; concentration mmol/l) was added and the kinetics of nitroanilide formation measured by absorbance at nm (a ) over time (t, minutes). the catalytic concentration of the samples (b in units per milliliters) were calculated as b = × a /t. puromycin ( mmol/l) was added to negative control samples. the relative activities toward leuenkephalin and ala-na were compared in each s and p sample by using competition assays. the km was determined for each substrate. in the first set of competition studies, leu-enkephalin was added at its km concentration along with a wide range of ala-na concentrations to determine the effect of leu-enkephalin on the ala-na assay. similarly, ala-na was added at its km to the leu-enkephalin assays to determine their degree of interaction. rat brain-soluble ps-ap was purified as described. [ ] [ ] [ ] a female goat was injected subcutaneously with this enzyme, then boosted with µg of protein in incomplete freund's adjuvant and weeks later. after phlebotomy, the igg fraction was obtained by precipitation with % ammonium sulfate and dialyzed against pbs. nasal extracts were treated with native sample buffer without sds or mercaptoethanol. - samples ( µl) and buffer (negative control) were applied to % tris-glycine polyacrylamide gels (novex experimental technology, san diego, calif), electrophoresed, then stained for protein with coomassie blue (pharmacia lkb, piscataway, nj). enzyme activity was detected by placing gels in . mmol/l l-alanyl- -methoxy- -naphthylamide (l-ala- after electrophoresis, proteins were transferred from the native gel to nitrocellulose membranes by unilateral electrodiffusion blotting at v for minutes. [ ] [ ] [ ] membranes were blocked with % nonimmune rabbit serum, . % bsa in tris buffer ( . % nacl, mmol/l tris hcl, ph . ) at room temperature for minutes, rinsed twice in tris buffer, then incubated at °c for hours in ps-ap polyclonal antibody diluted : in . % bsa in tris buffer. after -minute washes in tris buffer, the membranes were incubated with phosphataselabeled rabbit anti-goat igg (kirkegaard & perry laboratories, gaithersburg, md) diluted : in . % bsa in tris buffer at room temperature for minutes. after washing times in tris buffer, membranes were placed in bcip/nbt phosphatase substrate (kirkegaard & perry) for minutes at room temperature. the color reactions were terminated by washing in large volumes of distilled water. for western blots, samples were concentrated through centricon microconcentrators (amicon, danvers, mass). s preparations were concentrated to approximately mg/ml, p to approximately mg/ml, and nasal lavages to approximately mg/ml. fresh tissues (n = ) were fixed with % paraformaldehyde in pbs (ph . ) at °c for hours and stored in pbs before being embedded in paraffin. [ ] [ ] [ ] six-micron-thick paraffin sections were cleared in xylene and graded alcohols, washed in water and pbs, and blocked in % bsa in pbs. sections were incubated with goat polyclonal anti-rat brain-soluble ps-ap ( : dilution in % bsa, pbs) for hours at °c in a humidified chamber, washed in pbs, and incubated with colloidal gold-labeled rabbit anti-goat igg (biocell, ted pella, redding, calif; : dilution with % bsa in pbs) at room temperature for minutes. after washing times in pbs for minutes each and times in distilled water for minutes each at room temperature, silver enhancing solution (intense, janssen) was added. the development of the stained slides was monitored under light microscopy. the slides were washed in distilled water and counterstained with nuclear fast red. cryostat sections were cut from frozen tissues (n = ) and allowed to dry at room temperature before fixation in % analytical grade acetone for minutes at °c followed by brief drying in air. aminopeptidase activity was demonstrated with the use of the simultaneous azo-coupling technique with l-ala-mna (final con- centration . mmol/l) and fast blue bb salt (sigma chemical co, final concentration mg/ml) in . % dimethylformamide and . % trismalate buffer (ph . ). slides were incubated in this substrate for minutes at °c, washed for to minutes in distilled water, fixed for minutes in % aqueous formalin, rinsed briefly in distilled water, and mounted in glycerol gelatin. the sites of lala-mna catabolism were identified by red staining of the tissue. a total of adult subjects ( male, female) between the ages of and years were studied. subjects were excluded if they had had an upper respiratory tract infection in the past weeks, any nasal symptoms at the time of the study, or were taking any medications in the previous hours. atopic subjects (n = ) with perennial (n = ) or seasonal (n = ) symptoms were defined by at least positive allergy prick skin test (wheal > mm than glycerol negative control) from a panel of relevant aeroallergens. seasonally atopic subjects were studied out of their allergy season. nonatopic subjects (n = ) had no allergic symptoms and had negative skin test results. nasal challenges were performed as previously reported. - , nasal lavage fluid was collected through f rubber catheters. after the nasal cavity was washed with -ml saline prewashes, the challenge protocols were begun. nasal challenge solutions prepared in normal saline were applied to one or both sides of the nasal cavity from a nebulizer bottle. each -minute challenge period was followed by a -ml saline lavage. in the first protocol, subjects were challenged sequentially with saline followed by mg methacholine. in the second protocol, saline challenge was followed by challenge with mg histamine. in the third protocol, subjects were sequentially challenged with µg atropine and then mg methacholine. finally, patients received an allergen challenges as previously described. baseline secretions were collected minutes after saline challenge. solutions of relevant allergen ( pnu) were instilled, then lavages were collected every minutes for the first hour and then every minutes for the second hour. leu-ap was assessed as described except that nasal lavage samples ( µl) were incubated with µl of [ h]leuenkephalin ( . nmol/l final concentration) and leuenkephalin ( µmol/l final concentration) in mmol/l tris hcl (ph . ). lactoferrin was measured by a modified noncompetitive elisa with an assay range of to ng/ml. , albumin was measured by a direct, competitive elisa with an assay range of to µg/ml. , igg was measured by a direct, noncompetitive elisa with an assay range of to ng/ml. , total protein was measured by the lowry method with bsa as standard. , leuap activity was detected in both the cytosolic (s ) fraction and membrane-rich (p ) fractions from each of adult human nasal turbinates. the specific activity was significantly higher in p fractions ( . ± . pmol/min/mg protein, range . to . ) than s fractions ( . ± . pmol/min/mg protein, range . to . ) (p < . ). in all samples, the ki values for puromycin were higher than for bestatin (fig ) . puromycin completely inhibited leu-ap activity in all samples, but only at very high concentrations. in the s fraction, components were discernible, being a puromycin-sensitive fraction representing % of the total ed-ap activity, and exhibiting a ki of . µmol/l. this behavior was consistent with this fraction being soluble ps-ap. the remaining % of the activity had a ki of µmol/l and thus represented a novel aminopeptidase activity. all of the activity in the p fraction exhibited a high ki for puromycin of µmol/l. thus no ps-ap was detected in the membrane fraction. bestatin inhibited all leu-ap activity in s (ki = µmol/l) and p (ki = . µmol/l). ala-ap activity was detected in both s and p fractions. the activity in s fractions was . ± . mu/mg protein (range . to . ). p fractions had higher activities of . ± . mu/mg protein (range . to . ). the km for leu-enkephalin was . mmol/l in s and . mmol/l in p (fig ) . when ala-na was the substrate, km was mmol/l with s and mmol/l with p . the addition of mmol/l ala-na to s samples shifted the leu-enkephalin degradation curve -fold to the right. in the p fraction, addition of mmol/l ala-na increased km by -fold. the addition of . mmol/l leu-enkephalin to s fractions shifted the km for ala-na to mmol/l, a -fold increase. similarly, the km for ala-na was increased -fold by leu-enkephalin in p fractions. these data indicate that leu-enkephalin and ala-na measure the same aminopeptidase activities. electrophoresis of the dilute and concentrated s ( mg/ml) and p ( . mg/ml) fractions followed by l-ala-nma azocoupling zymography demonstrated a single band in both preparations with the same mobility (fig ) . the differences between p and s concentrations suggest a higher specific activity for aminopeptidase in the membrane-rich p fraction. ps-ap may have had too low an activity to be detectable by these methods. soluble ps-ap-immunoreactive material was localized to the epithelium, submucosal glands, and endothelium in human nasal turbinate mucosa (figs and ) . the most intense staining was concentrated in the outer layer of the epithelium in all the specimens. the immunoreactivity was identified in both the columnar and basal cells but not in the goblet cell vacuoles (fig , a) . in the submucosal glands, immunoreactivity was seen in both serous and mucous cells (fig , c) , but the serous cell immunoreactivity was more intense (fig , a) . glandular staining was inconsistent between samples, suggesting variable expression or loss after exocytosis or other processes. immunoreactivity was seen in both small (fig , a) and large vein endothelial cells (fig , a) . enzyme histochemistry was used to localize l-ala-mna-cleaving aminopeptidase to the epithelium and submucosal glands (fig , a and c) . these l-ala-mna histozymograms were inhibited by preincubation with µmol/l puromycin for minutes at °c (fig , b and d) . the glandular staining was most concentrated on the lumenal surface of acinar cells, making it difficult to distinguish serous cells from mucous cells. there was almost no l-ala-mna activity in either small or large vessels. leu-ap activity after saline challenge was . ± . pmol/min/ml (range . to . ). methacholine provocation increased leu-ap activity to . ± . pmol/min/ml (range . to . ), indicating active secretion. pretreatment with atropine blocked the methacholine-induced leu-ap release ( . ± . pmol/min/ml, range . to . ). histamine increased leu-ap to . ± . pmol/min/ml (range . to . ). methacholine stimulated glandular exocytosis and secretion, whereas histamine caused vascular leak with extravasation of plasma proteins plus stimulation of nociceptive nerves and recruitment of cholinergic reflexes that cause glandular exocytosis. [ ] [ ] [ ] , these secretory data suggest that cholinergic stimulation of glands by direct and reflex actions was the predominant mechanism regulating leu-ap secretion in human nasal mucosa in vivo. to determine if leu-ap secretion occurred in association with glandular and/or vascular proteins, leu-ap activity in the secretions was compared with the total protein, lactoferrin (a specific marker of serous cell secretion), and albumin (a plasma protein marker reflecting vascular permeability) (fig ) . after methacholine provocations (fig , a, b, and c) , leu-ap was highly correlated with lactoferrin (r = . , p < . ) and total protein (r = . , p < . ), whereas leu-ap and albumin secretion were not correlated (r = . , not significant). after histamine provocation (fig , d, e, and f) , leu-ap was correlated with total protein (r = . , p < . ), lactoferrin (r = . , p < . ) and albumin (r = . , p < . ). these data are in agreement with other studies of histamine provocations that show parallel stimulation of both vascular permeability and cholinergic reflex-mediated glandular secretion. [ ] [ ] [ ] , thus both methacholine and histamine provocation systems indicate that leu-ap secretion correlated most closely with glandular secretion. repeated saline challenges ( to minutes, fig ) had no effect on the leu-ap content of nasal lavage fluids. allergen given at minutes induced sneezing, rhinorrhea, nasal pruritus, and congestion in all subjects within minutes of challenge. nasal lavages were enriched with total protein, albumin, igg, and lactoferrin. leu-ap increased minutes after challenge ( minutes), reached maximum concentration after minutes ( -minute time point), and returned to baseline minutes after allergen. leu-ap levels from the first hour after allergen ( to minutes) were significantly higher than levels from saline ( to minutes) and the second ( to minutes) lavage fluids (p < . , analysis of variance for multiple comparisons with bonferroni correction). there were no significant differences between individual time points because of the wide variances in leu-ap values. leu-ap release between and minutes correlated with lactoferrin (r = . , p < . ), igg (r = . , p < . ) and total protein (r = . , p < . ) (fig ) . these experiments indicate that leu-ap secretion was increased during the immediate phase of an allergic reaction. identification of ap activities in human nasal mucosa has been difficult because of the diverse assays, substrates, and inhibitors required. despite these limitations, aminopeptidase activities were discovered. the majority of aminopeptidase activity (approximately %) was found in the membrane-rich (p ) tissue fraction. this puromycin-resistant/bestatin-sensitive aminopeptidase probably represented epithelial, glandular, and other cell surface apm activity. the soluble s fraction contained the remaining approximately % of total aminopeptidase activity. soluble aminopeptidase activities have also been detected in urine, , bile acid, serum, and nasal lavage fluid. approximately % of the s activity ( % of total extract aminopeptidase activity) can be attributed to ps-ap. the ps-ap-immunoreactive material was localized to epithelium, glands, and endothelium with the use of anti-rat brain-soluble ps-ap. release of this soluble enzyme from exocrine cells may have been under cholinergic control. the bulk of the s activity ( % of s , % of the total aminopeptidase activity) was a soluble puromycininsensitive/bestatin sensitive aminopeptidase. this activity may correspond to the novel aminopeptidase recently described by erbeznik and hersh. their "ap " had a ki for puromycin of mmol/l and . mmol/l for bestatin. their -kd macromolecule may have been a dimer because high salt led to dissociation into one kd electrophoretic band. this may correspond to the ala-ap activity detected in epithelium and gland cells by zymography (fig , b) . the nasal provocation experiments demonstrated that methacholine, antigen, and histamine could each induce aminopeptidase secretion that correlated with exocytosis of markers of glandular secretion but not markers of vas-cular permeability. [ ] [ ] [ ] , because glandular exocytosis is regulated by cholinergic reflexes, parasympathetic pathways may regulate aminopeptidase activity in secreted mucus. benefits of secreting aminopeptidases into airway mucus may include their antiviral activity and capacity to generate bradykinin. cell surface aminopeptidase acts as a binding site for coronaviruses. it is conceivable that mucus aminopeptidase may bind to these virions to prevent epithelial cell binding, uptake, and infection. virion destruction could be mediated by plasma proteins because aminopeptidases generate bradykinin from one of its precursors, lysyl-bradykinin (kallidin). , , bradykinin potently stimulates vascular permeability that causes flooding of the airway lumen by immunoglobulin-containing, complement-containing, and fibrin-containing plasma. vasodilation, nociceptive sensory nerve activation that conveys sensations of burning pain, and arachidonic acid metabolism that promotes additional glandular exocytosis, also occur. bradykinin is elevated in secretions during the common cold and after allergen provocation. however, bradykinin antagonists have had little effect in the common cold, and inhibition of aminopeptidase with bestatin does not accentuate the vascular permeability induced by tobacco in the hamster cheek pouch model. these findings suggest an alternative hypothesis: secretion of glandular aminopeptidase along with kininogens of vascular and glandular origin into the airway mucus leads to bradykinin production that is only the consequence and marker of their parallel secretion rather than the primary cause of mucosal inflammation. these competing hypotheses require additional testing that should include human nasal provocations with aminopeptidase inhibitors such as bestatin. human nasal mucosal neutral endopeptidase (nep): location, quantitation, and secretion angiotensin converting enzyme in human nasal mucosa human nasal mucosal carboxypeptidase: activity, location, and release characterization membrane bound aminopeptidases from rat brain: identification of the enkephalin degrading aminopeptidase kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis dipeptidyl(amino)peptidase iv and aminopeptidase m metabolize circulating substance p in vivo metabolism of vasoactive peptides by plasma and purified renal aminopeptidase m the colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases molecular cloning and amino acid sequence of rat kidney aminopeptidase m: a member of super family of zinc metallohydrolase human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n purification and characterization of arylamidase from monkey brain solubilization and characterization of two rat brain membrane-bound aminopeptidases active on met-enkephalin the relationship between enkephalin degradation and opioid receptor occupancy cloning of the human puromycin-sensitive aminopeptidase and evidence for expression in neurons an aminopeptidase from bovine brain which catalyzes the hydrolysis of enkephalin characterization membrane bound aminopeptidases from rat brain: identification of the enkephalin degrading aminopeptidase isolation and characterization of enkephalin degrading aminopeptidase from rat brain determination of l-alanylpeptide hydrolase in urine isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium sulfolobus solfataricus pathophysiology of rhinitis : assessment of the source of proteins in histamine induced nasal secretions effects of nedocromil sodium on allergen induced rhinitis in humans urinary excretion of n-acetyl-ß-d-glucosaminidase and alanine aminopeptidase in patients receiving amikacin or cis-platinum aminopeptidase m from human liver, ii: kinetic analysis of inhibition of the enzyme by bile acid elektrophoretische varianten der alanineaminopeptidase in serum bei hepatobiliaren erkrankungen a novel mammalian high molecular weight aminopeptidase virus-receptor interactions in the enteric tract: virus-receptor interactions kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen an enzyme in human blood plasma that inactivates bradykinin and kallidins comparative nasal effects of bradykinin, kallidin, and [des-arg ]-bradykinin in atopic rhinitic and normal volunteers angiotensin converting enzyme and neutral endopeptidase modulate smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa in the hamster cheek pouch model key: cord- - v zfue authors: hamilos, daniel l. title: host-microbial interactions in patients with chronic rhinosinusitis date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: v zfue there has been considerable investigation of host-microbial interactions in patients with chronic rhinosinusitis (crs) in hopes of elucidating mechanisms of disease and better treatment. most attention has been paid to bacterial infection and potential underlying defects in innate immunity. bacterial biofilm is present in most patients with crs undergoing surgical intervention, and its presence is associated with more severe disease and worse surgical outcomes. a role for viral or fungal infection in patients with crs is less clear. there is no evidence for a primary defect in mucociliary clearance in most patients with crs. decreased levels of certain antimicrobial proteins, most notably lactoferrin, have been found in sinus secretions, whereas levels of other antimicrobial proteins have been found to be normal. no primary defects in toll-like receptors have been found in patients with crs, although a % reduced expression of toll-like receptor was reported in patients with recalcitrant nasal polyps. a polymorphism in a bitter taste receptor was recently associated with refractory crs and persistent pseudomonas aeruginosa infection. a downregulation of innate immunity by maladaptive t(h) tissue inflammation has also been described in patients with recalcitrant nasal polyps, suggesting a link to persistent infection. to date, an effective means of restoring host-microbial balance and mitigating disease in patients with crs remains elusive. there has been considerable investigation of host-microbial interactions in patients with chronic rhinosinusitis (crs) in hopes of elucidating mechanisms of disease and better treatment. most attention has been paid to bacterial infection and potential underlying defects in innate immunity. bacterial biofilm is present in most patients with crs undergoing surgical intervention, and its presence is associated with more severe disease and worse surgical outcomes. a role for viral or fungal infection in patients with crs is less clear. there is no evidence for a primary defect in mucociliary clearance in most patients with crs. decreased levels of certain antimicrobial proteins, most notably lactoferrin, have been found in sinus secretions, whereas levels of other antimicrobial proteins have been found to be normal. no primary defects in toll-like receptors have been found in patients with crs, although a % reduced expression of toll-like receptor was reported in patients with recalcitrant nasal polyps. a polymorphism in a bitter taste receptor was recently associated with refractory crs and persistent pseudomonas aeruginosa infection. a downregulation of innate immunity by maladaptive t h tissue inflammation has also been described in patients with recalcitrant nasal polyps, suggesting a link to persistent infection. to date, an effective means of restoring host-microbial balance and mitigating disease in patients with crs remains elusive. (j allergy clin immunol ; : - .) key words: chronic rhinosinusitis, host, microbial, biofilm, innate, immunity, antimicrobial discuss this article on the jaci journal club blog: www.jacionline.blogspot.com. host-microbial interactions play a critical role in crs disease initiation and perpetuation. this article aims to summarize knowledge of host-microbial interactions elucidated in relation to normal sinus physiology and pathology of patients with chronic rhinosinusitis (crs), including the subsets regarded as chronic rhinosinusitis without nasal polyps (crssnp), chronic rhinosinusitis with nasal polyposis (crswnp), and allergic fungal rhinosinusitis (afrs). most studies of innate immunity and host-microbial interactions in patients with crs have focused on patients with ''refractory'' or ''recalcitrant'' disease. refractory crs has been defined on the basis of failure to stabilize after surgery, antibiotics, saline rinses, and topical steroid treatment. somewhat differently, ''recalcitrant crs'' has been defined based on recurrence of nasal polyps (nps) after polyp surgery. these definitions are noteworthy because patients with refractory polyposis, for example, might have more evidence of infection, whereas patients with recalcitrant polyposis might have little or no evidence of infection but more evidence for maladaptive t h -biased mucosal inflammation. viral upper respiratory tract infections are potentially highly relevant to crs. the average healthy adult person experiences to common colds per year (http://www.niaid.nih.gov/topics/ commoncold/pages/overview.aspx). in healthy subjects the onset and time course of cold symptoms and levels of viral mrna detectable in nasal secretions over days have been mapped out after experimental rhinovirus infection. , patients with asthma or chronic obstructive pulmonary disease manifest a significantly higher peak rhinovirus viral load and duration of symptoms. asthmatic patients also manifest a corresponding -fold decreased induction of type i (b) and type iii (l and l / ) interferons. given the similarities between asthma and crs at the tissue level and the fact that many crs exacerbations occur during the viral season, it is plausible that a similar defect exists in patients with crs. however, experimental rhinovirus infection has not been studied in patients with crs. the innate antiviral response to rhinovirus infection involves activation of type i interferons through interferon-regulatory factor gene activation, an increase in nitric oxide (no) production, and epithelial production of human b-defensin (hbd) , il- , and rantes. , il- a was found to augment production of hbd- and il- but downregulate production of rantes in this model. the chemokine cxcl (interferon-inducible protein ) is also induced. there has not been a study of the incidence of rhinovirus infection in patients with crs. a study by jang et al reported that % of patients with crs have detectable rhinovirus infection. this study examined nasal lavage fluid and turbinate epithelial cells (collected with a rhino-probe mucosal curette; rhino-probe, arlington scientific, arlington, tex) from patients with crs and healthy control subjects (hcs). using an rt-pcr-based assay, they found that lavage fluid from all patients with crs and hcs and turbinate epithelial cells from hcs were negative for picornavirus, whereas ( %) of epithelial cell samples from patients with crs were positive. further examination revealed that all patients with crs with positive results had positive results for rhinovirus. it is unclear whether these represented subclinical infections because patients were studied at only time point. in an in vitro experiment wang et al infected nps and nasal turbinate epithelial cells from patients with crswnp and sphenoid sinus and turbinate epithelial cells from hcs with rhinovirus (rhinovirus ) . no significant differences in rates of infection or induction of il- or il- were found. our group found that cultured airway epithelial cells from patients with crssnp had an exaggerated response to stimulation with the combination of double-stranded rna (a toll-like receptor [tlr] agonist and surrogate for viral infection) plus cigarette smoke extract, with exaggerated production of rantes and hbd- . finally, although studies are quite limited, there is a lack of evidence for persistence of viral infection in patients with crs. again using pcr methodology, wood et al , using special techniques to optimize recovery of anaerobic bacteria identified these bacteria in roughly % of children with crs. prospective studies in adults identified a positive bacterial culture in a variable percentage of patients with crs (reviewed by meltzer et al ). coagulase-negative staphylococcus species was the most common aerobic isolate in several studies, often accompanied by staphylococcus aureus and viridians streptococci. organisms associated with acute bacterial rhinosinusitis were cultured in some cases. in several studies gram-negative enteric rods, including pseudomonas aeruginosa, klebsiella pneumoniae, proteus mirabilis, enterobacter species, and escherichia coli were also isolated. these organisms are rarely found in middle meatus cultures from healthy subjects. more recent studies of intraoperative sinus cultures with simultaneous analysis of cultures and biofilm reported positive cultures in . % to % of cases, with a predominance of s aureus and p aeruginosa in the isolates. [ ] [ ] [ ] the frequency with which anaerobic organisms have been recovered from adults with crs has varied widely, with anaerobes found mainly by investigators using special techniques to optimize their recovery. , several species, including pigmented prevotella, fusobacterium, bacteroides, and peptostreptococcus species, were isolated. in support of a role for anaerobic bacteria in chronic maxillary sinusitis, finegold et al found recurrence of signs and symptoms to be twice as frequent when cultures yielding anaerobic bacterial counts of greater than cfu/ml. further supportive evidence came from the detection of igg antibodies to anaerobic organisms commonly recovered from sinus aspirates, namely fusobacterium nucleatum and prevotella intermedia. antibody levels to these organisms decreased in the patients who responded to therapy but did not decrease in those in whom therapy failed. recent studies with molecular techniques have shed new light on the potential role of anaerobes in patients with crs (see below). role of atypical bacterial infection in patients with crs. studies with conventional culture techniques or molecular techniques to overcome issues of detection of nonculturable bacteria suggest that atypical mycobacterial infection is rare in patients with crs but should be sought in patients with refractory crs (see additional text in this article's online repository at www.jacionline.org). bacterial biofilm in patients with crs. biofilm formation is an important survival mechanism for microorganisms through attachment to surfaces. formation of biofilm is a complex process controlled by different genetic pathways depending on growth conditions and exposure to membrane-targeting antibiotics. furthermore, biofilm-associated bacteria are known to have enhanced resistance to antimicrobial agents relative to floating (planktonic) bacteria. biofilm formation on sinonasal mucosal surfaces was first described in and later in several other studies. [ ] [ ] [ ] [ ] [ ] [ ] multiple techniques for biofilm detection have been described and are discussed in the additional text in this article's online repository. table e in this article's online repository at www. jacionline.org summarizes the results of several studies of biofilm (including fungal biofilm) in patients with crs, including the techniques used for biofilm identification. most studies have not subcategorized patients as having crssnp, crswnp, afrs, or eosinophilic mucin chronic rhinosinusitis (emcrs). one study found biofilm to be present in only of patients with nps, a lower prevalence than reported in most other studies of crs. when the studies using scanning electron microscopy (sem), transmission electron microscopy (tem), or confocal scanning laser microscopy (cslm) are taken in total (excluding the study confined to patients with nps), the prevalence of biofilm in the crs case series summarized in table e was . %. is the presence of biofilm of prognostic value? the presence of bacterial biofilm, as determined by using cslm, was found to be associated with more severe sinus disease preoperatively (worse radiologic and symptom scoring) and worse sinus symptom and nasal endoscopy scores months after surgery. , single-organism h influenzae biofilm was associated with mild clinical and radiographic disease and normalization of sinus mucosa a short time after surgery. in contrast, polymicrobial biofilm or biofilm containing s aureus was associated with more severe disease and a poorer postoperative course. the presence of bacterial biofilm was strongly associated with persistent mucosal inflammation after endoscopic sinus surgery ( % of biofilm-positive patients vs % of biofilm-negative patients). one study showed that the likelihood of detecting bacteria with biofilm-forming capacity increases in relation to prior endoscopic sinus surgery, possibly reflecting the severity of their disease. similarly, zhang et al found an association of biofilm forming-capacity in vitro from clinical samples with prior sinus surgeries and nasal steroid use in the month before sample collection but no association with the presence of nps, allergy, or samter triad and an association with positive culture results at least suggesting that biofilm might be of greater relevance in patients with nonpolypoid crs. in summary, these studies suggest that that mucosal biofilm is a marker of more severe mucosal disease and a predictor of poorer outcome after sinus surgery. on the basis of animal models of rhinosinusitis, biofilm formation might reflect the severity, chronicity, or both of sinus infection. intracellular bacteria in patients with crs. intracellular intraepithelial staphylococcus aureus (iesa) was first reported in sinus epithelium by corriveau et al using a peptide nucleic acid fluorescence in situ hybridization (fish) assay. in this study iesa was found in the epithelium of patients with crs but was also seen in some hcs, raising the question of its pathologic significance. in a recent study using a similar s aureus fish probe with propidium iodide counterstain and cslm, iesa was detected in % of patients with crs undergoing sinus surgery but none of hcs. simultaneous analysis revealed the presence of s aureus biofilm in % of the iesa-positive tissues and % of the tissue from iesa-negative patients with crs. the cslm-fish/propidium iodide technique has the advantage of being able to detect iesa and s aureus biofilm in the same tissue sample. , presently, it is unclear whether the presence of iesa has any prognostic or pathologic significance beyond that afforded by the presence of biofilm, and there are no studies linking the presence of iesa to any specific defects in epithelial innate immunity. bacteriology of patients with crs using molecular techniques. the evolving view of bacterial involvement in patients with crs has expanded beyond that of infection with individual pathogens to considerations of increased bacterial burden, biofilm formation and alterations in the microbial community (ie, the microbiome). the microbiome is the universe of culturable and nonculturable microorganisms present in a specific ecologic niche, such as the gastrointestinal tract or the sinus mucosa. analysis of the microbiome has gained much recent attention as a novel means of studying host-microbial relations in various organs, including the respiratory tract and sinuses. this has been made possible by the advent of broad-platform molecular techniques, including microarrays and sequencing methods, allowing for identification of the full microbiome based on microbial rna. the most commonly used technique is pyrosequencing to identify bacterial species based on the conserved s eubacterial s ribosomal gene. techniques to study the microbial community are rapidly emerging but include methods to quantify bacterial diversity, representation of specific bacterial species, and bacterial ''load'' or ''burden.' ' stephenson et al used pyrosequencing with modified s eubacterial primers to amplify the -bp region of s rrna genes. eighteen patients with crs ( % with nps) were compared with control subjects. conventional cultures identified bacterial growth in % of patients with crs (the percentage positive in control subjects was not mentioned), with an average of . isolates per sample. in contrast, the results of pyrosequencing were positive in %, with a mean of and a range of to isolates per sample. the most prevalent organisms in patients with crs were anaerobic bacteria, diaphorobacter and peptoniphilus species, whereas coagulase-negative staphylococcus species were identified in % of samples. s aureus and corynebacterium and propionibacterium species were also identified in control subjects. feazel et al obtained middle meatus swabs and compared bacterial culture and pyrosequencing results in patients with crs (including with nps) versus hcs. standard bacteriologic cultures were positive for all patients with crs and hcs, with an average of . isolates reported per subject (range, - isolates). no statistically significant differences were found between the number of isolates obtained from patients with crs and control subjects. the most commonly cultured organisms were coagulase-negative staphylococci ( %), s aureus ( %), and propionibacterium acnes ( %). by using pyrosequencing, the most prevalent dna sequence types detected were coagulase-negative staphylococci ( %), corynebacterium species ( . %), p acnes ( . %), and s aureus ( . %). in general, cultured organisms comprised less than % of the organisms identified by means of sequencing, but bacteria identified by using culture were identified by means of pyrosequencing in most cases. some of the nonspecific crs culture results, such as ''mixed gram-negative rods,'' were associated with pyrosequencing detection of moderate mixed anaerobes representing % of the total sequences identified. this confirms a long-held contention of some investigators that studies reporting a low prevalence of anaerobes in crs samples might have had methodologic flaws related to sample collection, bacterial culture, or both. in the study by feazel et al, s aureus was selectively enriched in patients with crs by means of culture ( / patients with crs vs / hcs). dna sequences representative of s aureus were detected in a similar percentage of patients with crs ( / [ %]) and hcs ( / [ %]), but a greater abundance (bacterial burden) of s aureus sequences was found in patients with crs. in this study both antibiotic exposure and asthma were associated with reduced microbial diversity and increased s aureus abundance. using a special technique of ''mucus preservation'' (sample collection on saline-moistened nonstick gauze to prevent adsorption of mucus into gauze and prompt transport of samples to the laboratory for staining), ponikau et al reported that % of patients with crs had detectable fungal hyphae in mucus obtained from diseased sinuses. virtually all patients with crs and hcs also had a positive fungal culture of nasal secretions, with a broad array of fungi isolated. the latter finding was confirmed by an independent group of investigators who found a positive fungal stain or culture in % of patients with crs and . % of hcs. gosepath et al analyzed tissue specimens from patients with crs using a highly sensitive pcr assay and detected fungal dna in all patients with crs with both a universal fungal primer and a primer specific for alternaria species. in contrast, although the universal primer detected fungal dna in of hcs, none of these subjects had positive results with the alternaria species-specific primer. fungal biofilm has also been described in patients with crs. healy et al used specific bacterial fish dna probes and a panfungal probe to detect biofilm in mucosal samples from patients with emcrs and afrs or crs. three mucosal samples from control subjects were also collected. samples were also analyzed by using epifluorescent microscopy. bacterial biofilm was demonstrated in of samples and of control specimens, and fungal elements were detected in association with bacterial biofilm in the majority of cases. foreman et al analyzed patients with crs and control subjects for the simultaneous presence of bacterial and fungal biofilm by using bacteria-specific fish probes and a universal probe for fungus. they identified bacterial biofilm in % and fungal biofilm in % of patients with crs. in this study there were too few patients to compare the prevalence of fungal biofilm in patients with emcrs, afrs, and crs. epithelial barrier function is important for maintaining mucosal hydration and preventing penetration of foreign particles, including microbes, into the subepithelial layer. defective epithelial barrier function has been found to be a key risk factor for development of atopic dermatitis, leading to increased transepidermal water loss and possibly contributing directly to increased susceptibility to s aureus skin infection. , tight junction proteins are essential to normal epithelial barrier function. they are composed of a branching network of sealing strands, with each strand acting independently. the key tight junction proteins are the claudins and the occludins. in the intestine tight junction proteins efficiently restrict most microbes from penetrating into the subepithelial tissue. acute reversible changes in barrier function have been associated with intestinal viral or bacterial infections, including rotavirus and enteropathogenic e coli. whether primary defects in epithelial barrier function increase susceptibility to inflammatory bowel disease (ibd) is not presently established. no primary defects in epithelial tight junction proteins have been described in patients with crs or nps. soyka at al found that nps have decreased transtissue resistance and an irregular, patchy decreased expression of the tight junction proteins occludin and zonula occludens . these effects are likely secondary to tissue inflammation because it was shown that in vitro culture of hcs or np epithelial cells in the presence of il- resulted in downregulation of occludin and zonula occludens expression, whereas culturing with ifn-g caused an upregulation of these proteins. unlike tight junctions, less is known of the role of adherens junction proteins and desmosomal proteins in epithelial barrier function. decreased expression of the epithelial adherens junction protein e-cadherin was described in nps and linked to other manifestations of epithelial-mesenchymal transition. decreased expression of the desmosomal junction proteins desmoglein and desmoglein was reported. in the latter study culture of human bronchial epithelial cells with tnf-a or il- downregulated desmoglein expression, suggesting that the defect in desmoglein expression in nps might be secondary to the underlying inflammatory process. mucociliary clearance is an essential process in normal sinus function. impairments in mucociliary clearance lead to mucostasis, bacterial colonization, biofilm formation, and crs. genetically engineered mice that lack normal cilia spontaneously have bacterial sinusitis. , the classic example of abnormal ciliary function is primary ciliary dyskinesia (pcd; also known as immotile cilia syndrome). pcd is a rare, usually autosomal recessive disorder characterized by sinopulmonary disease, laterality defects (eg, situs inversus), and male infertility. pcd has traditionally been diagnosed based on ultrastructural defects in cilia, which are identified in about % of patients with pcd and involve the outer dynein arms, inner dynein arms, or both. mutational analyses have discovered that % of patients with pcd carry mutations of the dynein genes dnai and dnah . because it is estimated that normal ciliary function involves more than gene products, additional mutations accounting for the remaining % of patients with pcd are likely to be uncovered. recently, screening of unrelated pcd families identified patients in families with reduced outer dynein arms carrying mutations in ccdc . the ccdc gene product might function as a tightly bound, axoneme-associated protein. a somewhat similar clinical syndrome known as young syndrome is characterized by obstructive azoospermia with normal sperm structure and function and recurrent sinopulmonary infections. unlike patients with pcd, no mutations in ciliary structural genes have been identified in patients with young syndrome, and the problem has been presumed to be caused by an abnormality in mucus. several studies have established that crs is associated with an acquired reduction in mucociliary clearance. [ ] [ ] [ ] [ ] [ ] mucociliary clearance typically normalizes after clearance of infection and restoration of normal sinus drainage. , , this has been confirmed by in vitro studies showing that sinonasal mucosal explants from patients with crs show blunted ciliary beat frequency responses to cholinergic and adrenergic stimulation but normal responses within hours of culturing. lai et al reported increased expression of the centrosomal protein (cp ) in inflamed sinus tissue from patients with crs. cp is known to prevent the terminal step in ciliary maturation. they further showed that cp could be induced in vitro by proinflammatory cytokines in cultured sinus epithelial cells with concomitant inhibition of ciliogenesis. in contrast, there is no evidence for a primary defect in mucociliary clearance to account for crs. to date, genetic association studies of patients with refractory crs have not uncovered any gene polymorphisms that would suggest a primary defect in mucociliary clearance. composition of sinus mucosal secretions. fig , - illustrates the general structure of the sinus epithelium, submucosal mixed seromucinous glands, and stroma and summarizes the many proteins and peptides produced by sinus mucosal cells. submucosal seromucinous mixed glands comprise a series of ducts with interconnecting serous and mucous tubules that terminate in acini. alcian blue can be used to stain acidic mucoglycoproteins within epithelial goblet cells and submucosal glands, whereas serous glands are not stained. serous glands are reported to be the major site of production of antimicrobial peptides, although many of the antimicrobial peptides illustrated are produced by both surface epithelial cells and submucosal glands. protease-activated receptor has been demonstrated on submucosal glands, and a heightened glandular secretory response to protease-activated receptor agonists, including house dust mite extract, has been described in patients with crs. lysozyme. lysozyme catalyzes the breakdown of bacterial cell wall peptidoglycan by cleaving b [ ] [ ] [ ] [ ] glycosidic linkages between n-acetylmuramic acid and n-acetylglucosamine (http:// www.ncbi.nlm.nih.gov/gene/ ). lysozyme localizes primarily to serous cells of submucosal mixed glands and neutrophils, with weak staining of the mucus cells of submucosal mixed glands and goblet cells. , decreased immunostaining for lysozyme was reported in longstanding cases of crs ; however, more recent studies reported increased rather than decreased immunostaining in patients with crs. , , in the study by woods et al, increased immunoreactivity for lysozyme was found in mucosal biopsy specimens of both patients with crssnp and those with crswnp, including low-level immunostaining in the epithelium. lactoferrin. similar to lysozyme, lactoferrin is detected primarily in serous cells of submucosal glands. although zhang et al reported an increase in lactoferrin immunostaining in patients with crs, psaltis et al reported a decrease in lactoferrin at both the mrna and protein levels in patients with crs and an even further decrease in expression in the presence of bacterial biofilm. secretory leukocyte proteinase inhibitor. secretory leukocyte proteinase inhibitor (slpi) protects epithelial tissues from serine proteases, including trypsin, leukocyte elastase, and cathepsin g (http://www.ncbi.nlm.nih.gov/gene/ ). it is a cationic protein with a very high affinity for leukocyte elastase. it also has antibacterial and other immunomodulatory properties and is the third most abundant antimicrobial protein in the upper airways. the antibacterial properties of slpi are thought to reside in the n-terminal domain of the protein, where its activity could be mediated by its cationic charge. slpi has antibacterial activity against paeruginosa, s aureus, staphylococcus epidermidis, and candida albicans. the role of slpi in innate immunity and control of inflammation is complex. furthermore, it has been shown that the decreased levels of slpi found in pseudomonas species-infected lungs of patients with cystic fibrosis is due to degradation by neutrophil elastase. slpi has been recovered from purulent maxillary sinus secretions in patients with maxillary sinusitis, but there have been no studies linking abnormalities of slpi to crs. antimicrobial peptides defensins, cathelicidins, dmbt- , and s- proteins. defensins and cathelicidins are the main families of antimicrobial peptides present in airway secretions and expressed by the airway epithelium. they represent small ( - kda) cationic peptides that are either produced constitutively or induced by microbial products (including tlr ligands), cytokines, or growth factors. the a-defensins are produced mainly by neutrophils and intestinal paneth cells, whereas hbd- , hbd- , hbd- and hbd- are primarily produced by epithelial cells. in epithelial cells hbd- production is constitutive, whereas hbd- , hbd- , and hbd- production is inducible. negligible expression of hbd- and hbd- was found in normal nasal mucosa. reduced expression of hbd- was found in epithelial cells isolated from nps. in human subjects only cathelicidin (hcap- ) is expressed. in neutrophils the cathelicidin proform hcap- is proteolytically processed by proteinase , resulting in the release of ll- . in skin serine proteinases control cleavage of hcap- to ll- . ll- was detected in epithelial cells, submucosal glands, and inflammatory cells in nasal tissue explants and induced by culture with fungal allergens from aspergillus and alternaria species. , no clear pattern of deficiency in the production of ll- in sinus tissue has been demonstrated in patients with crs. bactericidal/permeability-increasing protein and plunc family proteins. a family of proteins, including bactericidal/permeability-increasing protein (bpi) and lpsbinding protein (lbp), has antimicrobial effects against gramnegative bacteria. both bind to bacterial lps, causing growth arrest and inhibition of lps-induced activation of inflammatory responses. , although bpi and lbp are found in airway secretions, there has been little study of their levels in nasal or sinus secretions. plunc proteins, members of the palate lung and nasal epithelium clone (plunc) family, are structurally related to lbp and bpi. recent studies suggest physiologically relevant concentrations of plunc inhibit p aeruginosa biofilm formation in vitro without acting directly as a bactericide. , this finding suggests that plunc protein might have relevance to patients with refractory crs. splunc and lplunc were found to be differentially expressed in serous and mucous cells, respectively, of submucosal glands in uncinate tissue. decreased splunc and lplunc levels were found in nps relative to those seen in healthy control uncinate tissues, likely reflecting the decreased number of submucosal glands in nps because their levels correlated with decreased lactoferrin levels. similarly, wu et al found plunc protein levels to be reduced in nps and somewhat increased in patients with crs relative to hcs, which is consistent with other studies showing an increased number of submucosal glands in patients with crssnp relative to hcs. dmbt (gp- ). the deleted in malignant brain tumor (dmbt ) gene encodes alternatively spliced proteins referred to as gp- (dmbt gp ) and salivary agglutinin (dmbt sag ). the former is secreted in bronchoalveolar lining fluid, whereas the latter is present in saliva, but the molecules are identical. dmbt interacts with and agglutinates several gram-negative and gram-positive bacteria, including streptococcus mutans, a bacterium responsible for dental caries. dmbt interacts with surfactant protein a (sp-a) and surfactant protein d (sp-d) and a variety of other host proteins involved in innate immunity or wound healing. a precise role for dmbt in human disease has yet to be elucidated. dmbt- is overexpressed in nps mainly in submucosal glands. ficolins and collectins (sp-a, sp-d, conglutinin, and mannose-binding lectin). ficolins and collectins (''collagenous lectins'') are soluble innate pattern recognition receptors that have an innate function resembling that of antibodies in adaptive immunity. subunits of ficolins and collectins recognize carbohydrate arrays of their microbial targets through globular trimeric carbohydrate-recognition domains. sp-a, ficolins, and mannosebinding lectin (mbl) also share structural similarity with c q, and ficolins and mbl (but not sp-a) activate complement. whether collectins interact with immune cells through receptors is unclear. there are no studies of ficolin in the upper airway. sp-a expression was reported to be increased in sinus mucosal biopsy specimens in patients with crssnp and also present in nps. however, woodworth et al found comparable expression of sp-a and sp-d in hcs and patients with crswnp. in these studies sp-a and sp-d immunoreactivity was found in epithelial cells and submucosal seromucinous glands. one other study reported reduced immunostaining for sp-d in nasal submucosal glands and a lack of induction of sp-d by aspergillus and alternaria species allergens in cultured nasal explants from patients with nonallergic fungal eosinophilic sinusitis. in contrast, these allergens induced sp-d production in hcs and patients with crs. ramanathan et al showed that culturing human sinonasal epithelial cells in the presence of the t h cytokines il- or il- for hours reduced expression of antimicrobial innate immune genes by using real-time pcr, elisa, and flow cytometry, including tlr , hbd- , and sp-a. in these experiments il- and il- reduced expression of these innate factors in both hcs and patients with crs. however, given that mucosal sp-a expression is increased in patients with crswnp, the significance of in vitro suppression of sp-a by il- /il- is unclear. mbl, also referred to as mannose-binding protein, is a calcium-dependent serum protein that binds carbohydrate determinants on the surfaces of a wide range of pathogens (viruses, bacteria, fungi, and protozoa), thereby activating the complement cascade or acting directly as an opsonin. mbl is a member of the collectin family of proteins and has structural similarity to complement component c . mbl deficiency is associated with an increased incidence of upper respiratory tract infections in children, presumably because their adaptive immune systems are still immature. however, a study of mbl deficiency (defined by expression of an mbl deficiency allele) in danish adults found no significant differences in infectious disease prevalence or mortality in mbl-deficient subjects versus control subjects. however, there has been a paucity of studies of mbl levels or genotype in a population of patients with crs. one study found increased serum levels of both c and mbl in patients with crssnp and those with crswnp. complement components. the complement component c and serum amyloid a protein are produced by sinonasal epithelial cells. serum amyloid a functions as an acute-phase opsonin against gram-negative bacteria, including e coli and p aeruginosa, through an outer membrane protein a family member. no differences were found in expression of c or serum amyloid a in patients with crs compared with that seen in hcs. increased expression of factor b, c , and c mrna was found in tissues from patients with afrs and those with crswnp compared with that seen in hcs, but the functional significance of this is unclear. no. no is constitutively produced at high levels in sinus epithelium by virtue of high constitutive levels of inducible nitric oxide synthase (or nos a). the no concentration in a healthy maxillary sinus ( . . ppm) exceeds that necessary for antibacterial effects in vitro and is vastly higher than that produced in the nose or lungs (normal exhaled no value, < ppb). the antimicrobial effects of no in the sinuses might also relate to stimulation of increased ciliary beat frequency and complex reactivities between no radical superoxide, metals, and thiols. no production is also downstream of innate signaling through bitter taste receptors (see below). recent experiments have demonstrated an no-responsive quorum-sensing mechanism in vibrio harveyi and shewanella oneidensis that regulates biofilm formation. in these bacteria no stimulates biofilm formation by controlling levels of the bacterial cyclic diguanosine monophosphate. whether a similar mechanism exists in other pathogens remains to be explored, but it has been reported that low no levels ( . - . mmol/l) stimulate biofilm formation in s aureus. multiple innate pattern recognition receptor pathways are either known or potentially relevant to sinus epithelium, including transmembrane tlrs and intracellular nucleotide-binding oligomerization domain (nod) receptors, , dectin receptors, and bitter taste receptors. table e in this article's online repository at www.jacionline.org summarizes the pattern recognition receptors involved in microbial recognition by airway epithelial cells, their microbial ligands, and abnormalities described in patients with crs. fig , , summarizes some of the salient features of host-microbial interactions involved in triggering innate immune responses in patients with crs. sinonasal epithelial cells express tlrs through . , tlr , tlr , tlr , and tlr signaling has been demonstrated in sinonasal epithelial cells. , tlr ligation in airway epithelial cells results in activation of specific intracellular signaling pathways (reviewed by bals and hiemstra ), leading to production of ( ) innate antimicrobial peptides and ( ) cytokines and chemokines that amplify innate responses (eg, neutrophil infiltration) and activate adaptive immune responses. the tlr receptor has the greatest diversity of ligands and recognizes a wide array of gram-positive and gram-negative bacteria, as well as fungi, in part because of formation of heterodimers with tlr or tlr . the tlr pathway is extremely important in host responses to gram-negative bacterial infection in the airway, and polymorphisms in tlr have been associated with more gram-negative bacterial infections in patients in an intensive care unit. the response of human tracheobronchial epithelium to lps requires the tlr ''coreceptor'' cd . studies of tlr signaling pathways have focused on patients with ''refractory'' crs unresponsive to medical management or those with ''recalcitrant'' nps. increased expression of tlr was found in patients with recalcitrant crs. an exaggerated response to tlr plus cigarette smoke extract was found in cultured epithelial cells from patients with crssnp, with excess production of rantes and hbd- . reduced baseline expression of tlr was found in cultured epithelial cells from patients with refractory nps, and this might be linked to the effects of locally produced t h cytokines. dectin- is a type ii transmembrane protein with a c-type lectin-like carbohydrate recognition domain, a transmembrane region, and a cytoplasmic tail containing an immunoreceptor tyrosinase activation motif. dectin- binds specifically to b- , glucans and induces intracellular signaling. dectin- and tlr /tlr signaling combine to enhance the responses triggered by each receptor. , dectin- deficiency has been associated with mucocutaneous candidiasis. although dectin receptors are regarded as receptors on myeloid cells, sun et al recently showed that dectin- receptors could be induced on airway epithelial cells by alternaria species in a tlr dependent manner with induction of tnf-a, gm-csf, il- , hbd- , and hbd- . although fungal colonization and mucosal responses to fungi have received much attention, dectin- has not yet been studied in patients with crs. nod is a member of the nod-like receptor protein family that initiate inflammatory responses when exposed to ligands derived from bacterial components intracellularly. , polymorphisms in nod (card ) lead to impaired nod function and increased susceptibility to crohn disease (cd), a condition marked by excessive inflammatory responses to normal bacterial flora. the ligand for nod is muramyl dipeptide, the ''minimal bioactive peptidoglycan motif common to all bacteria.'' nod ordinarily downregulates responses to tlr stimulation, and it has been suggested that nod polymorphisms in patients with cd result in a decrease in negative regulation of tlr responses and a pathologic increase in responses to normal gut flora. alternatively, it has been suggested that impaired activation of the nod pathway might facilitate invasion of the intestinal epithelial cells through commensal or pathogenic bacteria. nod defects in patients with ibd are expressed widely in bodily tissues, raising the question as to whether there is any association between ibd and crs. one study found a prevalence of chronic ''sinonasal disease'' of % in patients with ibd, with a higher prevalence in patients with cd ( %) versus those with ulcerative colitis ( %) and an even higher prevalence of chronic sinonasal disease in patients with cd with obstructive bowel complications ( % vs %), with % of these patients reporting crs. another study that examined this association found a similar prevalence of crs in patients with ibd as in the general population, although the proportion of patients with crs having nasal polyposis was higher among patients with ibd. there are no studies linking genetic polymorphisms in nod , nod , or nod pathway genes with crs. lee et al recently discovered that one of the bitter taste receptors, t r , is activated by a quorum-sensing molecule from p aeruginosa associated with biofilm formation. bitter taste receptors are a family of g protein-coupled receptors that signal by inducing a transient intracellular calcium flux and stimulating ciliary beat frequency. activation of the receptor induces production of no and increases ciliary beat frequency in sinus epithelial cells. a common polymorphism (tas r variant) was discovered that is associated with reduced signaling, no production, and ciliary beat frequency and increased growth of p aeruginosa in air-liquid cultures of human airway epithelial cells. the effect of tas r on the killing of p aeruginosa was shown to be no dependent. the tas r genotype correlated with the presence of sinonasal gram-negative infection in patients with crs, suggesting a mechanistic link between a deficiency in innate signaling and increased bacterial infection. adaptive immune antimicrobial signaling mechanisms: il- a and il- signaling pathways il- a and il- are ''signature'' cytokines of t h cells involved in host defense against extracellular pathogens, including fungi, bacteria, and some parasites. il- is considered an ''essential guardian of mucosal immunity against bitter taste receptor is activated by a quorum-sensing molecule from p aeruginosa and stimulates production of no, which then stimulates mucocociliary clearance and has direct antimicrobial effects. depiction of the intracellular tlr signaling pathways was adapted from the iavi report (http://www.iavireport.org/back-issues/pages/iavi-report- ( )-tollbridgetoimmunity.aspx). upper right inset (left panel), sem of bacterial biofilm showing characteristic glycocalyx and water channels. the photograph was used with permission from sanclement et al. right panel, cslm image ( magnification) of a patient with crs stained with the baclight live/dead kit (invitrogen, molecular probes, carlsbad, calif) demonstrating a bacterial biofilm comprised of many intensely fluorescing live bacteria organized in clusters (large arrow). small arrows designate the larger live and dead epithelial cells. used with permission from psaltis et al. the bitter taste receptor depicted in the small middle inset was adapted from fenech c, patrikainen l, kerr ds, grall s, liu z, laugerette f, et al. ric- a, a ga protein guanine nucleotide exchange factor potentiates taste receptor signaling. front cell neurosci ; : . j allergy clin immunol volume , number extracellular bacteria in the lung and gut.'' in intestinal epithelial cells il- stimulates the production of a wide variety of antibacterial proteins, stimulates mucin production under inflammatory conditions, and enhances epithelial regeneration with goblet cell restitution. il- deficiency in mice is associated with a more severe form of gram-negative enteric infection with citrobacter rodentium, with increased intestinal epithelial damage, systemic bacterial burden, and mortality. in this model il- mediates production of reg family antimicrobial proteins, including regiiib and regiiic, by colonic epithelial cells. both t cells, including cd cells and innate lymphoid cells, and dendritic cells produce il- . - a subset of cd helper t cells that produce il- but not il- has been identified in human subjects and termed t h cells. il- also protects the lungs from gram-negative klebsiella pneumonia infection. in conjunction with il- a or il- f, il- synergistically induces expression of hbd- and s a and additively enhances the expression of s a and s a . il- -mediated effects on airway epithelial cells require signaling through the il- receptor (il- r) and activation of signal transducer and activator of transcription . il- , produced mainly by phagocytes and dendritic cells, is a crucial upstream regulator of il- and il- production. , although il- and il- a act synergistically or additively to promote the expression of genes involved in mucosal defense, t h and t h cells likely serve different roles in mucosal immunity and autoimmunity. , il- a and il- also have an established role in antifungal immunity at mucosal surfaces, which would suggest that defects in the signaling pathways of these cytokines in the sinus mucosa might be associated with difficulty handling fungal infection. however, this has not been studied. to date, no definite defect in il- a or il- signaling has been identified in patients with crs. using a genome-wide association study, endam et al identified a polymorphism in il- r in association with refractory crs, but no functional studies were performed to explore this relationship. reduced il- r expression in sinus mucosal epithelial cells was found in patients with recalcitrant nps, and a relationship was found between reduced expression of il- r and a higher rate of recurrence of nps after sinus surgery. it has long been recognized that crs is a disease in which the local tissue inflammatory response might be strongly biased toward t h inflammation despite a lack of systemic evidence for allergic disease. this is particularly true in patients with crswnp but also true, to a lesser degree, in patients with crssnp. [ ] [ ] [ ] [ ] there is evidence that links colonizing microorganisms to this maladaptive t h ''local allergic'' response in patients with crswnp (schematized in fig ) . this has been shown in cultured airway epithelial cells and dispersed t lymphocytes from nps. fungi are commonly detected in the attached mucus of sinus tissues in patients with crs , and can induce eosinophil activation and degranulation. certain fungi, particularly alternaria and candida species, were shown to induce production of il- and il- , as well as ifn-g, in peripheral blood lymphocytes from patients with crs. it was hypothesized that this maladaptive response would account for an eosinophilic mixed t h /t h mucosal immune response. fungal allergens also elicit modest production of il- and il- from dispersed np t lymphocytes. however, a recent study did not demonstrate consistent il- production in response to alternaria species in patients with crs from utah. a small randomized placebo-controlled trial of intranasal amphotericin b reported improvement in radiographic mucosal thickening, nasal endoscopy, and eosinophilic inflammation. however, other randomized placebocontrolled trials with intranasal antifungal treatment did not demonstrate a significant clinical benefit. , in addition, antifungal treatment did not alter the cytokine or chemokine production profiles of nasal inflammatory cells. mucosal colonization with s aureus has been found in % of patients with crswnp compared with roughly % of healthy subjects or patients with crssnp. in a study of patients with massive polyposis, % of patients were found to have enterotoxin-producing s aureus in the nasal mucus adjacent to polyps. it was further shown that t lymphocytes isolated from the polyps showed a skewing of vb use with enrichment for vb known to respond to staphylococcal superantigens. this finding was later confirmed by another group. , ige antibodies directed against staphylococcal superantigens were found in np homogenates in . % of patients with nps and . % of patients with nps with coexisting asthma. finally, staphylococcal enterotoxin b (seb) was found to induce robust production of il- and il- in dispersed np t lymphocytes. these studies suggest that colonizing s aureus might be a major driver of the local t h inflammatory response in patients with crswnp. staphylococcal superantigens, such as seb, have been shown to impair oral tolerance and promote allergy in a murine model of food allergy. seb has also been shown to augment allergic inflammation in a murine model of eosinophilic crs, leading to formation of nasal polyposis. given the strong adaptive t h -type chronic inflammation characteristic of patients with crswnp, investigations were undertaken to ascertain whether t h -type inflammation modulated innate immune function. ramanathan et al first demonstrated that cultured primary nasal epithelial cells (pnecs) from hcs and patients with crswnp express surface tlr and respond to cpg (a tlr agonist) by increasing production of hbd- and il- . they found that tlr expression on pnecs from patients with crswnp was reduced by % compared to control pnecs. culturing control pnecs in the presence of the t h cytokine ifn-g increased tlr expression by %, whereas culturing in the presence of the t h cytokines il- or il- decreased tlr expression by . %. because this group previously reported reduced epithelial expression of il- r in patients with recalcitrant nps, it would be interesting to know whether t h cytokines would induce this effect. is there an inappropriate or heightened response to colonizing ''commensal'' organisms in patients with crs? epithelial surfaces have adapted specialized mechanisms for coping with potential intruders. these not only protect the host from infection but also provide beneficial effects in terms of digestion (in the gut) and immune maturation (gut and respiratory tract). mechanisms have also evolved to cope with ''commensals'' in the gut and presumably also in the oral mucosa and nasal epithelium, where bacterial colonization is normal. it has been suggested that crs might have a proinflammatory response to common colonizing organisms, such as coagulase-negative staphylococcus species. could it be that the ''proinflammatory'' state of crs relates to a lack of tolerance to seemingly innocuous ''commensal'' organisms? we do not know, but this represents an exciting area for future investigation. there is limited information indicating that patients with crs have an increased incidence of rhinovirus infection and some suggestion that they might have an exaggerated response to viral infection, but there is no evidence for persistent viral infection in the majority of cases. it would be instructive to map the time course and innate immune response to experimental rhinovirus infection and ascertain whether patients with crs have increased susceptibility, increased viral burden, or an inappropriate innate immune response. a role for bacterial infection or colonization in crs pathogenesis is well established. bacteria are cultured and bacterial biofilm is present in the majority of patients undergoing surgical intervention. diseased sinus tissue also has an increased bacterial burden, most convincingly demonstrated for s aureus, and bacterial biofilm with other organisms, including p aeruginosa, is often present. the majority of patients with crs also have fungal hyphae detectable in mucus extracted from diseased sinuses, and fungal biofilm is detectable in some cases, usually in association with bacterial biofilm. the presence of bacterial biofilm, particularly polymicrobial biofilm or biofilm containing s aureus, has prognostic value and is associated with more severe sinus disease preoperatively and worse symptom and nasal endoscopy scores after surgery. mucociliary clearance is essential for normal sinus function. active disease is associated with a reduction in mucociliary clearance but normalization of mucociliary clearance after clearance of infection and restoration of normal sinus. there is no evidence for a primary defect in mucociliary clearance to account for crs, except in the distinct clinical syndrome of pcd. a defect in local host innate immunity has long been suggested as a pathogenic mechanism of crs to account for crs, especially because systemic immune function is normal in the vast majority of cases. decreased levels of the antimicrobial proteins, most notably lactoferrin, have been found most often in sinus secretions, whereas levels of other antimicrobial proteins and peptides have been reported to be normal. multiple innate pattern recognition receptor pathways, notably tlrs and intracellular nod receptors and bitter taste receptors, are critical in host/microbial interactions in the sinuses. no primary defects have been found in these pathways to account for refractory crs, although a % reduction in the expression of tlr has been found in patients with refractory nps. a downregulation of epithelial innate immunity by maladaptive t h tissue inflammation has been demonstrated in patients with recalcitrant crswnp. in vitro studies have shown that il- and il- decrease tlr expression on cultured epithelial cells by nearly % and also reduce expression of hbd- and sp-a. colonizing fungi might play a role in maladaptive t h responses and thereby promote tissue eosinophilia. colonization with enterotoxin-producing s aureus is a driver of the local t h response in patients with crswnp because it induces a local expansion of t lymphocytes and production of enterotoxinspecific ige antibodies. to date, an effective means of restoring host-microbial balance and mitigating disease in patients with crs remains elusive. there are few studies on the effectiveness of medical treatment alone at eradicating bacterial infection and bacterial biofilm in patients with crs. clinical trials of antifungal rinses have been generally unsuccessful. surgical removal of diseased sinus tissue with restoration of sinus ventilation and use of culture-directed antibiotics remain the best approaches to treatment. there is hope that further elucidation of the genetic underpinnings of crs and the host-microbial interactions present will provide greater insight into disease pathogenesis and more effective treatment. one study using sensitive molecular techniques (pcr) found no evidence for mycoplasma pneumoniae, chlamydia pneumoniae, or legionella pneumophila in mucosal samples from patients with crs. e recent studies used pyrosequencing to potentially overcome issues of detection of nonculturable bacteria in mucosal samples from patients with crs; did not detect mycoplasma, ureaplasma, and chlamydia species; and found legionella, mycobacterium, and nocardia species in only rare cases. e , e tichenor et al e identified patients with crs with refractory symptoms who had positive cultures for atypical mycobacteria over a -year period. mycobacterium avium molecular fingerprints clonally related to the patients' isolates were also identified in the plumbing of of households sampled, suggesting that at least in these cases, indoor plumbing and sinus irrigations might have been responsible for the infection. solyar et al e reported on a series of patients with refractory crs who had a positive mycobacterial culture over a -year period. the most frequent isolates were mycobacterium abscessus ( . %), mycobacterium avium-intracellulare complex ( . %), and mycobacterium chelonae ( . %). it was reported that . % of outpatient-acquired and . % of inpatient-acquired cultures were positive for atypical mycobacteria. some evidence of improvement in crs was noted after treatment with antimycobacterial antibiotics. these studies suggest that atypical mycobacterial infection is rare in patients with crs but should be sought in patients with refractory crs. see table e for a summary of studies of bacterial or fungal biofilm in patients with crs. sem and tem have the advantage of high magnification and are the only techniques that provide ultrastructural confirmation of biofilm presence. technical artifacts can arise because of sample dehydration and surface distortion. disadvantages of sem include small sample size, potentially introducing sampling artifacts. cslm has the advantage that specimens can be imaged without fixation or dehydration, and specific bacteria or fungi can be stained with fluorescent markers. the fish assay uses either universal bacterial probes, such as eub , e or species-specific primers based on unique sequences in the s ribosomal rna gene. the live/dead baclight bacterial viability kit (invitrogen, carlsbad, calif) uses nucleic acid stains: green-fluorescent syto stain, which labels both live and dead bacteria, and redfluorescent propidium iodide, which penetrates only bacteria or cells with damaged membranes. cslm is used to confirm the presence of biofilm on the mucosal surface. no bacteria-specific probes are used. a direct comparison of techniques in a sheep model of rhinosinusitis concluded that cslm is more objective for documenting biofilm presence than sem or tem because of the inherent flaws, sampling error, and subjectivity involved in sem and tem. e another study determined that the baclight method was more specific than cslm/fish for biofilm detection in sinus tissues. e , e hochstim et al e reported that bacterial biofilm can be detected by using routine hematoxylin-eosin staining, with good agreement with fish/cslm as the control standard. however, this technique is suspect because of its reliance on identification of small basophilic bacterial clusters on the epithelial surface. a concern is whether any of the techniques other than sem or tem are sufficiently rigorous to define biofilm because they do not provide ultrastructural confirmation of the presence of biofilm. the calgary biofilm assay is an in vitro test used to assess the ability of a bacterial isolate to form biofilm. the bacteria are cultured on a -well plate with round pegs positioned over the plate. a semiquantitative analysis using uptake of crystal violet in the biofilm has been developed. e the live/dead baclight bacterial viability kit (see above) can also be used to assess biofilm formation in vitro on -well chamber slides. e prince et al e used the calgary biofilm detection assay to examine in patients with crs with mucopurulence and found that . % of patients had biofilm-forming capacity. polymicrobial cultures, p aeruginosa, and/or s aureus comprised % of samples. postsurgical cases had a higher prevalence of biofilmforming capacity than surgery-naive patients ( . % vs %). ie-dap d-lactyl-l-ala-g-glu-meso-dap-gly heptanolyl-g-glu-meso-dap-ala gm-tripeptide adapted from bals and heimstra, e ooi et al, e and roeder et al. e àbacterial dna containing unmethylated cpg dinucleotides. rhinosinusitis: establishing definitions for clinical research and patient care refractory chronic rhinosinusitis: pathophysiology and management of chronic rhinosinusitis persisting after endoscopic sinus surgery th cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells quantitative and qualitative analysis of rhinovirus infection in bronchial tissues experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation role of deficient type iii interferon-lambda production in asthma exacerbations acute exacerbations of chronic rhinosinusitis occur in a distinct seasonal pattern role of nasal nitric oxide in the resolution of experimental rhinovirus infection interleukin- a modulates human airway epithelial responses to human rhinovirus infection selective transcriptional down-regulation of human rhinovirus-induced production of cxcl from airway epithelial cells via the mek pathway detection of rhinovirus in turbinate epithelial cells of chronic sinusitis infection rate and virus-induced cytokine secretion in experimental rhinovirus infection in mucosal organ culture: comparison between specimens from patients with chronic rhinosinusitis with nasal polyps and those from normal subjects cigarette smoke combined with toll-like receptor signaling triggers exaggerated epithelial regulated upon activation, normal t-cell expressed and secreted/ ccl expression in chronic rhinosinusitis is chronic rhinosinusitis caused by persistent respiratory virus infection? the role of respiratory virus infections in childhood asthma inception bacteriologic features of chronic sinusitis in children aerobic and anaerobic bacteriology of concurrent chronic otitis media with effusion and chronic sinusitis in children confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis evaluation of bacterial biofilms in chronic rhinosinusitis molecular characterization of the polymicrobial flora in chronic rhinosinusitis correlation between microbiology and previous sinus surgery in patients with chronic maxillary sinusitis bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults immune response to fusobacterium nucleatum and prevotella intermedia in patients with chronic maxillary sinusitis biofilms: survival mechanisms of clinically relevant microorganisms the metabolically active subpopulation in pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms bacterial resistance to antibiotics: the role of biofilms evidence of bacterial biofilms in human chronic sinusitis demonstration of biofilm in human bacterial chronic rhinosinusitis bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis the impact of biofilms on outcomes after endoscopic sinus surgery biofilm and persistent inflammation in endoscopic sinus surgery evidence of bacterial biofilms on frontal recess stents in patients with chronic rhinosinusitis biofilms and chronic rhinosinusitis: systematic review of evidence, current concepts and directions for research evidence of bacterial biofilms in nasal polyposis the effect of bacterial biofilms on post-sinus surgical outcomes different biofilms, different disease? a clinical outcomes study prevalence of biofilm-forming bacteria in chronic rhinosinusitis clinical factors associated with bacterial biofilm formation in chronic rhinosinusitis a sheep model for the study of biofilms in rhinosinusitis detection of staphylococcus aureus in nasal tissue with peptide nucleic acid-fluorescence in situ hybridization the multiplicity of staphylococcus aureus in chronic rhinosinusitis: correlating surface biofilm and intracellular residence identifying intracellular staphylococcus aureus in chronic rhinosinusitis: a direct comparison of techniques pyrosequencing for microbial typing microbiome complexity and staphylococcus aureus in chronic rhinosinusitis the diagnosis and incidence of allergic fungal sinusitis incidence and detection of fungi and eosinophilic granulocytes in chronic rhinosinusitis fungal dna is present in tissue specimens of patients with chronic rhinosinusitis biofilms with fungi in chronic rhinosinusitis role of bacterial and fungal biofilms in chronic rhinosinusitis the antimicrobial skin barrier in patients with atopic dermatitis filaggrin in atopic dermatitis tight junctions as targets of infectious agents epithelial barriers in homeostasis and disease defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by ifn-gamma and il- hypoxiainducible factor mediates nasal polypogenesis by inducing epithelial-tomesenchymal transition pathophysiology of nasal polyposis: the role of desmosomal junctions forkhead transcription factor hfh- expression is temporally related to ciliogenesis conditional deletion of dnaic in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis genetic defects in ciliary structure and function ccdc mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms young's syndrome. obstructive azoospermia and chronic sinopulmonary infections mucociliary function, ciliary ultrastructure, and ciliary orientation in young's syndrome the effect of endoscopic sinus surgery on mucociliary clearance in patients with chronic sinusitis alterations of nasal mucociliary transport in patients with hypertrophy of the inferior turbinates, deviations of the nasal septum and chronic sinusitis mucociliary function of the maxillary sinuses after restoring ventilation: a radioisotopic study of the maxillary sinus pre-and postoperative mucociliary clearance in functional endoscopic sinus surgery mucociliary function in the early weeks after nasal surgery reversal of chronic rhinosinusitis-associated sinonasal ciliary dysfunction inflammation-mediated upregulation of centrosomal protein , a negative modulator of ciliogenesis, in patients with chronic rhinosinusitis genetics of rhinosinusitis lysozyme and lactoferrin in human maxillary sinus mucosa during chronic sinusitis. an immunohistochemical study immunolocalization of surfactant protein a and d in sinonasal mucosa serum amyloid a, properdin, complement , and toll-like receptors are expressed locally in human sinonasal tissue innate immunity of the sinonasal cavity: expression of messenger rna for complement cascade components and toll-like receptors innate antimicrobial activity of nasal secretions identification of human nasal mucous proteins using proteomics epithelium: at the interface of innate and adaptive immune responses evidence of a role for b cell-activating factor of the tnf family in the pathogenesis of chronic rhinosinusitis with nasal polyps epithelial genes in chronic rhinosinusitis with and without nasal polyps role of acidic mammalian chitinase and chitotriosidase in nasal polyps chronic rhinosinusitis with and without nasal polyps is associated with decreased expression of glucocorticoid-induced leucine zipper evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis the cytokine-driven regulation of secretoglobins in normal human upper airway and their expression, particularly that of uteroglobin-related protein , in chronic rhinosinusitis expression of neutrophil gelatinase-associated lipocalin in nasal polyps glandular gene expression of sinus mucosa in chronic rhinosinusitis with and without cystic fibrosis density of middle turbinate subepithelial mucous glands in patients with chronic rhinosinusitis protease-activated receptor -dependent fluid secretion from airway submucosal glands by house dust mite extract an immunohistological study of nasal and paranasal mucosa of patients with relapsing chronic sinusitis lysozyme expression is increased in the sinus mucosa of patients with chronic rhinosinusitis nasal mucosa expression of lactoferrin in patients with chronic rhinosinusitis reduced levels of lactoferrin in biofilmassociated chronic rhinosinusitis isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase innate host defense functions of secretory leucoprotease inhibitor decreased levels of secretory leucoprotease inhibitor in the pseudomonas-infected cystic fibrosis lung are due to neutrophil elastase degradation antileukoprotease in patients with maxillary sinusitis the role of epithelial beta-defensins and cathelicidins in host defense of the lung human beta-defensins and toll-like receptors in the upper airway human cathelicidin, hcap- , is processed to the antimicrobial peptide ll- by extracellular cleavage with proteinase kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin fungal allergens induce cathelicidin ll- expression in chronic rhinosinusitis patients in a nasal explant model human cathelicidin antimicrobial peptide is up-regulated in the eosinophilic mucus subgroup of chronic rhinosinusitis patients structure and functions of endotoxin-binding peptides derived from cap the bactericidal/permeability-increasing protein (bpi) in antibacterial host defense plunc is a novel airway surfactant protein with anti-biofilm activity plunc: a multifunctional surfactant of the airways reduced expression of antimicrobial plunc proteins in nasal polyp tissues of patients with chronic rhinosinusitis expression and clinical significance of plunc protein in nasal polyp and chronic sinusitis tissue the new histologic classification of chronic rhinosinusitis gene expression profiles in human nasal polyp tissues studied by means of dna microarray antibody equivalent molecules of the innate immune system: parallels between innate and adaptive immune proteins upregulation of surfactant protein a in chronic rhinosinusitis expression and significance of surfactant a in nasal polyps of chronic rhinosinusitis surfactant protein d expression in chronic rhinosinusitis patients and immune responses in vitro to aspergillus and alternaria in a nasal explant model acute respiratory tract infections and mannose-binding lectin insufficiency during early childhood a population-based study of morbidity and mortality in mannose-binding lectin deficiency increased serum complement component and mannose-binding lectin levels in adult chinese patients with chronic rhinosinusitis serum amyloid a is an innate immune opsonin for gram-negative bacteria alterations in gene expression of complement components in chronic rhinosinusitis high nitric oxide production in human paranasal sinuses nitric oxide and the paranasal sinuses no inhibitions: antimicrobial properties of nitric oxide discovery of a nitric oxide responsive quorum sensing circuit in vibrio harveyi nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-gmp signaling network the effects of nitric oxide on staphylococcus aureus biofilm growth and its implications in chronic rhinosinusitis innate immune recognition recognition of microorganisms and activation of the immune response ric- a, a ga protein guanine nucleotide exchange factor potentiates taste receptor signaling expression of tlr and tlr messenger rna in the epithelial cells of the nasal airway sinonasal epithelial cell expression of toll-like receptor is decreased in chronic rhinosinusitis with polyps innate immunity in the lung: how epithelial cells fight against respiratory pathogens heterodimerization of tlr with tlr or tlr expands the ligand spectrum but does not lead to differential signaling human toll-like receptor mutations but not cd polymorphisms are associated with an increased risk of gram-negative infections cd -dependent lipopolysaccharide-induced beta-defensin- expression in human tracheobronchial epithelium altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps dectin- mediates the biological effects of beta-glucans collaborative induction of inflammatory responses by dectin- and toll-like receptor the role of the beta-glucan receptor dectin- in control of fungal infection mendelian traits causing susceptibility to mucocutaneous fungal infections in human subjects dectin- is inducible and plays a crucial role in aspergillus-induced innate immune responses in human bronchial epithelial cells the molecular basis of nod susceptibility mutations in crohn's disease pattern recognition receptors -molecular orchestrators of inflammation in inflammatory bowel disease. cytokine nod is a general sensor of peptidoglycan through muramyl dipeptide (mdp) detection chronic sinonasal disease in patients with inflammatory bowel disease chronic rhinosinusitis in the setting of other chronic inflammatory diseases t r taste receptor polymorphisms underlie susceptibility to upper respiratory infection interleukin- : a sheep in wolf's clothing healing of intestinal inflammation by il- interleukin- mediates early host defense against attaching and effacing bacterial pathogens il- is produced by innate lymphoid cells and limits inflammation in allergic airway disease th cells are an important source of il- for host protection against enteropathogenic bacteria il- mediates mucosal host defense against gram-negative bacterial pneumonia interleukin (il)- and il- are coexpressed by th cells and cooperatively enhance expression of antimicrobial peptides border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by il- il- and il- : master regulators of innate and adaptive immunity novel p protein engages il- p to form a cytokine, il- , with biological activities similar as well as distinct from il- il- in antifungal immunity polymorphisms in the interleukin- receptor alpha- gene are associated with severe chronic rhinosinusitis chronic rhinosinusitis with nasal polyps is associated with decreased expression of mucosal interleukin receptor evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis a clinical and pathologic study of chronic sinusitis: the role of the eosinophil assessment of inflammation in noninfectious chronic maxillary sinusitis eosinophilia in chronic childhood sinusitis features of airway remodeling and eosinophilic inflammation in chronic rhinosinusitis: is the histopathology similar to asthma? chronic rhinosinusitis: an enhanced immune response to ubiquitous airborne fungi nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils role of fungal antigens in eosinophilia-associated cellular responses in nasal polyps: a comparison with enterotoxin immunologic response to fungus is not universally associated with rhinosinusitis treatment of chronic rhinosinusitis with intranasal amphotericin b: a randomized, placebo-controlled, double-blind pilot trial topical antifungal treatment of chronic rhinosinusitis with nasal polyps: a randomized, double-blind clinical trial amphotericin b nasal lavages: not a solution for patients with chronic rhinosinusitis the effect of topical amphotericin b on inflammatory markers in patients with chronic rhinosinusitis: a multicenter randomized controlled study staphylococcus aureus colonization and ige antibody formation to enterotoxins is increased in nasal polyposis a superantigen hypothesis for the pathogenesis of chronic hyperplastic sinusitis with massive nasal polyposis staphylococcal exotoxins and nasal polyposis: analysis of systemic and local responses superantigens and chronic rhinosinusitis ii: analysis of t-cell receptor v beta domains in nasal polyps impairing oral tolerance promotes allergy and anaphylaxis: a new murine food allergy model proinflammatory impact of staphylococcus epidermidis on the nasal epithelium quantified by il- and gro-alpha responses in primary human nasal epithelial cells the role of atypical bacteria in chronic rhinosinusitis molecular characterization of the polymicrobial flora in chronic rhinosinusitis microbiome complexity and staphylococcus aureus in chronic rhinosinusitis nontuberculous mycobacteria in household plumbing as possible cause of chronic rhinosinusitis atypical mycobacterium detection in refractory chronic rhinosinusitis biofilm detection with hematoxylin-eosin staining a sheep model for the study of biofilms in rhinosinusitis confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis targeted imaging modality selection for bacterial biofilms in chronic rhinosinusitis svabic-vlahovic m. a modified microtiter-plate test for quantification of staphylococcal biofilm formation in vitro biofilm formation in an -well chamber slide prevalence of biofilm-forming bacteria in chronic rhinosinusitis evidence of bacterial biofilms in human chronic sinusitis demonstration of biofilm in human bacterial chronic rhinosinusitis bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis characterization of bacterial and fungal biofilms in chronic rhinosinusitis biofilms correlate with th inflammation in the sinonasal tissue of patients with chronic rhinosinusitis damage to ciliated epithelium in chronic rhinosinusitis: what is the role of bacterial biofilms? the impact of biofilms on outcomes after endoscopic sinus surgery evidence of bacterial biofilms in nasal polyposis altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps cigarette smoke combined with toll-like receptor signaling triggers exaggerated epithelial regulated upon activation, normal t-cell expressed and secreted/ ccl expression in chronic rhinosinusitis production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type ii epithelial cells: implications for local defense to bacterial endotoxins toll-like receptors and innate antifungal responses differential expression of toll-like receptor pathway genes in chronic rhinosinusitis with or without nasal polyps bacterial dna evokes epithelial il- production by a mapk-dependent, nf-kappab-independent pathway th cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells t r taste receptor polymorphisms underlie susceptibility to upper respiratory infection innate immunity in the lung: how epithelial cells fight against respiratory pathogens innate immunity in the paranasal sinuses: a review of nasal host defenses method of biofilm detection key: cord- - zjghwkq authors: ronit, andreas; berg, ronan m.g.; bay, jakob t.; haugaard, anna k.; ahlström, magnus g.; burgdorf, kristoffer s.; ullum, henrik; rørvig, sara b.; tjelle, klaus; foss, nicolai b.; benfield, thomas; marquart, hanne vibeke; plovsing, ronni r. title: compartmental immunophenotyping in covid- ards: a case series date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: zjghwkq background: severe immunopathology may drive the deleterious manifestations that are observed in the advanced stages of coronavirus disease (covid- ) but are poorly understood. objective: our aim was to phenotype leukocyte subpopulations and the cytokine milieu in the lungs and blood of critically ill patients with covid- acute respiratory distress syndrome (ards). methods: we consecutively included patients less than hours after intubation following informed consent from their next of kin. bronchoalveolar lavage fluid was evaluated by microscopy; bronchoalveolar lavage fluid and blood were assessed by -color flow cytometry and a multiplex cytokine panel. results: four mechanically ventilated patients (aged - years) with moderate-to-severe covid- ards were included. immature neutrophils dominated in both blood and lungs, whereas cd and cd t-cell lymphopenia was observed in the compartments. however, regulatory t cells and t(h) cells were found in higher fractions in the lung. lung cd and cd t cells and macrophages expressed an even higher upregulation of activation markers than in blood. a wide range of cytokines were expressed at high levels both in the blood and in the lungs, most notably, il- ra, il- , il- , ip- , and monocyte chemoattactant protein- , consistent with hyperinflammation. conclusion: covid- ards exhibits a distinct immunologic profile in the lungs, with a depleted and exhausted cd and cd t-cell population that resides within a heavily hyperinflammatory milieu. cd t cells and macrophages expressed an even higher upregulation of activation markers than in blood. a wide range of cytokines were expressed at high levels both in the blood and in the lungs, most notably, il- ra, il- , il- , ip- , and monocyte chemoattactant protein- , consistent with hyperinflammation. conclusion: covid- ards exhibits a distinct immunologic profile in the lungs, with a depleted and exhausted cd and cd t-cell population that resides within a heavily hyperinflammatory milieu. (j allergy clin immunol ;nnn:nnn-nnn.) key words: acute respiratory distress syndrome, bronchoalveolar lavage, covid- , cytokines, flow cytometry in december , a cluster of pneumonia cases with unknown cause was reported in wuhan in the hubei province of china. a novel coronavirus was identified in january as severe acute respiratory syndrome coronavirus (sars-cov- ), and as of july , , nearly million cases of corona virus disease (covid- ) with more than , deaths had been confirmed worldwide. although recent results support the use of an rna polymerase inhibitor (remdesivir) and low-dose dexamethasone in hospitalized patients requiring oxygen therapy, , the mortality of critically ill patients with covid- remains high. , a subgroup of patients with covid- progress to severe respiratory failure mimicking acute respiratory distress syndrome (ards). , these patients tend to be older and have more comorbidity than their nonprogressing counterparts, but the underlying pathways leading to increased disease severity are incompletely understood. an important clinical hallmark of invasive disease is lymphopenia, and it has thus been suggested that a severe course of disease is triggered by a dysregulated innate and adaptive immune response and hyperinflammation. clinical trials of immunomodulatory treatment are currently pending, and to aid this agenda a better understanding of the host immune response to sars-cov- is warranted. although the available evidence favors the presence of severe immunopathology during covid- ards, the compartmental transmission of immunoinflammatory processes between blood and lungs remains unexplored, and characterization of distinct leukocyte subpopulations and their cytokine mediators could thus potentially reveal both novel therapeutic targets and guide the timing of treatment. in the present study, we immunophenotyped bronchoalveolar lavage fluid (balf) and blood of mechanically ventilated patients with moderate-to-severe covid- ards. myeloid cell population as well as t-and b-cell subsets were characterized with focus on cellular differentiation and activation. we further studied a wide spectrum of inflammatory mediators and performed a detailed microbiologic analysis. for inclusion in this cross-sectional study, participants had to be adults (> _ years) with sars-cov- confirmed by pcr, with presence of ards determined according to the berlin criteria and less than hours of mechanical ventilation after admittance to the intensive care unit (icu). all patients were sedated and unable to provide informed consent, which was obtained from their next of kin. the study was approved by the regional ethics committee of copenhagen (h- ) and the knowledge center for data review of copenhagen (p- - ) and registered at clinicaltrials.gov (nct ). a consolidated standards of reporting trials checklist and flow diagram are provided in table e and fig e ( both of which are available in the online repository at www.jacionline.org), respectively. approximately patients with covid- have been hospitalized in denmark ( . million inhabitants), with requiring treatment in an icu as of june , . at our hospital (hvidovre hospital), a total of patients with severe covid- were treated in the icu, with all except requiring mechanical ventilation. of the eligible patients admitted during the study inclusion period, were included in our study. additional details on study inclusion are provided in the online data supplement in this article's online repository (available at www.jacionline.org). we originally intended to include a total of patients, but because of rapidly decreasing admission rates and as this was a descriptive immunopathologic study, we chose to disseminate the findings already at hand. arterial blood samples were drawn immediately before the bronchoalveolar lavage procedure, which was performed in a standardized fashion according to current guidelines ; the procedure and processing of samples are described in further detail in the online data supplement. in addition, balf supernatant and edta-stabilized plasma were separated and stored at c for cytokine analyses. a total of healthy controls ( men and women with a median age of years [range - years]) were used for comparison of flow cytometry results in blood, and healthy nonsmoking volunteers (all of them men, with a median age years [range - years]) from a previous study were used for comparison of cytokines in the blood and balf. the duration of the bronchoalveolar lavage procedure was between and minutes and there were no procedure-related complications. bronchoscopy revealed slight erythema and friability of the mucosa and mild-to-moderate edema but no visible secretions. balf return volumes ranged between and ml, and vol% recovered ranged between % and %. in all patients, the initial return was colorless and then progressively turned pinker with subsequent aliquots. none of the samples were yellowish or lactescent in color, and no clumps or debris were observed. five cytospins (each containing to drops of balf) from each of the patients were made. two of the cytospins were stained with may-gr€ unwald-giemsa stain. for flow cytometry, we used a -color antibody panel consisting of prefabricated customized duraclone tubes from beckman coulter (beckman coulter, brea, calif) with freeze-dried antibodies. additional details on flow cytometry, including use of antibodies (see table e in this article's online repository at www.jacionline.org) and flow characterization of cell populations (see table e in this article's online repository at www.jacionline. org), are provided in the online data supplement. we identified key cell populations to be included in the study a priori. the msd v-plex human biomarker plex kit (meso scale discovery platform, [meso scale diagnostics, rockville, md]) was used for cytokine and chemokine quantification and run ( ml of balf supernatant and ml of plasma) according to the manufacturer's protocol, with lower limits of detection ranging between . and pg/ml. balf and blood cytokines from patients with covid- and controls were analyzed on the same plates. sars-cov- rna was determined by real-time rt-pcr targeting the orf a/b nonstructural region that is unique to sars-cov- . in addition, a biofire filmarray pneumonia plus panel (see table e in this article's online repository at www.jacionline.org) was used for detection of respiratory pathogens, and s and s pcr were performed on all balf samples. a more detailed description of these analyses is provided in the online data supplement. we decided to limit the use of summary statistics and instead present data as case by case. we further decided against generating p values because of the small sample size. figures were generated by using r software, version . . . patient characteristics, laboratory data, and specific icu therapies are depicted in tables i and ii . all patients had moderate-tosevere impairment of oxygenation at icu admission that deteriorated before the study procedure, and they were treated with broad-spectrum antibiotics (piperacillin/tazobactam or meropenem) at the time of inclusion. none was treated with corticosteroids, antivirals, or immunomodulatory drugs off protocol, but of the were included in a randomized clinical trial before icu admission (table i) . microbiologic evaluation of balf revealed detectable sars-cov- rna at the time of the procedure in all patients. patient was found to be infected with methicillin-sensitive staphylococcus aureus; the other identified pathogens are listed in table e (in this article's online repository at www.jacionline.org). we found no evidence of coinfection with viral or fungal pathogens. radar plots of the leukocyte subsets in the blood (and balf) are provided in fig . lymphopenia was observed in all cases (ranging from . to . /l), with normal (in of the patients) or increased (in of the patients) neutrophil counts (table ii) . two of the patients had eosinopenia (table ii) . b-cell counts were within the normal range ( . - . /l [ fig ] ), but reduced isotype switching (< % of the b cells) was observed in patients; patients had increased plasmablast concentrations in their blood, and in of these patients, mainly non-isotype-switched plasmablasts were present (see fig e in this article's online repository at www.jacionline.org). t-cell levels were low, with cd t-cell counts ranging from . to . /l (normal range . - . /l) and cd t-cell counts ranging from . to . /l (normal range . - . /l) (fig ) . all patients had low cd recent thymic emigrant (rte) counts (ranging from . to . /l), indicating reduced thymic output. the distribution of cd t-cell subsets was directed toward central memory, whereas cd t-cell subsets were generally directed toward terminal differentiation; patient had severely reduced cd rte counts and a high fraction of both cd and cd terminal differentiation (fig ) . regulatory t (treg) cell levels were elevated in all cases, whereas t h levels were comparable to those found in healthy controls (see fig may-gr€ unwald-giemsa-stained balf cytocentifugates were characterized by a background of erythrocytes and a variable number of neutrophils, macrophages, small lymphocytes, and (to a lesser extent) eosinophils. we did not detect any hemosiderinladen alveolar macrophages. in line with this, flow cytometry identified the major components in balf as neutrophils, monocytes/macrophages, and eosinophils but noticeably few lymphocytes (fig ) . further characterization of the lymphocyte subpopulations revealed that the fraction of cd and cd t cells was specifically reduced (fig ) but the proportion of both treg cells and t h cells was nonetheless higher in balf than in blood (see fig e ) . neutrophils were primarily cd -(see fig e ) . in balf, cd monocytes showed marked upregulation of cd and hla-dr expression compared with blood monocytes. in addition, a distinct §patients and were referred to another icu with extracorporeal membrane oxygenation (ecmo) facilities within days after the procedure. patient was discharged from the hospital after days of ecmo therapy followed by days of hospital admission. patient was weaned off ecmo but later died due to refractory hypoxemia; patient died within hours after the procedure as a result of cardiogenic shock. patient was discharged from the icu but was still hospitalized as of june , . feu, fibrinogen equivalent unit; vv-ecmo, venovenous extracorporeal membrane oxygenation. *laboratory data and chest radiography before the bronchoscopy procedure. adjuvant therapies applied during the icu stay. àspecific characteristics of mechanical ventilation immediately before the bronchoscopy procedure. cd subpopulation with very bright hla-dr expression and increased autofluorescence identifying the macrophages showed increased expression of cd and cd and (to a lesser extent) increased expression of cd b (see fig e ) . furthermore, as in blood, the concentration of dendritic cell populations was low in balf (see fig e ) . cd and cd t cells and natural killer cells in balf expressed very high levels of cd activation (fig and see fig e ) , and ctla- was also markedly upregulated on both cd (ranging from % to %) and cd (ranging from % to %) t cells (see fig e ) . albumin was detected in balf from all patients (table ii) . the levels of several cytokines and chemokines in both blood and balf were markedly elevated; this was most pronounced balf is dominated by myeloid cells. in healthy adults, monocytes comprise . % to . %, lymphocytes comprise . % to . %, neutrocytes comprise . % to . %, eosinophils comprise . % to . %, and basophils comprise . % to . % of leukocytes in balf. cd cells comprise % to %, cd cells comprise % to %, b cells comprise . % to . %, and natural killer cells comprise % to % of the leukocytes. gd, gd. j allergy clin immunol volume nnn, number nn except for the concentration of il- , which was higher in balf than in blood, the concentrations of the inflammatory mediators seemed to be elevated to the same extent in the compartments. a heatmap depicting the correlation between cytokines in the lung and systemic compartment and crp dynamics before and after study inclusion have been included as supplementary material (see figs e and e , respectively, in this article's online repository at www.jacionline.org). the clinical management of critically ill patients with covid- is hampered by a limited understanding of the underlying pathophysiologic mechanisms, and so far, only a handful of studies have focused on in vivo immunopathology, primarily by analyzing the immune system at a transcriptional level in either blood or the lungs. in the present study, we analyzed simultaneously obtained blood and balf from patients with covid- ards, thus allowing for comparison of the distinct immunologic compartments. our main findings are that although certain aspects of the immune response (notably, the dominance of immature neutrophils and vast increases in a wide array of cytokines) are similar in blood and the lungs, several changes within the t-cell population are markedly compartmentalized. thus, the depleted t-cell population in the lungs encompassed elevated treg cells and t h cells and exhibited a prominent upregulation of activation markers. in all patients we observed macroscopic signs of inflammation but no visible secretions, the latter being in contrast with what was recently found in a larger population of patients with table e . cm, central memory; em, effector memory. covid- who underwent bronchoscopy. accordingly, we did not experience macroscopic bleeding and the yield of more erythrocyte-rich balf with successive aliquots and detectable albumin most likely reflects fragile lung parenchyma and vascular leakage in the more distal airways. no hemosiderin-laden macrophages were found, which suggests diffuse alveolar damage rather than diffuse alveolar hemorrhage, a finding that is supported by autopsy studies in patients with covid- . , a consistent finding in the present study was the lymphopenia driven by low t cells counts in blood, which appears to be pathognomonic for severe covid- . , furthermore, of the patients were eosinopenic, which some authors have highlighted as a putative hallmark of covid- . correspondingly, the proportion of lymphocytes in balf was low, with a concomitant reduction in both cd and cd t-cell fractions compared with the normal values. a recent single-cell rna sequencing balf study in patients with covid- showed a similarly low proportion of t cells in the balf of patients who were critically ill compared with the proportion in those with moderate disease severity. this has important pathophysiologic implications; assisted by cd t cells, both cd t and b cells play an important role in viral clearance, and systemic depletion of cd and cd t cells has previously been shown to be associated with adverse outcomes in sars-cov infection. another consistent finding in the present study was a higher fraction of treg cells. a high fraction of treg cells in balf has previously been shown in non-covid- ards, where treg cells appear to be involved in resolution of lung injury by induction of neutrophil apoptosis, macrophage efferocytosis, and decreasing fibrocyte recruitment. the cd and cd cd t cells seemed to be markedly elevated in balf compared with blood. t h cells may mediate tissue destruction by recruitment of neutrophils to the lungs. however, these cells have also been found to be elevated in community-acquired pneumonia, and this finding may thus not be specific for covid- . however, t h cells were characterized by using the surrogate surface markers cd and cd without intracellular staining, which may impede correct identification of these cells. the mechanisms explaining the reduction in circulating cd and cd t cells in severe covid- remain to be elucidated, but may share similarities with that observed during sepsis-induced immune cell apoptosis. , because of the remarkably low cd and cd fractions in balf, this is not likely caused by extravasation (ie, compartmentalization to the alveoli). rather, reduced t-cell production (as indicated by the low cd rte cell count in blood) is likely at play. several mediators may promote thymic dysfunction/involution, including systemic hyperinflammation per se , ; specifically, the presence of high levels of tnf-a and il- in severe covid- could potentially lead to both reduced t-cell production and accelerated t-cell apoptosis. finally, the increased frequency of treg cells and t h cells in blood may be explained by a decrease in the number of naive t cells in combination with strong immune activation and differentiation in lung. we observed a significant upregulation of activation markers on both lung and blood leukocytes, including cd and hla-dr. notably, the immune checkpoint molecule ctla- was upregulated on both foxp -expressing cd treg cells and cd t cells in blood, and it was even more pronounced on t cells from balf. ctla- is constitutively expressed in treg cells but upregulated on the cell surface only after activation, a phenomenon that is particularly notable in cancers. this finding in covid- ards is likely secondary to massive stimulation of the immune system caused by the viral invasion, thus resulting in an inexpedient exhaustion of the immune system. exhausted t cells are generally defined as pd -and cd -expressing cd t cells. thus, the marked pd upregulation found not only in patient blood cd t cells but also in cd t cells likely indicates a more general pronounced exhaustion in patients with covid- . although activation markers were expected to be upregulated in the lungs compared with in the blood, these expression levels far exceeded what has previously been reported in balf for healthy controls, and they were also higher in the blood of patients with covid- ards than in the blood of healthy controls. another consistent finding within the balf cellular compartment was the dominance of neutrophils and (to a lesser extent) monocytes and macrophages (cd with very bright hla-dr expression and increased autofluorescence), which also displayed pronounced upregulation of surface markers related to activation (cd , cd , hla-dr, cd b, and cd ). in comparison, blood monocytes showed moderate signs of activation by upregulation of cd and cd and slight decrease in hla-dr. previous studies in patients with severe sars-cov found high levels of proinflammatory cytokines (il- , il- , and il- ), t h cytokine inf-g, neutrophil chemotactic factor (il- ), mcp- , and t h chemokine ifn-d-inducible protein- (ip- ). [ ] [ ] [ ] a recent study performed genome-wide transcriptome sequencing of rna obtained from balf in patients with covid- with unknown disease severity; it reported different expression of genes, including a high expression of cytokines such as mcp- , ip- , mip- a, and mip- b. in comparison, another transcriptomic analysis of patients with covid- suggested increased expression of proinflammatory genes, including interferon-stimulated gene. in the present study, we found nearly all cytokines to be elevated in the balf and blood of the patients with covid- compared with in healthy volunteers. importantly, the chemokines il- , ip- , and mcp- were found in very high concentrations in the lungs and accordingly may elicit and perpetuate local inflammation. of these, il- exhibited a notable compartmentalized response within the lungs, which when considering the cellular immune response reported here, is consistent with the well-established role of il- in the recruitment of neutrophils to the lungs during acute pulmonary inflammation. the observed cytokine and chemokine levels in both blood and balf point toward an extensive hyperinflammatory phenotype in severe covid- , a phenomenon that some authors have coined a ''cytokine storm.'' however, in addition to proinflammatory cytokines, several anti-inflammatory cytokines (eg, il- ) were also detected at high levels in balf. this may be a beneficial response that helps regulate local inflammation, although excess production of anti-inflammatory mediators could potentially also compromise the host's ability to clear sars-cov- . the current hope is that existing, approved therapies aimed at dampening or inhibiting inflammation, including il- , il- , and il- receptor blockade, will increase survival. a list of registered off-label trials with respect to the cytokines evaluated in this study is presented in table e (in this article's online repository at www.jacionline.org). in addition, recent proteomic studies have revealed that key protein kinases (eg, p mitogen-activated protein and casein kinase ii) are involved in covid- pathogenesis, which could be targeted by using existing protein kinase inhibitors. our study has strengths and limitations. we performed a comprehensive protocoled study with uniform balf and blood sampling in patients with covid- ards. this provided a unique material for in-depth characterization of the leukocyte and cytokine milieu in the lungs and blood, and it allowed for direct intraindividual and interindividual comparison of the compartments. we intended to include additional patients, but the course of the covid- epidemic in denmark did not allow us to do so. hence, major limitations of our study are the small sample size and the explorative design. moreover, we had a control group only for flow cytometry comparison in blood and not for balf. our study was observational and cross-sectional, and because of the nature of the design, we are not able to infer causation. in conclusion, our study provides novel phenotypic insight into the cell composition and inflammatory mediators simultaneously present in the lungs and blood in patients with covid- ards. our findings indicate that although both compartments exhibit a severely hyperinflammatory milieu, in which activated lymphocytes are present in low numbers, the t cells within the lungs are remarkably activated, and dominated by subtypes that function to govern both reparative and destructive processes. future studies should determine how these findings are related to the clinical course and how protocoled anti-inflammatory therapies may affect the immunophenotype at both the local and systemic level. world health organization. coronavirus disease (covid- ) pandemic european centre for disease prevention and control. covid- situation update worldwide, as of remdesivir for the treatment of covid- -preliminary report dexamethasone in hospitalized patients with covid- -preliminary report baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area covid- pneumonia: ards or not? covid- does not lead to a ''typical'' acute respiratory distress syndrome clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan acute respiratory distress syndrome: the berlin definition statens serum institut. tal og overv agning: covid- technical aspects of bronchoalveolar lavage: recommendations for a standard procedure transcompartmental inflammatory responses in humans: iv versus endobronchial administration of endotoxin bronchoscopy in covid- patients with invasive mechanical ventilation: a center experience autopsy findings and venous thromboembolism in patients with covid- : a prospective cohort study pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- clinical course and outcomes of intensive care patients with covid- covid- with different severities: a multicenter study of clinical features eosinophil responses during covid- infections and coronavirus vaccination bronchoalveolar lavage cell pattern from healthy human lung single-cell landscape of bronchoalveolar immune cells in patients with covid- haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis blt -dependent alveolar recruitment of cd ( )cd ( ) foxp ( ) regulatory t cells is important for resolution of acute lung injury t helper cells are involved in the local and systemic inflammatory response in community-acquired pneumonia sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy increased percentage of cd cd regulatory t cells during septic shock is due to the decrease of cd cd -lymphocytes altered bone marrow lymphopoiesis and interleukin- -dependent inhibition of thymocyte differentiation contribute to thymic atrophy during trypanosoma cruzi infection acute endotoxin-induced thymic atrophy is characterized by intrathymic inflammatory and wound healing responses increased tnf-alpha-induced apoptosis in lymphocytes from aged humans: changes in tnf-alpha receptor expression and activation of caspases disruption of ctla- expression on peripheral blood cd t cell enhances anti-tumor efficacy in bladder cancer expression of lymphocyte activation surface antigens in bronchoalveolar lavage and peripheral blood cells from young healthy subjects plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome temporal changes in cytokine/ chemokine profiles and pulmonary involvement in severe acute respiratory syndrome persistence of lung inflammation and lung cytokines with high-resolution ct abnormalities during recovery from sars transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients heightened innate immune responses in the respiratory tract of covid- patients interleukin- (il- ): the major neutrophil chemotactic factor in the lung prevention of the cytokine storm in covid- the global phosphorylation landscape of sars-cov- infection we thank all the patients with covid- and their families for their participation. we also thank anna louise sørensen, ann-charlotte lundstedt, unni nielsen, and the staff at the department of pathology, microbiology and clinical biochemistry for their outstanding laboratory assistance. we also thank the clinical staff and nurses at the department of anesthesiology and intensive care for their dedicated contribution. d the identification of a distinct pulmonary immunologic phenotype in covid- ards is potentially of use for identification of targets for future anti-inflammatory therapies.d the cellular immune response of covid- ards is dominated by immature neutrophils in both the blood and the lungs, with concomitant cd and cd t-cell lymphopenia and elevated inflammatory chemokine and cytokine levels.d the t-cell profile in the lungs is substantially different from that in blood, with expression of much higher levels of activation and higher frequency of treg cells and t h cells. key: cord- - fn ei f authors: hanania, nicola a.; king, monroe j.; braman, sidney s.; saltoun, carol; wise, robert a.; enright, paul; falsey, ann r.; mathur, sameer k.; ramsdell, joe w.; rogers, linda; stempel, david a.; lima, john j.; fish, james e.; wilson, sandra r.; boyd, cynthia; patel, kushang v.; irvin, charles g.; yawn, barbara p.; halm, ethan a.; wasserman, stephen i.; sands, mark f.; ershler, william b.; ledford, dennis k. title: asthma in the elderly: current understanding and future research needs—a report of a national institute on aging (nia) workshop date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: fn ei f asthma in the elderly is underdiagnosed and undertreated, and there is a paucity of knowledge on the subject. the national institute on aging convened this workshop to identify what is known and what gaps in knowledge remain and suggest research directions needed to improve the understanding and care of asthma in the elderly. asthma presenting at an advanced age often has similar clinical and physiologic consequences as seen with younger patients, but comorbid illnesses and the psychosocial effects of aging might affect the diagnosis, clinical presentation, and care of asthma in this population. at least phenotypes exist among elderly patients with asthma; those with longstanding asthma have more severe airflow limitation and less complete reversibility than those with late-onset asthma. many challenges exist in the recognition and treatment of asthma in the elderly. furthermore, the pathophysiologic mechanisms of asthma in the elderly are likely to be different from those seen in young asthmatic patients, and these differences might influence the clinical course and outcomes of asthma in this population. states is currently about % but is projected to grow from about million in to more than million by , accounting for % of the population. the age group with the largest growth will be those older than years, which is estimated to be more than million by . , in , the us prevalence of asthma for those years or older was %, with , , reporting an asthma attack in the previous months. older asthmatic patients are more likely to be underdiagnosed, undertreated, , and hospitalized than younger asthmatic patients. they also have the highest death rate ( . per million persons) of any other age group. older women are hospitalized more than twice as often as older men. asthma in older adults is superimposed on a background of aging-related changes in respiratory and immune physiology and often on multiple diseases and conditions common in older age. recognizing the paucity of research, the many challenges that exist in the recognition and treatment of asthma in older adults, and the opportunity to bridge geriatrics and the clinical specialties that focus on asthma, the national institute on aging (nia) sponsored a workshop on asthma in the elderly in herndon, virginia, on september and , . the workshop was planned by a committee of physician-scientists from us academic institutions or from the division of geriatrics and clinical gerontology in the nia. the planning committee selected speakers and participants for their expertise in asthma, pulmonology, allergy/immunology, primary care, emergency medicine, geriatrics, and/or gerontologic science (see the list of participants in appendix ). the immediate goals of this workshop were to summarize the current understanding of the mechanisms of asthma in older persons and to identify knowledge gaps and research opportunities leading to improved medical care and health outcomes for older persons with asthma. these research opportunities are discussed in the body of this report and summarized in table i . [ ] [ ] [ ] in addition, the nia, in collaboration with the national heart, lung, and blood institute and the national institute of allergy and infectious diseases, recently issued a set of program announcements inviting research proposals on asthma in older adults (http:// grants.nih.gov/grants/guide/pa-files/pa- - .html, http://grants. nih.gov/grants/guide/pa-files/pa- - .html, and http://grants. nih.gov/grants/guide/pa-files/pa- - .html). it is a central principle of gerontology that aging itself is not a disease. yet there are physiological changes within organs, tissues, and cells that result in diminished functional reserve and thereby increased susceptibility to stressors, disease, or both. a second principle is that these aging changes are highly variable and account for the great constitutional heterogeneity among older persons from very ''fit'' to very ''frail.'' in fact, the concept of frailty, both its causes and consequences, has become a focus of concentrated gerontologic investigation. at the root of age-associated physiological changes are a number of genetic, epigenetic, and environmental factors. molecular damage accumulates over time, and the capacity for dna repair decreases. cellular senescence, which is believed to be the consequence of accumulated dna and protein damage and reduced proliferative capacity, is becoming increasingly understood at the molecular level. however, just how this correlates with the phenotypic changes of advanced age remains incompletely understood. there has been much written about cellular senescence and the events that lead to cell death. [ ] [ ] [ ] after a finite number of divisions, normal somatic cells invariably enter a state of irreversibly arrested growth, a process termed replicative senescence. in fact, it has been proposed that escape from the regulators of senescence is the antecedent of malignant transformation. however, the role of replicative senescence as an explanation of organismal aging remains the subject of vigorous debate. the controversy relates, in part, to the fact that certain organisms (eg, drosophila species and caenorhabditis elegans) undergo an aging process, yet all of their adult cells are postreplicative. what is clear is that the loss of the proliferative capacity of human cells in culture is intrinsic to the cells and not dependent on environmental factors or even culture conditions. unless transformation occurs, cells age with each successive division. the number of divisions turns out to be more important than the actual amount of time passed. thus cells held in a quiescent state for months, when allowed back into a proliferative environment, will continue approximately the same number of divisions as those that were allowed to proliferate without a quiescent period. the question remains whether this in vitro phenomenon is relevant to animal aging. one suggestive observation is that fibroblasts cultured from samples of old skin undergo fewer cycles of replication than those from young skin. furthermore, when various species are compared, replicative potential is directly and significantly related to lifespan. an unusual b-galactosidase with activity peaks at ph has proved to be a useful biomarker of in vitro senescence because it is expressed by senescent but not presenescent or quiescent fibroblasts. this particular b-galactosidase isoform was found to have the predicted pattern of expression in skin from young and old donors, with measurably increased levels in dermal fibroblasts and epidermal keratinocytes with advancing age. the nature of the expression of this in vivo biomarker of aging in other tissues will be important to discern. for clinical investigators, frailty has proved hard to define primarily because of the seemingly insurmountable heterogeneity inherent in geriatric populations on the basis of these variable rates of organ system decrease and the presence or absence of or more diseases. yet, regardless of the pathway taken to frailty, the clinical picture has common features, including a reduction in lean body mass (sarcopenia), loss of bone mass (osteopenia), cognitive impairment, functional decline, and anemia. on the basis of data derived from large cohorts of elderly patients, fried et al have offered an operational definition of frailty incorporating an assessment of specific characteristics to ascribe a frailty index. on this -point scale, a score of or more has been shown to be independently predictive of a range of adverse clinical outcomes, including acute illness, falls, hospitalization, nursing home placement, and early mortality. [ ] [ ] [ ] furthermore, simple performance measures, such as the assessment of walking speed, are predictive of important outcomes, including survival. with the phenotype better defined, attention has shifted to pathophysiology. although frailty can occur in the absence of a diagnosable illness, the fact that some become frail and others do not suggests an inherent or acquired variability in homeostatic pathways. recent evidence from observational studies has raised suspicion that dysregulated inflammatory processes are involved in, if not central to, the variable patterns of aging. increased serum levels of certain proinflammatory cytokines, most notably il- , are increasingly present with advancing age and to a greater extent with frailty. , furthermore, the appearance of this and other inflammatory markers has been associated with a number of adverse clinical outcomes, including decreased strength and mobility, falls, dementia, and mortality. life expectancy, lifespan, and maximum survival from the perspective of those who study aging, there is an important distinction made between median (life expectancy) and maximum lifespan. over the past several decades, with the advent of modern sanitation, refrigeration, and other public health measures, including vaccination and antibiotics, there has been a dramatic increase in median survival. early deaths have been diminished, and more patients are reaching old age. in the united states today, life expectancy now approaches years. median survival is what concerns public health officials and health care providers, but for those studying the biology of aging, it is maximum survival that is the focus of greatest attention. it is worthwhile to note that it has been estimated that if atherosclerosis and cancer were eliminated from the population as a cause of death, about years would be added to the average lifespan, yet there would be no change in maximum lifespan. although several theories have been proposed, none suffice to account for the complexities of aging. lifespan is finite and varies generally from species to species and much less so within species. mice live, on average, ½ years, monkeys years, and human subjects about years. among species, larger animals generally live longer than smaller animals, but within species, smaller animals are likely to live longer. it is clear that aging is not entirely explained by dna sequence. for example, mice and bats have only . % difference in their primary dna sequence, but bats live for years, times longer than mice. a commonly held notion is that regulation of gene expression accounts for a longevity difference between species. the aging lung large, longitudinal, and more complete studies to determine the effects of aging on the function of the respiratory system improved knowledge about lung structure-function relationships in older age using techniques of imaging and measures of lung function not requiring effort (eg, high-resolution computed tomographic scanning and forced oscillation) improved assessment of lung processes underlying airflow limitation attributable to aging versus copd or asthma, especially in asthmatic patients who smoke studies to examine the effects of aging in ethnic groups and the role of gender epidemiology, effect, diagnosis, and management determine the true prevalence and cost of asthma in the older population develop a uniform definition of asthma to be applied to health care records that will distinguish asthma from copd and mixed asthma/copd evaluate evidence-based treatment algorithms for older asthmatic patients, such as those developed by the national heart, lung, and blood institute and global initiative for asthma guidelines assess the effect of asthma treatment, including direct medical costs of care, indirect costs of care, and value of treatment in improving quality of life , assess the effect of comorbid conditions, especially copd and congestive heart failure, on asthma characterize phenotypes of elderly asthma with regard to responses to therapy and long-term outcomes based on age of onset, duration of disease, and environmental triggers develop algorithms for electronic medical record systems that are asthma-specific evaluate effects of current asthma medications in older patients compared with younger patients identify pharmacogenetic determinants of response to asthma medications in older adults identify simpler and safer drug delivery systems and schedules for older adults develop simple methods to differentiate copd from asthma exacerbations in older adults understand how environmental or aging-related factors affect epigenetic changes in asthma in older adults identify differences between older and younger asthmatic patients or between lsa and loa with regard to inflammation, remodeling, intracellular mechanisms, responses to environmental pollutants, and allergy sensitization and their effects on the metabolism and action of asthma drugs identify naturally occurring age-related changes in airway cellular patterns develop animal models of age-related airway inflammation understand the significance of allergy sensitization associated with asthma in older adults (eg, through larger prospective studies) identify the utility of allergy tests, either skin tests or serum specific ige measurement, in reflecting allergy sensitization in older adults identify the role of the microbiome in patients with loa understand the role of non-ige mechanisms in older adults' inflammatory responses to inhalant allergens or pollutants (eg, t h lymphocytes producing il- or protease receptor responses to molds and dust mites) determine the roles of adaptive versus innate immune mechanisms on asthma development, progression, and response to treatment in older adults determine whether there are environmental pollutants peculiar to institutional settings identify viruses and other microbiological agents responsible for, and the mechanisms by which they cause, asthma exacerbations in older adults, which might lead to the development of vaccine-or antiviral drug-based interventions determine effects of asthma medications, viral or bacterial load, or allergy status on susceptibility to exacerbations in older patients define rates of infection and specific pathogens in older asthmatic patients distinguish roles of innate immunity in eosinophilic versus neutrophilic asthma it is now clearly established that certain specific genes can alter lifespan, at least in lower animals, but whether these same genes regulate ''aging'' is still in question. for example, transgenic drosophila species expressing increased copies of the free radical scavenging enzymes superoxide dismutase and catalase live on average a third longer than the appropriate controls. in even lower species (eg, yeast and nematodes) the identification of specific genes that influence lifespan , has led to the optimistic impression that analogous genes in higher organisms will lead greater insights into the aging process. yet the identification and functional analysis of analogous genes in human subjects remains elusive. the oldest human being alive today is approximately years old. what is intriguing is that the record has remained stable and unchanged by the public health initiatives mentioned above. in fact, there has been some recent data presented that the maximum survival is actually decreasing in the united states. , what is interesting is that, unlike the public health initiatives in human subjects in which median but not maximum survival has been enhanced, experimental interventions in lower species have resulted in prolongation of maximum survival. as mentioned above, transgenic drosophila species producing extra copies of superoxide dismutase and catalase survived about % longer than controls, and similarly, the maximum survival in c bl/ mice fed a calorically restricted diet enhanced by % or more. , the true mechanisms of aging might well be uncovered with a better understanding of how these interventions affect longer survival. future research in aging should attempt to improve our understanding of the basic biology of aging and interventions that retard the aging process. there is a need for the development and application of a standardized definition of frailty for future clinical investigation. investigations directed at the role of comorbidities in accelerating the aging process are important. furthermore, future research should focus on the development of cellular and animal models of typical, delayed, and accelerated aging and of large collaborative networks in which populations and resources can be shared to study aging and frailty. leveraging on well-characterized existing cohorts, when possible, is recommended. the lungs, like other organs, age and exhibit continued loss of function as a person grows old. lung function is traditionally assessed by means of a number of standardized methods. the most common measurement used is spirometry with the determination of fev and forced vital capacity (fvc). fev and fvc both show continuous decreases of between and ml with each year of life after about age years. the cause of this decrease is usually attributed to the loss of the driving forces for airflow as a result of reduced respiratory muscle performance, loss of static elastic recoil, or both. , the decrease in fev in asthmatic patients is largely a function of the decrease in fvc because of the increase in residual volume. stiffening of the chest wall and reduced respiratory muscle performance result in a decrease in total lung capacity and an increase in residual volume because of ever-increasing closing volume. accordingly, these aging processes lead to airflow limitation that might be hard to distinguish from an active disease process. not all older persons are able to perform spirometry, especially those with decreased cognition, coordination, and frailty. in addition, spirometry is effort dependent, and the very old can tire quickly. techniques of imaging and measures of lung function not requiring effort (ie, forced oscillation) should be used in future studies to extend our knowledge about lung structure-function relationships at the very end of life. bronchodilator responses are known to be less marked in the elderly, perhaps as a consequence of the aging effects attributed to the emphysema-like state of the senile lung ; however, this would not explain the slow temporal response to bronchodilators. other studies do not find such age-related bronchodilator differences. furthermore, although methacholine responsiveness has been reported to increase with aging, the exact mechanism for this is not apparent. increased incidence and prevalence of many lung diseases occur with age. alterations in immune function increase the risk of many of these diseases. studies of systemic immunity suggest that sustained antigenic stress over a lifetime leads to a decrease in naive t-cell numbers, an accumulation of memory t cells, and a decrease in t-cell repertoire and b-cell functions but a lesser decrease in innate immunity. , little is known about what happens to the immune/inflammatory pathways in older asthmatic patients. the immune system changes seen with aging will be discussed in more detail in the section on pathophysiology. in the united states the national health interview survey asks questions regarding lifetime history of asthma, current asthma prevalence, and asthma attacks in the last months. for all age groups, asthma prevalence has been steadily increasing since . for the years and older age group, asthma is consistently more prevalent in female than male subjects. the national center for health statistics tracks data on physician encounters for asthma. the national ambulatory medical care survey reported that those years or older have the second-highest rate of outpatient office visits after those aged to years. those years and older did not have significantly different emergency department visits than the other adult groups. the years and older age group accounts for a greater proportion of hospitalizations ( %) than the size of its population ( %) would indicate. not surprisingly, the elderly population is a high user of medical resources for the treatment of asthma. hospitalizations and emergency department visits are more common for these patients than for other adult cohorts. some of the increased costs are related to comorbid disease. for example, the presence of comorbid chronic obstructive pulmonary disease (copd) increases the risk of an asthma-related hospitalization in medicare patients . -fold, respiratory medical costs almost -fold, and total medical costs -fold. elderly female subjects appear at greater risk than elderly male subjects. [ ] [ ] [ ] [ ] asthma mortality increased steadily from until it peaked in . the highest mortality rates for asthma occur in the years and older group. in fact, the increase in asthma mortality between and was primarily driven by the years and older group. in addition, the decrease in asthma mortality between and was most evident in this age group. elderly women with asthma tend to have higher mortality rates than elderly men with asthma. one reason for the increasing prevalence of asthma in the elderly might be the improved longevity of the population. also, increased office visits for asthma in the elderly might be responsible for fewer attacks. increasing hospital admissions might account for decreased mortality. by continuing to gather surveillance data on asthma, reasons for these trends could become clearer. in addition, surveillance data help to focus intervention efforts in areas of greatest need. in the cardiovascular health study, a large community-based cohort of subjects older than years, questions were asked that were relevant to asthma and provided more insight into the prevalence and effect of asthma in this population. , , definite asthma was defined as a positive response to the questions indicating that the patient had current asthma and that a physician confirmed the diagnosis. probable asthma was defined as a history of wheezing in the past year associated with chest tightness or breathlessness. excluding smokers and those with a diagnosis of congestive heart failure, % of subjects had definite asthma, and % had probable asthma. among those who smoked, % had definite asthma, and % had probable asthma. among nonsmokers, subjects were identified who had definite or probable asthma; % were women, and % were older than years. the age of asthma onset was spread approximately evenly among decades. twenty-seven percent had late onset of disease after age years, and % had onset of disease before age years. as expected, respiratory symptoms in the older asthmatic subjects were more prevalent, with a -to -fold increase in cough, phlegm, wheezing, and dyspnea. dyspnea on exertion was . -fold more likely to be present in asthmatic patients than in those without the diagnosis. lung function was reduced in those with a diagnosis of asthma. mean fev was % of predicted value in those with definite asthma and % of predicted value in those with probable asthma compared with % in those who did not have asthma. forty-one percent of those with a diagnosis of asthma had airflow obstruction below the fifth percentile for the age group, and peak flow lability was increased. elderly asthmatic patients reported the most common trigger was a viral infection in % compared with animal allergies in %. two thirds reported seasonal worsening. asthma had a significant effect on quality of life, with % of patients with definite or probable asthma reporting a fair or poor health status compared with % of elderly patients without asthma. sixty percent of patients with definite asthma reported seasonal allergic rhinitis compared with only % in the nonasthma group. despite the high prevalence and morbidity of asthma in this population, inadequate treatment was common. only % of those with definite asthma had a rescue albuterol inhaler, and only % had inhaled corticosteroid use. , , , the pathophysiology of asthma in the older adult is poorly understood and understudied. many questions about this issue remain: is asthma the same disease in older adults as it is in children and younger adults? is late-onset asthma (loa; asthma that starts in middle age or older) different from longstanding asthma (lsa; asthma of early onset that has persisted into older adulthood)? if loa and lsa are the same disease, then the diagnosis and treatment should be similar. however, if loa and lsa are different phenotypes or at least have a different cause and pathophysiology, then the diagnosis and treatment might differ (table ii) . the traditional view of disease susceptibility has been expanded to include epigenetics to account for the influence of environmental factors and aging on the genomic blueprint. epigenetics is defined as heritable changes in gene expression that occur without alterations in dna sequence. it is the process by which genotype interacts with environment to produce a phenotype and explains differences between cells, tissues, and organs despite identical genetic information. genes function in a milieu determined by the developmental and environmental history of the cell, which constitutes the epigenotype. , epigenetic changes or marks can play a major role in human disease. , the most common examples of epigenetic marks are dna methylation of cpg islands by dna methyltransferases and chromatin modification of histone proteins, particularly acetylation by histone acetyltransferases and histone deacetylases. , the function of epigenetic changes is to regulate gene expression. epigenetic changes are known to contribute to cancer and autoimmune disease and are thought to contribute to common diseases, including cardiovascular disease, diabetes, and the loss of response to stress caused by aging. asthma is a markedly heterogeneous disease, and recent evidence suggests that environmentally induced epigenetic changes contribute to asthma phenotypes and that airway inflammation in patients with asthma and copd might involve epigenetic regulation. , , methylation patterns and chromatin structure change with age and are thought to contribute to the increase in the incidence of common diseases that begin in middle age. , the incidence of asthma in the elderly resembles the incidence of common diseases. moreover, characteristics and asthma drug response in the elderly asthmatic patient differ from those seen in childhood asthma. compared with younger cohorts, elderly asthmatic patients have a higher prevalence, higher rates of bronchial hyperreactivity, more severe asthma, and a lower prevalence of atopy. the symptoms of elderly asthmatic patients are more difficult to control with drug therapy, and these patients have steroid resistance and might respond better to leukotriene receptor antagonists compared with inhaled corticosteroids. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the contribution of epigenetics to differences observed between elderly asthmatic patients and younger cohorts is unknown. unlike genetic variants that contribute to disease, epigenetic changes can be reversed and therefore represent potential drug targets. older asthmatic patients are less responsive to albuterol treatments given in the emergency department and are more frequently admitted for hospitalization. thus it appears that the responsiveness to treatment is diminished and the severity of asthma exacerbations is greater. the exact reason for these disparities is not known. immune cell function decreases with aging, a property often termed immunosenescence. one often-confusing aspect of immunosenescence is the observation that aging might be associated with opposing immunologic effects. for example, t-cell secretion of il- , il- , or ifn-g has been shown to be both decreased with aging and also increased with aging. it is likely that both phenomena are correct but are dependent on the context of the immune function. thus the effect of aging on t-cell function in the context of allergen stimulation might be different than the effects of aging on t-cell function in the context of viral infection. given that asthma is an inflammatory disorder of the airway, it is of interest to determine whether asthmatic airway inflammation of the elderly might differ from that of younger asthmatic patients and thus represent a distinct phenotype of asthma. these changes might have implications for susceptibility to exacerbations because of viruses or other pathogens, as well as response to treatment. the aging process has been shown to exhibit changes in airway inflammation. an examination of the cellular composition of bronchoalveolar lavage fluid from -to -year-old subjects without a history of allergies, pulmonary disease, or gastroesophageal reflux showed increased airway neutrophilia, as well as increased numbers of cd t cells. , the t cells also appeared to be more activated in the elderly, with increased expression of hla-dr and cd . the increase in airway neutrophils with aging has also been observed in asthmatic patients. because there is a phenotype of severe asthma characterized by a predominantly neutrophilic airway inflammation, the question arises as to whether the increased presence of neutrophils contributes to greater asthma severity in the elderly. some of the prominent inflammatory cells recruited into the airway in asthmatic patients are eosinophils, neutrophils, and t cells, which are capable of secreting numerous inflammatory mediators, including leukotrienes and cytokines. it is not known whether immunosenescence affects the production of these mediators in elderly asthmatic patients, either at baseline or during an exacerbation of symptoms. furthermore, it is not known whether age-related changes in their production would have any implication for the clinical presentation or management of asthma in the elderly. peripheral blood eosinophils were isolated from younger ( - years old) and older ( - years old) subjects for in vitro functional assays. the eosinophil effector functions of degranulation and superoxide production were diminished in the older compared with the younger asthmatic patients. in another study examining the expression of neutrophil mediators in younger and older asthmatic patients, there was decreased baseline expression of leukotriene b in the sputum of older asthmatic patients despite greater numbers of neutrophils. whether these findings have implications during an asthma exacerbation has yet to be determined. nevertheless, the results demonstrate agerelated changes in the function of an inflammatory cell considered pathognomonic for allergic asthma and raise the question of whether additional effects of immunosenescence are relevant to airway inflammation in asthmatic patients. there have been several studies of animal models to address age-related changes in the airway inflammation induced by allergen challenge of sensitized aged animals (see experimental approaches section below). these studies yielded conflicting results, and there is concern that the animal models do not accurately represent the chronic features of human asthma with seasonal allergen exposure and intermittent exacerbations. the aged animals were both sensitized and challenged at old age, which is in contrast to the typical elderly human asthmatic patient who might be exposed to allergens for several decades. typically, nasal and ocular symptoms on exposure to allergens diminish with age. allergen-triggered asthma symptoms also diminish with age. the epidemiology and natural history of asthma (tenor) study examined the natural history of asthma in older (> years old) compared with younger patients and found that older asthmatic patients had lower total ige levels, fewer positive skin prick test responses, and less concomitant allergic rhinitis or atopic dermatitis. several studies have demonstrated age-related decreases in total ige and allergen-specific ige levels, [ ] [ ] [ ] [ ] [ ] [ ] suggesting that this might be the explanation for the decrease in allergy symptoms. there is also evidence for an age-related decrease in skin prick test responses to allergens. however, the relationship between total ige levels and allergic disease persists in the elderly, such that subjects with greater ige levels remain more likely to have allergic rhinitis or asthma. , given the changes in allergic inflammation with aging, one might conclude that asthma in the elderly should be milder. however, there are several other common triggers for exacerbations of asthma, including irritants (eg, cold air) and respiratory tract infections. estimates suggest that up to % of asthma exacerbations in adults are caused by viral upper respiratory tract infections. the role of environmental exposures and allergy in older asthmatic patients is largely unknown. in the general population, evidence regarding the effect of indoor pollution on asthma is summarized in ''clearing the air: asthma and indoor air exposure'' by the institute of medicine for the environmental protection agency published in . evidence was reported for asthma development related to house dust mites and for asthma development associated with environmental tobacco smoke in preschool children. the report also showed evidence for causation of asthma exacerbations for house dust mite, environmental tobacco smoke (in preschool children), cat, and cockroach; an association with exacerbations was found for dogs, fungi, formaldehyde, and rhinovirus. in addition, evidence associating exacerbation of asthma-related symptoms with self-reported damp air was reported in a review of damp indoor spaces and health. , there are only a few studies that have evaluated the role of atopy in elderly patients with asthma. one large national study of allergy skin tests that included older adults and several small studies of allergy skin tests in older adults with asthma , - j allergy clin immunol volume , number were reviewed. allergy skin test results were positive in % to % of all older adults. the prevalence of positive skin test results or specific ige levels to at least allergen in older adults with asthma ranged from % to %. those whose asthma had an unknown age of onset ranged from % to %, those with onset before age years ranged from % to %, and those with onset greater than age years ranged from % to % (table iii) . , , although there were no studies of allergen room exposure or bronchial challenge in older adults, neither prick-puncture skin test results nor specific ige levels predicted the nasal challenge response to dust mites. safety concerns for allergen challenges in older adults are unresolved. technical limitations of allergens, environmental measurements, and age-specific norms and cutoff levels for laboratory and physiologic tests are needed. a few epidemiologic studies suggest an association between outdoor environmental exposures and emergency department or hospital admissions in older adults. , in summary, studies of the general population suggest a causation or association between indoor air pollutants and allergy exposure and asthma. there are several small studies suggesting higher levels of positive allergy test results in older adults with asthma than in the general population of older adults. when age of onset is considered, asthma with an early onset (< years of age) has a much higher association with positive allergy test results than late-onset asthma. viral respiratory tract infections are common precipitants of asthma exacerbations during childhood. in approximately % of children with acute asthma exacerbations, a respiratory tract virus can be detected, with rhinovirus being the most frequent pathogen identified. although it is likely that viruses also lead to exacerbations of asthma in older adults, comprehensive studies regarding the rates and specific pathogens are lacking. several issues have made defining the role of viruses in adult asthmatic patients problematic and include difficulty distinguishing copd from asthma, lack of sensitive diagnostic tests, and issues with asymptomatic infection. a number of investigators have explored the incidence of viral infection in adult asthmatic patients. , , older studies using viral culture and serology for diagnosis demonstrated infection rates of % to %. in contrast, more recent studies, which include rt-pcr, have shown significantly higher infection rates of % to %. , similar to results found in children, rhinoviruses are the most frequently detected pathogen. few older persons were included in these studies, in which the mean ages of subjects were to years. the incidence of acute respiratory tract infections decreases steadily with advancing age, and rates of viral infection in older adults are influenced by place of residence. among community-dwelling older adults, rates of acute respiratory tract infections are roughly % to % per year, whereas rates in senior day care centers and long-term care facilities are substantially higher at % to %. in addition, the epidemiology of respiratory tract infections can be quite complex in these semiclosed populations, with multiple pathogens circulating simultaneously. influenza a, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) are the most commonly identified viruses among older persons hospitalized with acute cardiopulmonary conditions. , most patients who require hospitalization during a viral respiratory tract infection have underlying heart and lung conditions. although studies to date have largely focused on the role of viruses in copd exacerbations, it is reasonable to extrapolate infection rates and specific pathogens from these studies to older adults with asthma. johnston, in ontario, canada, found a seasonal peak in emergency department visits for all acute respiratory tract infections, as well as exacerbations of both copd and asthma, for persons younger and older than years. all the common respiratory tract viruses have been associated with copd exacerbations, and depending on the methodology and season of study, the specific rates of influenza, rsv, parainfluenza viruses, coronaviruses, hmpv, and rhinoviruses vary. , wheezing appears to be a common symptom in older adults infected with any of the respiratory tract viruses, particularly rsv and hmpv, and % of adults hospitalized with rsv will have a discharge diagnosis of asthma. , because most adult infections represent reinfection, the viral load in respiratory secretions tends to be low, making detection with conventional techniques difficult. viral culture and rapid antigen testing, which can be used successfully in children, have poor sensitivity in older adults. the use of molecular diagnostics has vastly improved the ability to detect a number of viruses, such as rsv, parainfluenza, and rhinoviruses, and allows the detection of hitherto uncultivable agents, such as hmpv and coronaviruses. viral infections appear to aggravate reactive airway disease through a number of different mechanisms. it has been postulated that viral infection disrupts the negative feedback loop of acetylcholine on the m receptor, leading to increased levels of acetylcholine and increased constriction of bronchiolar smooth muscle. infection of the respiratory epithelium also induces chemokines, cytokines, and immune and growth factors, which result in a proinflammatory state. , immunosenescence might affect the ability of older adults to clear viruses efficiently, and thus greater and more prolonged inflammation can result. in summary, respiratory viral tract infections are common among older adults and are likely precipitants of acute asthma exacerbations. furthermore, viral respiratory tract infections might likely precipitate the onset of loa, although this needs to be further examined. comprehensive studies regarding the rates and specific pathogens are lacking in older adults. distinguishing copd from asthma, lack of sensitive diagnostic tests, and issues with asymptomatic infections make it difficult to define the role of infections in older adults. classic symptoms of asthma in the elderly are mostly similar to those seen in younger asthmatic patients. , data on the clinical features of asthma in the elderly have been derived from both longitudinal community surveys and case studies. , , , , [ ] [ ] [ ] most patients complain of episodic wheezing, shortness of breath, and chest tightness. these symptoms are often worse at night and with exertion and, like those in younger asthmatic patients, are often precipitated by an upper respiratory tract infection. in fact, the majority of elderly patients who have asthma after age years have their first asthmatic symptom preceded immediately by or concomitant with an upper respiratory tract infection. asthma can often be triggered by environmental exposures, such as aeroallergens, irritants (cigarette smoke, household aerosols, and paints), strong odors (perfumes), and inhalation of metabisulfites (found in beer, wine, and food preservatives). asthmatic symptoms can also be triggered by medications, such as aspirin, nonsteroidal anti-inflammatory agents, angiotensin-converting enzyme inhibitors, or b-blockers, which are commonly used by this patient population. this emphasizes the need for the physician to perform a comprehensive review of medications taken by the older asthmatic patient. studies have consistently shown that elderly patients and their physicians frequently overlook symptoms caused by asthma. , , several factors contribute to the underdiagnosis and misdiagnosis of asthma. one reason, as shown in large community studies, is that most patients first have asthma in childhood or adolescence, and many physicians have had the misconception that asthma is a childhood disease. another important reason is that the symptoms of asthma are more commonly associated with other diseases seen in this age group. the symptoms of asthma in the elderly are therefore nonspecific and might be caused by conditions that mimic asthma. the differential diagnosis of asthma in the elderly is greater than seen in younger asthmatic patients and includes congestive heart failure, emphysema and chronic bronchitis (copd), chronic aspiration, gastroesophageal reflux disease (gerd), and tracheobronchial tumors. comorbid illnesses and the psychosocial effects of aging might also profoundly affect the diagnosis, clinical presentation, and care of asthma in the elderly. one particular diagnosis that is often difficult to detect and frequently overlooked by the patient and physician until the condition is advanced is upper airway obstruction, including the extrathoracic and intrathoracic central airways. common causes of upper airway obstruction include malignancy, infection, inflammatory disorders, trauma, and extrinsic compression related to enlargement of adjacent structures (eg, an enlarged thyroid gland). it appears that malignancy and benign strictures related to airway instrumentation (eg, endotracheal intubation and tracheostomy) are becoming increasingly more prevalent in the older age group. distinguishing chronic asthma from copd can be very challenging, and in some patients asthma cannot be distinguished from copd with widely available diagnostic tests. the management of these patients might have similarities to that of asthma. the distinction between loa and copd can be difficult to define precisely. the lung health study showed that methacholine-induced airways reactivity is present in many patients with mild-to-moderate copd (ie, % of men and % of women). approximately % of patients with tobacco-related copd demonstrate bronchodilator reversibility at least once on repeated testing sessions. the distinction between copd and asthma can be confounded by either the coexistence of the common disease entities, the progression of common pathobiologic mechanisms induced by different environmental agents, or different disease mechanisms leading to an overlapping clinical syndrome. it has been known for more than a century that early-morning wheezing is a prominent symptom of congestive heart failure. it has been called cardiac asthma because it can mimic the clinical picture of typical asthma. the usual symptoms of gastroesophageal reflux in the elderly, such as vomiting and heartburn, might be absent. in a study of elderly patients with esophageal reflux proved by means of intraesophageal ph monitoring, chronic cough, hoarseness, and wheezing were present in % of patients. in addition to causing asthma-like symptoms, there is also evidence that gerd might be a cause of worsening asthma. shortness of breath is a common symptom in the elderly and is most commonly caused by heart or lung diseases. it is usually experienced during exertion. shortness of breath at rest is not typical of heart disease or lung diseases, such as copd or interstitial lung disease, except in advanced stages. when present, it should prompt an investigation for asthma because sudden bronchospasm can cause respiratory distress at rest or exercise. paroxysmal nocturnal dyspnea, which is typical of congestive heart failure, is found in a smaller number of elderly patients with asthma. many elderly patients limit their activity to avoid experiencing dyspnea, and others assume that their dyspnea results from their aging process and thus avoid seeking medical attention early in their disease process. however, aging per se does not cause dyspnea, and a cause needs to be always pursued in assessing an elderly patient who complains of breathlessness. there are several other reasons why the diagnosis of asthma in the elderly might be delayed or not made at all. elderly patients have been shown to have a reduced perception of bronchoconstriction, and this might delay medical intervention. many elderly patients are fearful of having an illness and dying and are reluctant to admit they are having symptoms. underreporting of symptoms in the elderly might have many causes, including depression, cognitive impairment, social isolation, denial, and confusing symptoms with those of other comorbid illnesses. cough is a very prominent symptom and might occasionally be the only presenting symptom. wheezing, on the other hand, might not be as prominent, and its presence is not very specific and does not correlate with severity of obstruction. physical examination in elderly patients with asthma is usually nonspecific and might misguide the diagnosis: a negative examination result does not rule out asthma, and wheezing can be found in a number of conditions, such as copd, recurrent aspiration, and ''cardiac asthma'' (congestive heart failure). two distinct clinical presentations have been described for asthma in the elderly. these are based on the onset and duration of the disease state. , , patients with loa start having asthma symptoms for the first time when they are years of age or older (some studies have suggested middle age or older). some studies of elderly asthmatic patients have shown that, as a group, as many as % will have their first attack after the age of years. patients belonging to this group tend to have fewer atopic manifestations, higher baseline fev , and a more pronounced bronchodilator response than those with lsa. patients with lsa start having asthma symptoms early in life. patients belonging to this group tend to have a higher incidence of atopic diseases, more severe and irreversible or partially reversible airway obstruction, and more hyperinflation. the duration of the disease in this group is an important determinant of severity and of the development of irreversible airflow obstruction. longitudinal studies of asthmatic populations, whether new onset or long standing, have shown that remission from asthma is uncommon in older groups, occurring in less than % of patients. this contrasts with asthma in children and adolescents, in whom remission of asthma symptoms is common, especially in the second decade of life, and might be seen in as many as % to % of patients. objective measures to confirm the diagnosis of asthma are uncommonly performed in primary care settings. inhalers are prescribed for patients who are evaluated for asthma-like symptoms, and during a follow-up visit, the patient is asked whether the controller inhaler reduced the frequency of asthma symptoms or whether the albuterol inhaler quickly relieved the symptoms. such an empiric approach might work most of the time for young patients with mild asthma but is more likely to result in an incorrect diagnosis, poorly efficacious treatment, or unnecessary medication side effects in older patients. the onset of wheezing, shortness of breath, and cough in an elderly patient is likely to cause concern. although the adage ''all that wheezes is not asthma'' is true at any age, it is especially true in the elderly. diagnosis based on objective measures is essential. moreover, lung function testing, even in the presence of minimal symptoms, is especially important in this age group because there is thought to be an age-related reduction in the perception of exertional dyspnea in the elderly. an older patient with chronic, untreated, severe airway obstruction caused by asthma might reduce activity to avoid dyspnea and stoically deny impairment of activity. this might reflect either neurocognitive function or changes in lifestyle that favor sedentary activities. there exist some barriers to lung function testing in the elderly. spirometry might be difficult to perform in some situations because of physical or cognitive impairments. however, % to % of elderly persons are able to perform goodquality spirometry when tested by skilled technologists. [ ] [ ] [ ] [ ] [ ] the global initiative for obstructive lung disease guidelines for diagnosing the airway obstruction of copd by using a fixed fev /fvc ratio of less than . caused a high misclassification rate in older persons. however, almost all computerized spirometers automatically calculate the appropriate lower limit of the normal range for fev /fvc ratio and for fev by using race-specific national health and nutrition examination survey iii reference equations. in addition, it is hard to define the lower limits of predicted normal values in this age group. although complete reversibility of airflow obstruction is frequently seen with young asthmatic patients, most elderly asthmatic patients show incomplete reversibility despite continuous intense therapy, and many show fixed airflow obstruction as if they have copd. however, objective measures of lung function, such as spirometric and peak flow measurements, are generally underused in elderly patients, and this also contributes to the delay or absence of diagnosis. , lung function testing is especially important in this age group because of the age-related reduction in the perception of dyspnea seen in the elderly. spirometry is easily performed to determine that fev and fev /fvc ratio are demonstrated with the timed vital capacity maneuver. the flow-volume loop, which also measures inspiratory flow, is especially useful when the cause of respiratory tract symptoms is not known and an upper airway obstruction is in the differential diagnosis. although it might be difficult to perform spirometry in the elderly in some situations because of physical and poor cognitive impairment, studies have demonstrated that between % and % of elderly patients are able to perform the test properly. [ ] [ ] [ ] [ ] [ ] on the other hand, it might be more difficult to define the lower limits of predicted normal values in this age group. traditionally, an fev /fvc ratio of less than % increases the probability of asthma in an elderly patient with asthma symptoms, but this ratio normally decreases with age because of a decrease in elastic recoil, and a ratio lower that % might be a normal finding. a brisk response to a short-acting bronchodilator might demonstrate the second cardinal feature of asthma: reversible airflow obstruction (ie, ''a responder''). when airflow obstruction is found in an elderly patient, attempts should be made to demonstrate reversibility after the inhalation of a short-acting b-adrenergic agent, such as albuterol. evidence of reversibility (postbronchodilator fev or fvc increases of > % and ml) increases the probability of a diagnosis of asthma. elderly asthmatic patients, however, might have an impaired b-agonist bronchodilator response because the number of b-adrenergic receptors on smooth airway muscles is decreased with aging. although the bronchodilator response to inhaled b-agonists decreases with age, this is not the case with anticholinergic agents. airway obstruction might be absent at the time of testing, and further testing might be needed to facilitate the diagnosis. bronchoprovocation testing with a methacholine challenge can be useful, and it is a safe and effective method to uncover asthma in older adults. , a negative test result will rule out asthma; a positive test result must be interpreted and include an assessment of pretest probability. in addition, some studies have shown that bronchial responsiveness is heightened in older adults, and therefore aging might be an independent factor that influences airway responsiveness. there is a relationship between the degree of bronchial hyperresponsiveness and prechallenge pulmonary function; a low fev predicts heightened responsiveness. other factors that might contribute to heightened airway responsiveness in the older population are atopy and current or previous smoking history. peak expiratory flow variability might be helpful in the diagnosis and follow-up of younger patients with asthma, but poor coordination and muscle weakness in some elderly patients might lead to an inaccurate reading. , a prospective study did not demonstrate any advantage of peak flow monitoring over symptom monitoring as an asthma management strategy for older adults with moderate-to-severe asthma when used in a comprehensive asthma management program. other tests, such as measuring the carbon monoxide diffusing capacity of the lung, have been advocated to distinguish between asthma and copd because the diffusing capacity of the lung is reduced by parenchymal destruction found with emphysema. however, studies have shown that differences in lung function tests, although statistically significant, cannot be used clinically to separate the groups of subjects because of a large overlap. there is growing evidence that the airway function of young and middle-aged asthmatic patients decreases at a greater rate than that of healthy subjects. [ ] [ ] [ ] the rate of decrease increases with increasing age and in those who smoke cigarettes. , in patients with loa, there is evidence that lung function is reduced even before a diagnosis is made and decreases rapidly shortly after diagnosis. , thereafter, it remains fairly stable. although the effect on older asthmatic patients with lsa is variable, in a random survey of elderly asthmatic patients older than years, only in patients had normal pulmonary function (fev > % of predicted value), whereas a similar number showed moderate-to-severe airflow obstruction (fev < % of predicted value) after an inhaled short-acting bronchodilator. because structural changes of emphysema are minimal in elderly asthmatic patients, except if they are previous smokers, airway remodeling is thought to be the main cause of fixed airflow obstruction. nitric oxide (no) is a gas generated by the action of no synthase from the substrates molecular oxygen and arginine. it was originally identified as a biologically important signaling molecule with the properties of an activity previously described as endothelial-derived relaxing factor. this molecule is important in regulating vascular integrity and blood flow and is thought to be a regulator of vascular smooth muscle relaxation. more recently, it has been found that no can be generated by a variety of inflammatory cells, including polymorphonuclear leukocytes, mononuclear cells, and, importantly, eosinophils. this finding led to the identification of no as a molecule present in exhaled breath. studies of no exhalation have found that it is increased in infection and inflammation of the airway. although high levels of no are found in nasally expired air, studies in pulmonary inflammation have avoided this by redirecting airflow through the oral airway. it has been found that exhaled no reflects airways inflammation and particularly eosinophilic inflammation. exhaled no levels are increased during the allergy season in atopic subjects. inhaled glucocorticoids promptly suppress exhaled no and do so in conjunction with suppression of eosinophilic inflammatory infiltrates. studies have demonstrated that monitoring exhaled no might permit better regulation of asthmatic symptoms, exacerbations, and total steroid use than treatments based on guidelines or symptoms. furthermore, increases in exhaled no levels might predict asthma exacerbations. it is of interest that no levels in expired air decrease after bronchoconstrictive stimulation of asthmatic airways. little is known of the effects of age on no levels in the expired air. it appears that no production and vascular responses to no might be diminished in the elderly, but that effect might be overcome by exercise to increase fitness. an unanswered question in airways biology is whether no is causative of airways dysfunction, a marker for this dysfunction, or an ineffective homeostatic response to airways constriction. [ ] [ ] [ ] [ ] [ ] challenges in defining asthma in the elderly there is agreement that asthma is both a common and underrecognized health problem for the elderly that leads to impairments of lung function and quality of health and life. the first question that needs to be addressed is why we need to make such a diagnosis rather than just treat the symptoms. there are reasons that physicians must strive to assign a diagnosis to a patient with a symptom complex. the patient is given relief by letting him or her know what is wrong by giving the illness a name, which implies a cause, establishes a prognosis, and initiates a treatment plan. moreover, advancement of the understanding of epidemiology, natural history, pathobiology, and treatment require a definable disease entity. whether the threshold for diagnostic criteria is set at a high level of sensitivity, a high level of specificity, or a high level of accuracy depends entirely on the costs and benefits of an incorrect diagnosis versus a missed diagnosis. for example, enumeration of a disease might require a high level of accuracy, whereas diagnosis of an uncommon and difficult-to-treat disease (eg, metastatic cancer) ought to be highly specific. the diagnosis of a common and easily treatable disease (eg, vitamin deficiency) ought to be highly sensitive, even if there is a risk of overdiagnosis. asthma tends to be one of those disorders that is relatively easy (although not inexpensive) to treat and has morbid consequences if left untreated, suggesting that the diagnostic criteria ought to be highly sensitive. although medical students are taught the rigorous discipline of data collection, differential diagnosis, and test confirmation, most physicians do not practice this way. in practice, physicians typically rely on a constellation of signs and symptoms along with demographic characteristics and recent experiences to establish diagnoses through the process of pattern recognition. there are no shortages of official definitions of asthma, and modifications seem to be added every year. most of these definitions involve the definition of a clinical syndrome (episodic cough, wheezing, and dyspnea), an underlying pathophysiology (airway hyperresponsiveness, variable, and reversible airflow obstruction), an underlying biological process (chronic eosinophilic or neutrophilic inflammation of the airways), and an associated morbid anatomy (basement membrane thickening, smooth muscle hypertrophy, and mucus cell metaplasia). given this, why is it so challenging to diagnose asthma in the elderly? first, the syndrome of asthma is often confused with other common diseases in the elderly, such as copd, congestive heart failure, paroxysmal arrhythmias, pulmonary emboli, recurrent aspiration, and gerd. second, asthma can often coexist with these other conditions, and it can be impossible to determine which of the conditions is responsible for the patient's ill health. this diagnostic confusion can be amplified by the different manifestations of asthma in the elderly. elderly asthmatic patients can be insensitive to exertional dyspnea because of a sedentary lifestyle. they tend to be less atopic and have an incomplete response to bronchodilators. the elderly without asthma tend to show some signs suggestive of asthma: slower emptying of the lung during forced expiration, decreased lung elastic recoil, and a higher prevalence of nonspecific airways reactivity. the hope that formal testing of airways reactivity would prove useful in diagnosing asthma has led to disappointment. in young adults a history of asthma, wheeze, or treatment for asthma plus a positive methacholine challenge test result is highly specific for asthma ( %) but misses about half of the asthmatic population. in epidemiologic studies that have examined various criteria for diagnosing asthma, it turns out that the solution is relatively j allergy clin immunol volume , number simple. patients who answer yes to the question ''have you ever had asthma?'' have nearly % specificity and % to % sensitivity when compared with those receiving an independent expert's diagnosis. , the problem of diagnosing asthma in the elderly is more complicated because of the overlap with copd. asthma is typically considered a disease of onset in youth driven by atopy and eosinophilic inflammation causing reversible airflow limitation. copd, in contrast, is considered to be a disease of onset in middle age driven by cigarette smoking and neutrophilic inflammation and leading to irreversible airflow limitation. as evidence presented in this workshop has shown, asthma in the elderly displays many of the features of copd. the disease can have its symptomatic onset late in life, often is only partially reversible, and is associated with neutrophilic inflammation. moreover, the current cohort of elderly patients has a high prevalence of past smoking, reflecting the health habits in the united states in the s and s. the failure to deal with the population of elderly patients who have overlapping signs of asthma and copd is not just a matter of classification of disease. it has significant health consequences in that such patients are systematically eliminated from clinical trials and are not covered by treatment guidelines. little is known about how best to treat the elderly patient with asthma who smokes or the elderly patient with copd who has reversible airflow limitation. this confusion is manifest by diagnostic coding in older medicaid patients. of those who were hospitalized with an initial diagnosis of copd, % had an asthma diagnosis within years. of those who had an initial hospital diagnosis of asthma, % had a diagnosis of copd within years. price et al attempted to develop a discriminant function using clinical and demographic information that would separate patients with copd from those with asthma by using strict physiologic criteria. although several discriminating characteristics were found, the best diagnostic criteria were only % sensitive and only % specific. we need to ask whether it really is important to make the distinction between asthma and copd in the elderly in terms of prognosis or treatment. one study by hansen et al suggests that regardless of whether a person is given a diagnosis of asthma or copd, the prognosis is mostly determined by the impairment in fev . there are a number of ways to measure the effect of asthma in both young and elderly patients. assessments of symptoms, functional limitations, quality-of-life measures, and risk of adverse events are several that have been suggested by current asthma guidelines. in addition, measuring a patient's satisfaction with his or her asthma symptom control and overall asthma care has been advocated. the use of objective measures of asthma control and satisfaction can be especially important in the elderly because the perception of symptoms might be impaired with advancing age. in addition, many elderly patients unconsciously accommodate to their symptoms or assume that the symptoms are a function of the aging process itself. because the number of unscheduled ambulatory visits, emergency department visits, and hospitalizations are high in elderly asthmatic patients, , and quality-of-life scores are low in elderly patients with persistent asthma when compared with those with mild asthma or no asthma at all, careful assessment of asthma control is essential in this age group. despite severe symptoms and physiologic impairment, most elderly patients with asthma can lead active productive lives if their asthma is appropriately managed. in fact, when elderly patients with severe or difficult-to-treat asthma have been identified by a physician's assessment, they appear to do better than younger patients. in the tenor study, despite lower lung function, older asthmatic patients (mean age, years) had lower rates of unscheduled office visits, emergency department visits, and corticosteroid bursts. patients reported in the tenor study received more aggressive care than younger adults, including higher use of inhaled and oral corticosteroids, and this undoubtedly had an effect on outcomes. the tools to measure asthma outcomes include questionnaires and other self-report tools, such as diaries and standardized medical history forms. standardized questionnaires that assess asthma impairment include the asthma control test, , the asthma control questionnaire, , the asthma therapy assessment questionnaire, , and others. [ ] [ ] [ ] [ ] there are many tools available to clinicians to assess the quality of life of asthmatic patients. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] unfortunately, these psychometric instruments that claim to measure the same outcomes might produce disparate results, and none have been targeted for the elderly. in general, results that measure several domains are more accurate when a composite score is derived rather than when subscores of specific domains are compared. medical, administrative, and pharmacy records have also been used, especially to study larger asthmatic populations; these have proved useful for the assessment of a patient's change over time and to measure group differences. clinical trials of asthma therapy and educational, selfmanagement, and health services interventions have used psychometric instruments to assess elderly patients with asthma. in most of these studies, however, the majority of subjects are younger. there are no studies that have specifically determined the reliability and validity of these instruments in elderly persons. this is true of patient-satisfaction measures that have been used to assess asthma care. [ ] [ ] [ ] this is much needed because using lung function testing to measure outcomes has potential limitations in this age group. there are difficulties in defining normal predicted values at a very advanced age, and many patients with physical or cognitive impairment cannot reliably perform these tests. it is hopeful that newer biomarkers of lung inflammation have a particular role to play in the assessment of asthma control in the elderly. tools assessing physical function: self-reported and objective a major goal of geriatric and gerontologic research is to reduce the decrease in cognitive and physical function and prevent disability among older adults. accordingly, many functional status measures have been developed and used to understand the disabling process, as well as to evaluate interventions to prevent functional decline. it is useful to identify instruments that measure functional limitations and disability to investigate the functional consequences of asthma in older adults and to understand the pathway from asthma to disability. functional limitations are restrictions in performing basic physical and mental actions at the whole-person level (eg, walking or climbing stairs), whereas disability refers to limitations or difficulty in performing socially defined roles or tasks of everyday living in a given environmental context (eg, grocery shopping or bathing). , both self-reported and objective measures can be used to measure these different stages of disablement. self-reported measures can provide an indication of how well a patient is functioning in daily life and provide an assessment of care needs. these measures incorporate self-perception of function and can assess adaptations made to compensate for decrements in function. for disability assessment, self-reported difficulty or inability to perform basic activities of daily living (adl) is commonly used. for example, a composite score of adl items has been used as an outcome to evaluate the efficacy of a program to prevent functional decline in frail older adults. other composite scores assessing difficulty in ambulation, stair climbing, transferring, upper extremity function, and basic and instrumental adls have been developed. these comprehensive instruments of function and disability are amenable to computer adaptive testing. several objective measures of physical performance are used in studies of older adults and in disease-specific patient populations. tests of physical performance eliminate subjective attitudinal differences in the patient's reporting of physical function limitations. they have the advantage of providing an objective measure for comparisons across populations. these tests are sensitive to change over time and can detect decrements in function that might not be observed with self-reported instruments. many studies in older adults have used physical performance tests as predictors of adverse health events, as well as outcomes. for example, the short physical performance battery, which consists of timed balance, walking, and chair-rise tasks, is a powerful predictor of disability, nursing home admission, and mortality. , the short physical performance battery was also used as a screening instrument to identify functionally limited older adults and as an outcome in a randomized controlled trial of exercise. increasingly, objective measures of physical function are used to summarize the effect of total disease burden, including subclinical conditions and impairments, and to identify physiologic reserve that might help some older adults cope with disease burden. clinically meaningful differences have been established for commonly used performance measures. the goals of asthma therapy in elderly patients are not different from those for younger asthmatic patients. they are to treat acute symptoms, prevent chronic symptoms, decrease emergency department visits and hospitalizations, preserve normal activity level, and optimize pulmonary function with a minimal adverse effect from medications. , , optimal management should also focus on improving health status (quality of life) in these patients, which is often complicated by depressive symptoms and side effects from the drugs commonly used for asthma. unlike many younger adults who might require no medication or just asneeded b-agonist therapy for occasional symptoms, most older asthmatic patients need continuous treatment programs to control their disease. at a time when memory loss is common and financial resources are often limited, many older patients require complicated and frequent dosing with multiple expensive drugs. unfortunately, this has led to a significant rate of noncompliance among the elderly population in general. sex, socioeconomic factors, educational level, marital status, and severity of disease do not seem to be good predictors of compliance in elderly asthmatic patients. in summary, there are many challenges in the treatment of asthma in the elderly, which include a greater propensity to experience adverse events from medication use, as well as potential drug interactions with medications used for the treatment of comorbidities, , and thus it is particularly important to treat any disease in the elderly, including asthma, with a minimum of therapy while attaining maximum efficacy. a thorough understanding is required regarding which medications will be most effective in the treatment of asthma in the elderly to achieve this balance. because many current therapeutic options and those in development for asthma focus on specific inflammatory cells and mediators, any age-related changes in the airway inflammatory milieu will likely affect their therapeutic efficacy. therefore a rigorous characterization of age-related changes in airway inflammation will facilitate the management of asthma in the elderly. the therapeutic approach to asthma in elderly patients does not differ from what is recommended for young patients. statements on the standard of care for treating asthma have been published by the national institutes of health and are widely used as guidelines. , treatment protocols use step-care pharmacologic therapy based on the intensity of asthma symptoms and the clinical response to these interventions. as symptoms and lung function worsen, step-up or add-on therapy is given. as symptoms improve, therapy can be stepped down. in this age group special attention should also be given to the potential adverse effects of commonly used medications. corticosteroids are capable of reducing airway inflammation, thereby improving lung function, decreasing bronchial hyperreactivity, reducing symptoms, and improving overall quality of life. oral corticosteroids should be avoided if possible because they place the patient at risk for bone fracture and increased likelihood of cataracts, muscle weakness, back pain, bruising, and oral candidiasis. many studies have shown that inhaled corticosteroids are safe and effective treatment for persistent asthma, but none have specifically targeted the elderly population. long-term use of inhaled corticosteroids has been associated with a good safety profile, but higher doses of inhaled steroids (eg, > mg/d) are capable of causing hypothalamicpituitary-adrenal axis suppression. local adverse effects, such as hoarseness, dysphonia, cough, and oral candidiasis, do occur but can usually be avoided by the use of a spacer or holding chamber with the metered-dose inhaler and by rinsing the mouth after each use. despite the pivotal role of inhaled corticosteroids in asthma, many elderly patients are undertreated with this group of medications. , leukotriene-modifying agents (ltms) are also asthma controllers. these agents have been shown to be effective in preventing allergen-induced asthma, exercise-induced asthma, and aspirin-induced bronchospasm. studies on their use in the elderly are limited. when compared with ltms, low-dose inhaled corticosteroids have favored the latter. the ltms might also reduce asthma exacerbation rates and the need for steroid bursts. the ltms are generally very safe. , b-adrenergic agents are important medications in the acute and chronic management of asthma. elderly patients with asthma j allergy clin immunol volume , number might be less responsive to certain bronchodilators compared with younger patients. , inhaled short-acting b -adrenergic agonists are the treatment of choice for the acute exacerbation of asthma symptoms. despite the minimal systemic absorption seen with these agents, slight tachycardia might be observed. this is presumably because of vasodilatation, which results from the stimulation of b -receptors in vascular smooth muscle. tremor can also occur and is especially troublesome in the geriatric patient. tremor is thought to be caused by stimulation of b -receptors in skeletal muscle. in general, they have been proven to be safe and effective in all age groups. however, b-agonists can cause ( ) a dose-dependent decrease in serum potassium levels and ( ) a dose-dependent increase in the qt interval on electrocardiography. because sudden death from ventricular arrhythmia can be caused by both of these mechanisms, as well as being a complication of ischemic heart disease, the use of b-agonists in the elderly should be closely monitored. short-acting b -agonists should be used for rescue of symptoms, whereas long-acting agents should be used as maintenance medications only as an add-on to inhaled corticosteroids and never as stand-alone therapy. anticholinergics, such as inhaled ipratropium, a short-acting bronchodilator, and tiotropium, a bronchodilator with -hour action, have an excellent safety profile in the elderly. they should be considered when additional bronchodilator therapy is necessary; however, their role in long-term maintenance of asthma in the elderly has not been established. theophylline is an effective bronchodilator and has some antiinflammatory properties. however, its use has been greatly reduced over the past decade because of safety concerns, especially in the elderly. the narrow therapeutic range of theophylline, the frequency of concomitant illnesses that alter theophylline kinetics, and many drug interactions that affect the clearance of theophylline make it essential to closely monitor blood theophylline levels in older asthmatic patients. theophylline toxicity can cause seizures and cardiac arrhythmias, such as atrial fibrillation, supraventricular tachycardia, ventricular ectopy, and ventricular tachycardia. the most common cause for theophylline toxicity is a self-administered increase in medication. controlling triggers. measures should be taken to avoid triggers that can cause worsening of symptoms. as with asthma at any age, education concerning avoidance of aggravating factors that can lead to severe bronchospasm is very useful. although aeroallergens are less important in provoking symptoms in the elderly than in young patients, a program implementing environmental control measures, such as avoiding or minimizing aeroallergen exposure, should be instituted in patients with documented sensitivity to specific allergens. however, such programs might not be successful in all cases, especially because lifestyle changes in the elderly population might be difficult. the most important provocative factors include viral respiratory tract infections and irritants, such as cigarette smoke, paints, varnish, and household aerosols. pharmacologic agents that are often prescribed for concomitant illnesses (ischemic heart disease and hypertension), such as b-adrenoreceptor antagonists (b-blockers), can also provoke bronchospasm. this includes both noncardioselective agents (propranolol, pindolol, and timolol) and, to a lesser extent, cardioselective agents (metoprolol and acebutolol). topical b-blockers are also widely used in the elderly to reduce intraocular pressure in wide-angle glaucoma. with such treatment, sufficient systemic absorption might cause fatal status asthmaticus. the severity of b-blocker-induced bronchoconstriction correlates with the severity of underlying airflow obstruction and the degree of bronchial reactivity and might be reduced by the use of a cardioselective topical b-blocking agent, such as betaxolol. aspirin and nonsteroidal anti-inflammatory agents might precipitate acute bronchospasm in certain asthmatic patients, and angiotensin-converting enzyme inhibitors might cause dry cough in some, worsening the symptoms of asthma. gerd should also be considered a cause of worsening asthma symptoms. asthma education. the complexity of the prescription regimen (number and frequency of medications), coupled with the memory loss and cognitive dysfunction that might be present in this group of patients, contribute partially to poor compliance with therapy. , patient education is an effective tool and should be an integral part in the management of asthma. active participation by a patient and family members in monitoring lung function, avoidance of provocative agents, and decisions regarding medications provide asthma management skills that give that patient the confidence to control his or her own disease. mastering the technique of an inhaled medication delivery device is a challenging problem in elderly patients, and the great majority of elderly patients are unable to properly use the metered-dose inhaler, even after proper instruction. [ ] [ ] [ ] [ ] use of dry powder devices, although simpler, requires the generation of an adequate inspiratory flow that might be suboptimal in frail patients and those with severe airway obstruction. in such situations the use of spacer devices or nebulizers might be beneficial. patients should recognize the rationale behind using the different medications, the correct way to use them, and their side effects, and polypharmacy should also be avoided. asthma in the elderly can be effectively managed, and despite severe symptoms and physiologic impairment, most patients can lead active and productive lives. a demographic study of low-income elderly persons in chicago found that ( %) without a previous diagnosis of asthma or emphysema had symptoms compatible with those of obstructive lung disease. of patients with a previous diagnosis, only % were compliant with medications, and this was largely due to the cost of medications. in addition, health care use was high in this population. telephone intervention offers a simple option in the management of elderly patients with asthma. it has been shown that asthma care by means of telephone triage of adult asthmatic patients can lead to a higher percentage of asthmatic patients being reviewed at less cost per patient and without loss of asthma control when compared with usual routine care in the outpatient clinic. however, it has not been determined whether such an intervention could improve asthma care specifically in persons aged years or older. the following study was designed to evaluate this question. fifty-two elderly asthmatic patients who used their rescue inhalers more than twice a week and had at least emergency department or urgent care visit in the previous year were randomized to an intervention or control group. all patients received telephone calls over a -month period. the intervention group received an asthma-specific questionnaire, and the control group received a general health questionnaire. medication use and health care use were evaluated at the beginning and end of a -month period. the study was completed by control and intervention subjects. baseline data were similar in both groups. after months, % (n ) of the intervention group was taking an inhaled corticosteroid compared with % (n ) of the control group. the intervention group had fewer emergency department visits when compared with the control group. sixtyfour percent (n ) of the intervention group had an asthma action plan compared with % (n ) of the control group. this study provides evidence that using a simple telephone questionnaire can successfully improve asthma care in the elderly. by empowering the elderly with the appropriate knowledge regarding their asthma, an appropriate discussion about their asthma care can be initiated with their primary care physicians. pulmonary rehabilitation. although pulmonary rehabilitation is recommended as the standard of care for patients with copd, there are only a few studies that evaluate the benefit of rehabilitation for asthmatic patients, and none of these consider elderly asthmatic patients. one study looked at the effects of a -week outpatient rehabilitation program for asthmatic patients after years. [ ] [ ] [ ] they found that of subjects continued to exercise regularly all years; there was a decreased number of emergency department visits and a decrease in asthma symptoms. further studies are needed to assess empowerment strategies for elderly patients with asthma, as well as the potential benefits of pulmonary rehabilitation on morbidity and mortality. asthma pathogenesis is complex and incompletely understood. research into the pathophysiologic mechanisms is made more difficult by multiple factors, including the heterogeneity of the disease itself, variable presentations in different stages of life, and the lack of highly relevant animal models. [ ] [ ] [ ] [ ] [ ] in the last decade, increasing interest in asthma in the elderly has triggered more intensive investigation in both human and animal systems by using ever more sophisticated immunologic methodologies. early investigation with rats revealed a lack of total and allergenspecific ige in response to ovalbumin. this was born out by several later in vivo studies. [ ] [ ] [ ] igg subset analysis (igg vs igg ) provided further support for this phenomenon. igg , correlating in the mouse to a t h response (vs igg [t h ]) was shown to follow a similar pattern. , recent studies [ ] [ ] [ ] of cytokine profiles in aged rodents compared with young control animals enhanced the paradigm that age resulted in less robust t h cytokines, particularly il- , il- , and il- , in favor of t h gene and protein expression. , this pattern was not fully supported in a recent chronic murine asthma model wherein il- was greater in aged sensitized mice, making the picture more complex. ifn-g, a key t h cytokine, has been consistently overexpressed in aged versus young rodents. [ ] [ ] [ ] [ ] eosinophilia, which is considered a key component of (allergic) asthma, was more pronounced in younger versus older animals (bronchoalveolar lavage fluid, lung tissue, or both) after most, [ ] [ ] [ ] although not all, sensitization paradigms. molecular genetics and t-cell subset analysis has allowed further insight into possible mechanisms underlying the waning t h response observed in most models. [ ] [ ] [ ] specifically, elderly mice appear to have more memory t cells, less activated cd t h cells, and less activated monocytes. , resident goblet cells also appear to express upregulation of mucin and mucin gene expression. a key to the impaired t h response was recently found in the gata pathway. elderly mice do not phosphorylate components of the extracellular signal-regulated protein kinase/mitogen-activated protein kinase pathway, resulting in lack of downstream signaling with gata , with subsequent impairment of promoter regions for key t h cytokines, including il- . this could be an overarching explanation for many findings in the elderly asthmatic patient, including less ige (il- and il- are needed for opening switch regions for ige production); il- and il- are highly associated with airway hyperreactivity, and il- is associated with eosinophil activation, survival, and, to a lesser extent, trafficking. finally, airway hyperreactivity has been universally found to be greater in young versus aged animals. [ ] [ ] [ ] [ ] the mechanisms might be complex, including both an altered key cytokine milieu and alterations in muscle function at the muscarinic receptor level. [ ] [ ] [ ] clinical and translational research research into the pathogenesis of asthma in recent years has led to the discovery of a number of novel, potentially important targets for the development of new treatment options. much of this research has focused on t h lymphocyte-driven processes underlying allergic asthma and its characteristic eosinophilic airway inflammation. abundant information supporting this research has been derived from bronchial biopsy and bronchoalveolar lavage studies largely carried out in a young adult population. it is recognized, however, that the role of allergy and allergic triggers in asthma diminishes with age. , in addition, loa is often less reversible, more severe, and frequently occurs in response to a viral respiratory tract infection. a distinct asthma phenotype characterized by normal airway eosinophil numbers has been described. moreover, normal airway eosinophilia might also be associated with abnormal sputum neutrophilia. , recent studies have shown that neutrophilic asthma might be associated with activation of innate immune pathways in contrast to the adaptive immune response associated with t h -mediated allergic asthma. thus alternative immune pathways involving natural killer t or t h lymphocyte subtypes have been hypothesized as being potentially important in the pathogenesis of asthma, particularly in adult-onset asthma. , just as the discovery of t h -related pathways has led to important leads in drug discovery for allergic asthma, further clinical research into these alternative pathways should be carried out with the goal of identifying new and exciting targets for future drug discovery. this research should focus not only on the discovery of new molecular targets but also on the identification of noninvasive biomarkers that will help predict the success of any new therapy in an individual patient. asthma is an important disease in the older adult, affecting % of the population older than years, which is understudied and frequently underdiagnosed. there are data to suggest that asthma in older adults is phenotypically different from that in young patients, with a potential effect on the diagnosis, assessment, and management in this population. this workshop brought together many disciplines to further our current understanding, resolve gaps in knowledge, and explore future areas of research and education. table i lists specific areas in need of research and study. the coming acceleration of global population ageing the census bureau on prospects for us population growth in the twenty-first century. population and development review national surveillance for asthma-united states asthma in the elderly: current knowledge and future directions underdiagnosis and undertreatment of asthma in the elderly. cardiovascular health study research group increasing u.s. asthma mortality rates: who is really dying? quality of care for older adults with chronic obstructive pulmonary disease and asthma based on comparisons to practice guidelines and smoking status economic burden in direct costs of concomitant chronic obstructive pulmonary disease and asthma in a medicare advantage population report of the national institute on aging task force on comorbidity a painful interface between normal aging and disease epidemiology of aging correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species executing cell senescence a systematic look at an old problem overview of biological mechanism of aging molecular biology of aging replicative senescence: a critical review the limited in vitro lifetime of human diploid cell strains cell culture aging: insights for cell aging in vivo? the relationship between in vitro cellular aging and in vivo human age evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo a biomarker that identifies senescent human cells in culture and in aging skin in vivo frailty in older adults: evidence for a phenotype phenotype of frailty: characterization in the women's health and aging studies from bedside to bench: research agenda for frailty gait speed and survival in older adults age-associated increased interleukin- gene expression, late-life diseases, and frailty decreased cell proliferation and altered cytokine production in frail older adults determinants of longevity: genetic, environmental and medical factors united states life tables gains in life expectancy after elimination of major causes of death: revised estimates taking into account the effect of competing causes extension of life-span by overexpression of superoxide dismutase and catalase in drosophila melanogaster a genetic pathway conferring life extension and resistance to uv stress in caenorhabditis elegans divergent roles of ras and ras in yeast longevity can an improved environment cause maximum lifespan to decrease? comments on lifespan criteria and longitudinal gompertzian analysis longitudinal gompertzian analysis of stroke mortality in the u.s., - : declining stroke mortality is the natural consequence of competitive deterministic mortality dynamics nutritional interventions in aging and ageassociated diseases caloric restriction and aging physiological changes in respiratory function associated with ageing sex and age differences in pulmonary mechanics in normal nonsmoking subjects elasticity of human lungs in relation to age flow and age dependence of airway closure and dynamic compliance human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space inflamm-aging. an evolutionary perspective on immunosenescence patient factors and medication guideline adherence among older women with asthma refill adherence by the elderly for asthma/chronic obstructive pulmonary disease drugs dispensed over a -year period deaths: final data for asthma costs and utilization in a managed care organization asthma and its association with cardiovascular disease in the elderly. the cardiovascular health study research group the cardiovascular health study: design and rationale prevalence and correlates of respiratory symptoms and disease in the elderly. cardiovascular health study correlates of peak expiratory flow lability in elderly persons intra-individual change over time in dna methylation with familial clustering epigenetic reprogramming and imprinting in origins of disease aging by epigenetics-a consequence of chromatin damage? epigenetics at the epicenter of modern medicine the role of histone deacetylases in asthma and allergic diseases environmental epigenetics and asthma: current concepts and call for studies epigenetics, disease, and therapeutic interventions epigenetic regulation of airway inflammation epigenetic differences arise during the lifetime of monozygotic twins predictors of loss of lung function in the elderly: the cardiovascular health study overcoming gaps in the management of asthma in older patients: new insights allergic respiratory disease in the elderly the diagnosis and management of asthma is much tougher in older patients effect of age on response to zafirlukast in patients with asthma in the accolate clinical experience and pharmacoepidemiology trial (accept) is asthma in the elderly really different? diagnosis and severity of asthma in the elderly: results of a large survey in , asthmatics recruited by lung specialists asthma in the elderly asthma medications and their potential adverse effects in the elderly: recommendations for prescribing interleukin genetics, inflammatory mechanisms, and nutrigenetic opportunities to modulate diseases of aging trichostatin a attenuates airway inflammation in mouse asthma model prospective multicenter study of acute asthma in younger versus older adults presenting to the emergency department immunosenescence: emerging challenges for an ageing population pathways to a robust immune response in the elderly variation of bronchoalveolar lymphocyte phenotypes with age in the physiologically normal human lung neutrophils and low-grade inflammation in the seemingly normal aging human lung age-related changes in eosinophil function in human subjects asthma: defining of the persistent adult phenotypes characterization of leukotrienes in a pilot study of older asthma subjects asthma in older adults: observations from the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) study ige mediated hypersensitivity in ageing influence of ageing on ige-mediated reactions in allergic patients the association of age, gender and smoking with total ige and specific ige total and specific serum ige levels in adults: relationship to sex, age and environmental factors aging and serum immunoglobulin e levels, immediate skin tests, rast age-related serum immunoglobulin e levels in healthy subjects and in patients with allergic disease longitudinal changes in allergen skin test reactivity in a community population sample chronic respiratory symptoms and airway responsiveness to methacholine are associated with eosinophilia in older men: the normative aging study serum total ige and specific ige to dermatophagoides pteronyssinus, but not eosinophil cationic protein, are more likely to be elevated in elderly asthmatic patients respiratory viruses and exacerbations of asthma in adults clearing the air: asthma and indoor air exposures meta-analyses of the associations of respiratory health effects with dampness and mold in homes damp indoor spaces /media/files/report% files/ /damp-indoor-spaces-and-health/dampin door pagerforpdf.pdf. accessed is allergen skin test reactivity a predictor of mortality? findings from a national cohort asthma in older adults asthma in the elderly. a comparison between patients with recently acquired and long-standing disease characteristics of asthma among elderly adults in a sample of the general population asthma severity, atopic status, allergen exposure, and quality of life in elderly persons asthma in the elderly sensitization to cat allergen is associated with asthma in older men and predicts newonset airway hyperresponsiveness. the normative aging study the role of allergy and airway inflammation asthma in the elderly: cockroach sensitization and severity of airway obstruction in elderly nonsmokers prick puncture skin tests and serum specific ige as predictors of nasal challenge response to dermatophagoides pteronyssinus in older adults particulate air pollution and hospital admissions for cardiorespiratory diseases: are the elderly at greater risk? traffic and outdoor air pollution levels near residences and poorly controlled asthma in adults community study of role of viral infections in exacerbations of asthma in - year old children respiratory tract viral infections in inner-city asthmatic adults the incidence of respiratory tract infection in adults requiring hospitalization for asthma acute respiratory illness in an american community. the tecumseh study acute respiratory tract infection in daycare centers for older persons long-term care facilities: a cornucopia of viral pathogens respiratory syncytial virus infection in elderly and high-risk adults human metapneumovirus infections in adults: another piece of the puzzle the similarities and differences of epidemic cycles of chronic obstructive pulmonary disease and asthma exacerbations a community-based, time-matched, case-control study of respiratory viruses and exacerbations of copd viral infections in patients with chronic obstructive pulmonary disease respiratory syncytial virus infection in elderly adults virus-induced asthma attacks how viral infections cause exacerbation of airway diseases role of viral infections in asthma and chronic obstructive pulmonary disease incidence and outcomes of asthma in the elderly. a population-based study in rochester, minnesota the clinical outcome of asthma in the elderly: a -year follow-up study characteristics of asthma in the elderly asthma in the elderly: underperceived, underdiagnosed and undertreated; a community survey determinants of symptoms suggestive of gastroesophageal reflux disease in the elderly reduced subjective awareness of bronchoconstriction provoked by methacholine in elderly asthmatic and normal subjects as measured on a simple awareness scale duration of asthma and physiologic outcomes in elderly nonsmokers asthma in the elderly features of asthma in the elderly factors determining performance of bronchodilator reversibility tests in middle-aged and elderly quality of spirometric performance in older people study of respiratory function in the elderly with different nutritional and cognitive status and functional ability assessed by plethysmographic and spirometric parameters quality control of spirometry in the elderly. the sa.r.a. study. salute respiration nell'anziano respiratory health in the elderly aging on quality of spirometry reversible and irreversible airflow obstruction as predictor of overall mortality in asthma and chronic obstructive pulmonary disease differential changes of autonomic nervous system function with age in man impaired bronchodilator response to albuterol in healthy elderly men and women influence of age on response to ipratropium and salbutamol in asthma bronchoprovocation testing an assessment of methacholine inhalation tests in elderly asthmatics guidelines for methacholine and exercise challenge testing- . this official statement of the american thoracic society was adopted by the ats board of directors airway hyperresponsiveness in the elderly: prevalence and clinical implications normal range of methacholine responsiveness in relation to prechallenge pulmonary function. the normative aging study peak flow lability: association with asthma and spirometry in an older cohort a randomized clinical trial of peak flow versus symptom monitoring in older adults with asthma differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease are chronic wheezing and asthma-like attacks related to fev decline? the cracow study rate of decline of lung function in subjects with asthma decline of lung function in adults with bronchial asthma effects of domestic gas cooking and passive smoking on chronic respiratory symptoms and asthma in elderly women findings before diagnoses of asthma among the elderly in a longitudinal study of a general population sample the natural history of asthma in adults: the problem of irreversibility physical activity prevents age-related impairment in nitric oxide availability in elderly athletes effect of natural grass pollen exposure on exhaled nitric oxide in asthmatic children exhaled nitric oxide (no) is reduced shortly after bronchoconstriction to direct and indirect stimuli in asthma height, age, and atopy are associated with fraction of exhaled nitric oxide in a large adult general population sample use of exhaled nitric oxide measurements to guide treatment in chronic asthma exhaled nitric oxide: the effects of age, gender and body size defining asthma in epidemiological studies symptom-based questionnaire for identifying copd in smokers asthma and asthma-like symptoms in adults assessed by questionnaires. a literature review national heart, lung and blood institute national institute of health. national asthma education and prevention program: expert panel report -guidelines for the diagnosis and management of asthma. bethesda: national heart, lung, and blood institute asthma in older patients: factors associated with hospitalization asthma exacerbations in north american adults: who are the ''frequent fliers'' in the emergency department? development of the asthma control test: a survey for assessing asthma control validity of the asthma control test completed at home development and validation of a questionnaire to measure asthma control identifying 'well-controlled' and 'not well-controlled' asthma using the asthma control questionnaire association of asthma control with health care utilization and quality of life association of asthma control with health care utilization: a prospective evaluation a new tool for monitoring asthma outcomes: the itg asthma short form perceived control of asthma: development and validation of a questionnaire a -item brief measure for assessing perceived control of asthma in culturally diverse patients how should we quantify asthma control? a proposal reliability and validity of the asthma quality of life questionnaire-marks in a sample of adult asthmatic patients in the united states the marks asthma quality of life questionnaire: further validation and examination of responsiveness to change an evaluation of an asthma quality of life questionnaire as a measure of change in adults with asthma a scale for the measurement of quality of life in adults with asthma validation of a standardized version of the asthma quality of life questionnaire development and validation of the mini asthma quality of life questionnaire american translation, modification, and validation of the st. george's respiratory questionnaire a self-complete measure of health status for chronic airflow limitation. the st. george's respiratory questionnaire patient characteristics relevant to effective self-management: scales for assessing attitudes of adults toward asthma applicability of the asthma opinion survey in the spanish population: distribution and relationship with sociodemographic and clinical variables a new treatment satisfaction measure for asthmatics: a validation study an epidemiology of disability among adults in the united states. milbank mem fund q the disablement process assessing the building blocks of function: utilizing measures of functional limitation preclinical mobility disability predicts incident mobility disability in older women a program to prevent functional decline in physically frail, elderly persons who live at home a randomized trial comparing aerobic exercise and resistance exercise with a health education program in older adults with knee osteoarthritis. the fitness arthritis and seniors trial (fast) creating a computer adaptive test version of the late-life function and disability instrument variation in thresholds for reporting mobility disability between national population subgroups and studies a short physical performance battery assessing lower extremity function: association with self-reported disability and prediction of mortality and nursing home admission lower-extremity function in persons over the age of years as a predictor of subsequent disability effects of a physical activity intervention on measures of physical performance: results of the lifestyle interventions and independence for elders pilot (life-p) study meaningful change and responsiveness in common physical performance measures in older adults naepp working group: consideration for diagnosing and managing asthma in the elderly. bethesda: national institutes of health asthma: a six-part strategy for managing older patients drug treatment of asthma in the elderly adverse effects of oral corticosteroids in relation to dose in patients with lung disease underuse of inhaled steroid therapy in elderly patients with asthma loss of response to treatment with leukotriene receptor antagonists but not inhaled corticosteroids in patients over years of age are there any detrimental effects of the use of inhaled long-acting beta -agonists in the treatment of asthma? beta-adrenergic-blocking agents in bronchospastic diseases: a therapeutic dilemma respiratory arrest following first dose of timolol ophthalmic solution the effect of topical ophthalmic instillation of timolol and betaxolol on lung function in asthmatic subjects asthma in the elderly: the importance of patient education a comparison of breath-actuated and conventional metered-dose inhaler inhalation techniques in elderly subjects acquisition and short-term retention of inhaler techniques require intact executive function in elderly subjects what determines whether an elderly patient can use a metered dose inhaler correctly? asthma in the elderly. a diagnostic and management challenge prevalence of obstructive airways disease in the disadvantaged elderly of chicago targeted routine asthma care in general practice using telephone triage improving asthma care for the elderly: a randomized controlled trial using a simple telephone intervention asthmatic patients' views of a comprehensive asthma rehabilitation programme: a three-year follow-up a -year follow-up of asthmatic patients participating in a -week rehabilitation program with emphasis on physical training high-intensity physical training in adults with asthma. a -week rehabilitation program animal models of asthma usefulness and optimization of mouse models of allergic airway disease murine models of asthma modeling allergic asthma in mice: pitfalls and opportunities promise and pitfalls in animal-based asthma research: building a better mousetrap van der straeten m. the effect of age on ige production in rats decreased expression of th type cytokine mrna contributes to the lack of allergic bronchial inflammation in aged rats induction and maintenance of airway responsiveness to allergen challenge are determined at the age of initial sensitization impaired gata -dependent chromatin remodeling and th cell differentiation leading to attenuated allergic airway inflammation in aging mice effect of ageing on pulmonary inflammation, airway hyperresponsiveness and t and b cell responses in antigen-sensitized and -challenged mice failure of aged rats to accumulate eosinophils in allergic inflammation of the airway age differences in cholinergic airway responsiveness in relation with muscarinic receptor subtypes effect of ageing on nicotine-induced contraction of guinea-pig bronchial preparation effects of age on muscarinic agonist-induced contraction and ip accumulation in airway smooth muscle total serum ige is associated with asthma independently of specific ige levels. the spanish group of the european study of asthma analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin- innate immune activation in neutrophilic asthma and bronchiectasis persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease il- is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines fla co-chair: nicola a. hanania, md, ms section of pulmonary and critical care medicine asthma clinical research center baylor college of medicine houston, tex members sidney s. braman, md warren alpert medical school, brown university division of pulmonary, critical care, and sleep medicine rhode island hospital providence, ri carol saltoun we thank the nia for recognizing the need for this workshop, especially susan nayfield, md, who convened the workshop and provided tremendous support in moving this research field forward; evan hadley, md, the director of the division of geriatrics and clinical gerontology; and basil eldadah, md, who continued the work of dr nayfield and contributed valuable advice and encouragement to the authors in completing these proceedings. key: cord- -hj s ipp authors: agostoni, angelo; aygören-pürsün, emel; binkley, karen e.; blanch, alvaro; bork, konrad; bouillet, laurence; bucher, christoph; castaldo, anthony j; cicardi, marco; davis, alvin e; de carolis, caterina; drouet, christian; duponchel, christiane; farkas, henriette; fáy, kálmán; fekete, béla; fischer, bettina; fontana, luigi; füst, george; giacomelli, roberto; gröner, albrecht; erik hack, c.; harmat, george; jakenfelds, john; juers, mathias; kalmár, lajos; kaposi, pál n.; karádi, istván; kitzinger, arianna; kollár, tímea; kreuz, wolfhart; lakatos, peter; longhurst, hilary j.; lopez-trascasa, margarita; martinez-saguer, inmaculada; monnier, nicole; nagy, istván; németh, Éva; nielsen, erik waage; nuijens, jan h.; o'grady, caroline; pappalardo, emanuela; penna, vincenzo; perricone, carlo; perricone, roberto; rauch, ursula; roche, olga; rusicke, eva; späth, peter j; szendei, george; takács, edit; tordai, attila; truedsson, lennart; varga, lilian; visy, beáta; williams, kayla; zanichelli, andrea; zingale, lorenza title: hereditary and acquired angioedema: problems and progress: proceedings of the third c esterase inhibitor deficiency workshop and beyond date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: hj s ipp hereditary angioedema (hae), a rare but life-threatening condition, manifests as acute attacks of facial, laryngeal, genital, or peripheral swelling or abdominal pain secondary to intra-abdominal edema. resulting from mutations affecting c esterase inhibitor (c -inh), inhibitor of the first complement system component, attacks are not histamine-mediated and do not respond to antihistamines or corticosteroids. low awareness and resemblance to other disorders often delay diagnosis; despite availability of c -inh replacement in some countries, no approved, safe acute attack therapy exists in the united states. the biennial c esterase inhibitor deficiency workshops resulted from a european initiative for better knowledge and treatment of hae and related diseases. this supplement contains work presented at the third workshop and expanded content toward a definitive picture of angioedema in the absence of allergy. most notably, it includes cumulative genetic investigations; multinational laboratory diagnosis recommendations; current pathogenesis hypotheses; suggested prophylaxis and acute attack treatment, including home treatment; future treatment options; and analysis of patient subpopulations, including pediatric patients and patients whose angioedema worsened during pregnancy or hormone administration. causes and management of acquired angioedema and a new type of angioedema with normal c -inh are also discussed. collaborative patient and physician efforts, crucial in rare diseases, are emphasized. this supplement seeks to raise awareness and aid diagnosis of hae, optimize treatment for all patients, and provide a platform for further research in this rare, partially understood disorder. this supplement, like the c esterase inhibitor deficiency workshop and the many patient and physician remain little known in clinical practice and thus frequently misdiagnosed and inappropriately treated, often resulting in unnecessary suffering. similarities to allergic conditions and inappropriate framing as part of the urticariaangioedema syndrome frequently lead patients with hae to be considered allergic and treated with antihistamines and corticosteroids, ineffective in this disorder. abdominal edema may so closely resemble an acute abdomen that some patients with hae have undergone unnecessary surgical explorations, often more than once. because untreated edema of the larynx may be fatal, inappropriate management may result in death. for many, hae and aae present an ongoing clinical challenge. despite the recurrent nature of angioedema attacks, their acute treatment is often suboptimal, sometimes delayed, and often requires lengthy hospital stays. in some countries, including the united states, no safe and effective acute attack therapy is available. even the prophylactic management of these disorders is inconsistent across centers and nations, and, because of the side effects of antifibrinolytics and steroids currently in use, requires a lifelong, individualized calculation of benefits and risks. these drawbacks are well known to the small community of physicians who deal frequently with these diseases and are a feature of life for those patients who suffer frequent or severe attacks. nonallergic angioedema as a model for the treatment of rare diseases in recognition of these challenges, several national and international physician and patient initiatives have begun in the past decades. in many ways, the field of nonallergic angioedema, and especially hae, is becoming an exemplar for the understanding and management of rare diseases. the estimated frequency of hae is : , . as in many uncommon conditions, hae's infrequent incidence fosters collaboration, forcing clinicians and researchers to pool their anecdotal experiences and data to attain statistical significance. nonetheless, hae is an attractive field because it offers doctors a chance to improve the lives of their patients dramatically through study but also via educated case management. as such, it has brought together a group of motivated and compassionate physicians. the pharmaceutical industry has also been welcomed to the c esterase inhibitor deficiency workshop and other hae initiatives, fostering free exchanges between academia, industry, and patients. indeed, perhaps the most distinctive feature of hae physician initiatives is their inclusion of patients with hae, not only in a traditional capacity of raising awareness and research funding but also as ethical advisors and welcomed guests for the presentation of scientific abstracts and talks. the first c esterase inhibitor deficiency workshop, held in hungary in , was the earliest meeting to follow this model. since then, the subsequent workshops and other patient-association gatherings in the united states and canada have followed its inclusive precedent. such a high level of patient involvement reflects not only the close relationship between knowledgeable physicians and their patients but also regional shortcomings in diagnosis and treatment. because of the incapacitating and life-threatening aspects of the disease, patients and their families from areas where hae is largely unknown have been forced to become educated enough to explain the disorder to strangers and, often, emergency department personnel to obtain the proper treatment. even patients whose cases are managed by a competent local practitioner may have attacks while traveling or when their doctor is unavailable and thus may need to articulate their condition to someone entirely unfamiliar with the disease. by incontrovertible necessity, patients with hae are one of the best-educated patient populations, and this is especially true in areas where satisfactory therapy for acute attacks is unavailable. for patients and physicians alike, the internet facilitates increasingly more communication, both personal and scientific. for patients with hae, it can help to reduce the isolation of having a rare disease. many patients first contact their national patient association online and use email to stay in touch with fellow patients. the internet is also being used by physicians and scientists to support a private patient registry and a public, constantly updated human c -inh gene (c nh) mutation database. through this online contact and regular meetings open to all, information about nonallergic angioedema is shared rapidly among a small, concerned group. nonetheless, the need to educate more physicians and the general public remains. the rarity of nonallergic angioedema increases the likelihood that clinicians, especially general practitioners or emergency department personnel, may never have seen a case. patient organizations and other groups have thus worked to create emergency passports for patients with known hae to carry and educational materials to distribute to emergency departments. scientific opportunities and current areas of controversy (kayla williams, bs, ma, mfa, cambridge, mass) nonallergic angioedema is a puzzle with relatively welldefined borders: many specific c nh mutations resulting in hae have been identified, and the symptomatic results are known. however, several central pieces are missing. despite recognition of functional c -inh deficiency as the cause of most forms of nonallergic angioedema, the specific mechanism of attack generation has not been definitively described. likewise, symptoms similar to those of nonallergic angioedema have now been reported in patients with normal amounts of functional c -inh. multiple pathways have been proposed for the chemical cause of angioedema attacks. the murine hae model developed by han et al shares similarities with the human form of the disease but diverges from typical hae in the triggering of angioedema. despite homozygous c -inh deficiency, the mice, with few exceptions, have not been observed to have typical angioedema attacks. attacks, manifesting solely as local increases in vascular permeability, could be provoked by the application of mustard oil. rather than representing a shortcoming of the mouse model, such a high threshold for attacks might parallel the course of those human heterozygotes, identified via a family member with active hae, who nonetheless never have an attack (for documentation of such patients, see agostoni and cicardi ). the absence of spontaneous attacks despite profound c -inh deficiency suggests that multiple biological events must transpire for angioedema to manifest. equally fascinating is the range of human disorders associated with functional c -inh deficiency. on the mild end of the spectrum, the american physicians luong and nguyen have reported a group of apparently unrelated vietnamese women presenting to their california clinic with lower extremities discomfort of unknown etiology. all of these women were found to have reduced amounts of serum c -inh, and danazol treatment resolved both the c -inh deficiency and the discomfort. at the opposite end of the c -inh deficiency spectrum, some patients with hae have periods of weekly or nearcontinuous angioedema attacks. in the most severe cases, laryngeal attacks may extend far enough into the thorax that even tracheostomy cannot maintain airway patency. it is unclear whether discerning the mechanism of some forms of hae, aae, and c -inh deficiency-associated disorders may elucidate others, but the attraction of a unified theory is obvious. however, among other factors, the inhibitory promiscuity of the c -inh molecule and its predisposition to mutation may not lend themselves to a simple answer. nonetheless, given the many proposed pathways for attack generation, information gained toward a full understanding of nonallergic angioedema attacks may lead to a greater knowledge of or more chemical cascades, including the classical complement pathway, kinin generation, and the intrinsic coagulation pathway. the areas of greatest controversy include which vasoactive peptide is ultimately responsible for the increased vascular permeability that results in angioedema. bradykinin and second component of the complement cascade (c )-kinin have been proposed, , with recent research contributing evidence to the importance of bradykinin. [ ] [ ] [ ] [ ] nonetheless, within the current understanding of coagulation, kinin, and complement pathways, neither peptide seems to perfectly explain all of the symptoms of angioedema. although bradykinin is the only candidate mediator for which there is direct clinical evidence, it is possible that yet another system, intermediary, or molecule may be involved in edema-generating vascular leakage. specific triggers for vasoactive peptide release are also unknown. it is proposed that the activation of factor xii is crucial to attack generation, and that factor xii activation may be a result of phospholipids released from damaged or apoptotic cells. recently, endothelial cells have been implicated in the generation, via kallikrein, of bradykinin, both in the presence and absence , of factor xii. these hypotheses explain how illness or localized tissue damage may precipitate attacks but do not account for other triggers, which are themselves not well defined. in large part, triggers seem to vary from patient to patient and, in several attacks, may not be apparent. of these, the most scientifically documented and explored are hormonal triggers, made all the more interesting by relatively recent reports of patients with normal c -inh concentrations and hae-like symptoms provoked or exacerbated by increased levels of estrogen. the importance of hormones in the regulation of nonallergic angioedema has long been acknowledged via its prophylaxis with androgens. increasingly, the effects of estrogen, progesterone, and other sex hormones are being explored. in some women, estrogen results in an increased frequency of angioedema attacks, , but others appear unaffected. depending on the patient and trimester, pregnancy may reduce or increase the number and severity of attacks. , in this supplement, the role of progesterone is debated, with visy et al finding a positive correlation between serum progesterone values and attack frequency, whereas bork et al note no increase in attack frequency among patients whose oral contraceptive (oc) contained progesterone and estrogen compared with those receiving estrogen alone. indeed, bork et al refer to several published works in which progestins were used, with varying success, to ameliorate hae symptoms. a- a in contrast, danazol, a common prophylaxis, alters multiple biological mechanisms but is known to block progesterone receptors and increase progesterone's metabolic clearance. given these conflicting findings, the influence of progesterone seems a likely area for further study. karen binkley graciously shared her work on very short notice and dr. alvin davis iii provided a valuable review; dr. shih-wen huang's contribution to the us hae association newsletter informed me of the full range of c -inh deficiencies, dr. alvin schmaier explained the mystery of angiotensin ii receptor blocker-associated angioedema, and dr. erik nielsen, both thorough his online hereditary angioedema thesis and quick correspondence, provided information and inspiration. many thanks go to dr. ineke bos, whose model of the c esterase inhibitor molecule graces our cover; chrystal mcdonald who worked tirelessly to secure reprint permissions; dr. brunello wüthrich who provided images of hae attacks; and drs. werner müller and georg dewald for their additions to the text. i would also like to recognize mr. anthony castaldo of the us hae association for his review of text pertaining to the patient experience and his indomitable, sustaining sense of humor. lastly, i would like to dedicate this supplement to its many contributing authors and all the hae, aae, and non-allergic angioedema patients they strive to help. in the first part of this section, cicardi and zingale describe the varied ways in which hae can manifest and discuss other diseases that published case reports and their clinical case series have associated with hae. the symptoms of hae are caused by the extravasation of plasma into the deeper cutaneous or mucosal layers as a result of or more locally released vasoactive peptides. the edema in hae is nonwhealing, nonpruritic, and generally unrelieved by antihistamines, suggesting that histamine is not involved in its induction. the biological characteristics of the vasoactive peptides released in c -inh-deficient sera indicate that the peptides belong to the kinin family. however, the discussion is not entirely closed on whether bradykinin, released because of contact system activation, or a peptide originated from c on classical complement pathway activation and the generation of plasmin, is the main mediator of symptoms in patients with hae. , nonetheless, recent lines of evidence coming from c -inh knockout mice, studies in patients' plasma, and analysis of c -inh mutants from patients with hae support the bradykinin hypothesis. , , kinin peptides participate in inflammatory processes and increase vascular permeability, activating intracellular pathways that lead to the release of nitric oxide. , vascular leakage can occur without anatomical damage and rapidly revert when the release of mediator molecules ceases. hence, edema usually resolves within hours. in some cases, it may resolve within hours, but in others, it may persist as long as days. urticaria, a condition analogous to angioedema but with plasma leakage into the upper cutaneous layers, is typically absent or minimal and short-lasting in patients with hae. typical symptoms. the recurrence of cutaneous angioedema, abdominal pain, and asphyxia caused by laryngeal edema is the full clinical pattern of hae, present in about % of adult patients. attacks usually evolve within a single site, but it is not uncommon for some patients to have simultaneous or closely spaced cutaneous and abdominal involvement. most patients recognize several hours in advance that an attack is coming. they may have sudden mood changes, anxiety, or complete exhaustion. cutaneous symptoms. skin edema is nonpitting and nonerythematous, with ill-defined margins. it typically affects the face (fig ) , extremities, and genitals (fig ) . it usually spreads to disfigure the affected site, temporarily depriving it of function. most often, a single site is affected by an extended edema that grows and then regresses within to days. alternatively, edema may persist, although reduced in size, and migrate to different cutaneous locations. in contrast with edema of other etiologies, edema associated with hae does not principally manifest in the perioral region. edema can localize subcutaneously in any body part, including the trunk. abdominal symptoms. recurrent abdominal pain, a consequence of gastrointestinal wall edema, is reported by % to % of patients with hae. , , this is a distinguishing feature of c -inh deficiency because abdominal involvement is rarely seen in angioedema of other origins. it presents with symptoms that may vary from mild discomfort to severe, intractable pain accompanied by vomiting and/or diarrhea. in this setting, hypovolemia can result from a combination of fluid loss, plasma extravasation, and vasodilation and can progress to hypovolemic shock. , ascites resulting from extravasation into the peritoneal cavity, edema of the bowel wall, or changes in splenoportal axis caliber have been described during abdominal attacks as detected by ultrasounds or computed tomography. [ ] [ ] [ ] [ ] [ ] [ ] gastrointestinal endoscopy performed during an abdominal attack revealed gastric involvement. interestingly, during the healing process after a prominent gastric edema, several small nodules and raised erosions developed over the entire gastric mucosal surface. within days, the gastric mucosa had returned to normal. the similarity between bowel angioedema and surgical emergencies is confirmed by the fact that approximately e of patients with undiagnosed hae undergo unnecessary surgery during abdominal attacks. however, even after a diagnosis of hae has been established, differentiating angioedema of the bowel from a surgical emergency remains a critical task for the physician. the physical examination can show the presence of an abdominal defense reaction. moderate or sometimes even marked leukocytosis can be part of an angioedema attack. abdominal ultrasounds and computer-assisted tomogra-phy scans demonstrate the presence of free peritoneal fluid and edematous intestinal mucosa. , , however, all of these signs are clearly not specific to angioedema. the authors note that this symptomatic generality should be borne in mind to avoid the situation that occurred with a patient in their case series. surgery was inappropriately delayed when acute appendicitis was mistaken for intestinal angioedema. the efficacy of c -inh plasma concentrate in resolving symptoms may help to distinguish angioedema from a true surgical emergency. laryngeal symptoms. laryngeal edema is the most dramatic clinical event for patients with hae. half of them have it at least once in their lives, but a history of recurrent episodes of suffocation caused by laryngeal edema is not uncommon, and deaths still occur as a result. in the past, % to % of patients with hae died from laryngeal edema. this percentage has dramatically dropped for patients who are appropriately diagnosed because of the availability of effective treatments in several countries. nevertheless, because of previous life-threatening experiences, some patients with hae still carry permanent tracheal cannulae, allowing them to breathe by bypassing the larynx when edema occurs. as mentioned, angioedema without urticaria is the hallmark of c -inh deficiency. however, a discrete number of patients, % in a survey by frank et al, have erythematous mottling, erythema multiforme, or erythema marginatum, always mild and transient, that inconstantly heralds or attends their angioedema. some patients recognize this symptom as announcing an attack, and when on prophylactic treatment, can still have a rash not followed by swelling. fig depicts several erythematous rashes experienced by patients before or during attacks of angioedema. unusual symptoms. reports in the literature suggest that edema caused by c -inh deficiency could occur in locations other than the characteristic sites of manifestation. [ ] [ ] [ ] [ ] frank et al reported transient pleuritic symptoms with pleural effusion in patients. local cerebral edema has been considered responsible for transient seizures and hemiparesis seldom described in patients with hae. , this assumption, despite its attraction and its occurrence in other forms of angioedema, has not been confirmed so far. neurologic disorders and the potential manifestation of cerebral edema remain a rarity in patients with hae. although atypical, urinary symptoms mimicking an infection have been described, and in patient, the presence of bladder edema was documented by endoscopy and biopsy. , pulmonary edema as a consequence of c -inh deficiency has occasionally been suggested but never clearly demonstrated. in the authors' experience, such an event was never observed to accompany an angioedema attack. they suggest that the high efficiency of the pulmonary vascular tree in the inactivation of bradykinin accounts for the lungs' protection from its effects. age of onset and frequency of symptoms. c -inh deficiency is present at birth, and a minority can have perinatal angioedema symptoms. most commonly, symptoms begin at school age. half of patients with hae had symptoms within the first decade of life, and another third had symptoms by the second decade. asymptomatic adults carrying a c nh mutation, detected because of the presence of offspring with clinically overt disease, have been described and are estimated to account for % of all patients with hae. the frequency at which bouts of angioedema recur is extremely variable among subjects and may vary in the same individual during different stages of life. a survey of the italian case list showed that slightly less than e of untreated patients with hae have more than angioedema attack per month, % have to swellings per year, and the remaining % are infrequently symptomatic or completely symptom-free. this range of phenotypic expression has no significant correlation with plasma concentrations of c -inh and is usually inconsistent among family groups. it should therefore be concluded that factors other than c -inh deficiency intervene to determine a subject's tendency to develop angioedema. these factors might be genetic or environmental. the hypothesis that symptom frequency correlates with specific functional polymorphisms of some of the proteins involved in pathogenesis is attractive but thus far unproven. an initial report suggesting that a polymorphism within the bradykinin receptor could distinguish oligosymptomatic from polysymptomatic patients has not been confirmed. farkas et al found that patients with hae infected with helicobacter pylori are more susceptible to symptoms than uninfected patients, and that eradication of the infection reduces the frequency and severity of swellings, particularly angioedema of the bowel. if confirmed in a larger group of patients, these findings could support those of several groups suggesting that infections increase susceptibility to angioedema in the general population as well as in patients with hae. , , [ ] [ ] [ ] clinical and laboratory criteria for diagnosis are provided in table i ; a severity scale for the evaluation of nonallergic angioedema is provided in table ii . these tools are based on contributions elaborated from experts from european countries who received a grant from the european commission for a project called novel methods for predicting, preventing, and treating attacks in patients with hereditary angioedema (prehaeat), consisting of a concerted action in the framework of the specific research and a technologic development program, quality of life and management of living resources, designed to improve the lives of patients with hae. diseases associated with c -inh deficiency. most often, patients with hae are substantially healthy apart from problems associated with swelling. however, there are several reports of autoimmune diseases in patients with hae, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and systemic lupus, in particular, has been described rather often. in a systematic study, of patients with hae had some kind of autoimmune disorder. moreover, patients with hae, because of defective control of the classical pathway of complement activation, have a deficiency of the fourth component of the complement cascade (c ) and c , a condition that increases the risk of autoimmune diseases. a large epidemiologic study in based on major autoimmune diseases estimated the prevalence of autoimmune diseases in americans to be in ( . %). given that all autoimmune diseases were not evaluated in this general population study, one cannot definitively conclude that patients with hae have a higher risk of autoimmune disease, but it appears likely. the association of hae with other inherited and noninherited conditions has occasionally been reported, but these observations remain isolated. [ ] [ ] [ ] [ ] last, patients with hae can be exposed to risk through needed treatments. several cases of hepatitis c virus (hcv) in the italian case series were a result of receiving plasma-derived products. these cases occurred before the introduction of viral inactivating procedures for plasma products. no cases of hiv were reported, but because of hcv, approximately % of their patients now have liverrelated problems. in this section, harmat et al describe the results of a study of hungarian patients with hae in whom ultrasonography was used to evaluate acute abdominal attacks of hae. background and rationale. ascites can result from diverse causes. the most common etiology, found in approximately % of cases, is the decompensated liver (cirrhosis). the remaining % result from other pathologies, such as malignancy in the abdomen ( %); various inflammatory diseases and other disorders, such as nephrotic syndrome, exudative enteropathy, chylous ascites, and mesenteric thrombosis; and others. however, hae is very seldom mentioned as a cause of ascites. this is a real problem, because ascites are a significant diagnostic sign of this uncommon but serious disease. the most common symptoms of hae appear in the form of ascites that cause acute abdominal attacks. for diagnosing this state, ultrasonography is the most potent tool. , , methods. ultrasonographic assessment is especially well suited to investigating the cause of abdominal symptoms. this study was performed to evaluate the usefulness of ultrasonographic diagnosis and included patients ( pediatric) from the hungarian hae center database. of these, had hae type i and had hae type ii. the male to female ratio was : , and patient age ranged from . to years. patient follow-up continued for a decade. in addition to biochemical studies, ultrasound investigations were performed at -month intervals. patients with typical symptoms of hae were hospitalized if the presence of other pathologies could be ruled out and if the manifestation was associated with hypovolemia and included recurrent paroxysms of acute colicky pain, nausea and vomiting, or profuse diarrhea, not responding to symptomatic therapy. all hospitalized patients underwent ultrasonography. during each abdominal attack, ultrasound examinations were performed before treatment and repeated at and hours post-treatment. , ultrasonographic investigations were performed by using a hitachi , a hitachi eub (hitachi medical systems, zug, switzerland), or an aloka ssd- diagnostic system (aloka co, ltd, tokyo, japan), with a . -mhz or -mhz convex transducer or a linear . -mhz transducer. subdiaphragmatic and pelvic regions were scanned with the patient in a supine position. kidneys were explored and the presence of free peritoneal or retroperitoneal fluid was ascertained with the patient in the supine and lateral positions or, when necessary, standing. free fluid, when detected, was classified into of categories, as follows: ( ) small-volume free peritoneal fluid was visible only in the subhepatic or subsplenic space, and in every case, in the douglas cul-de-sac. clinical criteria major ( ) self-limiting, noninflammatory subcutaneous angioedema without major urticarial rash, often recurrent and often lasting more than hours ( ) self-remitting abdominal pain without clear organic etiology, often recurrent and often lasting more than hours ( ) recurrent laryngeal edema minor ( ) family history of recurrent angioedema and/or abdominal pain and/or laryngeal edema laboratory criteria ( ) c inhibitor antigenic levels < % of normal at separate determinations with patient in basal condition and after the first year of age ( ) c inhibitor functional levels < % of normal at separate determinations with patient in basal condition and after the first year of age ( ) mutation in c inhibitor gene altering protein synthesis and/or function diagnosis can be established in presence of major ( - ) clinical criterion and laboratory criterion ( ) moderate-volume ascites, in addition to ascites found in these regions, included those identified in the sublienal space and among the intestinal loops. the intestinal walls were also swollen (thickness in excess of mm ). results. an ultrasound image taken during an acute abdominal attack (fig ) clearly illustrates the abdominal manifestations of hae. in this medial sagittal section of the pelvic area, a large amount of free peritoneal fluid can be observed in the douglas cul-de-sac, distal to and well separated from the urinary bladder. a floating intestinal loop can be seen. during the attack, an edematous thickening of the intestinal wall and a thin, echo-free fluid layer around the bowels also could be observed, as illustrated in edema of the portal veins, biliary ducts, and cholecyst wall, causing gross structural changes in the liver, was also observed. the liver parenchyma generally appeared less echogenic, whereas the walls of the portal vein radicles displayed increased echogenicity, resulting in a so-called starry sky texture that could be observed during the acute phase. because of local edema, the pancreatic region also displayed an increased echogenicity (fig ) . in addition to the hepatic portal vein, the wall of the cholecyst was also echogenic (fig ) . after treatment with c -inh concentrate, the former brightness disappeared, and the echo pattern of the liver returned to normal (sonogram not shown). discussion. early recognition of acute abdominal attacks is of utmost importance because incorrect or delayed diagnosis often leads to unnecessary surgical intervention. in undiagnosed patients, ultrasound examination can be a differential diagnostic means for recognizing hae in the abdominal organs because of its ability to detect nonspecific but sensitive clues such as thickening of the intestinal wall, free peritoneal fluid, intestinal hypermotility or hypomotility, and echo pattern fig . sagittal and transverse sonograms during an hae attack before and after treatment. sagittal sections are shown above, transverse below. a, a large amount of free peritoneal fluid has accumulated in the pouch of douglas and, in the sagittal section, a floating intestinal loop is visible. the urinary bladder appears below. b, soon after treatment with c -inh concentrate, the amount of peritoneal fluid is somewhat decreased. c, only a minimal amount of fluid is present in the pouch of douglas hours after c -inh treatment. several sonograms have previously been published in slightly different format. , transverse panels b and c reprinted with permission from acta paediatrica. sagittal panels a-c reprinted with permission from the european journal of gastroenterology and hepatology. fig . transverse sonogram during an abdominal hae attack: liver and pancreas. increased hepatic reflection (starry sky liver) and thickened, echogenic portal veins (arrow); the pancreatic region is also hyperechoic (double arrows). reprinted with permission from acta paediatrica. changes of the liver and pancreas. ultrasound examination has therefore proven very useful as a complementary, quick, and painless tool for recognizing the early phase symptoms of hae. patients presenting with skin symptoms (erythema marginatum) or acute pains, nausea, vomiting, or profuse diarrhea of unknown origins should be immediately hospitalized and investigated with ultrasound. ultrasound follow-up in known cases of hae is also capable of proving the efficacy and expeditiousness of acute attack treatment. in rare cases in which patients with known hae present with abdominal symptoms unresponsive to c -inh concentrate, ultrasonography may help distinguish between a refractory hae attack and an unrelated surgical emergency. abdominal and pelvic ultrasound examination is a highly reproducible and informative diagnostic tool and thus is indicated during acute abdominal attacks of hae unresponsive to c -inh concentrate. conversely, a search for hae is warranted when the typical sonographic features are ascertained in a patient with abdominal symptoms. angioedema may be caused by reasons as various as allergies, inherited or acquired deficiencies of c -inh, or drug reactions. , for the life of a patient presenting with unexplained airway swelling, the most important etiologic distinction is that between angioedema of allergic, histaminergic origins and the far rarer c -inh-associated or nonallergic angioedema. when allergic angioedema has been ruled out, nonallergic angioedema is next determined to be hereditary or acquired, and subclassification is pursued. allergic angioedema, with histamine as its major mediator, may best be defined by its clinical response to antiallergic drugs such as antihistamines and cortico-steroids. in this type of angioedema, reaction of specific ige antibodies with an allergen induces the release of histamine and other mediators from mast cells. it is often associated with urticaria. in contrast, angioedema caused by c -inh deficiency is not known to be triggered by an allergic reaction, is not usually associated with hives, and likely has bradykinin as its principal mediator. current systems for classifying hae and aae describe the disorders in terms of c -inh deficiency type. although observed convention supports the classification of major types, some further classifications, such as aae types, are more fluid. in the case of the more recently described estrogen-sensitive angioedema, a new formal description is suggested here. in the interest of both definition and the elucidation of mechanism that these differences imply, the divisions of hae and aae type are presented. for an example of prevalence, table iii presents agostoni's -patient angioedema case series by type. hereditary angioedema related to c inhibitor deficiency is a well-defined autosomal dominant trait. its variants include types i (hae-i) and ii (hae-ii), associated with mutations of the c inhibitor gene (c nh or serping ), , and a newly described type not associated with c -inh deficiency , , further defined and discussed in another section. the disease results from a large variety of mutations of the c nh gene, located in the q -q . subregion of chromosome . according to the relative concentrations of antigenic and functional c -inh, types of hae have traditionally been described. the defective gene produces either no c -inh (hae-i) or a dysfunctional c -inh (hae-ii). , , , in either case, it is associated with low functional activity of c -inh, low levels of c , and normal levels of the third component of the complement in hae-i (; % of patients with c -inh-associated hae), defective expression of allele results in low antigenic and functional concentrations of c -inh. in hae-ii (; % of patients with c -inh-associated hae) , concentrations of functional c -inh are low, but c -inh antigenic levels are normal or increased, with the presence of a dysfunctional mutant protein. for both, c -inh function is usually % to % of normal, instead of the % expected if the single normal allele were fully expressed. this difference is ascribed to permanent c and contact phase activation, with subsequent c -inh consumption in the periphery. , interestingly, the description of low levels of nonfunctional c -inh mutants in patients with hae-i has demonstrated that the distinction between hae-i and hae-ii is not absolute. this finding occurred in patients with mutations to exon at the carboxy terminus of the c nh gene, thought to be responsible for the proper folding necessary for transport outside of the cell and exposure of the reactive site loop. thus, although these patients with low antigenic concentrations of c -inh appear to have hae-i, they are in fact expressing nonfunctional c -inh that cannot efficiently exit the cell. estrogen-dependent and estrogen-associated inherited angioedema (previously hae type iii) (karen binkley, md, frcpc, and alvin e. davis iii, md, toronto, canada, and boston, mass) a type of angioedema, to date manifest only in women, has recently been described. , , its symptoms closely resemble those associated with functional c -inh deficiency but occur in the presence of normal c -inh concentrations; the genetic defect responsible is currently unknown. although this type of angioedema has been referred to as hae type iii (online mendelian inheritance in man [omim] ), others have argued that this designation is both redundant and misleading. the following piece by binkley and davis explores their work in a kindred with estrogen-dependent inherited angioedema, more fully describes estrogen-sensitive forms of inherited angioedema, and proposes a rational system of nomenclature. overview. the authors investigated a family with symptoms of angioedema restricted to conditions of high estrogen levels. although this investigation was undertaken to provide better care for the affected family members, it also presented a unique opportunity to better understand the effects of estrogen and androgens on c -inh. however, instead of altered hormonal regulation of c -inh, this family seemed to possess a completely novel abnormality, as suggested by the absence of identifiable mutations in either the coding or the # regulatory regions of the c nh gene and normal c -inh function and activity in a pregnant, symptomatic family member. the exact mechanisms responsible for angioedema in these patients have yet to be identified. the importance of kinin degradation pathways and aminopeptidase p (app) in the control of angioedema generation has been independently recognized in studies of angiotensin-converting enzyme (ace) inhibitor-related angioedema. bradykinin and its active metabolite, des-arg-bradykinin, are metabolized largely by enzymes, ace and app. [ ] [ ] [ ] with ace inhibitor administration, app becomes the primary enzyme responsible for inactivating bradykinin and des-arg-bradykinin. in fact, individuals with low plasma concentrations of app appear to be predisposed to developing angioedema during ace inhibitor treatment, when neither ace nor app is available to inactivate these kinins. kinin inactivation pathways might also modulate clinical symptoms in classic hae. for example, decreasing kinin inactivation in patients with hae with the use of ace inhibitors can result in exacerbation of angioedema. [ ] [ ] [ ] [ ] given the important contribution of kinin inactivation pathways to the control of angioedema, this may be an avenue for further investigation. case histories and investigation of the index family. the index family presented with histories of episodic, hae-like angioedema. these episodes occurred only during pregnancy, oc use, or estrogen replacement therapy. symptoms began to days after conception, or within to days of starting endogenous hormones. no episodes occurred in the postpartum period. one patient's description was particularly compelling: ''my period was just a day or two late, but when one side of my face swelled up, i knew i must be pregnant, because this is just like what happened to my mother and sisters every time they were pregnant.'' in affected individuals, symptoms occurred in all pregnancies and with each course of estrogen therapy. unaffected individuals had no symptoms at any time. there were affected women in generations and obligate male carrier. transmission was consistent with an autosomal dominant inheritance. complement values, c -inh, c -inh function, prekallikrein, factor xii, and high molecular weight kininogen were normal in patients during asymptomatic periods. genetic investigations were undertaken for the following reasons: ( ) the patients were asymptomatic at the time of presentation, ( ) baseline biochemical investigations were unremarkable, and ( ) exposing patients to estrogens for the purpose of detecting resultant biochemical abnormalities was unethical in light of the risk of laryngeal edema. the striking clinical similarity to classic hae focused initial investigations on the c nh gene. however, no abnormalities in the coding sequences of the c nh gene or in the # regulatory region were detected. when patient iii- became pregnant and developed recurrent angioedema, biochemical investigations were undertaken. c -inh antigen and function were both normal. the mechanism by which increased estrogens precipitate symptoms thus remains under investigation. related phenotypes: hae with normal c inhibitor activity in women. most of the women with angioedema in families reported by bork et al appeared to have a phenotype different than that of estrogen-dependent angioedema, because only of patients had attacks exclusively during pregnancy. in of patients, attacks occurred more frequently during oc use but were not limited to these periods. by extrapolation, of these patients had angioedema apparently unrelated to use of ocs or pregnancy. age of onset of symptoms in the patients of bork et al was variable and was not reported as directly correlating with onset of exogenous estrogen use or pregnancy. symptoms in at least patient started as early as year of age, before significant hormonal effects were likely as the authors note. these features are in sharp contrast with those of patients with estrogen-dependent inherited angioedema, in whom episodes of angioedema occurred exclusively during pregnancy or exogenous estrogen therapy, and suggest that a different underlying defect might be responsible for the different phenotypes. in the women described by bork et al, c -inh and c levels were normal in the affected individuals without symptoms. normal measurements of c and c -inh during symptomatic periods were also obtained in some individuals. other pedigrees have also been reported. nomenclature. until further biochemical and molecular genetic studies elucidate the underlying defects in these pedigrees, it remains unclear whether the different pedigrees represent subtle abnormalities in the same underlying pathway or distinct biochemical and clinical entities. therefore, affected patients can currently be classified only on the basis of phenotype, without reference to the underlying defect. this has implications for the nomenclature applied to these conditions. the term hae type iii may be misleading because it implies that these patients have a defect similar to hae-i (inadequate c -inh concentration) and hae-ii (inadequate c -inh function). this is clearly not the case, because c -inh concentration and function are normal in several pedigrees. , further confusion arises because the term hae type iii had been previously suggested to apply to a form of angioedema resulting from inadequate c -inh function caused by a mutation resulting in inappropriate binding to albumin. , although hae type iv was suggested for the patients of bork et al to address this latter concern, the term still erroneously implies a defect in c -inh function. the authors thus suggest that patients should be categorized on the basis of their phenotype and recommend the terms estrogen-dependent inherited angioedema and estrogenassociated inherited angioedema , until molecular studies suggest an alternate, rational nomenclature. clinical implications. further studies are required to identify the factors that contribute to angioedema in patients with estrogen-dependent angioedema. unaffected family members might then be identified through biochemical or genetic assays so that they might use ocs or plan pregnancies freely. identification of affected family members would allow these individuals to avoid ocs, bypassing a trial of therapy and the attendant risk of laryngeal edema. should effective treatment became available, affected individuals wishing to use ocs or become pregnant could begin treatment prophylactically or, at least, ensure its availability beforehand. if a particular factor is conclusively shown to be reduced in these patients, symptomatic individuals might be treated by replacing the missing factor. other possible treatments include novel strategies to reduce kinin formation or enhance kinin inactivation. prenatal diagnosis of fetal status (affected or unaffected) might also be relevant to the management of pregnancy in these individuals. the reported kindred showed significant variation in symptom severity during pregnancy, with some individuals experiencing relatively mild symptoms. in at least affected individual, it is likely that symptoms during a pregnancy with an affected fetus (identified as such only later in life) were accurately recalled as being particularly severe (binkley, unpublished data, march ) . it is interesting to speculate that an affected fetus would not provide the missing factor to the affected pregnant mother, and this might explain the severity of the symptoms. conversely, an unaffected fetus might act as a source of the otherwise missing factor during pregnancy and might mitigate symptoms in an affected pregnant mother. if pregnancies could be identified early as being at high or low risk for severe angioedema on the basis of fetal status, follow-up and management could be guided accordingly. at least direction for further study of the mechanisms responsible for symptoms in patients with estrogenassociated angioedema is suggested by the reduced kinin inactivation in ace inhibitor-associated forms of angioedema. elucidation of the defect responsible for this phenotype would allow better diagnosis and possibly specific treatment. general strategies to reduce kinin formation and/or enhance inactivation might also be helpful for the amelioration of symptoms. concerning hae-i and hae-ii, just as variations in serum concentrations of app appear to determine which individuals in a normal population develop angioedema with a second perturbation of kinin metabolism, such as the use of ace inhibitors, it could be speculated that variations in either kinin activation or inactivation pathways might contribute to the differences in severity of angioedema in individuals with a pre-existing perturbation in kinin metabolism, such as a mutation in c -inh (as occurs in hae). thus, it is possible that some of the variation in symptom severity seen between different members of the same family, carrying the same c -inh mutation, comes from variation in other kinin pathways. identification of the defects in estrogen-dependent and estrogen-associated angioedema might illuminate potential candidate factors. knowledge of kinin production and inactivation pathways and how they are influenced by sex hormones may also offer insight into some perplexing issues regarding the effects of sex hormones on c -inh values and angioedema symptoms in hae. androgens are effective in reducing episodes of angioedema and are used clinically for this purpose in hae. , although androgens increase plasma concentrations of c -inh, the amount of c -inh increase does not correlate well with symptom diminution. it is tempting to speculate that androgens may also increase kinin inactivation pathways, and this, perhaps in combination with slightly higher amounts of c -inh, contributes to the observed reduction in angioedema with androgen therapy. further studies will be necessary to explore this possibility as well. use of estrogen therapies typically results in some lowering of plasma c -inh concentration in normal individuals, and use of estrogen therapy tends to exacerbate angioedema in patients with hae. however, during pregnancy, estrogen concentrations are high, c -inh concentrations decrease, [ ] [ ] [ ] and paradoxically, episodes of angioedema may decrease, especially in late pregnancy. these puzzling observations have long suggested that a second mechanism is important in controlling angioedema. kinin inactivation pathways may be one such mechanism. speculation about possible mechanisms of symptom reduction in pregnancy suggests potential fruitful areas for further study. for example, are there hormonal factors in pregnancy, not operative during estrogen therapy, that increase kinin inactivation or other factors and reduce angioedema, despite an estrogeninduced lowering of c -inh? is the fetus or placenta a source of kinin inactivation factors or other factors that mitigate the effects of estrogen-induced lowering of c -inh? does variation in fetal production of kinin-inactivators or other factors underlie any variation in angioedema symptoms between pregnancies in the same individual, or between individuals in the same families, all with the same c -inh mutation? acquired angioedema is typically caused by ace inhibitor treatment, and less commonly is caused by autoantibodies directed at c -inh. general strategies to reduce kinin formation or/and increase kinin inactivation, identified through characterization of the elements of these pathways as well as their regulation, may be applicable to these patients as well. moving ahead. the discoveries of estrogen-dependent and estrogen-associated inherited angioedema are likely to focus attention on mechanisms other than c -inh that control angioedema. pathways involving kinin production and inactivation may be fruitful areas of further study in these conditions, a better understanding of which might provide new therapeutic opportunities potentially relevant to all types of angioedema. angioedema may be acquired, mainly in association with lymphoproliferative disorders or occasionally with autoimmune, neoplastic, or infectious diseases. aae also includes various other types of secondary c -inh deficiency, angioedema caused by certain antihypertensive medications, urticaria-associated angioedema, and idiopathic angioedema. , in the laboratory, aae is characterized by low functional c -inh, low amounts of c , and normal amounts of c . concentrations of c q are often very low. angioedema caused by acquired c -inh deficiency: type i and type ii distinguished. (marco cicardi, md*, andrea zanichelli, laurence bouillet, md, cca,* and emel aygören-pürsün, md, milan, italy, grenoble, france, and frankfurt, germany) in this section, cicardi et al review the current classifications of aae and discuss the possible pathogenic mechanisms on which these distinctions are ostensibly based. angioedema caused by acquired deficiency of the inhibitor of the first component of human complement (c -inh), usually referred to as acquired angioedema, is a rare, life-threatening disease first described by caldwell et al. characteristic of acquired c -inh deficiency are the increased consumption of c -inh and the hyperactivation of the classical pathway of human complement. as a consequence, these patients have almost undetectable serum levels and/or activity of c -inh, c , c , and c q, r, and s. usually, these abnormalities are constantly present, but temporary normalization of or more of these parameters has been reported. the clinical manifestations of the disease mimic those of the inherited defect of c -inh and include subcutaneous, nonpruritic swelling without accompanying urticaria; involvement of the upper respiratory tract manifested as dysphagia, voice change, or respiratory stridor; and partial obstruction of the gastrointestinal tract presenting as colicky abdominal pain. angioedema caused by acquired c -inh deficiency differs from hae by the absence of a family history of angioedema and a late onset of symptoms (in the fourth decade of life or later). response to treatment varies compared with hae caused by the c -inh hypercatabolism characteristic of acquired c -inh deficiency. acquired c -inh deficiency is frequently reported in association with b lymphoproliferative diseases. different forms of b lymphoproliferation can occur, ranging from benign monoclonal gammopathies of undetermined significance (mgus) to true malignancies. in , autoantibodies inactivating c -inh were first detected in patients with acquired c -inh deficiency. initially, autoantibodies inactivating c -inh were identified in otherwise healthy patients. on the basis of this observation, it was proposed that separate forms of acquired c -inh deficiency existed: type i, paraneo-plastic, mainly associated with lymphatic malignancies; and type ii, autoimmune, caused by autoantibodies to c -inh. the latter form appeared to be characterized further by elevated serum levels of cleaved c -inh. , because cleaved c -inh was not invariably found to be present in the serum of patients with so-called autoimmune acquired c -inh deficiency, this division has been questioned. , furthermore, autoantibodies to c -inh were later described in patients with associated diseases. these autoantibodies were found to be common in patients with mgus and frequently exhibit the same isotype of the m component. , , autoantibodies to c -inh impair c -inh function. although the exact mechanism for such impairment remains controversial, , the majority of these autoantibodies appear to enhance c -inh cleavage by target proteases, preventing their inactivation. a recent article on patients with acquired c -inh deficiency followed for as long as years (median, years) demonstrated that half of the patients with malignancies also had autoantibodies to c -inh, either at the time of onset of angioedema or later in the course of disease, indicating that autoimmune acquired c -inh deficiency is not distinct from the acquired c -inh deficiency that occurs in the setting of malignancies or other diseases. detection of autoantibodies to c -inh in a patient with acquired c -inh deficiency should not decrease the importance of considering the possibility of an associated pathologic condition. compared with the general population, patients with acquired c -inh deficiency presented higher risk for b-cell malignancies. in patients with acquired c -inh deficiency, the risk for progression of mgus to malignancy was not higher than in other patients with mgus. (angelo agostoni, md, milan, italy) new causes of aae, especially drug-related aae, are still being discovered, posing the question whether all types of aae share a common biomechanism if not a common etiology. in the descriptive sections that follow, agostoni surveys several classes of aae by cause. idiopathic nonhistaminergic angioedema. cicardi et al describe a subset of angioedema patients having normal complement values, no history of provoking drug treatment, and who are unresponsive to antihistamines. this condition, with a clinical presentation similar to that of c -inh deficiency, is deemed idiopathic nonhistaminergic angioedema. it is possible that this classification might overlap, at least in part, with that of estrogensensitive angioedema. ace inhibitor-related angioedema. angioedema may be a consequence of an adverse drug reaction not induced by an allergic or parallergic mechanism. ace inhibitor-related angioedema occurs in . % to . % of patients taking the drug. decreased bradykinin degradation is implicated because ace, also known as kinase ii, activates both angiotensin i and bradykinin. ace inhibitor-related angioedema may be an underestimated side effect because it can appear after years of ace inhibitor use, thus obscuring its relationship with the drug. unlike patients with c -inh deficiency, patients who develop ace inhibitor-related angioedema show no evidence of the cleavage products of high molecular weight kininogen (hk) in their plasma, despite high plasma concentrations of bradykinin. because the cleavage of hk generates bradykinin, the pathogenic mechanism of ace inhibitor-related angioedema probably resides in the catabolic side of bradykinin metabolism instead. when ace is inhibited, app plays a major role in plasma bradykinin catabolism. to identify patients at risk of developing angioedema during ace inhibitor treatment, adam et al evaluated blood concentrations of app. their results indicated lower plasma concentrations of app in patients who had previously had ace inhibitorassociated angioedema, suggesting an inverse relationship between app concentration and the tendency to develop angioedema. it is evident that ace inhibitor use should be avoided in patients with hereditary or acquired c -inh deficiency. angioedema related to other drugs. rare instances of angioedema have been reported with angiotensin ii (at ) receptor antagonsists, although this adverse effect seems to occur less frequently with at receptor antagonists than with ace inhibitors. it is unknown whether the adverse drug reactions share the same mechanism. scattered reports have suggested the possibility of angioedema associated with the use of estrogens, fibrinolytic agents, psychotropic agents, and antihypertensives other than ace inhibitors. the molecular diagnosis of angioedema is primarily based on evidence of the decrease or lack of c -inh function, which is routinely stated by its control capacity toward the target protease c s in spectrophotometric assays. molecular sizing of circulating c -inh can subsequently be assessed by sds-page and immunoblot. as discussed in greater detail in the pathogenesis and pathobiology of hae and aae section, c -inh controls several proteases, including c r and c s, the mannosebinding protein associated serine protease (masp) system, kallikrein, coagulation factors xiia and xia, plasmin, and tissue plasminogen activator. [ ] [ ] [ ] [ ] [ ] [ ] therefore, c -inh plays a key role in regulating the early steps of complement and the contact system of kinin formation. this broad inhibitory ability ensues from a property unique to the serpin class: highly efficient complex formation with target proteases. thus, mutations of the c nh gene typically affect many pathways. to add further complexity, many different c nh mutations resulting in hae have been discovered. through the study of these mutations, it is hoped that more complete knowledge of the many functions of c -inh can be gained, ultimately contributing to a better biochemical knowledge of hae. mutation analysis of the c nh gene in this section, drouet et al review the methods currently available for detecting c nh mutations and describe the powerful online mutation database that has grown out of such efforts. c nh gene. the c nh gene maps to chromosome . theriault et al, by using in situ hybridization in , localized it to q -q . ; a year later, janson et al mapped it to q -q . . it consists of exons distributed over a dna stretch of kb, with introns containing repetitive alu sequences (fig ) . the structural abnormalities in the c nh gene in patients with hae have been found to be very heterogeneous. illustrative examples explain the generation of c nh gene defects: large deletions or, less frequently, partial duplications involving alu repeats distributed along the c nh gene , ; deletions resulting from a peculiar consensus sequence or an alternative secondary structure ; and mutations based on cytosine-phosphateguanine (cpg) methylation and subsequent cytosine deamination to thymine. , as shown in table iv , more than mutations have been reported in unrelated patients, with pathogenic amino acid substitutions distributed over the entire length of the coding sequence. , in addition, the frequent de novo mutations in the c nh gene underline the presence of multiple hot spots, including those containing a cpg dinucleotide. all of these lead to an apparent failure to synthesize or secrete functional c -inh protein. in the context of a normal steady-state c -inh mrna content in almost all cases of hae-i and hae-ii, defective expression has been related to impaired protein secretion, , transinhibition of c -inh translation, or extensive consumption. the consequences of several missense mutations have been determined at the functional and intracellular processing level by transfection of an in vitro mutagenized construct into cos cells. point mutations help map amino acid residues critical for proper molecular folding and processing, with subsequent conversion of the serpin as a substrate, determination of target protease specificity, , , or spontaneous multimerization. , , hence, many different mutations can lead to dysfunctional c -inh, as recently reviewed. , strategies for mutation analyses of the c nh gene. molecular genetic analysis of c nh gene anomalies in patients serves as a supplementary diagnostic tool for accurate diagnosis at the molecular level. it contributes to the understanding of dna mutagenesis processes, protein folding and processing, and the structure-function relationships of the c -inh serpin. , , , such studies may also help the collection of population distribution data, potentially deepening understanding of the relationship between de novo mutation formation and distinct, independent founder effects in different geographical locations. c -inh deficiency is heterogeneous at the gene level and is caused by subtle changes affecting or several nucleotides, large deletions, or duplications. , , this heterogeneity prompted the authors to develop suitable methods for the detection not only of mutations previously identified in probands but also of as yet unknown mutations. de novo c nh mutations, including exon deletions, account for nearly % of cases of angioedema. this finding has implications relevant to the genetic epidemiology of and genetic counseling for this disease. the following sections describe the principles, advantages, critical parameters, and drawbacks of some technical strategies, including successful developments in the detection of c nh gene anomalies. scanning methods for point mutations or small deletions or insertions. methods based on heteroduplex dna and single-strand conformation analyses are of proven efficiency in the screening of large segments of genomic or complementary dna. , specific pcr amplification is first performed on exons and flanking intronic sequences or cdna. any variation detected is then sequenced to characterize the molecular change. in the following text, whenever possible, methods are accompanied by citations for reviews describing their advantages and limits. chemical cleavage of mismatches (ccm) technology is based on selective reactions of mismatched thymine and cytosine with oso and hydroxylamine, respectively. the modified mismatched bases are subjected to piperidine cleavage reactions, and the resulting fragments are separated and identified by gel electrophoresis. this method is, in principle, well suited to detect mutations independent of the length and sequence composition of the examined region and capable of detecting nearly all single-base mismatches. it has been successfully developed in a large number of studies (reviewed in ellis et al ) , including those of the c nh gene to detect pathogenic mutations and analyze its polymorphism. even if all mutations could potentially be detected, the efficiency of chemical modification and cleavage of the ccm depends on type of the mispairing and the stability of the adjacent sequences. in consequence, some mismatches are poorly cleaved, with their mutations subsequently undetectable via ccm. the need for a method suited to identifying most anomalies in the c nh gene, including point mutations and short and medium-sized deletions or insertions, prompted some groups to take advantage of ccm and fluorescent probes with the development of fluorescenceassisted mismatch analysis. , , denaturing hplc (dhplc) uses an alkylated nonporous poly(styrene-divinylbenzene) matrix and an amphiphilic ion gradient enabling separation of homoduplexes and heteroduplexes by means of ion-pair reversephase liquid chromatography (reviewed in xiao and oefner ) . illustrative examples of its application in c nh gene studies are given in fig . the most important advantage of the dhplc method is its easy automation by using a mechanical sampler. denaturing gradient gel electrophoresis is based on the migration of double-stranded dna molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent (reviewed in fodde and losekoot ). c nh-specific primers have been designed to amplify genomic dna segments ranging from to bp. the amplified fragments identified as forming heteroduplexes were directly sequenced. single-stranded conformational analysis (ssca) is an electrophoretic method using a nondenaturing gel, in which the mobility of heat-denatured single strands is dependent on their folding according to individual secondary structure formation. since its introduction in , it has gained popularity for its technical simplicity, low cost, and high sensitivity sufficient to detect most mutations (reviewed in nataraj et al ) . the method has been used in c nh gene studies , , (fig ) . analysis of dna rearrangements. because large genomic rearrangements account for nearly % of the total spectrum of the observed changes (reviewed in tosi ), methods adapted to analyzing dna rearrangements are required every time the aforementioned strategies are unsuccessful. southern blot analysis. southern blot analysis was first used for c nh gene analysis by groups who compared dna from multiple members of families with hae against dna from unrelated patients, yielding the first indication that a defective structural gene was responsible for the disease. , digestion of genomic dna with bcli can detect most deletion/insertion boundaries, because the -kb-long c nh gene lies within a -kb bcli fragment (fig ) . to localize gene alterations more precisely, other enzymes have been used in single or multiple digestions: bamhi, bglii, ecori, hindiii, saci, sali, pvui, and psti. , quantitative exon multiplex pcr. southern analyses are difficult to set up and time consuming. to ensure complete molecular characterization of c -inh deficien-cies, a fluorescent multiplex assay has been constructed to amplify exons of c nh and exon of the brca gene simultaneously. in brief, after c nh exon amplification, the fluorescence intensities of c nh exons are compared quantitatively with those of a control exon (eg, brca exon ) under conditions in which template concentration is rate limiting. the method has been further validated in exploring large deletions and insertions. reliable estimates of relative gene dosage can be obtained by comparing peak levels in the test dna with those of appropriate controls in deletion/insertion situations (fig ) . c nh promoter sequence analysis. the promoter of the c nh gene is unusual because it contains no tata sequence, but instead contains a tdt-like initiator element at nucleotides to and a polypurine-polypyrimidine tract between nucleotides and . only a few mutations have been reported to affect promoter sequence transcriptional activity, and among them, an interesting promoter variant (a c-to-t transition at position ) was found in an exceptional family with recessive trans- mission of the disease. however, subsequent transcriptional alteration remains to be demonstrated. these anomalies have been successfully detected via mismatch scanning (fluorescence-assisted mismatch analysis) in reactions with external and internal primers specific for the promoter sequence. sequencing and expression of sequence data. the most widespread, gold standard technique for mutation detection is direct sequencing. this technique is now commonly available with a high degree of automation and a parallel decrease in labor and cost. the principle is based on dideoxy chain termination reactions using fluorescent dideoxy nucleotides followed by automated gel or capillary electrophoresis. direct sequencing identifies the complete nucleotide sequence of a selected and pcr-amplified dna fragment and detects heterozygosity. the resulting data must be carefully interpreted. any newly discovered missense mutation has to meet the following criteria: ( ) the complete coding sequence and splice sites of the c nh gene were screened/sequenced; ( ) the new mutation is not present in general population chromosomes; and ( ) the new mutation segregates with the disease, ie, affected members carry the mutations whereas healthy relatives do not. novel interactive, locus-specific mutation database of the c nh gene. published c nh gene mutations were periodically summarized in reviews, , but such collections became quickly outdated when several new studies described a multitude of novel mutations. , , , , c nh gene mutations are also represented in large universal databases (omim ; human gene mutation database ), but these databases update their contents infrequently, with poor interactions and heavy requests. one of the goals of the european concerted action (prehaeat) is to perform systematic analyses of c nh gene mutations in several laboratories for structure-function relationships and consequence on disease expression. to achieve this goal, a mutation database (http://hae.biomembrane.hu) was created with the following purposes: ( ) to help the comprehensive collection of information on genetic alterations of the c nh gene, ( ) to create a database in which data can be searched and compared, and ( ) to provide additional help in deciding whether a new mutation segregates with the disease. c -inh serpin function defect: contributions from understanding c nh mutations. the need to ascertain the consequence of c nh mutation on c -inh expression and function prompted investigators to develop experimental systems adapted to both transcription and protein expression studies. patient peripheral blood monocytes and fibroblasts were successfully developed to study c -inh synthesis in pathological conditions. , [ ] [ ] [ ] it then became interesting to correlate c nh gene anomalies with transcription and translation defects, specifically with reference to serpin function, without the need for patient cells. pathological mutations can now be introduced in c -inh cdna expression systems and transfected into cell lines suitable for biosynthesis experiments. , , [ ] [ ] [ ] [ ] serpin function is routinely assessed in plasma samples (see varga et al in the following laboratory diagnosis section). some type ii mutant c -inh proteins have been described with respect to their atypical interactions with the target protease. a more complete model of nonfunctional c -inh mutants can yield insights into the c -inh inhibitory mechanism and aid in the development of a relevant dynamic -dimensional model of the c -inh molecule. knowledge of these type ii mutant c -inh proteins allows every mutation to be associated with its corresponding serpin control failure, as proposed in a category classification scheme : class i includes mutations that lead to altered exposure of the active site, with consequences on protease specificity (denoted § in table iv) ; class ii mutations convert c -inh protein into a substrate, with subsequent inefficient protease trapping (denoted à and k in table iv) ; and class iii encompasses mutations with a spontaneous insertion of the reactive loop, either into the same molecule or another molecule entirely (denoted k and { in table iv) . finally, as posited by cumming et al, it is important to correlate mutations with disease expression. however, on the basis of most clinical data, disease expression cannot be attributed to specific mutant proteins. variable clinical presentation is thought to result from genetic or nongenetic elements distinct from the c nh gene. in laboratory practice. every index case characterized by impaired c -inh serpin function and ruled not to be of an acquired origin can be submitted to a genetic analysis. to ensure an economical and reliable strategy, an algorithm is proposed in fig . currently, to be exhaustive, the conjunction of scanning methods and southern hybridization or exon multiplex technology is required to detect most mispaired or unpaired sequences and rearrangements, respectively. every nucleotide change detected from the index case has to be analyzed in the family members, keeping in mind that de novo mutations are not uncommon. as stated, and in particular when detected in introns, every new mutation without a known significance has to be distinguished from a polymorphism in the c nh gene after establishing its absence in the relevant healthy population (from at least chromosomes) and its segregation with the disease. the interpretation of biochemical data on c -inh serpin function and of c nh gene anomalies is of great importance to establish a better knowledge of the pathogenic effects of c nh gene mutations. moreover, the possibility of c -inh polymorphisms affecting disease expression should be considered. generating a c -inh molecular model should be of great help in the understanding and treatment of hae type ii and discerning which c -inh residues are essential for efficient control of all its target proteases. the uniform resource locators for data related to this section are as follows: from a pathogenetic perspective, the form of angioedema most extensively studied is that resulting from c -inh deficiency. nonetheless, the pathogenesis of this clinical condition remains incompletely understood. in the following section, hack briefly reviews the biochemistry and biology of c -inh as well as the biochemical changes that occur in persons with c -inh deficiency during both attacks and symptom-free periods. on the basis of these reviewed data, hack then discusses the potential pathogenesis of c -inh deficiency angioedema. often, hae results from a deficiency of c -inh. an interesting feature, revisited in the previous clinical manifestation and diagnosis section, is the great variance of the disease's clinical course. some patients are virtually free of attacks even in the absence of treatment, whereas others, despite therapy, have attacks nearly every week. angioedema attacks result from the extravasation of fluid caused by increased vasopermeability. unlike angioedema of allergic origins, angioedema caused by hae typically does not itch. pain is often not a primary feature of such angioedema, although it may manifest secondary to the localization of the angioedema, eg, severe pain caused by obstruction of the bowel lumen. in spite of decades of research, the pathogenesis of hae attacks is still unclear. c -inh biochemistry and biology. c -inh belongs to the family of serine protease inhibitors, or serpins, which also includes proteins such as a -antitrypsin and antithrombin iii [ ] [ ] [ ] [ ] (fig ) . most serpins have only or a few target proteases, but c -inh is exceptional in this respect. it is the major inhibitor of several proteases, including ( ) c s and c r-two serine proteases that, together with c q, constitute the c complex of the classical pathway of complement, ( ) the mannan-binding lectin (mbl)-associated serine proteases or masps, and ( ) the contact system proteases factor xia, factor xiia, and kallikrein. in addition, c -inh may interact with several proteases such as thrombin, plasmin, and tissue-type plasminogen activator (tpa). thus, c -inh regulates the activity of several inflammatory, clotting, and fibrinolytic proteases and is therefore an inhibitor of several pathways of inflammation. in comparison with other serpins, c -inh is a relatively weak inhibitor, approximately times less effective than other serpins. however, the inhibitory activity of c -inh toward c r, c s, and factor xia can be remarkably potentiated by heparin and other glycosaminoglycans. , the inhibitory activity of c -inh to factor xiia and kallikrein cannot be potentiated. dextran sulfate is the most effective glycosaminoglycan, enhancing c -inh function -fold in vitro and -fold in plasma. however, the in vivo effect of dextran sulfate is transient, probably because of dissociation of the dextran sulfate-c -inh complex and the subsequent rapid clearance of dextran sulfate. mature c -inh consists of amino acids and is heavily glycosylated (approximately % by weight). although on sds-page gels it migrates with an apparent molecular weight of kd, its calculated molecular weight is kd. the c -inh molecule has at least glycosylation sites and possibly glycosylation sites, part of them linked to threonine residues. most carbohydrate groups are located at the n-terminal region; their function is largely unknown. the c -inh molecule is composed of an n-terminal domain of amino acids and a serpin domain of amino acids. although the function of the n-terminal domain is unknown, recent evidence suggests that it helps to stabilize the central bsheet by forming disulfide bridges linking cysteine to cysteine , and cysteine to cysteine . the structure of the serpin domain is homologous to that of other serpins and is essential for the inhibitory capacity of the molecule. recently, a novel -dimensional model of c -inh was proposed on the basis of the crystal structure of other inhibitory serpins. similar to other serpins, c -inh inhibits proteases by binding to their active site via its reactive center. this reaction follows the equation in the first step, a reversible complex is formed between the target protease and c -inh; c -inh then undergoes conformational changes caused by insertion of part of its reactive center loop into a -stranded b-sheet. this results in the formation of modified c -inh (c -inh* in the equation) that is tightly bound to the target protease (step ). mutations of the residues at the p , p , and p positions in the reactive center interfere with efficient loop insertion and cause the reaction to proceed to step . in that case, c -inh has become a substrate for its target proteases. such mutations have been found in some patients with hae-ii (characterized by the presence of dysfunctional c -inh protein). complexes between modified c -inh* and proteases are very stable and only very slowly dissociate into an inactive, modified c -inh (with changed conformation caused by a completed insertion of the cleaved active site loop into the -stranded b-sheet) and an active target protease (step in the equation). most of the stable complexes will be removed from the circulation before dissociation via receptors specific for complexed serpins. this mechanism ensures efficient removal of biologically active proteases. c -inh is an acute phase protein, of which plasma concentrations may increase as much as -fold during uncomplicated infections. woo et al reported that the synthetic rate of c -inh may increase as much as . times the normal rate in patients with rheumatoid arthritis. cytokines such as interferon-g stimulate the synthesis of c -inh. in normal volunteers, the fractional catabolic rate of c -inh is . % of the plasma pool per hour, which results in an apparent plasma half-life time of clearance of about hours. the half-life time of clearance of human c -inh in rabbits is comparable, at hours, whereas that in rats is considerably shorter, at about . hours. the apparent half-life time of clearance is markedly longer in patients with hae, in whom it may exceed hours, as determined by assessing the course of plasma concentrations after the intravenous administration of exogenous c -inh. however, at low plasma concentrations of c -inh, such as occur in untreated patients with hae, c is autoactivated and consumes functional c -inh. at higher c -inh concentrations such as occur after the administration of exogenous c -inh, this autoactivation is inhibited, leading to a decreased consumption of c -inh. hence, monitoring of plasma concentrations after a therapeutic dose of c -inh may lead to an overestimation of the half-life caused by inhibition of consumption of endogenous c -inh. removal of sialic acids greatly enhances the clearance of c -inh from the circulation, yielding an apparent half-life of to minutes, presumably by binding to asialoglycoprotein receptors in the liver. subsequent removal of penultimate galactosyl residues largely restores the clearance rate to a value similar to that of normal c -inh. activation of cascade systems in hae. as mentioned, c -inh regulates the activation of at least different so-called plasma cascade systems, ie, the classical and mbl pathways of complement via its effect on c r, c s, and the masps; the contact system via its effects on kallikrein and activated factor xii (factor xiia); and the intrinsic pathway of coagulation via its effect on activated factor xi (factor xia). in addition, it may regulate coagulation to some extent via its effects on thrombin, and it may regulate fibrinolysis through its effect on tpa and plasmin. except for the mbl pathway, there is evidence for activation of all of these systems in patients with hae. the classical pathway of complement consists of c q, c r, and c s, together forming the c complex; c and c ; as well as the inhibitors c binding protein and factor i. typically, patients with hae have low c and c but normal c values even during attack-free periods. low c and c result from uncontrolled activation of activated c because the concentration of activation products of c , c b/c, or c d is elevated. , activation of c is typically low or absent, probably because of efficient control of activation at the level of c by c binding protein and factor i. this continuous activation of the classical pathway is attributed to enhanced autoactivation of c , because this autoactivation process is dependent on functional c -inh values. however, it is now clear that the classic pathway of complement may be activated by apoptotic cells through various mechanisms. [ ] [ ] [ ] hence, one could postulate that the excessive activation of the classic pathway in untreated patients with hae results from activation of this pathway by cell debris. indeed, familian and hack have observed increased amounts of complement activation products in patients with hae (unpublished data, november ), reflecting activation via the penetraxin serum amyloid p component, of the proteins that can bind to apoptotic cells. ongoing activation of the classical pathway in untreated patients continuously produces activated c . the contact system of coagulation consists of the proteins factor xii (also known as hageman factor), prekallikrein, and high molecular weight kininogen. the system is activated in vitro on contact with negatively charged surfaces, but the nature of potential activators in vivo is currently unknown. activation of the system yields factor xiia and kallikrein. c -inh inhibits both proteases. in addition, kallikrein can also be inhibited to some extent by a -macroglobulin. kallikrein can cleave hk, yielding cleaved hk and bradykinin. the latter nonapeptide is a potent inducer of vasodilation and increased vasopermeability. activation of this system is often assessed by measuring factor xiia-c -inh and kallikrein-c -inh complexes, although these parameters are unsuitable to assess activation in hae. alternatively, activation can be measured by detecting increased amounts of cleaved hk or decreased antigenic quantities of the contact system proteins. activation of the contact system in hae typically occurs during attacks and is hardly detectable during attack-free periods. in particular, increased amounts of cleaved hk are found in most, if not all, patients with attacks. , , remarkably, nearly all hk is often cleaved during attacks, such that native hk is hardly detectable. it is questionable whether all hk cleaved during an attack gives rise to bradykinin, given its extremely potent activity as a vasodilator. bradykinin is known to cause a significant drop in blood pressure even at low concentrations, yet hypotension is not characteristic for hae. however, there are no data supporting the cleavage of hk via other degradation pathways during hae attacks such as would generate minimal or no bradykinin. it should be noted that the measurement of bradykinin in plasma or other body fluids is hampered by its rapid degradation by carboxypeptidases and is only possible using samples collected in special inhibitor cocktails to prevent enzymatic degradation. few studies have attempted to measure bradykinin according to such protocols in hae, but these studies have shown increased bradykinin generation during attacks and not during symptom-free periods, particularly when samples are drawn from the site of the edematous swelling. , , c -inh is a major inhibitor of factor xia of the intrinsic pathway of the intrinsic pathway of coagulation , and also inhibits thrombin, although inhibition of this latter enzyme is likely physiologically inconsequential. the role of factor xi in coagulation is not yet resolved. originally, coagulation was considered to proceed via at least different pathways, the intrinsic and the extrinsic, merging at the level of factor x into a common pathway of thrombin generation. the extrinsic pathway is initiated by exposure of factor vii to tissue factor, whereas the intrinsic pathway is triggered by factor xii activation, eg, upon contact of blood with negatively charged surfaces such as glass. in this view, activation of factor xii led to thrombin generation via activation of factor xi, which then activated factor ix, which together with factor viiia activated factor x, which then together with factor va activated prothrombin. this traditional distinction between intrinsic and extrinsic pathway has been left behind, and the intrinsic pathway is now considered to amplify extrinsic pathway activation in that factor ix amplifies factor viia-induced activation of factor x, whereas factor xi, activated by thrombin, activates additional factor ix. according to this revised scheme of coagulation, the activation of factor xi is no longer mediated solely by factor xiia but also may occur with significant thrombin generation. in the traditional scheme of coagulation, c -inh is expected to have a major effect on intrinsic pathway activation because it blocks the activity of factor xiia, kallikrein, and factor xia, whereas according to the current understanding, c -inh has only a limited effect on coagulation, with its main effect the inhibition of factor xia-mediated amplification of factor ix activation. clinical observations in hae do not support that c -inh deficiency is a major risk for thromboembolic disease, although some generation of thrombin may occur during attacks. this mild effect of c -inh is easily understood through the revised scheme of coagulation. although c -inh inhibits plasmin and tpa in vitro, and to some extent in vivo, this inhibition is weak. nonetheless, significant plasmin formation, as measured by the formation of plasmin-a -antiplasmin complexes, occurs during attacks in patients with hae but hardly at all during symptom-free periods. at present there is no evidence for tpa or urokinase-type plasminogen activator involvement in the formation of plasmin during hae attacks. as such, this generation most likely results from factor xii activation. although debated in the literature, convincing in vivo evidence exists to support the activation of plasminogen in human beings by factor xiia. , angioedema attack pathogenesis: mediators of angioedema. angioedema is a frequently experienced side effect for patients receiving ace inhibitors. this angioedema does not itch, is not associated with urticarial lesions, and does not respond at all to corticosteroids or antihistamines, features also typical of angioedema associated with c -inh deficiency. ace inhibitors slow down the processing of c-terminal arginine residues of various vasoactive peptides such as angiotensin and bradykinin, thus prolonging the biological activity of these compounds. the association between ace inhibitors and angioedema is a strong argument that this condition may result from exposure of the endothelium to increased levels of vasoactive peptides. considering the similarities between ace inhibitor-related angioedema and that caused by c -inh deficiency, the latter form also likely results from enhanced exposure of the endothelium to vasoactive peptides. studies in a few patients indicate that bradykinin may be the vasoactive peptide involved. , , considering that contact activation in hae mainly, if not exclusively, occurs during attacks, and that activation of this system results in the liberation of bradykinin from hk, most investigators currently believe that c -inh deficiency angioedema results from the local generation of bradykinin caused by uncontrolled proteolytic activity of factor xiia and kallikrein (fig , bottom) . kallikrein has important effects in contact activation: it ( ) amplifies factor xii activation via so-called reciprocal activation, and ( ) releases bradykinin from hk. this mechanism of contact activation and bradykinin generation as the basis for angioedema is supported by observations in the murine model of c -inh deficiency, because the enhanced vasopermeability in this model can be blocked with bradykinin receptor antagonists. according to this mechanism, inhibiting kallikrein or blockading bradykinin receptors (bkr- ) would be therapeutic options; as discussed in the treatment section of this supplement, both are currently being investigated in clinical trials. activation of the contact system holds several attractions as an explanation for angioedema attacks. first, fig . different pathways of kinin generation in c -inhibitor deficiency state. low c -inhibitor leads to uncontrolled activation of factor xii, which generates kallikrein and plasmin. kallikrein liberates bradykinin from hk, whereas plasmin cleaves off c -kinin from activated c . activated c is continuously produced during baseline complement activation, which is increased as a result of insufficient control of autoactivation of c caused by c -inh deficiency. factor xii activation can be triggered by chemical activators and may well occur in bouts. second, bradykinin is a potent vasoactive peptide that can enhance vasopermeability. third, ace inhibitors have been shown to result in the delayed breakdown of bradykinin, and part of their hypotensive effects are actually blocked by bradykinin receptor antagonists. however, some strong arguments exist against this mechanism as the sole explanation for angioedema. first, bradykinin is a strong vasodilating agent and among the most hypotensive agents known, whereas hae attacks are not known to be associated with clear hypotensive reactions. nonetheless, one could argue that most bradykinin is generated locally, at the site of edema formation, rather than in the circulation. second, the local injection of bradykinin results in a strong pain sensation, whereas primary, localized pain is not typical for hae attacks. third, contact activation cannot explain the efficacy of antifibrinolytic agents such as tranexamic acid. it has been suggested that these agents are efficacious by reducing plasmin formation during attacks, which would lead to the reduced consumption of functional c -inh by plasmin. in spite of this, it is difficult to understand how active plasmin would be capable of mediating c -inh consumption, because it is rapidly inhibited by a potent plasmin inhibitor, ie, a -antiplasmin. moreover, in most patients with hae, rather than being elevated, the concentration of cleaved c -inh (free c -inh* in the equation) is fairly low. finally, although vasopermeability seems to be enhanced and dependent on bradykinin formation in c -inh-deficient mice, these permeability changes do not appear to occur in bouts and are otherwise atypical for angioedema. for all of these reasons, other mechanisms may be involved in hae attacks. some decades ago, another vasoactive peptide was thought to be involved in angioedema formation. this peptide can be generated by cleavage of the c b fragment, but not from native c , by plasmin, and is therefore called c -kinin. this c-terminal fragment of c b can be generated by c s or plasmin. mediation of attacks by this fragment requires activation of multiple plasma cascade systems: ( ) complement, to generate activated c ; ( ) the contact system, to generate factor xiia; and ( ) the fibrinolytic system, to generate the c -kinin generating enzyme, plasmin. according to this mechanism, attacks are triggered by factor xii activation that then yields plasmin, which in turn cleaves the c -kinin sequence from c b (fig , top) . the attractive aspects of this controversial mechanism include its explanation of the efficacy of antifibrinolytic agents. furthermore, one may postulate that c -kinin does not induce vasodilation, explaining why hae attacks are not associated with hypotension and may not induce sensations of pain, and why angioedema in general does not hurt. nevertheless, it should be noted that the biologic properties of c -kinin are not well established, and thus, it is not truly known whether this kinin's properties are consistent with the description. moreover, receptors for this kinin have not been identified, and it is therefore unknown whether their distribution correlates positively with the localization of attacks. in addition, there are no data regarding concentrations of c -kinin in patients with hae. finally, if correct, the c -kinin mechanism provides no explanation for the profound cleavage of hk and the apparent lack of biological activity of bradykinin. these mechanisms are not necessarily exclusive and may be simultaneously involved in attacks. furthermore, the involvement of other, as yet unknown vasoactive peptides in the pathogenesis of attacks cannot be excluded. triggers of angioedema attacks. clinically, hae attacks are often triggered by trauma or emotional stress. attacks of hae are specifically associated with activation of the fibrinolytic and contact systems, whereas complement activation occurs continuously, independent of clinical symptoms. activation of the fibrinolytic system can occur via plasminogen activators: tpa, urokinasetype plasminogen activator, and factor xii. there is no evidence that the former are involved in plasmin formation during attacks; hence, it likely results from factor xii activation. thus, attacks are triggered by activation of factor xii. though factor xii (hageman factor) was discovered in the s, physiological activators of this contact system protein have not yet been identified. in vitro, factor xii is easily activated by glass and other negatively charged compounds such as dextran sulfate. obviously, these compounds are not relevant as activators of the contact system in vivo. the author writes that recently, his group has studied the generation of thrombin by phospholipid microparticles released in vivo from activated platelets and damaged cells. by using various specific mabs against tissue factor, factor vii, factor xi, and factor xii, they noticed that a small but significant part of thrombin generation by microparticles purified from human plasma was dependent on factor xii. indeed, earlier studies have clearly indicated that factor xii can be activated by phospholipids in vitro. , apparently, factor xii can be activated by phospholipid microparticles in vivo. damaged or apoptotic cells generate such particles, and one may therefore postulate that these particles constitute triggers for contact activation. under normal conditions, ie, at normal concentrations of c -inh, this weak activation will never yield substantial contact activation. however, in the absence of sufficient amounts of this inhibitor, the system may become profoundly activated by even small amounts of microparticles via the principle of reciprocal activation (factor xii activates prekallikrein into kallikrein, which in turn activates additional factor xii, and so on). microparticles and damaged cells are likely generated during trauma, which may explain why this is a trigger for angioedema attacks. emotional stress as a trigger for attacks is more difficult to understand, although one may speculate that it predisposes patients to microtrauma. support for this hypothesis is that occasional patients have been described who, in spite of acquired c -inh deficiency, are free of symptoms. two such patients had detectable antibodies against phospholipids. these antibodies might interfere with the binding of factor xii to phospholipid microparticles or damaged cells. further studies are needed to provide evidence toward this hypothesis of hae attack generation. attack frequency. the angioedema attack rate in persons with c -inh deficiency may vary widely, ranging from no attacks to an attack every week. the molecular mechanism for the variation in attack rate is unknown. in the previous paragraph, mechanisms to explain the trigger for attacks are discussed. both mechanisms claim that attacks are initiated by activation of factor xii. therefore, one may speculate that the frequency of attacks is strongly linked to an individual's tendency toward factor xii activation. if true, attack rates might be influenced by the concentration of factor xii as well as by the amount of factors that compete with factor xii for binding to its activator molecules. as discussed, in the case of phospholipid microparticles, antiphospholipid bodies may prevent factor xii from binding to the particles, conferring protection against the activation of factor xii. older literature has revealed several proteins capable of competition with factor xii for binding to artificial activators. , the concentrations of these competing proteins might well determine whether factor xii may become activated on exposure to an activator and therefore influence hae attack frequency. future research will test this hypothesis. in summary. angioedema attacks caused by c -inh deficiency are likely mediated by the excessive release of vasoactive peptides such as bradykinin and c -kinin. this release is initiated by the uncontrolled activation of factor xii, leading to kallikrein and plasmin formation. the trigger for attacks may be phospholipids, released from damaged cells, that activate factor xii. factor xii's ease of activation may be determined by its concentration as well as the concentration of proteins that compete with it for binding to activators, and may explain the variable frequency of attacks in patients. others, assuming bradykinin to be the principal mediator of nonallergic angioedema attacks, have begun work toward bridging the gap between c -inh deficiency and local angioedema generation. bolstered by the increasing evidence that ace inhibitors [ ] [ ] [ ] can cause aae in a small percentage of individuals, investigators have sought to determine whether-independent of c nh gene sequence-an increased liberation of bradykinin, a decrease in its catabolism, or a combination of the may contribute to the angioedema experienced in hae, aae, and related disorders. phenotypic characteristics. mating of heterozygousdeficient mice produced the expected : : mendelian ratio of c -inh / :c -inh / :c -inh / mice. as observed in human heterozygotes, c -inh expression was less than % of normal in heterozygous mice; homozygous c -inh knockouts did not produce detectable plasma concentrations of c -inh. in male but not in female mice, carrying the defective c -inh gene correlated with a slight but statistically significant decrease in weight. the growth of both heterozygous-deficient and homozygous-deficient mice appeared normal, with no more newborn deaths observed than in wild-type littermates. limited analysis of serum c concentration in heterozygous-deficient and homozygous-deficient mice revealed that homozygotes had % to % as much c as their wild-type littermates, constituting a significant mean difference from wild-type; c in heterozygotes was more variable, ranging from % to % of wild-type. the group also reported that total hemolytic complement activity in the sera of null homozygotes was reduced compared with wild-type. although the c -inh / and c -inh / mice were not observed to have spontaneous attacks of skin edema, heterozygotes and homozygous knockouts developed spontaneous abdominal distension secondary to intestinal wall edema. because these mice were euthanized, it is unclear whether these episodes would have spontaneously resolved. no such episodes of abdominal edema were observed in wild-type littermates. measuring increased vascular permeability. evans blue dye was used to visualize differences in vascular permeability between the types of mice. within to minutes of injection into the tail vein of wild-type mice, the dye caused a faint blue coloration, particularly visible around the nose and eyes and on the feet. after similar injections, c -inh / and c -inh / mice showed a much more saturated blue color in these areas, with homozygous null mice producing the most saturated blue. these differences could be quantitated spectrophotometrically, as shown in fig . the mice were euthanized and the dye extracted from the area in question by using formamide, then read at an absorbance of nm. han et al noted that unlike in the feet and face, there were no apparent vascular permeability differences in the ears of mice of all genotypes after evans blue injection. however, when mustard oil, a local irritant, was applied to the ears of injected mice, a pronounced permeability increase correlating with c nh mutation could be spectrophotometrically demonstrated. this increase could be reversed if the mice were given human c -inh concentrate. intestinal vascular permeability was also investigated in a few mice; although of borderline statistical significance because of the sample size, intestinal permeability was also increased in c -inh / versus wild-type mice (fig ) . bradykinin type receptor assays. groups of heterozygous and homozygous c nh-deficient mice were treated with the plasma kallikrein inhibitor dx- , the bradykinin receptor antagonist hoe (icatibant; jerini, berlin, germany), or the ace inhibitor captopril (figs and ). both interference with plasma kallikrein (and thus contact system-mediated bradykinin generation) and disruption of the bradykinin receptor interaction with its ligand decreased vascular permeability. treatment with the bradykinin receptor antagonist des-arg ,[leu ]-bradykinin had no effect. treatment with captopril dramatically increased vascular permeability. when c nh knockout mice were crossed with bradykinin receptor knockout mice, the resulting offspring were overtly phenotypically normal. as shown in fig , absence of the bradykinin receptor was able to reverse the vascular permeability observed in both c -inh / and c -inh / mice. in conclusion, although this mouse model does not exclude the possibility of other contributing mechanisms, it strongly supports the hypothesis that bradykinin mediates angioedema. bradykinin-mediated angioedema? (kayla williams, bs, ma, mfa, cambridge, mass) in this section, williams reviews recent information on the role of bradykinin in both drug-related angioedema and hae and explores the contributing factors that may influence the generation of bradykinin-mediated angioedema. drug-related angioedema: a growing problem. as the use of ace inhibitors, at receptor antagonists (at blockers), and related, next-generation drugs increases, it is projected that the incidence of drug-related angioedema will also increase. , although angioedema is a relatively rare adverse effect of such medications, the medications are commonly and increasingly prescribed. the incidence of angioedema during ace inhibitor treatment has been estimated at between and per patients. these cases of drug-induced angioedema are interesting because, unlike other forms of nonallergic angioedema, the role of bradykinin in ace inhibitor-related angioedema is fairly well established. also of note and concern are reported racial differences in the response to this vasoactive peptide. bradykinin, race, and ace. in , gainer et al published the results of a clinical trial evaluating wheal response to bradykinin among african americans and white americans. in this randomized, double-blind study, increased bradykinin dose, hypertension, and african american race each correlated with an increased wheal response to bradykinin. further investigations by the same group compared the effects of short-term ace inhibition in normotensive and hypertensive individuals of both races. in this study, the hypotensive effects of the ace inhibitor captopril were shown to be greatly reduced in all subjects by concomitant administration of the bradykinin receptor antagonist icatibant. icatibant was also shown to significantly alter the change in plasma renin activity in response to ace inhibition. these results not only confirmed the importance of bradykinin in the short-term effects of ace inhibitors but also suggested that bradykinin contributes to the effects of ace inhibition on the renin-angiotensin system. in addition, when considered together, these results suggest that clinicians prescribing ace inhibitors for patients of afro-caribbean descent should be especially aware of the possibility for drug-related angioedema, a warning beginning to be supported by case analysis. , further antihypertensive-related angioedema. initially, at blockers were thought to be a safer alternative for patients who had angioedema associated with ace inhibitors. unlike ace inhibitors, at blockers have not been shown to increase bradykinin concentrations. nonetheless, further case reports documented angioedema after therapy with at blockers. , in a survey of the literature, % of these patients also had similar episodes in conjunction with ace inhibitor treatment. additional documented cases of at blocker-associated angioedema threw bradykinin's role in the pathogenesis of such episodes into doubt because increased concentrations of bradykinin are not associated with at blocker use. however, very recent work has shown that increased concentrations of at , such as would occur during a blockade of at receptors, are associated with an increase in the expression of bradykinin receptors that may be almost -fold. thus, increased opportunities for bradykinin binding may explain angioedema in the absence of extra bradykinin. the mediator for this receptor crosstalk effect has been shown to be the at receptor . (presumably, this phenomenon could occur in the presence of at receptor blockers only in the absence of a total blockade.) meanwhile, the new drug application for the vasopeptidase inhibitor omapatrilat, a dual inhibitor of both ace and neutral endopeptidase (nep), was voluntarily withdrawn when the food and drug administration raised questions regarding the comparative incidence of angioedema reported within the new drug application database. in response to these questions, a large ( , -patient) multinational trial was initiated to compare the efficacy and safety of omapatrilat versus the ace inhibitor enalapril in patients with heart failure. the results of this study have recently been reported. angioedema occurred in . % of subjects receiving omapatrilat compared with . % of subjects receiving an ace inhibitor alone. two of the dual inhibitor-treated subjects had angioedema with airway compromise. an editorial published after the trial results were released but before their publication noted that the rates of angioedema were much higher in african americans receiving the dual inhibitor ( . %) than in those receiving the ace inhibitor alone ( . %). of smokers receiving the dual inhibitor, . % had angioedema, versus . % of smokers receiving the ace inhibitor alone. the biologic activity of nep must account for this several-fold increase in angioedema. indeed, among its many possible functions, nep has been shown to metabolize bradykinin to an inactive form. a combination of ace and nep inhibition could thus be expected to prevent efficiently the degradation of bradykinin to inactive metabolites, thereby increasing the risk of angioedema. in certain subgroups with altered responses to bradykinin or altered pathways for its generation or degradation, these effects could be especially pronounced, and such predisposing factors may also be involved in the generation of c -inh deficiency-related angioedema. implications for c -inh deficiency angioedema. early investigations have confirmed that bradykinin may be at work in c -inh deficiency angioedema. the transience of bradykinin has complicated measurements of this nonapeptide; however, several small studies of plasma bradykinin concentrations in patients with angioedema have revealed that high concentrations of this vasoactive peptide occur during edematous attacks. , , this increase in plasma bradykinin was demonstrated both for patients with hae with c -inh deficiency and for patients with drug-associated angioedema who had an angioedematous attack in conjunction with ace inhibitor treatment. patients receiving ace inhibitors showed elevated plasma bradykinin concentrations throughout ace inhibitor therapy, both during attacks and during periods of remission. as documented in patient, this increase reverted to normal on discontinuation of ace inhibitor therapy. in contrast, patients with c -inh deficiency were shown to have high concentrations of bradykinin during their attacks but have been documented to have normal or marginally increased amounts of bradykinin during periods of remission. at least in c -inh deficiency, bradykinin increases may be highly localized; in previously studied c -inh-deficient patients, bradykinin concentrations in blood draining from an angioedematous site were and times as high as those measured in systemic circulation. thus, bradykinin appears to be important in various currently identified forms of nonallergic angioedema. following the example put forth in the large-scale trial of antihypertensive agents that showed ( ) african descent and, to a lesser extent, ( ) smoking to correlate with increased angioedema risk, clinicians and researchers may wish to investigate the interaction of these variables with c -inh deficiency. bradykinin magnified. even if bradykinin is the major mediator of nonallergic angioedema, many questions regarding the triggering and localization of angioedema attacks remain. why should patients with ace inhibitorinduced angioedema have this angioedema in bouts if bradykinin is constantly elevated? what is different about the small percentage of patients taking ace inhibitors who have angioedema? what regulatory pathways are involved? how, chemically, does one proceed from insufficient functional c -inh to localized, full-blown angioedema? the past decade of research indicates that the answers may reside in a surprisingly multifactorial system. bradykinin-mediated effects can result from increased amounts of bradykinin, the reduced breakdown of bradykinin to inactive metabolites, the increased conversion of bradykinin to active metabolites, and/or the increased uptake of these active metabolites. each of these mechanisms may be involved in at least form of nonallergic angioedema. pathways for the breakdown of bradykinin are depicted in fig . decreased or alternative catabolism. molinaro et al have demonstrated that in human plasma, bradykinin is metabolized mainly by metallopeptidases: ace, app, and kininase i (carboxypeptidase n). of these, ace and app, respectively, are the first and second major pathways of inactivation. kininase i represents a minor pathway unless ace is inhibited, whereupon kininase i transforms bradykinin into its active metabolite des-arg -bradykinin, which, in turn, is inactivated by app and ace, with app the major inactivating metallopeptidase. nep, as discussed in the context of vasopeptidase inhibition, likely represents another minor pathway. once formed, bradykinin and des-arg -bradykinin are short-lived and thus exert their effects locally at the site of their formation, stimulating b and b receptors, respectively. , during ace or vasopeptidase inhibition, patients with low app activity may be subject to angioedema. in fact, adam et al have found a correlation between low app activity with a history of angioedema among patients receiving ace inhibitors. likewise, in their estrogen-dependent and estrogen-associated inherited angioedema piece earlier in this supplement, binkley and davis propose reduced app activity as a contributing element in estrogen-sensitive inherited angioedema. in addition, they note that estrogen may decrease gene expression of ace. , to demonstrate further the delicate balance of enzymes and receptors, it should be noted that mathews et al observed angioedema in a subject with familial serum carboxypeptidase n deficiency. because the subject's sister had an equally depressed serum carboxypeptidase value but no reported episodes of angioedema, it is possible that another defective control mechanism, possibly app, contributed to the angioedema. pathways for bradykinin generation and metabolism. on the endothelium, bradykinin generation likely initiates independent of factor xii; however, once kallikrein is generated, it soon catalyzes factor xii activation, thus amplifying the response. increased anabolism. as hack discussed in the pathogenisis and pathobiology of hae and aae section, c -inh is known to inhibit not just c esterase but also other serine proteases, including factor xii (hageman factor). in addition, c -inh inhibits the serine protease kallikrein. without the controlling effects of c -inh, it is possible that both of these serine proteases may contribute to an increased generation of bradykinin. this reaction is thought to occur at the endothelial cell surface. the way in which this unchecked bradykinin generation starts has been debated, with recent evidence supporting the secondary role of factor xii, , and placing the conversion of prekallikrein to kallikrein, likely by an enzyme such as prolylcarboxypeptidase, , foremost, at least under resting physiologic conditions. it is thought that once kallikrein is present, it catalyzes the activation of factor xii. only in the presence of activated platelets, neutrophils, or lymphocytes can the high local concentration of free zinc ions necessary for factor xii to bind to the endothelial cell surface-and thus initiate bradykinin release-occur. this provides yet another example of how infection or tissue damage might aggravate an existing tendency to angioedema. in vitro, kallikrein and plasmin together have been shown to exert a synergistic effect on the release of kinins from hk. of interest, plasmin's digestion of hk in this model could be inhibited by e-aminocaproic acid. because c -inh is also known to inhibit plasmin, and antifibrinolytics show at least partial efficacy in the treatment of hae, this enhancing mechanism might well be operative in c -inh deficiency angioedema. in addition, because angioedema has been reported in patients treated for acute ischemic stroke who received recombinant tissue-type plasminogen activator (rtpa; often concomitantly with an ace inhibitor), molinaro et al undertook an in vitro comparison of human plasma incubated with and without rtpa in the presence of an ace inhibitor. their results showed that rtpa generates bradykinin that is further metabolized to des-arg -bradykinin; this effect could be recreated to a similar extent in the absence of rtpa by the addition of negatively charged glass beads, thus mimicking contact activation. joseph et al , have identified yet another possible mechanism active at the endothelial cell level, showing that the cytosol of endothelial cells contains a factor capable of converting prekallikrein to kallikrein in the absence of factor xii. they identified this factor as heat shock protein . perhaps this mechanism might account for angioedema attacks that occur in the wake of trauma or infection when ruptured cells spill their contents into local circulation. indeed, increased kallikrein may be a feature of mechanical trauma: an experiment conducted more than years ago demonstrated that mechanically generated blisters in of patients with hae contained active kallikrein that could be inactivated by c -inh concentrate or antibodies to plasma kallikrein. in contrast, blister fluids from of healthy subjects did not contain detectable amounts of plasma kallikrein. last, these anabolic effects might be created or magnified by hormonal interactions. as noted by binkley and davis, estrogens may increase the expression of factor xii , - and prekallikrein. increased uptake. various factors may increase gene expression of the b and b receptors. not normally expressed, the b receptor has a high affinity for kinin metabolites. expression of b receptors may be induced during inflammation and tissue damage. , although b receptors are constitutively expressed, their expression may be increased by estrogens and at receptor antagonists. in summary. despite increasing evidence that bradykinin is indeed the major mediator of nonallergic angioedema, this knowledge does not represent a simple answer given the many variables governing bradykinin and its metabolites. however, an understanding of this web of chemical interactions may yield more successful management algorithms for the various types of nonallergic angioedema. this recent knowledge also suggests a place for bradykinin receptor antagonists and kallikrein inhibitors for the control of nonallergic angioedema, especially when it occurs in the presence of functional c -inh. the current scientific understanding of the triggering of angioedema attacks ranges from the anecdotal to that supported by relatively large and rigorous studies. patients with hae, aae, or related forms of angioedema, especially patients who have angioedema attacks in the absence of a formal diagnosis, pay particular attention to the events that precede their angioedema attacks; however, even in the same patient, these events may vary among attacks. various patients have implicated exposure to cold; mechanical trauma sustained during routine activities such as gardening; prolonged sitting or standing; eating certain foods, such as eggs; exposure to pesticides or chemicals in new fabrics and clothing; infection or illness; or excitement or stress in triggering their attacks (k. williams, unpublished data, august ). however, as with other, better studied triggering events, these conditions or activities did not always predictably provoke attacks. ethical concerns and the small patient populations available to would-be investigators prevent the systematic investigation of most patient-reported angioedema attack triggers. those triggers more readily quantifiable via laboratory assays without necessitating patients' increased risk of developing attacks have received the most scientific attention, largely restricting the study of attack triggers to the possible role of hormones. (laurence bouillet, md, cca,* grenoble, france) in the first part of this section, bouillet reviews his group's clinical experience and other reports linking sex hormones and hae. on the basis of the literature, he proposes possible pathogenic mechanisms by which these influences might manifest. these relationships are discussed with a final view toward patient care. current literature. it is currently known that hae is influenced by the fluctuation of female hormones, but the effects sometimes appear to vary greatly among women. some patients seem to be estrogen-dependent: puberty, pregnancies, or estroprogestative contraception can precipitate attacks, whereas menopause impairs the disease. however, the course of some women's disease seems to be unaffected by estrogen. reports have highlighted the close relationship between female hormones and angioedema. yip and cunliffe described cases of hae occurring in a mother and her daughter whose symptoms appeared to be estrogen-influenced. their first attack happened around puberty; angioedema worsened premenstrually and when they took ocs. likewise, a woman with hae-ii associated with turner syndrome was reported as experiencing an increase in both the severity and the frequency of her angioedema attacks on beginning estrogen physiological replacement at age years. mcglinchey and mccluskey also described hae manifesting after the initiation of estrogen replacement therapy. bork et al, as discussed later in this supplement, reported their evaluation of the effect of oc use or hormone replacement therapy on the frequency and the severity of hae attacks, finding that % of women with hae taking these drugs presented with new or worsened angioedema. some kinin-mediated angioedema appears to be related to estrogen therapy. the first cases of angioedema during oral contraception were described in a report by warin et al a in which sisters presented with hae only after taking oral contraception and during pregnancy: c -inh concentration and complement components were normal. bouillet's group has also reported women whose angioedema attacks manifested after starting oral contraception. a study of c -inh biological function revealed a lowered c -inh activity with marked protein cleavage on the immunoblot. clinical and biological anomalies ceased after oral contraception was discontinued. hypoandrogenism has been associated with insufficient production of c -inh in women taking cyproterone acetate as well in men with hypogonadism. in , pichler et al reported the cases of women taking the contraceptive drug cyproterone acetate who developed clinical hae reversed by discontinuation of this contraceptive; also described were the cases of men with hypogonadism who had recurrent angioedema successfully cured by androgen treatment. as discussed in previous sections, reports described a new estrogen-influenced hae. , , only women had angioedema, and their attacks were precipitated when estrogen concentrations were increasing because of pregnancies, oral contraception, or hormone replacement therapy. no c -inh abnormalities have been reported in these cases. hormone-related pathogenesis. for these clinical events to be understood, they must be placed in a biochemical context. as discussed, the complement, contact, and fibrinolytic systems are involved in the genesis of angioedema attacks, , , and all are regulated, at least to some extent, by c -inh. bradykinin is thought by many to be the principal candidate mediator of angioedema genesis, and a mouse model demonstrated that angioedema is mediated by bradykinin via b receptor. in human beings, studies have shown local elevations in bradykinin concentration during hae attacks. , it is speculated that factor xiia, kallikrein, and thrombin may be increased as well. an elevation of plasmin-a antiplasmin complexes is also noted. in ovariectomized rats, studies showed that b-estradiol favored the increase of factor xii by stimulating its gene transcription. , [ ] [ ] [ ] this hormone also increased kininogen and kallikrein concentrations. in addition, estrogens regulate b receptor gene expression and function: when reduced in ovariectomized rats, the vasodepressor response to bradykinin and b receptor mrna levels could be restored by estrogen substitution. progesterone did not modify factor xii concentration but seemed to increase the amount of kallikrein cdna. in healthy women taking oral contraception, there is an increase of the fibrinolytic proteins: plasmin and factors vii, x, and ix are increased, whereas plasminogen activator inhibitor is decreased. , , these effects appear to be estrogen dose-dependent. plasma samples from these women showed enhanced in vitro fibrinolysis. the contact system is also altered: factor xii, prekallikrein, kallikrein, and hk increase. [ ] [ ] [ ] last, c -inh levels are decreased, correlating with the increase in factor xii. , , hormone replacement therapy (hrt), although using a weaker estrogen dose than oral contraception, appears to have the same effects. fibrinolytic proteins are also increased (plasminogen and tpa), whereas plasminogen activator inhibitor decreases. [ ] [ ] [ ] hrt increases factor xii, prekallikrein, c , and c concentrations. , , moreover, some studies have shown hrt to influence the bradykinin system: ace activity decreases, whereas bradykinin concentration increases. , , toward treatment. this imbalance in these systems appears to trigger angioedema attacks in some estrogensensitive women. each physician should identify these estrogen-sensitive patients to manage specifically their contraception needs, pregnancies, and choices regarding hrt. further studies to determine predictive biological, genetic, and clinical parameters would be of use. role of sex hormones in hae, part ii most reported studies have investigated the influence of exogenous sex hormones on the frequency and severity of hae attacks. here, visy et al very briefly summarize a study they undertook measuring serum concentrations of endogenous sex hormones in women with hae and correlating these results with the occurrence of hae attacks. this work has since been published in clinical endocrinology. objective. the fluctuations of sex hormone concentrations at the beginning of adolescence, in the perimenopausal period, and during pregnancy or oc use can precipitate hae attacks. edematous attacks usually disappear after the onset of menopause. the authors undertook their study to establish any relationship between serum concentrations of sex hormones and the incidence of hae attacks. patients and measurements. serum concentrations of luteinizing hormone (lh), follicle-stimulating hormone, progesterone, estradiol, testosterone, prolactin, and sex hormone binding globulin (shbg) were measured in patients (mean age, . years; range, - years) with hae. a questionnaire was used to explore the medical history of adult patients to characterize the evolution and properties of attacks. results. twenty-one patients had been symptom-free before menarche. symptoms in of patients first occurred during adolescence, whereas the remaining patients had no attacks in the pubertal period. edema during the perimenstrual period was reported by . %. pregnancy was associated with a higher incidence of attacks in %; edema formation was less common in %, and % had no change in the frequency of symptoms despite pregnancy. of the patients using ocs, reported an increase in the frequency and severity of edematous symptoms compared with the period before hormonal contraception had been initiated. serum concentrations of sex hormones were normal in the majority of patients; however, more than half of the subjects had progesterone values above the upper limit of the normal range. during the -year follow-up, the attack rate was times higher in female patients with high progesterone concentrations (above the menopausal threshold of nmol/l) than in women with normal or low serum concentrations. the eminent role of this hormone is also confirmed by the observation that only progesterone was higher in more than % of subjects than the serum level considered normal for sex, age, and stage of menstrual cycle. therefore, serum progesterone concentrations above the postmenopausal threshold are predictive of a higher incidence of edematous attacks in female patients with hae. in addition, multiple logistic regression analysis demonstrated a significantly lower attack frequency during -year follow-up in patients with higher ( nmol/l) shbg level (odds ratio, . confidence interval; . - . ; p = . ). this difference existed independently of age and danazol dose (fig ) . looking ahead. in view of these results, the monitoring of progesterone and shbg concentrations can prove useful in the prediction of hae attacks. further inves-tigations might address whether this increased progesterone is a cause or a result of frequent attacks, or whether progesterone itself exerts any direct effects on vascular permeability. last, progesterone's effects on relevant gene expression might represent another valuable avenue of exploration. influence of ocs or hrt on hereditary forms of recurrent angioedema (konrad bork, md,* and bettina fischer, md, mainz, germany) in this section, bork et al write of a systemic study undertaken after they noticed women in their clinic with new or worsened angioedema in response to oral contraception or hrt. their study crosses the barriers of c -inh deficiency and hae type classifications to investigate the effects of exogenous hormones on hae. the new type of inherited angioedema recently described by their group has clinical features highly similar to classic hae-i and hae-ii; however, it is not associated with a c -inh deficiency. currently, the genetic defect is unknown. until now, the disease has been observed exclusively in women , , ; however, in families the existence of clinically unaffected male carriers has been deduced. , in the following report, the authors show that estrogens play a similar role in hae-i and this new type of inherited angioedema, herein described as hae with normal c inhibitor (hae type iii) in accordance with the original report of this condition by bork et al. among their case series, the authors observed female patients with hae who had newly developed recurrent angioedema after receiving ocs or postmenopausal hrt. in addition, other women had a worsening of their preexisting angioedema after initiating ocs or hrt, suggested that these treatments might trigger hae. however, this possible relationship has not formally been investigated, nor is angioedema listed as an adverse effect of ocs or hrt. [ ] [ ] [ ] information regarding the relationship between hormone administration and the first occurrence or worsening of hae symptoms is limited to anecdotal reports. , - , , , , , , , , a, in this study, a systematic approach was used to obtain information about the relationship between the use of oc or hrt and the clinical manifestation of hae-i and hae with normal c -inh (hae type iii). methods. the case series by bork et al included women with recurrent angioedema who had taken oc, hrt, or both. of these, this study investigated women with hae-i and women with hae with normal c -inh (hae type iii). the ocs used by all patients were combinations of estrogen (ethinyl estradiol) and progestin. medications used for hrt contained only estrogens, as was standard medical practice in germany until recently. all patients underwent a thorough clinical examination, and a standardized medical and family history was taken. in particular, patients were questioned about the frequency, intensity, and organ involvement of their angioedema since the very first manifestation of their disease. the start date and duration of administration of sex hormones, with special emphasis on oc and hrt, were recorded. patients were asked whether their angioedema occurred shortly after beginning this hormonal medication. if yes, the time between starting oc or hrt and the onset of angioedema was recorded. patients who had recurrent angioedema before the intake of oc or hrt were asked whether their angioedema episodes were more frequent or severe after beginning treatment. concentrations of c -inh, c , and c q were assayed by radial immunodiffusion, and c -inh functional activity was determined by using the chromogenic substrate c h co-lys(e-cbo)-gly-arg-pna (immunochrom c -inh; immuno diagnostics, vienna, austria). , results. as shown in table v , oc, hrt, or both treatments were associated with angioedema attacks in % of women with hae-i and % of women with hae type iii. among the group of patients whose angioedema attacks were newly induced by estrogen-containing medications, there was a high preponderance of hae type iii, whereas in the patient group with an exacerbation of symptoms, there were more patients with hae-i (fig ) . in women- with hae-i and with hae type iii-occurrence of skin or gastrointestinal angioedema manifested for the first time after the administration of oc. because of the assumed intolerance, several women tried different oc formulations, and all trials were followed by recurrences of angioedema episodes so severe that all oc had to be discontinued. the interval between starting oc and episode occurrence ranged from to days (mean, days). in of the women, symptoms were limited to the period of oc administration and ended after discontinuation. all had hae type iii. in the other patients, angioedema attacks recurred despite oc discontinuation. episodes occurred less frequently and less severely in , whereas symptoms were unchanged in women. there was occurrence of angioedema for the first time after starting postmenopausal hrt. one woman initially developed recurrent angioedema after starting hrt with estradiol ( mg/d). the patient continued to take oral estradiol over a period of years. after a consultation in the outpatient clinic, hrt was discontinued. symptoms persisted but occurred less frequently and were milder. the patient's daughter had recurrent skin angioedema and gastrointestinal pain attacks since age years; therefore, with a normal c -inh concentration and activity in both mother and daughter, a diagnosis of hae type iii was assumed. in women, the administration of oc induced a severe exacerbation of their pre-existing angioedematous disease. because of this exacerbation, women switched to another oc, some trying many different preparations. all of these oc doses were followed by recurrences of severe angioedema such that oc had to be discontinued. five women with previous recurrent angioedema of the skin and the gastrointestinal tract developed an exacerbation of their disease after starting hrt. of these, only were eventually able to tolerate such therapy. in of the , their angioedema also had exacerbated when on oc several decades before. discussion. the results indicate that in % of women with hae-i and in % of women with hae type iii, the use of oc or hrt was associated with either the initial appearance of angioedema attacks or with a severe exacerbation of previously existing angioedema. correspondingly, although also taking oc or receiving hrt, % of women with hae-i and % of women with type iii tolerated these medications without any negative influence on their disease; their disease was neither estrogen-dependent nor estrogen-sensitive. the results demonstrate that estrogen sensitivity is not a feature specific to hae type iii because it occurs in a similar percentage of women with hae-i. results from several case reports have suggested that administration of estrogens may exacerbate clinical manifestations of hae-i. frank et al mentioned young women with an exacerbation of hae associated with the use of oc. similar effects of the administration of oc or hrt on the clinical condition of patients with hae-i have been documented by others. - , , , , a, as in the current study, initiation of hormonal therapy was associated with the onset of clinical disease or a marked increase in the frequency and severity of angioedema attacks. with respect to hae-ii, fletcher and weetman described a patient with hae-ii and coexisting turner syndrome who had a severe exacerbation of angioedema attacks after initiation of a hrt with a combination of conjugated estrogen and progesterone. another type of hae, not associated with a c -inh deficiency, has been recently described. , , this disease has been reported exclusively in women and was termed hae type iii by bork et al and estrogen-dependent inherited angioedema or estrogen-associated inherited angioedema by others. , ten of the women described as having hae type iii in a previous study by bork et al took estrogen-containing oc and reported either the first appearance of clinical symptoms or a severe exacerbation of the disease in association with this treatment. in additional families with this new type of hae, , of affected women, developed angioedema after starting oc, as did after initiating hrt. warin et al a described sisters with estrogen-associated urticaria/angioedema. with the exception of urticaria, the symptoms in these women closely resemble those of patients bork et al have described as having hae type iii. it is highly probable that the agent responsible for worsening of pre-existing angioedema or provocation of initial symptoms in the women studied was estrogen rather than progestin. six of the women evaluated in their study had new angioedema attacks or exacerbation of preexisting disease after receiving estrogens alone, and their symptoms did not differ from those of women who received estrogens plus progestins. moreover, progestins have even been used for treatment of hae caused by c -inh deficiency, and improvement of disease has been observed in at least some of the patients treated. [ ] [ ] [ ] most important in the current context, progestin treatment has not been associated with worsening of angioedema. the causative role of estrogens in disease exacerbation suspected in this and other reports is also supported by the fact that attenuated androgens are effective in preventing edema attacks in hae caused by c -inh deficiency. all of these observations are consistent with the view that estrogens and not progestins are responsible for provocation of angioedema attacks or worsening disease in women with inherited angioedema who take oc or receive hrt. the pathophysiological mechanisms underlying the occurrence of angioedema in association with estrogencontaining medications is not known. as stated, available evidence suggests that bradykinin is involved in the development of symptoms in patients with hae caused by c -inh deficiency. , , in addition to its inhibitory effects on c s and c r, c -inh blocks the activity of other plasma proteases including kallikrein and factor xiia/xiif, enzymes that participate in bradykinin formation. bradykinin is generated from hk by kallikrein; the activation of prekallikrein to kallikrein is mediated by activated factor xii. the degradation of bradykinin to des-arg bradykinin and finally vasoinactive peptides is mainly mediated by carboxypeptidase n (kininase i) and angiotensin i converting enzyme (kininase ii). estrogencontaining medications might favor edema formation by influencing both kinin generation and degradation. gordon et al showed that c -inh levels in the plasma of women taking oc are lowered. administration of estrogens also leads to increased factor xii levels, possibly via a functional estrogen response element in the promoter of the gene encoding factor xii. , , plasma concentrations of prekallikrein have also been shown to be increased in women using estrogen-containing oc. estrogens might also influence the degradation of bradykinin via their influence on ace. it has been demonstrated that hrt in postmenopausal women leads to a decrease in ace activity. , , nogawa et al also observed an increase in plasma bradykinin levels in such women, a finding consistent with the action of ace in the breakdown of bradykinin. the mediator responsible for edema formation in hae type iii is not known. however, many clinical similarities exist between hae-i and type iii, and the percentages of women whose disease is negatively affected by estrogencontaining medications is similar in the conditions. these facts could permit speculation that estrogen-associated effects in women with hae type iii may also be related to the kinin pathway. however, whether the influence of estrogen-containing oc or hrt on the kinin pathway actually plays a role in favoring angioedema formation in patients with inherited angioedema is far from being clear today. the current results suggest that among patients with underlying hae, administration of estrogen does not always result in the appearance or worsening of symptoms. for example, other factors that might predispose women with hae to new or exacerbated symptoms when treated with estrogens could include a functionally or quantitatively relevant genetic polymorphism in the kinin system. information about such polymorphisms might be important in differentiating women whose angioedema would be aggravated by administration of estrogen-containing medications. recent guidelines for urticaria and angioedema released by the british association of dermatologists define hae as angioedema without urticaria and highlight the need for a specific treatment for this condition. toward this end, the newly created european prehaeat network proposes a list of diagnostic criteria for angioedema caused by functional c -inh deficiency ( table i) . because of the tendency of nonallergic angioedema to mimic other disorders, these criteria rely on both clinical and laboratory assessments. in addition, in the case of asymptomatic or newborn family members of relatives with known hae, laboratory measures, and genetic analyses represent the sole means of learning whether they, too, have the disease and might have future attacks. these points underscore the importance of laboratory measures in the definitive diagnosis of hae and related disorders. laboratory findings not only permit more complete diagnosis and classification of angioedema caused by inadequate c -inh function but also form a key part of the evolving understanding of the genetic and biochemical aspects of these diseases. however, currently, no valuable laboratory criteria are available for the recently detected estrogen-dependent inherited angioedema. , , in the report that follows, varga et al, in an international collaboration arising out of the third c esterase inhibitor deficiency workshop, set forth guidelines for the proper laboratory investigation of suspected hae, aae, and related disorders. for diagnostic purposes, the c -inh protein phenotypes of the various types of angioedema without an allergic or pseudoallergic background are important (table vi) . mutations in the coding region of the c -inh gene leading to hae are all heterozygous conditions. (there is known exception, an italian family with a homozygous defect in the gene control region that might have evolved because of multiple consanguinity. however, even as a homozygous mutation, this defect did not lead to a complete deficiency of c -inh. , ) hae-i is defined as the presence of mutant c -inh gene whose gene product is undetectable in the circulation by routine methods. however, ultrasensitive methods might detect abnormal protein in some patients diagnosed with hae-i by routine analysis. in these patients, a reduced amount of normal c -inh is found (table vi) . hae-i may result from the production of a missing mutant protein, eg, from a mutation resulting in a stop codon, production of a mutant protein that is subsequently degraded intracellularly, production of a mutant protein that is not secreted and that might accumulate intracellularly, or production of a mutant protein that is released but rapidly catabolized or degraded in the circulation. hereditary angioedema type ii is defined as c -inh gene producing a dysfunctional protein that is released and coexists with the normal protein in patients' circulation. the current routine analytic tools for detecting c -inh antigen are unable to distinguish between normal and abnormal forms of circulating c -inh. as a consequence, normal or elevated concentrations of the protein are found (table vi) . notably, the distinction between hae-i and a transient rise in bradykinin can be measured during attacks. àat least patient with normal c q level was reported. §female patients and male patient with recurrent edema caused by androgen deficit. hae-ii is not absolute because some mutant protein can be detected in selected patients diagnosed with hae-i by using the most sensitive detection systems rather than routine methods. thus, the classification or diagnosis of hae-i or hae-ii might depend on the sensitivity of the analytic system used. independent of the type of hae, the affected heterozygous individuals show concentrations of native, functional c -inh far below % of normal. estrogen-dependent inherited angioedema patients (formerly hae type iii) show no abnormalities in c -inh concentration or function (table vi) . tissue swelling in the presence of normal c -inh concentration and function in this form of inherited angioedema might be understood through the assumption that attacks are related to transient increases in bradykinin concentration. for the classification of the acquired forms of angioedema, the presence of a pathogenic autoantibody to c -inh is relevant: type i shows no such pathogenic autoantibody, whereas in patients with type ii, such autoantibodies are detectable. however, the distinction between type i and type ii aae may become obsolete over a disease course, because patients might present with symptoms in the early stage of aae without detectable autoantibodies, whereas at later stages, autoantibodies can be detected. regarding the distinction between the types of aae, the diagnostic tools used and their sensitivities or accuracy of assessment might be crucial for classification. the presence of anti-c -inh autoantibodies causes hypercatabolism of c -inh; increased amounts of a cleaved c -inh with a reduced molecular weight are found in the circulation in patients with aae. finally, a few patients with recurrent, nonallergic angioedema in association with androgen deficit have been reported. the report mentions an affected male patient in addition to affected female patients; disease manifestation was reversible by appropriate correction of androgen levels. inheritance was not reported. it remains open whether this form fits best to aae. according to comprehensive family studies and case reports, the onset of hae-i can manifest during an affected child's first years of life. [ ] [ ] [ ] as early as , a high number of colicky babies were reported in families with hae. several authors have observed the onset of the disease beginning in children . years of age. , farkas et al found clinical manifestations of hae in children from . years. in a large hae-affected family in northern norway, children with the disease had their first typical symptoms at to years of age. however, in case, a mother noticed different phonation, edema of the mouth and tongue, and enlargement of the uvula in her -month-old child, arising during a period of primary teeth eruption. even if hae often makes it debut later, the statement in a review on the management of hae in pregnancy in the journal anesthesia that ''hae is always asymptomatic in infancy'' is misleading. early infancy is a likely age for the first manifestations, and physicians dealing with newborns with hae-affected parents should suspect an underlying c -inh deficiency as a potential cause of symptoms such as unspecific recurrent abdominal pain, diarrhea, upper respiratory disease, or skin rashes. therefore, a reliable diagnosis of children born to hae-affected parents is required as soon as possible. a c -inh concentration or functional activity < % of normal, in conjunction with low c concentration, supports a diagnosis of hae. tools for laboratory diagnosis. life-threatening laryngeal edema in association with inadequate c -inh function in untreated or poorly treated patients has been reported to have a lethality of % to %. , , because c -inh functional deficit might remain clinically silent for decades, the high lethality because of acute airway obstruction underscores the importance and significance of awareness of this syndrome and the availability of an expert laboratory to assess c -inh functional deficiency. laboratory analyses should be performed under at least minimal quality assurance conditions. test systems using calibration curves with only points must be considered as not meeting the standard; through points, every curve fitting is possible. the test system must also include quality control specimens: normal to high and low. a test is valid only if the quality control measurements are within a range of predefined values. it is advisable to assess the laboratory's own normal range. the number of normal sera/plasma used (in bern) for such a purpose is > . a standard specimen is prepared by pooling equal aliquots of each serum/plasma; next, testing individual plasma samples against the pool provides the normal range. material for analysis. for complement protein concentration measurements, edta-treated plasma is best suited. in the event that citrated plasma is used, normal ranges assessed with citrated plasma must be applied to correct for dilution by the citrate. for assessment of c -inh function, serum or citrated plasma is convenient; heparinized plasma should not be used. a diagnosis of any type of c -inh deficiency or dysfunction should be confirmed with at least assessments by using samples drawn to months apart with the patient in basal condition. for the early diagnosis of newborns born to parents with hae-i, sufficient blood can easily and painlessly be obtained from the placenta or the umbilical cord at birth. because newborns have a very high hematocrit of . and a consequently low plasma portion, the amount of protein will be diluted when using citrate as anticoagulant. this should be kept in mind when comparing reference values from newborns; citrate dilution of samples must also be considered with samples from adult patients. c -inh concentration. the concentration of c -inh protein can be assessed by several routine methods such as radial immunodiffusion, nephelometry, or elisa. the mean serum concentration in normal individuals is around . g/l. in the sera of newly diagnosed patients with hae-i or patients with hae-i patients who do not need medication because of mild symptoms, the mean concentration of c -inh protein is considerably reduced (table vii) . in patients with hae-ii, the serum c -inh concentration is approximately % or may be elevated to % to %. the latter excess is a consequence of an arg cys mutation of the reactive site, resulting in a free sulfhydryl group, enabling the mutated c -inh protein to bind to other serum proteins (table vii) . in the sera of patients with aae, the concentration of c -inh can be low or normal. normal antigenic amounts of c -inh can be detected if interaction with autoantibodies against the -kd c -inh protein results in a cleaved, nonfunctional -kd protein. c -inh function. c -inh function can be assessed by several methods. when analyzing samples from a patient with hae-i before and minutes after c -inh concentrate infusion, considerable differences emerge in rise of apparent c -inh function, depending on which method is applied (table viii) . c -inh function, when assessed by enzyme-based chromogenic assays, results in a linear relationship with the antigenic concentration of the c -inh protein. the chromogenic substrates used are etco-lys (e-cbo)-gly-arg-pna (formerly from immuno, vienna) or meco-lys (e-cbo)-gly-arg-pna (aventis behring, marburg, germany). some reports favor the assessment of c -inh function by loss of immunologically reactive c r. the method uses the natural substrate c r of c -inh as the indicator and works exclusively with endogenous complement. by using this inexpensive type of assay system, one can observe a functional threshold value for c -inh concentration and function. formation of c -inh-c r or c -inh-c s complexes can be assessed by an elisa test system that also uses the natural but exogenous substrate of c -inh as an indicator. by commercial elisa tests based on the detection of formed c -inh-c s complexes, the median functional concentration is less than % of normal at the time of diagnosis (table ix, (table ix) . this observation is somewhat discordant with numerous observations made in bern, reported in table viii . analysis of other complement components. to exclude or to confirm an acquired form of c -inh de- the concentration of c may be assessed by nephelometry or radial immunodiffusion by using polyclonal antibodies. a substantial part of the already diminished c actually consists of c split products, as can be measured with a monoclonal antibody specific for a neoepitope on c split-products. , the ratio of c splitproducts over c was . for a normal subject and . for the subject's cousin with hae. functionally active c can be determined by hemolytic assay. in aae with diminished c , the replacement of c must be considered, because the low c value may hamper c assessment. the antigenic or functional concentration of c is very low in almost all newly diagnosed or untreated cases of hae (table x) and aae. in hungary, a borderline c concentration was found in only of newly diagnosed patients with hae. thus, for an inexpensive follow-up, assessment of c concentration might suffice. indeed, increased c -inh concentration and function because of therapy that resulted in a reduction in edematous attacks was associated with a measurable increase of c concentration to nearly normal when using polyclonal antibodies to assess c (fig ) . it should be noted that such a test system is unable to distinguish between natural and cleaved c . a diminished antigenic level of c is extremely rare in hae, but in % to % of cases, even in of in newly diagnosed patients with hae, a slightly depressed concentration of c was measured in hungary. there is no correlation between the concentration of c q and c . detection of pathogenic anti-c -inh autoantibodies. the majority of patients with aae have autoantibodies to c -inh in their serum (aae type ii). these antibodies bind epitopes within or close to the reactive site of c -inh. anti-c -inh autoantibodies that bind to c-inh with different and generally low affinities can belong to each class (igg, iga, or igm). anti-c -inh autoantibodies are detected by elisa. diagnosis of c -inh deficiency in newborns at risk. in adults with angioedema caused by c -inh deficiency, determination of c and c -inh antigenic and functional concentrations nearly always allows a correct diagnosis. however, these complement components show considerable age-specific variations in children. the concentration of c -inh antigen measured in umbilical cord blood, as a rule, is approximately f the concentration found in adults. [ ] [ ] [ ] [ ] [ ] a reference range for the common functional c -inh test in newborn cord blood was published in . the percentiles are presented in table xi . functional assays even allow the prompt detection of the rare form of hae, ie, hae-ii, in which the antigenic concentration of c -inh is normal or even higher than normal, but mostly consists of a dysfunctional protein. whereas in the study the mean c -inh functional activity in healthy newborns was approximately % of the mean adult value, sonntag et al found a somewhat higher c -inh function (; %) in their study in citrated cord blood. during the child's first days, c -inh and c values rapidly rise and may reach . times values found in adults. andrew et al found that near-adult values are achieved for c -inh by months of life. in healthy premature infants, c -inh concentrations reached the normal adult range by week of age. norman et al found large variation in c , which in their opinion limits the diagnostic usefulness of quantitative c concentrations in children. diagnostic problems and strategy. several consequences result from the aforementioned experiences. to confirm or disprove a suspicion of clinical hae-i, measuring the concentration of c -inh protein is satisfactory; however, this is not adequate to detect hae-ii or aae type ii. assessment of c -inh function is therefore required. normal functional c -inh concentrations can be observed in patients with hae under therapy when measured by assays based on the formation of c -inh-c r/c s complexes. if the analyzing laboratory is unaware of this fact and receives a sample from a patient not known in the laboratory, a diagnosis of hae might be missed. in contrast, c -inh measurements using commercial enzyme-based chromogenic assays yielded a false diagnosis of hae in % of samples. the positive predictive value for a diagnosis of c -inh deficiency was only % with the chromogenic assay, whereas it was % with the c -inh-c s elisa test kit. the negative measurement of c concentration can be used as a screening marker when hae is suspected. however, low c is not sufficient for diagnosis, because this is seen in many conditions with classic pathway activation. if c and the antigenic concentration of c -inh are both normal, c -inh functional deficiency can be excluded. low c concentration is also a characteristic feature of aae. diminished amounts of c or its subcomponents, preferably c q, are indicative of an acquired c -inh deficiency. however, low c (q, r, s) together with low c is not a proof for aae (table vi) . a normal complement profile (normal antigenic c -inh, c q, and c ) was reported in patients with aae. , in the latter case, a transient improvement in c -inh function was associated with a transient loss of anti-c -inh autoantibodies. on the basis of the listed diagnostic possibilities, the authors suggest an algorithm for the diagnosis of any type of suspected c -inh deficiency, as shown in fig . the use of such an algorithm is strongly advised because, on follow-up of patients. according to the literature, c -inh concentrations in hae-i patients greater than % to % of normal are sufficient to provide inhibition of classical pathway activation, a finding that is confirmed by clinical observations. thus, in such patients, the dose of attenuated androgens should not be increased with the aim to reach antigenic c -inh levels approximately % of normal. ignorance of this fact might provoke higher and higher doses of, eg, attenuated androgens that bring no added benefit for c -inh function and might harm the patient's liver. enzyme-based chromogenic substrate assays are excellent for initial diagnosis of deficient c -inh function, but seem less well suited for monitoring during therapy (table viii) . measurement of c concentration is an inexpensive and generally useful parameter, even to follow up therapy, and especially in aae. concentrations of c close to the lower limit of normal, ie, at the lower % limit of confidence, have been found to indicate sufficient function of c -inh to allow patients to be attack-free (fig ) . in summation. for definitive diagnosis of angioedema associated with c -inh functional deficiency (hae-i, hae-ii, and aae), the assessment of c -inh concentration and/or function and, in some cases, the concentration of at least of the c subcomponents are the routine diagnostic tools. for laboratory diagnosis, measurements should be performed twice, at an interval of at least month, showing c -inh concentration and/or function < % of normal. laboratory diagnosis in newborns is difficult and needs special consideration. assessments of other complement parameters, such as c concentration, are to be considered surrogate markers, although excellent ones. no laboratory analyses to reveal estrogen-dependent inherited angioedema are thus far available. because purified c -inh concentrate is derived from human plasma, virus safety is critical. as mentioned earlier, in to , patients developed a non-a, non-b hepatitis related to the administration of c -inh concentrate, probably because of a contaminated batch. since the introduction of viral inactivation steps, transmissions of infectious agents have not been reported. side effects of c -inh concentrate are rare and include fever and headache. to date, the formation of autoantibodies to c -inh as a result of c -inh concentrate administration has not been observed in any patient with hae. c -inh concentrate is expensive because of high production costs, the low number of patients with hae, and the relatively low dose per patient compared with other blood products, such as those used in hemophilia. c -inh concentrate is now available in most countries, at least on a named-patient basis, but is not available in the united states. fresh frozen plasma. fresh frozen plasma (ffp) is also effective and can be used if c -inh concentrate cannot be obtained. because ffp is not virally inactivated, the risk of transmitting infectious agents is relatively high. the administered volume is larger and requires a longer infusion time, which might be critical in emergency situations. the use of ffp is associated with several adverse effects, including urticaria, anaphylactic shock, and hemolysis. other. corticosteroids and antihistamines are usually ineffective. attenuated androgens require at least a few days to become effective and therefore are usually not administered in acute attacks of hae. the same is typically true for antifibrinolytics; however, some patients with mild attacks respond to g tranexamic acid by mouth every to hours for to hours. for abdominal pain during attacks, spasmolytics such as butylscopolamine may be used symptomatically and are of benefit in milder attacks. future treatment options for acute edema episodes. kallikrein inhibitors. the candidate mediator of angioedema caused by c -inh deficiency is bradykinin, released upon kallikrein activation. more than years ago, aprotinin, a kallikrein inhibitor extracted from bovine lungs, was shown to be effective in treating hae attacks. because of the risk of fatal anaphylactic reactions, its use was discontinued after substitutive therapy with c -inh concentrate became available. a novel kallikrein inhibitor has recently been developed. it is a synthetic kunitz domain protein designed by phage display technology to bind to human kallikrein. it has a high affinity and is highly selective for kallikrein. it is effective in abating acute attacks of hae but is not yet commercially available. bradykinin antagonists. because bradykinin is assumed to be the key mediator of hae, blocking its b receptors might be therapeutically useful. icatibant is a synthetic peptide with a structure similar to bradykinin. however, it contains nonproteinogenic amino acids and is not degraded by the main bradykinin cleaving enzymes, carboxypeptidase and ace. a potent antagonist, icatibant has the same affinity for b receptors as bradykinin across several tested species, including human beings. it is specific for b bradykinin receptors and does not interact with receptors of other peptides. preliminary studies were performed in human beings with asthma and seasonal allergic rhinitis, , and icatibant was also shown effective in reversing increased vascular permeability in the murine hae model. recently, the results of a phase ii single-dose trial for the treatment of acute hae attacks in patients were reported. in this european trial conducted by bork et al. a icatibant appeared effective and well tolerated. recombinant c -inh. during recent years, a recombinant c -inh produced in the milk of transgenic rabbits has been developed. however, because of glycosylation differences, it has a shorter half-life than c -inh derived from human plasma. initial results in patients with hae concerning tolerability and safety are encouraging. the interim results of a phase ii trial conducted by levi et al a in acute attacks of hae appear positive. long-term prophylaxis. long-term prophylactic treatment is indicated for patients with many harmful and disturbing edema episodes, usually patients with more than attack per month. long-term prophylaxis may be performed with attenuated androgens, antifibrinolytic agents, and c -inh concentrate. all of these medications are associated with potential adverse effects, limiting their use as standard, life-long prophylactic treatment. attenuated androgens. in , spaulding reported the striking efficacy of methyltestosterone in hae, describing both a decrease in the severity and number of attacks. later, further androgens, fluoxymestrone in men and oxymetholone in women (with fewer virilizing effects than methyltestosterone), were tested. in , stanozolol was synthesized, having of the largest anabolic/androgenic ratios. in , danazol, a synthetic analogue of ethinyltestosterone and less virilizing than the early synthetic androgens, was developed. in , the attenuated androgen danazol was shown to considerably reduce the number of hae attacks. since then, attenuated androgens ( -a alkylated androgens), mostly danazol or stanozolol, have been widely used for long-term prophylaxis in hae. , although -a alkylated androgens are more efficient than other androgens, they are metabolized by the liver. thus, hepatotoxicity and the induction of liver cell adenoma or carcinoma are major concerns. androgens' mechanisms for exerting beneficial effects in patients with hae are unknown. major contraindications for treatment with androgens are pregnancy, lactation, childhood, and prostate cancer. however, as discussed later, there are a few reports on the successful use of androgen treatment in children. danazol. a widely used regimen is to start with an induction dose of to mg daily for weeks and then taper down to the lowest maintenance dose that provides symptomatic relief. most patients' hae is controlled with to mg daily or every other day. long-term administration may be associated with several adverse effects, including weight gain, amenorrhea, decreased libido, menstrual irregularities, virilization in women (hirsutism, deepening of the voice, and decreased breast size), acne, muscle cramps, myalgia, fatigue, headaches, hemorrhagic cystitis, arterial hypertension, and hepatic necrosis or cholestasis. [ ] [ ] [ ] [ ] recently, hepatocellular adenoma and liver cell carcinoma have been reported in patients with hae taking danazol for many years. [ ] [ ] [ ] laboratory changes may include decreased sex-hormone binding protein and thyroxine-binding protein, increased aminotransferases and cholesterol, and polyglobulia. stanozolol. stanozolol is used in initial doses of to mg per day for month, then tapered to the minimal effective dose ( . - mg daily). potential adverse effects may be similar to those of danazol but seem to occur less frequently. however, this needs further confirmation, because fewer patients treated with stanozolol have been reported in the literature compared with patients receiving danazol. other androgens. several patients have been treated with an oxandrolone. a small number of patients do not respond to androgens. antifibrinolytic agents. antifibrinolytic agents may also be used for the continuous prophylactic treatment of hae. however, their efficacy is lower than that of androgens. in about % of patients, attacks occur less frequently and are usually milder. a considerable reduction of the number and severity of attacks is reached in only approximately % of patients. if long-term prophylaxis is necessary in children, tranexamic acid may be preferable to androgen treatment because androgen treatment should be avoided in childhood. presumably, antifibrinolytic agents act through the inhibition of plasmin. all antifibrinolytics bear the risk of thromboembolic events. e-aminocaproic acid is effective and reduces the frequency of attacks. the daily dose is usually to g in equally divided doses. it is associated with various side effects such as thrombosis, postural hypotension, muscular pain and weakness associated with an increase in creatine kinase and aldolase, anal pruritus, and myositis. because of the many adverse effects associated with e-aminocaproic acid, tranexamic acid is the antifibrinolytic of choice. the daily tranexamic acid dose is usually to g per day. side effects are uncommon but include abdominal discomfort, mild transient diarrhea, nausea, headache, and anal pruritus. c -inh concentrate. not only an acute attack therapy, c -inh concentrate has been found effective in the longterm prophylaxis of hae. in , the first patients were treated with u c -inh concentrate or times weekly for year or more. a report of patients who received c -inh concentrate twice weekly was published in . short-term prophylaxis before surgery. because of the risk of developing angioedema and possibly experiencing life-threatening laryngeal edema after dental surgery, endoscopy, endotracheal intubation, and other surgical procedures, preoperative treatment has been attempted. to date, available information is limited to case reports or small patient series. furthermore, even in the absence of preoperative prophylaxis, not all patients with hae develop edema attacks after surgery. patients who undergo multiple dental surgeries may develop edema episodes after some tooth extractions but not others. it is therefore difficult to assess the efficacy of a recommended preoperative prophylaxis until studies with larger numbers of patients are available. such prophylaxis has been performed with ffp, antifibrinolytics, attenuated androgens, and c -inh concentrate. in several patients, ffp has been used prophylactically before dental surgery; in some patients, mild attacks occurred despite pretreatment with ffp. [ ] [ ] [ ] antifibrinolytics such as e-aminocaproic acid and tranexamic acid , have also been used for preoperative prophylaxis in some patients. because antifibrinolytics are less effective in long-term hae prophylaxis, preoperative treatment focuses on attenuated androgens and c -inh concentrate. in patients with known hae caused by c -inh deficiency, preoperative prophylaxis with attenuated androgens, either danazol or stanozolol, has been shown to be effective in many patients, , [ ] [ ] [ ] although some patients nonetheless developed swelling. , for short-term prophylaxis, attenuated androgens may be administered from days before to days after the event, danazol at a dose of mg/kg/d, maximum mg per day, or stanozolol at a dose of mg per day. currently, c -inh concentrate has been used as preoperative prophylaxis in only a few patients; for this purpose, the dose of c -inh concentrate was or u. , the risk of developing angioedema attacks secondary to dental surgery or other oral manipulations cannot be completely avoided by preoperative prophylaxis. as such, it is important to inform the patient that an angioedema attack might occur, to describe the clinical symptoms of laryngeal edema, and to educate the patient about what to do in case a laryngeal edema occurs. in this section, cicardi et al describe the typical treatment of aae and discuss the special considerations unique to this disorder. from pathogenesis to treatment. like other acquired forms of protein deficiencies, the course of acquired c -inh deficiency can be related to the course of the underlying disease. the possibility of reversing the biochemical and/or clinical abnormalities of acquired c -inh deficiency by curing the associated disease was reported by cohen et al and subsequently confirmed. , , [ ] [ ] [ ] however, the response can be temporary, even without evidence of relapse of the associated disease. analogous to hae, patients with acquired c -inh deficiency have received attenuated androgens for prophylaxis and c -inh concentrate to treat acute attacks. , nevertheless, these patients are frequently resistant to attenuated androgens, , whereas they tend to benefit from antifibrinolytic agents. , , antifibrinolytics are thus more effective for long-term prophylaxis in this population and represent the first choice for patients with acquired c -inh deficiency. replacement therapy with c -inh plasma concentrate is the treatment of choice for life-threatening laryngeal attacks. however, patients with aae partially resistant to this treatment have been reported. , , , the response to treatment differs from hae because of the rapid catabolism of c -inh that characterizes aae. higher doses of c -inh plasma concentrate were required in patients with a slow response to treatment. slow responsiveness is a high-risk condition because it seems to increase with subsequent treatment. a new peptide, dx- , a synthetic kallikrein inhibitor based on the same functional domain (kunitz domain) of aprotinin, is under study for treating angioedema in patients with hae. , by inhibiting kallikrein activation, this peptide can stop generation of the vasoactive peptide bradykinin from hk, for hae and related conditions, plasma-derived c -inh concentrate remains the acute attack therapy of choice. in this section, juers and gröner detail the steps that are taken to ensure the high margin of virus safety for berinert p, produced at aventis behring gmbh, a zlb behring company. before the advent of specific therapy, the hae mortality rate caused by laryngeal edema was as high as %; even today, rates as high as % mortality associated with hae have been published. , the introduction of c -inh concentrate (berinert in in germany) was followed by the german registration of a pasteurized c -inh concentrate (berinert p) in . these drugs significantly changed the treatment procedures for acute attacks in patients with hae, becoming the treatment standard in the countries where berinert p is available. in countries where berinert p or another c -inh concentrate was not available (for example, the united states), treatment of acute attacks was and sometimes still is usually performed with ffp. however, during the preparation of ffp, no reduction of potentially present viruses is achieved, in contrast with the plasma-derived c -inh concentrate berinert p. similarly, although solvent-detergent treated ffp might seem an alternative, this treatment does not inactivate nonenveloped viruses. the following sections describe the current status of virus safety of berinert p. methods. berinert p is produced from pooled human plasma, primarily from source plasma, ie, plasma collected by plasmapheresis. in general, plasma-derived products may potentially transmit infectious and/or pathogenic viruses. this risk is minimized by careful selection of donor centers with regard to virus marker rates (epidemiology) and careful selection of donors; each donor, including qualified donors (ie, repeat donors), is evaluated before each donation for vital signs including temperature and blood pressure and subjected to an intensive questionnaire. details have been described recently. furthermore, every donation is tested with serologic methods for hepatitis b surface antigen (hbsag) and antibodies against hcv, hiv- , and hiv- as well as elevated alanine aminotransferase levels. in addition, sample pools of donations are tested by using nucleic acid amplification technique (nat)/pcr for genomic material of hepatitis a virus (hav), hepatitis b virus (hbv), hcv, hiv- , and high titers of parvovirus b (b v). reactive donations are discarded. furthermore, the plasma pool for fractionation is tested and released for further processing only if the pool is nonreactive (negative) for hbsag and nonreactive for antibodies against hcv, and hiv- , and hiv- as well as nonreactive for hav rna, hbv dna, hcv rna, and hiv- rna, and for b v dna not exceeding iu/ml. the third main step ensuring virus safety is the elimination and inactivation of possible viral contaminants by the manufacturing procedure, whose capacity for virus reduction is tested in virus validation studies. the virus inactivation/removal steps used in the manufacture of berinert p use treatment in aqueous stabilized solution at °c for hours (pasteurization) and specific chromatography. results. in virus validation studies designed to assess the capacity of the manufacturing process to inactivate and/or eliminate intentionally added virus, high overall virus reduction factors were demonstrated (table xii) . these virus validation studies were performed in accordance with the applicable committee for proprietary medicinal products (cpmp) notes for guidance. viruses covering a wide range of physicochemical properties were used in these studies (hiv- and hav are relevant viruses, bovine viral diarrhea virus [bvdv] and canine parvovirus [cpv] are specific model viruses for hcv and b v, respectively, and pseudorabies virus (prv) is a nonspecific model virus for large enveloped dna viruses). the results demonstrate that the pasteurization and the manufacturing process of berinert p effectively inactivate and/or eliminate relevant human pathogenic viruses. to demonstrate that the manufacturing steps explored in the virus validation studies were very robust and that variations in the manufacturing procedure had no effect on the virus reduction capacity, specific studies covering parameters beyond production specification were performed. these studies showed that even under these conditions, a very effective virus inactivation occurred. regarding analysis of postmarketing surveillance for berinert p from until now, no cases of proven virus transmission, according to the assessment of the global pharmacovigilance department at aventis behring, were reported, although a total of almost million units of berinert p were administered. discussion. the outbreak of aids in the s and the resulting infections caused by blood transfusion and treatment with certain plasma derivatives, mainly factor viii concentrates, showed that virus safety in plasma proteins is a field of permanent attention, screening, and continuous research. since the former behringwerke ag introduced pasteurization in the s (developed by heimburger primarily for hemophilia products), no hiv transmission via the company's pasteurized products has occurred. [ ] [ ] [ ] this pasteurization procedure has been adapted to aventis behring's plasma products, including c -inh concentrate. the pasteurization procedure has been shown to have an excellent track record of virus safety, and the validity of the pasteurization for virus inactivation was confirmed in scientific articles. [ ] [ ] [ ] pasteurization is a very effective and robust virus inactivation method, but there are also limits. animal parvovirus, for example, is inactivated by pasteurization only to a certain degree. however, because of recent findings, it should be kept in mind that the human b v is heat-sensitive and can be effectively inactivated by pasteurization; preliminary data from the group's own experiments (data not shown) also confirm this observation for stabilized aqueous solutions. in addition, antibodies against b v in the plasma pool for fractionation and in intermediates derived from that pool will neutralize b v to a high degree. furthermore, regarding cpv, the specific chromatographic step was validated for its capacity to remove cpv, and a very effective and robust removal could be demonstrated. in conclusion, on the basis of the data from virus validation studies, independent effective virus reduction steps in the production procedure for berinert p, effective for a wide range of enveloped and nonenveloped viruses, could be demonstrated. as outlined in the cpmp guideline (cpmp/bwp/ / ), the aim of virus validation studies is ''(i) to provide evidence that the production process will effectively inactivate/remove viruses which are either known to contaminate the starting materials, or which could conceivably do so, and (ii) provide indirect evidence that the production process might inactivate/remove novel or unpredictable virus contamination.'' so far, all new emerging viruses or members of the same virus family, which may be of severe concern in a blood transfusion setting, were shown to be reduced through manufacturing procedures. this was especially demonstrated for west patients had medical, psychologic, and economic difficulties attributable to hae or aae. among patients with a home supply of c -inh concentrate, patients had avoidable adverse effects. one developed severe anxiety and depression requiring outpatient psychiatric care on being diagnosed with aae. another was unable to work as a baker on several occasions because of severe hand edema. the third had frequent angioedema and longstanding severe temperomandibular pain, which resolved when she was started on regular c -inh concentrate. this patient required frequent absences from work. of patients who did not have access to c -inh concentrate at home, patients had avoidable adverse effects. as fig illustrates, patients had avoidable admissions to hospital, attributable to c -inh concentrate treatment not being given or being delayed, resulting in suboptimal response to therapy. one man required intubation and a -day stay in the intensive care unit when no c -inh concentrate could be found in the department. the same man was listed for surgery (tracheotomy for laryngeal edema and laparotomy for acute abdominal pain and guarding) on occasions despite carrying a letter giving advice concerning the emergency management of his aae. on both occasions, after calls to his regular physician, he responded to treatment with c -inh concentrate, and surgery was canceled. in addition, patients were treated with ffp, a product that is likely to be less safe and possibly less efficacious than c -inh concentrate. , one woman received an average of units of ffp each month at her local emergency department, where personnel were unwilling to prescribe c -inh concentrate. one patient had severe anxiety and depression and was unable to work. she improved when started on the home therapy program. however, she had a severe exacerbation of her psychiatric condition when funding difficulties put the future of her home therapy in doubt. four additional patients had day or more per month of absence from work or school because of symptoms, and patient requested a private prescription of c -inh concentrate to enable him to travel abroad safely, despite being entitled to c -inh concentrate funded by the uk national health service. discussion. many uk patients have difficulty in accessing appropriate emergency treatment for hae or aae, with avoidable medical, psychiatric, and economic consequences. the events recorded here are the most serious. many patients have ongoing anxiety concerning access to treatment. some are reluctant to travel; many accept frequent painful attacks and disruption of education or work without complaint (price; uk hae network; personal communication, june ). this audit suggests that patients who carried their own supplies of c -inh concentrate were less likely to have difficulties in accessing appropriate care and were less likely to have ill effects. reasons for pcts' reluctance to fund home c -inh concentrate included lack of familiarity with the use of c -inh, reluctance to use a product not licensed in the uk, lack of national management guidelines for hae and aae, lack of agreement among uk specialists of the necessity for a home c -inh supply, expense of the product, and the mistaken belief that the hospital had funds to prescribe c -inh concentrate. on the basis of their experience, the author's group recommends that all patients with hae or aae should carry their own supply of c -inh concentrate and that their local hospital should stock additional supplies so that this can be replaced immediately after use. to improve access in the uk, the author recommends negotiation with uk primary care trusts to fund costs of hae or aae treatment for all patients, development of national and international guidelines concerning hae management and supply of c -inh concentrate, and improved education for medical staff. in this companion piece, longhurst and o'grady describe a program allowing patients with hae to selfinfuse c -inh concentrate and detail its outcome. the benefit and safety of home therapy is well established for many patient groups, including those requiring blood products such as igs for antibody deficiency and factor viii for hemophilia a. , in the uk, patients with hae are advised to attend their local emergency department for treatment of severe or potentially life-threatening angioedema attacks. patients with frequent attacks are also offered prophylaxis with attenuated androgens, such as danazol, or tranexamic acid. , despite prophylaxis, a minority of patients with c -inh deficiency require repeated emergency hospital visits for administration of c -inh concentrate to treat life-threatening laryngeal swelling and severe abdominal pain. waiting times in uk emergency departments can be several hours, and personnel may be unfamiliar with managing hae. as a result, hospital attendance is disruptive to the patient, who may be tempted to wait until the attack is severe, with potentially serious consequences. methods. the authors have introduced a home therapy program to improve access to treatment, thereby improving quality of life for people with severe hae and reducing the use of emergency services. patients entering the home therapy program must fulfill the following criteria: ( ) proven c -inh deficiency ( ) prophylactic therapy optimal ( ) required infusions of c -inh concentrate at least every months ( ) infusion partner available ( ) commitment to program of patients considered for the program, patients from the same family were already self-administering (under the care of their general practitioner) and received a refresher course. six further patients' symptoms were not controlled after optimizing prophylactic therapy. of these, patients met the criteria for inclusion, patient was refused funding, and patient did not wish to have home therapy. all patients were trained and successfully use c -inh home therapy. a minimum of hours training over a period of visits was followed by a formal assessment of patient and infusion partner. patients kept a record sheet of all attacks and contacted the specialist nurse if they infused. records were reviewed and infusion technique (using sterile saline) and knowledge were assessed at -month intervals. case reports. patient a, a -year-old female patient, was initially treated with danazol. this was stopped because of persistently abnormal liver function. tranexamic acid was only partially effective. she had attacks of severe angioedema affecting the skin, abdomen, and occasionally the larynx, requiring treatment with iu of c -inh concentrate at least once a week. intractable temperomandibular pain developed. she was started on c -inh concentrate (berinert p) iu twice weekly. her temperomandibular pain improved, and she had fewer angioedema attacks. patient b, a -year-old female patient, had discontinued danazol because of excessive weight gain and voice abnormalities. she had subsequently received depot methyltestosterone mg subcutaneously every months without side effects for many years, in combination with tranexamic acid g twice daily. however, she had cutaneous and severe abdominal angioedema every to days. her mother had died of hae despite knowledge of the diagnosis, and the patient developed severe anxiety, necessitating specialist psychiatric treatment. after inclusion on the home therapy program, the patient infused to iu c -inh concentrate approximately once a month, as required for severe symptoms, and reported an improvement in her quality of life. however, the threat of suspension of her home therapy because of funding problems led to an increase in her anxiety and the number of angioedema attacks, a well-recognized consequence of psychologic distress, such that iu of c -inh concentrate was required once or twice per week. resolution of the funding problems led to an immediate restoration of well-being and reduction in use of medical services, but she continued to require to infusions of iu c -inh concentrate per month. patient c, a -year-old female patient, had frequent abdominal hae attacks, requiring an average of days off work per month. she attended her local hospital emergency service approximately once per month and on each occasion received units of ffp. after inclusion in the home therapy program, she reported a greatly improved quality of life, no time off work, and no further exposure to ffp. she infused to iu of c -inh concentrate approximately once per month. patient d, a -year-old female patient who had infrequent attacks without prophylaxis, developed severe abdominal attacks, occurring once or twice a week from the th week of her first pregnancy. she was treated with iu of c -inh concentrate twice weekly from week until weeks after delivery of a healthy boy. she had only mild abdominal attack during her pregnancy and continued full time work until the th week. after delivery, her attacks were infrequent, despite discontinuation of c -inh concentrate. she chose to withdraw from the home therapy program but plans to retrain if necessary during any future pregnancies. family e, a -year-old mother, her sons ( and years), and her daughter ( years), had been trained in the use of c -inh concentrate by their family doctor, and had been using this for treatment of acute attacks for years without significant side effects. this family was the inspiration for the establishment of home therapy service. all members of this family have been able to complete tertiary education, pursue demanding professional careers, and travel extensively, including to regions where medical care is not readily available, despite, in case, severe hae. results. in all cases, home therapy resulted in improved quality of life as a result of improved access to treatment and reduced use of emergency services. no patient used c -inh concentrate inappropriately. no adverse incidents occurred. discussion. despite the existence of successful home therapy programs for ig and factor viii, patients also retain the option of hospital treatment if conditions for home therapy are not optimal. these results show that use of c -inh concentrate is appropriate and that, in general, c -inh concentrate usage is not increased by home therapy. use of emergency and routine medical services and time off work is reduced, and all patients report great improvements in their quality of life. (jan h. nuijens, md, phd, and c. erik hack, md, phd,* leiden and amsterdam, the netherlands) as discussed, recombinant c -inh is among the therapies currently being investigated for the future treatment of hae. in this section, nuijens and hack describe the potential advantages of such a therapy and the steps being taken toward realizing this means of treatment. functional deficiency of the plasma protein c -inh in patients with hae and aae accounts for the recurrent attacks of localized submucosal and/or subcutaneous swelling that can cause serious discomfort and are sometimes life-threatening. intravenous supplementation with human plasma-derived c -inh is the preferred treatment of severe attacks of angioedema, and this treatment appears very effective and well tolerated. , [ ] [ ] [ ] however, plasma-derived products are of limited availability for reasons related to their human blood origin, supply, safety concerns, and/or development costs. three different plasma-derived c -inh preparations are licensed by regulatory authorities in to european countries each. even where licensed and/or available, treatment with plasma-derived c -inh often is restricted to laryngeal attacks with risk for suffocation, severe abdominal attacks, and preoperative prophylactic use. these restrictions have grown out of viral safety concerns, although the risks of transmission of human pathogenic viruses may nowadays be considered minimal. long-term prophylactic treatment with plasmaderived c -inh, although apparently safe and efficacious, occurs only on a very small scale. plasma-derived c -inh has also been tried in several serious pathological conditions such as sepsis, vascular leakage syndromes, and acute myocardial infarction, ie, in conditions associated with excessive activation of the complement and contact systems and/or absolute or relative deficiency of functional c -inh. , although its application in these conditions appeared safe, its efficacy was not yet demonstrated unequivocally. rationale for production of recombinant human c -inh. until very recently, all animal and clinical studies with c -inh used human blood plasma-derived c -inh. although the advantage of this c -inh preparation is that it has normal clearance kinetics, normal specificity, and no antigenicity, the apparent disadvantages are that the source material is limited and potentially contaminated with blood-borne pathogens. in particular, the limited source is an obstacle to develop c -inh for indications other than hae because these indications require more c -inh than for the treatment of hae (eg, approximately - u/kg body weight for an hae attack vs u/kg for the treatment of myocardial infarction). hence, availability of recombinant c -inh is warranted. major advantages of production of human c -inh via recombinant dna technology are that the recombinant material is not derived from a human source, that the production can more easily be controlled, and that the production can be scaled up to meet the requirements for the investigational treatment of hae as well as other potential indications. on cloning of c -inh by bock et al, recombinant functional c -inh was expressed in cos- cells by eldering et al in the late s. the c -inh expressed in this way was fully active, but expression levels were low (approximately mg/l), thus allowing only structurefunction relationship studies. expression in escherichia coli was also attempted. though much larger quantities of protein were expressed, only a fraction (a few percent) was active, whereas the majority was denatured (hack et al, unpublished data, january ). similar observations have been made by lamark et al, although these investigators could obtain more functional c -inh preparations by using different vectors and different strains of e coli. hence, other expression systems are needed to produce recombinant c -inh for clinical applications. two such systems, expression in transgenic animals and a yeast system, are discussed here. generation of recombinant human c -inh transgenic rabbits and pharmaceutical development of transgenic recombinant human c -inh. expression of c inh via the mammary gland of transgenic animals was first attempted in rabbits, among others, because of rabbits' short generation time. in addition, a lactating rabbit may produce l milk per year; because of this, transgenic rabbits may yield sufficient quantities of recombinant human c -inh (rhc -inh) for the hae indication. a mammary gland-specific expression vector containing a bovine milk-specific promoter sequence (as casein) functionally linked to the gene encoding human c -inh was introduced into the rabbit germ line by microinjection of fertilized oocytes isolated from superovulated female donor animals. a production line was selected with a relatively low transgene copy number and that expressed rhc -inh in milk at levels exceeding g/l. to ensure continuous availability of a herd of transgenic animals from the selected founder line and to restrict potential rearrangements of the transgene through subsequent generations, a master transgenic bank and a manufacturing working transgenic bank have been established. production herds are housed in specially designed and validated facilities subject to stringent environmental monitoring. animal health is continuously monitored, and the rabbits are regularly tested for the presence of a wide range of rabbit and human pathogens. rabbits showing any abnormality are excluded from being milked. this health monitoring system ensures that the production herd is a specified pathogen-free rabbit population. milk is collected by using a specially designed rabbit-milking device. the raw milk is analyzed for compliance with internal criteria before release for downstream processing. the purification of rhc -inh from raw milk is composed of skimming the milk, cationexchange chromatography, viral inactivation by solventdetergent incubation, anion-exchange chromatography, zinc-chelating chromatography, virus removal by nanofiltration, ultrafiltration, final filtration, filling, and lyophilization. stability studies of vialed rhc -inh have indicated a shelf life of at least years. the downstream process of rhc -inh has been the subject of a virus removal or inactivation validation study using a panel of viruses representing different viral classes and sizes (enveloped and nonenveloped, single-stranded and double-stranded rna and dna viruses). characterization of transgenic rhc -inh protein. nonreduced and reduced sds-page of rhc -inh showed rather broad (single-chain) protein bands with an estimated relative molecular mass (mr) of approximately , . the observed heterogeneity in mr on sds-page of rhc -inh as well as the difference in mr (approximately , and , on nonreduced and reduced sds-page) of rhc -inh and plasma-derived c -inh most likely results from differential glycosylation. this follows from the observation that both the n-terminus and c-terminus of the protein are intact as determined by n-terminal and c-terminal amino acid sequence analyses. moreover, liquid chromatographymass spectrometry peptide mapping data revealed approximately % recovery of the amino acid sequence of rhc -inh and conformity with the expected amino acid sequence. the theoretical molecular mass of the polypeptide backbone of c -inh is approximately kd. the use of matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed a molecular mass of approximately kd and approximately kd for rhc -inh and plasma-derived c -inh, respectively, and suggests that approximately % and approximately % of the molecular mass of rhc -inh and plasmaderived c -inh can be ascribed to carbohydrate structures. monosaccharide analysis of the major monosaccharides in glycoproteins (fuc, galnac, glcnac, gal, man, and neu ac) confirmed that rhc -inh consists of approximately % carbohydrates. n-glycolyl neuraminic acid, a sialic acid that is not found in normal human tissue, as well as a-galactosyl were not detected on monosaccharide analysis of rhc -inh. mass spectrometric and nuclear magnetic resonance analyses of the glycans released from rhc -inh indeed showed the presence of at least different o-linked and at least different n-linked carbohydrate structures, including oligomannose and hybrid and complex-type carbohydrates. n-linked glycan profiling indicated that most of the n-linked complex structures of rhc -inh appeared to be monosialylated. the inhibitory potency of rhc -inh toward the target proteases c s, kallikrein, factor xia, and factor xiia was found to be comparable with that of plasma-derived c -inh. in addition, no difference in the inhibition of plasmin and thrombin was observed. physicochemical characterization to demonstrate identity, purity, potency, and consistency between different batches of rhc -inh revealed that it was consistently being produced as an active, intact molecule, with purity exceeding . %, containing no detectable amounts of cleaved rhc -inh, endogenous rabbit c -inh (less than ppm), and rabbit milk proteins (less than ppm). summary of experiments in animals with transgenic rhc -inh. the purpose of the preclinical safety program was to assess the initial safe dose in human beings and to identify safety parameters for clinical monitoring. the package contained safety pharmacology in dogs ( u/ kg), acute single-dose toxicity in rats and dogs ( - u/kg), -week subchronic toxicity in rats ( - u/kg), repeated dose toxicity in dogs ( u/kg/day for days), and local tolerance in rabbits (intravenous, perivenous, or intra-arterial microinfusions). overall, no adverse clinical signs were observed and no or negligible effects were reported on clinical laboratory parameters. on the macroscopic level, some minor findings were reported, but histopathological analyses revealed no treatment-related findings. rhc -inh was cleared more rapidly from the circulation of the rat and the dog in comparison with plasma-derived c -inh. in rat serum samples from animals in the highest dosage group (single-dose, u/kg; subchronic, u/kg), a mild specific immune response was noted. there was no evidence for the generation of neutralizing antibodies. on the basis of these studies, it was concluded that rhc -inh may be considered safe. phase i study of rhc -inh in asymptomatic subjects with hae. an open-label phase i study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending intravenous doses of rhc -inh ( . - u/kg) in asymptomatic subjects with hae was conducted at the centre for human drug research (leiden, the netherlands; cohen et al). the study subjects were invited to participate by levi (department of internal medicine, academic medical centre, amsterdam, the netherlands) in close communication with the dutch association of patients with hae and quincke's edema. all subjects were without angioedema symptoms at the time of the trial and received rhc -inh on occasions with an interval of at least weeks (table xiii) . the subjects were assessed clinically, and blood samples were taken to evaluate laboratory safety parameters and immunogenicity as well as the clearance and biological effects of rhc -inh. assays of pharmacokinetics and pharmacodynamics parameters were performed at sanquin research (hack, amsterdam, the netherlands). safety results were as follows. rhc -inh appeared safe and well tolerated: no probable product-related adverse events (eg, allergic reactions) or changes in electrocardiogram, vital signs, or routine laboratory parameters were observed. neither clinically significant increases in anti-c -inh or antirabbit milk protein nor evidence of c -inh neutralizing antibodies was noted. pharmacokinetics results were as follows. infusion of rhc -inh resulted in dose-dependent increases of functional c -inh response parameters maximum concentration (above baseline), area under effect curve above baseline, dose-normalized area under effect curve, and of time above . u/ml, whereas dose-normalized maximum concentration appeared constant (approximately . u/ml/u/kg). the profiles of functional c -inh showed a full initial recovery and a dose-dependent clearance of rhc -inh that indicated a saturable mechanism of elimination. after the infusion of rhc -inh at u/kg, a clearance of approximately ml/min, a half-life of about hours, a volume of distribution of about l, and an endogenous infusion rate of about u/min were observed by using a standard -compartment model of analysis. application of a -compartment model with michaelis-menten elimination provided dose-independent estimates of v max , k m , volume of distribution, and endogenous infusion rate of approximately u/min, approximately . u/ml, approximately l, and approximately u/min, respectively. thus, the half-life of rhc -inh in the subjects with hae appeared reduced compared with historical data of plasma-derived c -inh. , , this likely relates to the differential glycosylation and lower degree of sialylation in rhc -inh. the increased clearance of rhc -inh from the bloodstream likely occurs mainly through endocytic asialoglycoprotein and mannose receptors on hepato- cytes. after dosing with rhc -inh at u/kg, functional c -inh was at least -fold the normal level for about hours and remained above . u/ml for about hours. activation of the cascade systems releasing inflammatory mediators responsible for the generation of edema is thought to be sufficiently controlled with functional c -inh levels exceeding . u/ml. pharmacodynamics results were as follows. rhc -inh displayed biological activity in the subjects as evidenced by plasma c increases and inhibition of c cleavage. the dose-dependent increase in mean normalized c was highly variable within dosage groups and was approximately -fold increased after the infusion of rhc -inh at u/kg. c peak levels occurred at approximately hours postinfusion, similar to c responses after plasmaderived c -inh, and thereafter gradually declined to baseline. , an immediate dose-dependent effect of rhc -inh on plasma c b/c was observed. the magnitude and the duration of the decrease in c b/c appeared dependent on the dose of rhc -inh. cleavage of c appeared to occur once functional c -inh dropped below a level of approximately % of normal. taken together, the phase i results suggest that an adequate dosage of rhc -inh will be able to correct c -inh activity in blood for a sufficiently long period to halt the progression of a swelling episode and to allow the resolution of edema. that is, the results warrant clinical studies of the safety and efficacy of rhc -inh in symptomatic patients with hae. phase ii and phase ii/iii studies of transgenic rhc -inh for the treatment of acute attacks in patients with hae. a phase ii exploratory open-label study of the safety, tolerability, and efficacy as well as pharmacokinetics and pharmacodynamics of rhc -inh at u/kg in symptomatic patients with hae with severe attacks of angioedema in the abdomen, the facial-oro-pharyngeal region, and/or the genito-urinary region is currently being performed in the netherlands (levi) . on the basis of the initial encouraging results of the phase ii study, a multicenter phase ii/iii study of rhc -inh at sites in several european countries is being initiated. expression of wild-type c -inh in the yeast pichia pastoris. the yeast pichia pastoris is increasingly used as an expression system to produce recombinant proteins on a large scale. hence, recombinant c -inh has been expressed in this system as well. however, development of yeast-expressed rhc -inh for clinical use is still at an early stage. initial expression of c -inh was achieved by using the vectors ppiczaa and ppic . without optimization, expression of as much as g of recombinant protein per liter was observed. though some of the c -inh preparations produced in p pastoris were fully active, compared with plasma c -inh, other preparations were inactive because of proteolytic inactivation of the recombinant molecule by contaminating proteases. inactivation in particular was observed when the yeast was cultured for more than days. the recombinant protein was secreted in the culture supernatant and constituted at least %, in most cases > %, of the total amount of protein, which may make this system attractive for commercial production applications. recombinant c -inh was purified to > % homogeneity on sds-page on laboratory scale by using hplc cationexchange chromatography. plasma c -inh is a heavily glycosylated molecule, owing approximately % of its molecular mass to carbohydrate groups. in the original publication by bock et al describing the nucleotide sequence of cdna coding for human c -inh, glycosylation sites were identified. however, the number of potential glycosylation sites (n-linked and o-linked) may be as high as . the function of these carbohydrate groups is not completely clear, although there is conclusive evidence that incomplete glycosylation and/or undersialylation of the carbohydrate groups is involved in the clearance of the molecule in vivo. on the basis of migration patterns of yeast-expressed c -inh on sds-page as well as current knowledge of glycosylation by yeast (mainly mannose), it is expected that carbohydrate groups on yeast-expressed c -inh will need to be modified to achieve normal clearance in vivo. indeed, in a rat model, accelerated clearance of pichia-expressed c -inh was observed (half-life, minutes for pichia-c -inh vs . hours for plasma derived c -inh; bos et al). , expression of c -inh mutants. c -inh mutants have been made for different purposes, ie, to study the structure and function, to study glycosylation, and to modify function toward applicability in inflammatory conditions. regarding application in inflammatory conditions, c -inh, like many other serpins, can be catalytically inactivated by neutrophil elastase, which may limit its use in inflammatory diseases. to improve the potential performance of c -inh at sites of inflammation, it may be beneficial to enhance the resistance of the molecule to catalytic inactivation by elastase, because this would reduce the amount of inhibitor needed. c -inh mutants with amino acid replacements at the so-called p and p positions of the reactive center (this is the domain interacting with target and nontarget proteases) have been developed. these mutants have a nearly normal function but are much more resistant to inactivation by elastase than wild-type c -inh. until now, these mutants have never been tested in animal models to prove the principle that they indeed perform better in inflammatory conditions. as indicated, pichia-expressed c -inh is glycosylated differently (mainly mannose) than plasma-derived c -inh (mainly complex-type carbohydrate groups). most glycosylation sites are located in the n-terminal part of the molecule. the function of this part is currently unknown, although according to the latest data, the most c-terminal amino acids of this n-terminal part serve to stabilize the so-called central b-sheet of c -inh, which is essential for serpin function. to evaluate the role of the n-terminal portion in the functional activity of c -inh, deletion mutants lacking or amino acids of the n-terminus have been expressed in pichia. the functional activity of these mutants has yet to be studied in detail but at first glance looks normal. in contrast, a mutant lacking the complete n-terminal part was inactive. the mutant lacking amino acids is interesting in that it seems to have normal functional activity despite having only carbohydrate groups left. experiments with endoglycosidase h and concanavalin a reveal that these groups are n-linked (asn ; asn and asn ). for the future. recombinant protein technology not only offers the possibility of producing large amounts of protein at acceptable costs but also allows for mutation of the protein to adapt its function. expression of wildtype human c -inh has been successfully achieved in transgenic rabbits, and this recombinant inhibitor is now in phase ii clinical development for the treatment of acute attacks in patients with hae. pichia-expressed c -inh may constitute another alternative to plasmaderived c -inh but is still at a very early stage of development. kallikrein inhibition and nonallergic angioedema. the kallikrein inhibitor dx- was generated via phage display, a technique for rapidly identifying target-specific protein binders. the phage display process involves generating many possible binders specific for each desired target, in this case human plasma kallikrein, and then selecting the binder with the highest affinity. as such, this process resulted in a molecule with an extremely high ( pmol/l) binding affinity for human plasma kallikrein. kallikrein is known to catalyze the conversion of kininogen to bradykinin. assuming bradykinin is the major mediator of nonallergic angioedema, plasma kallikrein inhibition should be beneficial during angioedema attacks. kallikrein has also been shown to influence the renin-angiotensin system by promoting the conversion of prorenin to renin ; however, as discussed in the pathogenesis section of this supplement, the renin-angiotensin system likely plays a much lesser role in angioedema generation. phase i normal volunteer study. in a study conducted in scotland, dx- was administered to healthy male or female volunteers; pharmacokinetic and safety parameters were assessed. two patients each received or mg dx- , and patients each received or mg dx- . half-life measurements ranged from . to . hours. phase ii european study. an open-label study of dx- for the treatment of nonallergic angioedema attacks was conducted in centers in europe. of the treated patients, patients had hae, and had aae. patients presented with facial/lip, hand, genital, or abdominal attacks; patient was treated for simultaneous hand and abdominal edema. in this dose-ranging study, patients each received mg, mg, or mg intravenous dx- . one patient ( mg dx- ; genital edema) had a drugrelated anaphylactoid reaction. patient-reported times to the start of attack resolution ranged from to minutes (mean, minutes); patient-reported times to complete resolution ranged from to hours (mean, . hours). the authors note that dx- could potentially advance the treatment of hae by reducing patients' reliance on anabolic steroids or plasma-derived products. attenuated anabolic steroids are the most common prophylaxis for hae and associated disorders. although these agents are effective in many patients, they carry a wide range of side effects, many of which are undesirable. their mechanism of action in these diseases, and indeed the full extent of their systemic effects, is not entirely known. as illustrated by the following investigations, attenuated androgens such as danazol may have other, at least transiently positive effects in addition to their better-documented risks. attenuated androgens effectively prevent swelling attacks in hae. the attenuated anabolic steroid danazol is mainly used in the therapy of many estrogendependent diseases such as endometriosis, cystic fibrotic mastopathy, primary menorrhagia, primary pubertas praecox, and gynecomastia. , however, its other, less known clinical indication is the prophylaxis of hae. the high androgen and low estrogen levels caused by this drug , are responsible for some side effects. among these, seborrhea, acne, hirsutism, weight gain, hair loss, voice changes, clitoral hypertrophy, vaginitis, irregular menstruation or amenorrhea, and reduced breast mass are the most common. methods. this study investigated the effects of danazol on bone metabolism. low estrogen concentrations have previously been shown to play a role in the development and progression of osteoporosis. the authors' current investigation sought to discover how reduced estrogen production caused by danazol therapy influences bone turnover. according to available data, danazol's effects on bone metabolism have been investigated only in patients with endometriosis. [ ] [ ] [ ] [ ] these investigations gave contradictory results. in addition, the authors note that the danazol doses given to treat endometriosis in these studies far exceed the doses administered in hae. rock et al have reported a temporary increase in bone mineral density (bmd) lasting only for the weeks of treatment, which then decreased such that bmd values measured at the nd week were lower than the initial values. according to dodin et al, the lumbar bone mass slightly increased by the end of the third and sixth months. , dmowski et al found no significant change in bmd during the danazol treatment. the current investigation measured the degree to which the minimum effective dose of danazol for hae control influenced bone turnover. all patients cared for at the hungarian hae center were involved in this study. the control group contained patients not treated with danazol. concentrations of alkaline phosphatase, calcium, phosphate, parathyroid hormone, dehydroepiandrosterone sulfate, b-cross laps, and osteocalcin were measured in blood serum. dual-energy x-ray absorptiometry was also performed in patients older than years. results. significant correlations have been found by using multiple logistic regression analysis between the variable of b-cross laps, osteocalcin values, z score values detected by the femoral neck, and danazol treatment. b-cross laps and osteocalcin concentration (markers of bone resorption) have shown a negative correlation, whereas z score values (markers of bone formation) have shown a positive correlation with danazol treatment. in the authors' study, it was evident that b-cross lap concentration was lower in the danazol-treated group than in the control group. changes in serum osteocalcin concentrations, like those in b-cross lap values, showed a negative correlation. osteocalcin concentrations in patients treated with danazol were considerably decreased. however, the authors note that both osteocalcin and b-cross lap concentrations oscillated in the normal range. z scores measured by the femoral neck showed that positive results were much more frequently detected in the danazoltreated group, whereas values in the untreated control group were always between and . . thus, bone mineral density was significantly higher in the danazoltreated group. the authors propose that the increased androgen level may have compensated for the deleterious effect of decreased estrogens, citing that the anabolic effect of androgens on bone metabolism have been well documented in the literature. discussion. comparing the results of this study with those reported in the literature on the effects of high-dose danazol treatment in endometriosis, the authors suggest that the minimum effective dose treatment in hae does not cause osteoporosis and, indeed, may protect the bone from mineral loss. ovulation is the result of complex, interrelated mechanisms initiated by the surge of lh and characterized by resumption of meiosis and germinal vesicle breakdown, initiation of luteinization of the granulosa cells, rupture of the follicle wall, and release of a fertilizable ovum. hormonal control of ovulation and biochemical and morphologic changes of the preovulatory follicle, including collagenolysis and vascular changes, have been extensively studied. ovarian follicular fluid (ff) is an enzyme reservoir controlling the permeability of the capillaries of the antrum and proteolytically degrading collagen, thus leading to the rupture of the follicle wall. the plasminogen-activator-plasminogen hypothesis is the most likely explanation of the mechanism initiating the cascade that leads to follicular rupture. the preovulatory follicle, stimulated by gonadotropins, secretes urokinasetype plasminogen activator, which can convert plasminogen to plasmin. a latent collagenase activated by plasmin attacks collagen, and the resulting telopeptidefree collagen can be attacked and degraded by nonspecific proteases. the decrease of the tensile strength of the follicle wall then allows the rupture of the follicle by the action of the existing intrafollicular pressure. in addition to several multifactorial systems (eg, kinins and fibrinolysis) that have been demonstrated and characterized in human ff, the presence of active complement has also been demonstrated in amounts similar to those present in normal human serum. plasmin's activation of complement in ff is very important for the function of the enzymatic multifactorial mechanism of ovulation. after complement activation in ff, biologically active peptides such as c a, c a, and c a are released via the complement cascade. complement activation is also important for the assembly of the membrane attack complex on the follicle wall. complement activation and ovulation: consequences in hae. it has been clearly demonstrated that women of reproductive age with hae who were not under danazol treatment and off any therapy, including ocs, frequently had cystic ovaries with an ultrasound pattern of polycystic ovaries (pcos; approximately %) or of multifollicular ovaries (mfos; approximately %). although pcos in women not affected with hae are associated with increased lh and testosterone concentrations, and mfos with almost normal values of these hormones, in patients with hae, normal concentrations of lh and testosterone (and of prolactin, cortisol, and acth) are almost always demonstrable independently from the ultrasound pattern (pco, mfo, normal) but in the presence of significantly reduced follicle stimulating hormone and slightly increased lh:follicle stimulating hormone ratio. menstrual irregularities and oligomenorrhea are frequent features of the patients studied, and hirsutism is sometimes present in patients with hae both with pco and mfo. neuroendocrine connection? in hae, the role of c -inh deficiency in the development of clinical symptoms and in the management of the disease has been explored. nevertheless, some interesting questions remain. the proposed relationship between physical and/or psychologic stress and the onset of acute episodes has yet to be proven fully. likewise, the effects of repeated stressful situations, such as the attacks themselves, on patients' neuroendocrine systems have yet to be completely explained. however, some possible links between the neuroendocrine system and hae have been studied and reported. perricone et al have previously found high plasma bendorphin concentrations without simultaneous elevation of acth during hae attacks as well as during symptomfree periods. this provides evidence of a possible massive release of b-endorphin from the readily disposable pool present in the pituitary and/or for an increase in the turnover of the peptide as evaluated by b-endorphin: b-lipotropin ratios and, in turn, of a pituitary abnormality in the patients. also in the group of patients with hae affected with cystic ovaries (studied off therapy and in symptom-free periods), significantly high plasma b-endorphin concentrations and normal acth have been detected. the very high prevalence of pco and mfo in hae again emphasizes the possible presence of hypothalamic-pituitary abnormality in these patients. in fact, both pco and mfo are linked to hypothalamic-pituitary dysfunction, and in hae, of the features of such a dysfunction, ie, high plasma b-endorphin concentrations and normal acth, is present in many patients, including men, postmenopausal women, and girls who are not of reproductive age. influences on ovulation. the presence of both complement and ovarian abnormalities in patients with hae and the role of ff complement in ovulation induced the authors to study complement function in the ff of women of reproductive age affected with hae. they found that impaired complement function is present in ff obtained from female patients of reproductive age affected with hae. in fact, contrary to what had been observed both in previously studied women not affected with hae and in controls, hae patients' ff showed a dramatically decreased classic pathway activity as well as undetectable functional and antigenic c -inh, and very low c . in addition, the authors observed a slightly reduced or within normal serum range values for alternative complement pathway activity, including c and factor b. this complement profile was also detected in the patients' sera. it is well known that the multifactorial mechanism of ovulation involves relevant steps modulated by c -inh: ( ) the formation of plasmin, and ( ) the plasmin-dependent complement classical pathway activation. activation of ff complement is important for the correct continuation of the ovulatory process, and the lack of c -inh, which facilitates the formation of plasmin and plasmin-dependent complement activation, can, in theory, be beneficial. on the contrary, when the activation of complement is massive, as occurs during hae attacks, a significant release of biologically active complement products can induce consistent inflammation and, in turn, pain. abdominal pain is frequently encountered in female patients with hae during attacks and during ovulation as well. however, because complement is chronically consumed during remissions and dramatically consumed during hae attacks, the potential of complement activation in hae ff is indeed the result of complex interrelated phenomena. if significant consumption of complement components occurs as a result of hae attacks shortly before the periovulatory period, ff complement will represent a defective reservoir of components unable to support further activation during ovulation. such a situation occurred in patient: a few days before ovulation, an attack characterized by edema of the extremities and abdominal pain occurred; thereafter, the patient manifested neither abdominal pain nor detectable ff complement activities and c cleavage fragments during the periovulatory period. in another patient, the periovulatory period (as well as the time of the study) was far away from attacks ( days); at this time, abdominal pain (without other symptoms of the disease) in the presence of reduced but detectable ff complement activities and of c cleavage fragments occurred. notwithstanding the presence of some important mechanisms of proven efficacy that can compensate for the complement deficiency (eg, the activation of a latent collagenase and the action of lysosomal enzymes), it is possible that, in the presence of impaired complement function, the rupture of the follicle wall is more difficult. however, once significant time has elapsed since the last hae attack, complement function can ameliorate; if ovulation coincides with such a condition, it will meet an only mildly reduced complement that will be capable of participating in the rupture of the follicle wall. these considerations can explain the apparently undiminished reproductive capacity of female patients with hae. on the other hand, hae patients' ff complement supports a certain degree of activation by seminal plasma, possibly caused by the relative integrity of the alternative pathway supporting its participation in the phenomenon. it is nonetheless possible that the more difficult rupture of some follicles in hae and the consequent altered intraovaric regulation by atresic follicles can result in the pco and mfo frequently observed in the disease. it is known that in cystic ovaries, the rupture of the cysts is very painful; thus, the authors cannot exclude that in patients with hae, the rupture of ovarian cysts might contribute to the genesis of the dramatic abdominal pain during attacks. danazol and ovulation: conclusions. one of the mainstays in the management of hae is long-term therapy with danazol, a mild androgen capable of correcting low c -inh concentrations and of greatly reducing the typical symptoms of the disease. the authors therefore studied the effects of danazol on ovarian ff c -inh and on ovarian ultrasound pattern. six patients with hae affected with cystic ovaries ( with pco and with mfo) who had been off any therapy for at least year were treated with danazol ( mg daily). as a result of danazol treatment, hae clinical symptoms consistently improved, and c -inh serum concentrations increased. after months of therapy, these patients were monitored by pelvic ultrasound scanning, and their ovaries were found normal. serum c -inh and ff c -inh also increased. furthermore, when the ultrasound pattern of the ovaries was studied in another group of patients, women with hae of reproductive age who were usually under danazol treatment, cystic ovaries were found in only of patients ( %). taken together, these data suggest a further link between the neuroendocrine and immune systems, describing a pathology involving both hypothalamic-pituitary dysregulation and an immunologic disorder. recent research has contributed to a less anecdotal and more systematic appreciation of the differences between adult and pediatric hae and the special age-related concerns that attend hae from the first months of life through puberty. here, prospective follow-ups of pediatric case series are reported. most patients have the first manifestation of hae during childhood. unfortunately, treatment of acute attacks that manifest with circumscribed swelling of the skin and the subcutaneous tissues can be performed successfully only by administering c -inh concentrate. other medications that are usually helpful in edema of other origins (eg, antihistamines or corticoids) are not effective here. the authors report that f of their patients, when anamnestically well evaluated, had edema of the mucous membranes of the respiratory tract at least once; remarkably high lethality rates (as high as %) have been associated with this disease in the past. , , even recently, a mortality rate as high as . % was published in an article summarizing experiences with austrian, swiss, and german patients with hae. as previously discussed, bradykinin seems to be the major mediator of edema, but discussions have not yet been finalized. because this disease still bears unknown details thus far identified, the authors chose to examine the baseline parameters of patients with hae to broaden the general database available and to enhance the understanding of the medical and scientific community. methods. data from patients with hae have been collected in the database at the department for pediatric hematology, oncology and hemostaseology of the johann wolfgang goethe university, frankfurt/main, germany. as of this march analysis, this database contained a total of pediatric patients (age < years) with diagnosed hae. these patients had been followed up for a period of as long as . years. the baseline characteristics of a total of patients from different families with hae were analyzed. the authors further monitored c -inh plasma activity and c -inh plasma antigen and supplemented these values with analyses of c and c and measurement of the total hemolytic activity (ch ) in the patients' plasma. finally, the frequency and localization of attacks, the history of each patient, and the efficacy and safety of replacement therapy with c -inh concentrate (berinert p) were investigated. antigenic c -inh levels were determined by radial immunodiffusion with nor-partigen plates (dade behring, deerfield, ill). functional c -inh concentration was determined by chromogenic assay with berichrom c -inhibitor (dade behring). results and discussion. all patients showed low c -inh activity, with a median activity of % of normal (normal value, % to %; range, . % to %). all acute hae attacks were successfully treated with to units c -inh concentrate per kilogram body weight. in these pediatric hae patients, c -inh concentrate was effective and well tolerated. no side effects or seroconversions for hav, hbv, hcv, hepatitis g virus, hiv- , or hiv- were observed. the gold standard of treatment for acute attacks in pediatric patients with hae is the administration of a c -inh concentrate, preferably that is specifically virus-inactivated. the authors always used a pasteurized c -inh concentrate (berinert p) and did not observe any problems regarding safety or efficacy. in , the transmission of hepatitis g virus in patients with angioedema treated with a steam-heated concentrate of c -inh was reported. the authors have not observed a seroconversion with regard to hepatitis g virus in their patients treated with pasteurized c -inh concentrate. , as treatment of pediatric patients with hae with c -inh concentrate is not registered all over the world, some pediatric patients have to be treated with danazol therapy. introduced in by gelfand et al, this treatment seems to have a rationale in long-term prophylaxis of patients with hae. however, because this treatment sometimes loses efficacy over time despite increased doses of danazol and is often associated with severe side effects, especially in children, the authors recommend the treatment of pediatric hae exclusively with c -inh concentrate if possible on a named-patient basis through personal importation programs. in their experience, this approach should also be feasible for women of childbearing age and all other patients with hae who cannot tolerate the side effects associated with danazol treatment. methods. between and , ninety patients were registered in the hungarian hae center database. what follows is a review of the clinical data of pediatric patients from families. the male to female ratio was : , and the age at time of diagnosis ranged from . to years (mean, . years). ch total complement levels were measured by the standard hemolytic titration method, using sensitized sheep erythrocytes. the c complement fraction was determined by single radial immunodiffusion using anti-c antibodies (atab, stillwater, minn), and results were expressed as the percentage of standard, normal serum values. the c -inh concentration was also measured by radial immunodiffusion; c -inh activity was measured by kinetic assay using a commercially available diagnostic kit (behringwerke ag, marburg, germany). the activity of the c -inh in the serum was determined by photometry and expressed as the percentage of standard, normal values. in the follow-up protocol, the first occurrence, frequency, and localization of clinical manifestations, duration of the disease, as well as potential precipitating factors of edematous attacks were ascertained. d a pedigree analysis was performed in all cases. d long-term prophylaxis was elected if edematous attacks recurred frequently (! attack per month) or a life-threatening episode could be identified in the history. d the following tests were performed in patients receiving long-term danazol prophylaxis: -anthropometric assessment of growth (comparison of body height and weight development to agespecific normal values). -bone age determination (hand x-rays). -development of secondary sexual characteristics and time of puberty compared with control population. -mental development (performance at school). d short-term prophylaxis was administered before surgical or diagnostic procedures performed on the head and neck. d changes in serum complement fractions and clinical manifestations were monitored during therapy. potentially drug-related adverse effects of treatment were also recorded. d in patients undergoing long-term prophylaxis, laboratory tests (complete blood count, liver function tests, creatine kinase activity, and urinalysis) were repeated at -month to -month intervals, and abdominal ultrasound was performed semiannually during the first years of treatment. subsequently, asymptomatic individuals or patients with mild symptoms were checked every months to detect potential liver damage. d patients with an acute attack of submucosal (laryngeal or severe abdominal) edema were always hospitalized and abdominal ultrasound performed if the attack involved the gastrointestinal tract. hospitalization for an acute abdominal attack was considered justified when the presence of other acute abdominal pathologies could not be ruled out with certainty and/or manifestations were severe-that is, the attack was associated with obvious signs of hypovolemia (paleness of skin, prostration, dehydration, tachyarrhythmia) and included recurrent paroxysms of acute colicky pain unresponsive to symptomatic therapy, nausea and vomiting, or profuse diarrhea. d the c -inh concentrate used in this study was screened for hepatitis b and c by the manufacturer. nevertheless, in observance of the safety precautions pertinent to blood products, hbsag and hcv specific antibody were tested by using commercially available immunoassays in all patients who had received c -inh concentrate. acute edematous attacks were treated by administering c -inh concentrate (berinert p injection; centeon, vienna, austria), and e-aminocaproic acid (acepramin; pannon pharma, pecsvarad, hungary), tranexamic acid (exacyl; sanofi synthe labo-chinoin, budapest, hungary), and danazol (danoval; krka, novo mesto, slovenia) were used for prophylaxis. results. the initial manifestations of hae were observed within an age range of . to years (median, . years). six children were asymptomatic at the time of diagnosis, which was established only after hae was ascertained in their symptomatic parents. pedigree anal-ysis revealed familial occurrence of the disease in cases (from families) but failed to identify hae in firstdegree relatives of children; therefore, in these patients, c -inh deficiency must have resulted from a new c nh mutation (table xiv) . the localization of clinical signs was variable. in the majority of patients ( children; %), edema formation involved subcutaneous tissues, whereas gastrointestinal manifestations occurred in patients ( %) and laryngeal edema developed in patients ( %). subcutaneous edema of the extremities or genitals usually persisted for to days, then resolved spontaneously. edematous swelling of the skin was not accompanied by itching but caused an unpleasant sensation of distension within the involved region. in children, generalized, nonpruritic skin rash (erythema marginatum) was observed before and during edematous attacks of hae. the exploration of potential precipitating causes invariably identified mechanical trauma; however, upper airway infection was another important factor (table xv) . according to the results of serum complement studies, children had hae type i, whereas had hae type ii (table xvi) . all children with an acute edematous attack manifesting as laryngeal edema or severe acute abdominal complaints were hospitalized. emergency treatment was implemented by the parenteral administration of u c -inh concentrate exclusively (table xvii) . the injection accomplished substantial symptomatic relief within minutes to hour. this was accompanied by the regression of edematous swelling; however, complete resolution of clinical signs and symptoms took to hours. administration of an additional u dose (ie, a u cumulative dose) was necessary in patients ( with laryngeal and diffuse facial edema, with acute abdominal attack) to achieve a satisfactory rate of improvement. all patients hospitalized with an acute abdominal attack underwent abdominal ultrasound examination. ascites was invariably ascertained in all cases, whereas edema of the intestinal wall was detected in % of patients (fig , a) . as evidenced by follow-up ultrasound, appropriate treatment (c -inh concentrate) achieved complete disappearance of free peritoneal fluid and edematous swelling of the intestinal wall within hours. long-term drug prophylaxis with antifibrinolytic agents (tranexamic acid or e-aminocaproic acid) was initiated in cases because of frequent (weekly or monthly) recurrence of edematous attacks or the presence of life-threatening attacks in the history. complete remission was achieved in girls maintained on to g/d tranexamic acid; however, antifibrinolytic agents were ineffective in cases. transferring these patients to treatment with to mg danazol/d completely eliminated serious edematous attacks (table xvii) . to taper the cumulative dose, the dosage interval of danazol ( mg) was increased to to days after months of clinical remission. switching to this intermittent regimen (ie, doses repeated every other day or at -day intervals) from continuous dosage was successful in cases. in patients maintained on long-term prophylaxis, doubling the daily dose for several days aborted prodromal symptoms or prevented the progression of mild clinical manifestations to full-blown attacks. a similar protective effect was observed in cases exposed to a precipitating factor (eg, upper airway infection or mechanical trauma). no adverse effects potentially related to long-term prophylaxis necessitating the withdrawal of treatment were observed. the bone age, skeletal growth, and weight development were not substantially different from the agespecific averages of the hungarian pediatric population. the age at appearance of secondary sexual characteristics and the onset of puberty were also similar to the national average in children, but in girl, menarche was delayed (to the age of years), and her menstruation was irregular. hirsutism was not observed in patients receiving long-term danazol prophylaxis. monitoring of laboratory parameters revealed normal values in all children tested, and abdominal ultrasound showed no abnormalities. the efficacy of danazol treatment was demonstrated by the significant increases of c and c -inh concentrations in the serum (fig ) . nine children had only mild, subcutaneous symptoms, and even these resolved spontaneously within to days. accordingly, long-term prophylaxis was withheld, and treatment was administered only when prodromal symptoms and signs occurred. in patients, tranexamic acid ( g/d) given for to days reduced the severity and duration of subcutaneous manifestations. follow-up visits were scheduled at -month intervals for these children. short-term prophylaxis was necessary in boys (age and years) undergoing dental extraction, because both had facial and laryngeal edema precipitated by similar procedures in their history. danazol ( mg/d) initiated days before and continued days after dental treatment successfully prevented edema formation. the elimination of known precipitating factors also substantially reduced the incidence of edematous attacks. discussion. in % of hae cases, a known family trait of the disease facilitates diagnosis. in the remaining %, in which no afflicted relatives can be identified, hae results from a new gene mutation. in the hungarian pediatric population analyzed by the authors, the proportion of such cases was % and %, respectively. according to the literature, % of patients with hae have type i and % have type ii disease. the percentage distribution of type i and type ii cases was % and % in the hungarian series. in general, the clinical manifestations of hae first develop before years of age ; the median age of the hungarian study population was . years. however, no occurrences have been observed in newborns, and only infant ( -month-old) case has been reported in the literature. according to the experience of the authors, adolescence is often associated with substantial changes in the activity of the disease, particularly in girls, warranting closer follow-up during this period. in particular, cluster- ing of edematous attacks during the menstrual period was observed in girls. nowadays, contraception is initiated in early adolescence. however, ocs can precipitate acute attacks, and therefore, these agents are not recommended for girls with hae. the incidence and severity of characteristic manifestations show substantial interindividual variation. less than % of patients with hae are asymptomatic, and % had symptoms only sporadically. manifestations of the disease are common in % of patients and severe in % of this subpopulation. eleven of the children followed up in this study had frequent and severe attacks. usually, mechanical trauma is reported as the most common precipitating factor. through their experience, the authors confirm this and identify upper airway infection as another potential triggering factor. considering the importance of physical activity and sports on growth and development, restrictions should be determined carefully and individually. in children, as in adults, edema of the extremities is the dominant manifestation; furthermore, abdominal attacks are more common than laryngeal edema. , nevertheless, even rare occurrences of laryngeal edema should be considered real emergencies. because of the small diameter of upper airways in children, relatively mild swelling of the mucosal lining causes substantial obstruction, and suffocation can therefore rapidly ensue. in view of these risks, referral to intensive care is justified to monitor the clinical course and ascertain the efficacy of treatment. in some patients, repeated administration of c -inh concentrate may be necessary to achieve satisfactory control of edematous manifestations. this fact and the ineffectiveness of conventional agents (eg, antihistamines, corticosteroids, and epinephrine) account for the invariably high mortality of the disease and emphasize the importance of accurate diagnosis. the striking similarity between the clinical manifestations of abdominal hae attacks and surgical emergencies is an apparent diagnostic pitfall. failure to evade this trap often leads to unnecessary interventions on patients already prostrated by the consequences of the acute edematous attack. , the authors reviewed the medical history of their patients before diagnosis, revealing that children had undergone exploration or laparoscopy during an acute abdominal attack, of them twice. these interventions were clearly unwarranted, because no abnormality other than the edematous swelling of intestinal wall was ascertained. to avoid this diagnostic pitfall, the authors recommend close monitoring of the patient, volume replacement, parenteral medication, and assessment of therapeutic efficacy, all achievable by observing the patient for to hours. abdominal ultrasound is a noninvasive and readily accessible diagnostic test that yields reproducible results, features particularly advantageous in pediatric practice. the authors identify free peritoneal fluid and edema of the intestinal wall as invaluable sonographic clues for distinguishing abdominal hae attacks from other pathologies commonly associated with ascites formation (such as intra-abdominal inflammation, autoimmune disease, tumors, and so forth). they recommend that, when all other acute abdominal disorders had been ruled out, c -inh concentrate should be administered to afebrile patients without laboratory abnormalities, particularly to those with known hae. prompt symptomatic relief (or lack thereof) after c -inh concentrate administration can then confirm or refute the tentative diagnosis. during life-threatening hae attacks, appropriate measures can include the administration of ffp. this preparation contains c -inh; however, its use may be associated with alloimmunization or the transmission of infections. the most appropriate therapy is the intravenous administration of c -inh concentrate. its dosage is the same for children and adults (ie, - u). in this study, acute hae attacks were treated with c -inh concentrate exclusively. this approach was highly effective, resolving clinical symptoms within an hour, and was not associated with adverse effects. c -inh concentrate has been suggested as an ideal agent for permanent substitution, particularly for pediatric patients. unfortunately, the short ( -hour) half-life and high cost ($ per vial) of the concentrate preclude its use for continuous administration. furthermore, c -inh concentrate is a blood product, and the theoretical risk of blood-borne infections cannot be completely excluded. nevertheless, viral (hiv, hbv, hcv) safety of the concentrate is promoted by heat treatment as well as the establishment and continuous validation of safe plasma pools. , in the hungarian in pediatric patients, antifibrinolytic agents are the first choices for long-term prophylaxis because their safety profile is more favorable than that of attenuated androgens. , , , nevertheless, antifibrinolytic agents can cause adverse effects such as muscle weakness, myalgia, elevation of creatine kinase activity, vascular thrombosis, postural hypotension, and myonecrosis. the experience of the authors showed tranexamic acid to be better tolerated than e-aminocaproic acid, which often caused gastrointestinal discomfort. however, when these agents fail to achieve satisfactory improvement, treatment with attenuated androgens (eg, danazol, stanozolol) is necessary. numerous studies performed on adult patients have demonstrated the efficacy of a-ethyltestosterone (danazol) manifested by the substantial relief of symptoms as well as the elevation of serum c and c -inh concentrations. their mechanism of action has not yet been fully elucidated, and limited experience with their pediatric use has been obtained only in patients treated for idiopathic thrombocytopenic purpura. potential adverse effects of danazol include weight gain, myalgia, headache, tremor, libido changes, elevation of serum transaminase levels, microhematuria, menstruation irregularities, and hirsutism. in addition, reports have been published on hepatocellular adenoma formation observed in adults undergoing long-term, high-dose danazol therapy. nevertheless, using the lowest effective maintenance dose and intermittent dosage regimens can prevent adverse effects that would require the withdrawal of treatment. in the study population, the longest duration of danazol prophylaxis was years. no drug-related adverse reactions or impairment of growth was recorded in these pediatric patients, with the exception of case of delayed menarche with subsequent irregular menstruation probably caused by long-term treatment with mg/ d danazol. compared with age-specific percentiles of the hungarian population, somatomental development was normal in all cases. treatment prevented edematous attacks and elevated c and c -inh concentrations significantly. effective therapy eliminated the need for frequent hospitalizations for acute symptoms-along with their unfavorable psychologic sequelae-and thereby improved the patients' quality of life. the authors emphasize the essential importance of close follow-up, with appropriate tests performed at regular intervals. the diagnosis of hae can be made as early as at birth by assessing c -inh antigenicity and function. prenatal screening is not yet feasible. importantly, baseline complement concentrations are of no use for predicting the severity and frequency of future attacks; for example, some patients are asymptomatic during a patient's first years of long-term prophylaxis, laboratory tests should be performed every to months and abdominal ultrasounds scheduled at -month intervals. the clinical course of hae and reactions to treatment should be monitored to recognize potential adverse effects and ascertain the rate of somatomental development as well as the need for therapy modification. in view of their favorable experience, the authors suggest that danazol administered in the lowest effective dose seems to be well tolerated by pediatric patients and is probably appropriate for long-term therapy. nevertheless, adverse reactions can occur sporadically. in particular, case of delayed menarche and irregular menstruation was observed in this series; however, the abnormality was readily reversible on discontinuing danazol treatment. short-term prophylaxis is indicated for patients undergoing surgical or diagnostic interventions of the head and neck region. such procedures most commonly include dental procedures and tonsillectomy, but the potential consequences of endotracheal intubation for general anesthesia must also be emphasized. , although short-term prophylaxis is required less frequently for children than for adults, antifibrinolytic agents or attenuated androgens provide adequate protection if administered in higher than usual doses. nevertheless, prophylaxis with c -inh concentrate is the method of choice in patients with a history of severe attacks precipitated by similar procedures. in addition, follow-up should also include the education of patients and their parents, as well as advice on the most suitable means of lifestyle modification. the activity of patients' self-help groups can lend vigor to such efforts. early health education and meticulous follow-up initiated in childhood can successfully prevent social stigmatization and guarantee an improved the quality of life for adulthood. it is prudent to provide patients with hae with a medical information card summarizing essential knowledge and methods of emergency help in several languages. when justified by their history, patients should be supplied with c -inh concentrate to be kept at hand, ie, in the refrigerator at home, for emergencies. the data contained on the medical information card can prove an extremely useful aid for medical professionals relatively unfamiliar with hae and help to ensure patients' safety. this article first appeared in a slightly different form as although all patient and physician groups share the common goals of increased knowledge of hae and related disorders and optimized therapy, patient and physician experiences differ among countries. the following accounts demonstrate the range of perspectives, treatment options, and concerns found in europe and the united states, concluding with a short history of past c -inh deficiency workshops in which representatives from these and other countries have met to further their common aims. an international approach to patient registries (marco cicardi, md,* and john jakenfelds, md, milan, italy, and chalfont st. giles, uk) the concept of a european patient registry grew out of the first c -inh deficiency workshop. in this section, cicardi and jakenfelds describe the process of creating the european hae register and how the register will work. in , the european group of patients, clinicians, and scientists interested in hae met for the first c -inh workshop in visegrád, a hungarian town near budapest. workshop members identified the creation of a register containing the clinical and laboratory data of patients with hae as an obvious first step. there is a real need for a better insight into the full presentation of hae, the different ways it is managed, and the various outcomes (including clinical, functional, and economical variables) associated with different disease management strategies. currently, there are significant differences in the way patients with hae are treated in various hae expert centers. there is no consensus on the state-of-the-art treatment of hae, and there is a lack of data to develop uniform, evidence-based treatment guidelines. as a result, an enormous gap exists between resources already or potentially available and their exploitation. furthermore, the low awareness of this disease causes only a minority of patients to reach correct diagnosis and treatment. small and dispersed case lists and the lack of epidemiologic data lessen the value of clinical studies and discourage public and private investment in research aimed at developing new therapeutic strategies or tailoring existing ones to hae. given this situation, an international patient register could play a significant role in the development of a better understanding of hae and its management. first steps. two pharmaceutical companies, pharming and baxter, encouraged the establishment of an hae register in . baxter already had a product for the treatment of the disease (plasma-derived c -inh) in some european countries, and pharming was developing a recombinant version (rhc -inh). pharming became the major contributor until , when a group of hae experts from different european countries received a grant from the european commission for a project called prehaeat, consisting of a concerted action in the framework of the specific research and technologic development program, quality of life and management of living resources. establishing a european register for hae was the first work package of the project and was started accordingly by using the pharming-funded register as a starting point for further refinement. in this new register, physicians will enter completely anonymized patient data into a web-based data capture tool. princeton healthcare will be responsible for the central server and software, accessible by all sites and individuals wishing to enter data (fig ) . princeton is registered under the uk data protection act to store personal information as part of its ongoing work with clinical systems, which have been in use for several years. the register will work by using the internet. by connecting to the web and entering the site's name, the password-protected entry screen will be viewable (fig ) . on filling out details, the user will be taken to a menu of options, including the entry of new data or the viewing of reports covering anonymized patient records (either individual or for the group under treatment at the clinic). only the treating physician, by knowing a code, will be able to link an individual report to an individual patient. access initially will be restricted to the physicians participating in the concerted action and eventually will be open to any hae-treating physician on request. efforts will be taken to publicize maximally the existence of the register. access to data: ownership and management. each patient owns his or her own data. in order for the data to be entered into the register, patients must give consent to share their anonymized data. all clinicians contributing to the register will have access to their own data as individual or group reports. the board, consisting of the participants to the concerted action and of patient association representatives, will manage the anonymized database and may access aggregated reports but not individual data sets. no single member of the board can use or publish the data without the consent of the board and the contributors as a whole. the goal of the register is to develop a better understanding of the current benchmarks of care for hae and the outcomes that result to improve the ways in which patients are treated. for each patient, there will be an initial entry form and or more follow-up visit forms (fig ) . the initial entry form is structured to capture the natural course of hae in a patient. it will contain the past history before diagnosis of hae was recognized and/or specific treatment established. it will contain anagraphic data, information on characteristics and severity of symptoms, presence of associated diseases, and laboratory data at the time of diagnosis. the follow-up visit forms can be replicated indefinitely. they will collect the same data, updated according to the changes occurring in the course of the disease, and detailed data on the different therapies. specific care has been taken to record effectiveness and side effects of hae treatments. expected achievements and perspectives. this register is expected to become a reference database in hae. efforts have already begun to promote the harmonization of similar data collection systems on this topic around the world such that compatible formats will allow pooled data analysis. information describing the natural history of hae and the effect of treatment should be obtained. this database should also be used to provide policy makers and the pharmaceutical industry with data showing exactly how the disease affects the lives of patients who have it. the absence of an approved hae acute attack therapy in the united states of america represents a catastrophic unmet medical need. patients are suffering needlessly and in some cases are dying. indeed, the united states hae association (haea) is aware of hae-related deaths over the period of the past months. the most pressing near-term goal of the haea is to work with industry, the research community, and regulators to encourage usbased clinical trials that will result in licensure of an acute attack therapy. clearly, the lack of a therapy to treat an hae attack once it has begun poses a tragic and unnecessary risk to every us hae patient's life. fortunately, a phase ii clinical trial for an acute hae attack therapy sponsored by dyax corp is now up and running in the united states. the patient community is excited about the prospects for dyax's therapy on the basis of some promising safety and efficacy data developed during phase i and ii trials conducted in europe. the haea is working closely with dyax to recruit patients and complete this vital clinical trial. if the us phase ii results are as promising as results seen in phase i/ii european trials, the researchers hope that dyax will request an expedited licensing review by the food and drug administration. the haea is committed to ensuring that patients, industry, and the food and drug administration work together and strike an intelligent balance that permits an expeditious path to meeting a catastrophic unmet medical need while simultaneously protecting the public health and safety. meetings such as the one in budapest have provided the opportunity for patients with hae to interact and share information. this networking has spawned a movement that should soon result in the creation of a legally recognized international hae patient organization. clearly, an international hae organization can provide the world's hae groups with information, analysis, and guidance on key issues regarding hae management, diagnosis, and the direction of future research. indeed, researchers active in establishing an international hae group have already begun the process of examining the state-of-the-art for hae treatment in different parts of the world. this process has uncovered some strongly held views among the world's patients who are not satisfied with the current treatment options and standards. this drumbeat of concern is prompting international leaders to re-examine the hae treatment paradigm that almost always features androgens for prophylaxis, with acute attack therapy (c -inh concentrate) administered only for gastrointestinal and/or life-threatening attacks. there is some very exciting and innovative work going on in germany that quantifies patient dissatisfaction with the current treatment norm. specifically, the german data show that when given an option, many patients discontinue androgens and opt for on-demand (usually homebased) treatment with an acute attack therapy that they infuse during the attack prodrome. in sum, the treatment of hae in the united states is best described as medieval. without access to an acute attack therapy, patients must rely exclusively on prophylaxis with androgens. these drugs are highly toxic and have harsh side effects, particularly in women. the haea is working hard to assist companies interested in licensing an acute attack therapy in the united states. there is much hope that once formally established, an international hae organization will help to create and rationalize a partnership between the world's hae patients, researchers, regulators, and industry to ensure new therapies can be expeditiously tested, approved, and marketed. the hungarian experience: assisting the few to live a better life (istván nagy, arianna kitzinger, and henriette farkas, md, phd,* székesfehérvár, sopron, and budapest, hungary) in this section, hungarian physician farkas and patientresearchers nagy and kitzinger describe the importance of the hungarian hae center and its infrastructure, as well as their case series and approach to hae. creating an infrastructure. the hungarian complement laboratory in budapest was founded by prof. george füst in . the first article detailing hae patients' cases was published in . these achievements were followed by several milestones leading to the establishment of the hungarian hae center: d accumulating relevant knowledge from the international literature, adapting foreign experiences to domestic conditions, and developing hungarian protocols for clinical work-up and treatment d undertaking retrospective screening for patients by using archived laboratory test results (complement values) d communicating pertinent knowledge to medical professionals, graduate and postgraduate education programs, and others via publications d establishing a results-based referral system: if laboratory complement measurements are suggestive of hae, accessibility information for the hungarian hae center is printed on the laboratory test slip the development of research ran in parallel with the increasing number of patients diagnosed with hae. fig illustrates the rate of case accrual. currently, there are patients in hungary (total population: million people), of whom live in budapest and its suburbs. hungarian hae center. the headquarters of the hungarian hae center are located on the premises of semmelweis university in budapest. each facility has its assignments, as shown in fig . assignments of the allergy and angioedema outpatients' clinic at semmelweis university include the following: the complement laboratory functions as a unit of the hae center and is properly equipped to perform total complement profiling (ch , c , c q, c -inh concentration and functional activity, anti-c -inh antibodies, anti-c q antibodies). the molecular biology laboratory and the research laboratory of the third department of internal medicine conduct genetic testing and mutation analysis on all patients with hae. furthermore, the center participates in undergraduate and postgraduate education and also pursues scientific activities. case management standards. the hungarian algorithm for hae treatment, in the light of its symptoms, therapy, and management is as follows: d mild or infrequent subcutaneous edema does not require special treatment. self-management of symptoms will suffice, and medical monitoring can consist of yearly complement measurements. d severe or frequent subcutaneous and submucosal edematous symptoms are treated by either long-term prophylaxis (danazol, e-aminocaproic-acid, tranexamicacid) or short-term prophylaxis (danazol, e-aminocaproic-acid, tranexamic-acid, c -inh concentrate). such patients require regular medical and self-control. medical control involves liver function monitoring and blood cell and complement measurements twice yearly. d in case of acute abdominal or laryngeal edematous attacks, inpatient therapy with c -inh concentrate is needed. patients experiencing these symptoms also require regular medical and self-control. postattack follow-up is necessary. hungarian hae patients' association. established in , the hungarian hae patients' association organizes annual meetings at which patients can meet doctors and exchange views with each other. a foundation has been established to support patients with hae, and a hungarian hae web site is now being created. the hungarian hae patients' association recently collected patient diaries, drawing attention to topics important among the patient population. the association is now beginning to make a systematic survey of social background and its effect on the disease, which ideally will lead to a deeper understanding of hae and an improvement in patients' quality of life. the patients' association maintains fruitful relationships with foreign patients' self-help groups. as with all uncommon diseases, the elucidation of its epidemiologic features and pathomechanism, the establishment of current diagnostic algorithms, and the development of effective and safe treatment protocols all require international effort. this recognition led to the establishment of the european c -inh deficiency working group in . during its first conference held in rhodes, greece, this professional organization identified accomplishing these goals as its ultimate objective. since that time, the hungarian hae work group has been the main organizer of the european c -inh deficiency workshops, the basis for this supplement. much progress has been made since the first steps taken in the s. in addition to the results described, the association is glad to welcome several humanitarian changes. patients with the diagnosis of hae are now under regular medical control, in contrast with perpetual uncertainty of the past. as a result of patient education and counseling (ie, sufficient familiarity with the disease), patients are understood and helped more effectively by those in their environment. instead of isolation, patients feel integration, trust, and confidence: a major change and an impressive outcome in the case of this life-threatening disease. as shown in table xviii , all of this progress has helped patients to find an appropriate lifestyle. however, without doctors' professional knowledge and its perpetual updating, these developments in hungarian hae research could not have been achieved. many doctors regard their job as their hobby and also feel a commitment to help at both the domestic and international levels. their compassionate care for patients is both noticed and appreciated by those in need. portions of this article appeared in farkas h, varga l. the hungarian hae experience. transfus apheresis sci ; : - . they are reprinted with permission. the german experience: hae and selfmedication with berinert p (ursula rauch, aldenhoven/siersdorf, germany) in this section, rauch, a member of the german hae patient association, hae vereinigung e. v., writes of her experiences as she presented them at the c esterase inhibitor deficiency workshop in budapest. the german hae patient association's motto is nicht mehr allein, ''no longer alone.'' to introduce myself, i would like to tell you a little bit about my career as an hae patient. i think all the hae-affected here have had the same or similar experiences. so, here is the short version of my story. i am years old and have hae-i. i had my first attack at the age of ½, diagnosed as allergy; it was followed by tests and experiments with antiallergic remedies and corticoids, which were, of course, useless. there was an increase in my attacks during puberty, and i received the correct diagnosis at age . there was no remedy at that time, but it was a relief to know it was hae and to stop the antiallergy experiments. sometimes i went for months without an attack; sometimes i would spend weeks with a permanent attack; but to a certain degree i managed to arrange my life around my disease. but after my second pregnancy, the attacks greatly increased. i often needed help to care for my children. i became depressed and could not hold down a job because i was ill too often. not week went by without an attack, and i often had nonstop attacks that lasted to days. in , i finally found our support group, got a lot of information, and was told about the hae center in frankfurt. i made an appointment at the university hospital in frankfurt, where i received very good counseling, and weeks later i went to my first hae group meeting. with the help of the physicians of the university hospital in frankfurt and my practitioner, i learned to infuse myself in an emergency. so i was much more independent and felt much safer than before. after training with normal saline, when i tried my first injection, i went to my practitioner in order to have a supervisor-and it did work! and today? no endless pain, vomiting, and breakdowns of my circulation. no waiting for emergency medical personnel, especially on saturday or sunday, or at night. no more useless claims that corticoids always help, even though my emergency passport says differently. no hurrying to a hospital, breathless and in panic, because of a laryngeal edema. no waiting there for a tired physician who already has hours of work behind him. no enduring questions about whether my husband could have beaten me because my face looks just like a boxer's. i was able to participate in further professional education. i look after my children myself. i can again go on vacations, which always seemed too dangerous before. and i am active in the patient group as much as i can be. i learned how helpful and important it is to work together and fight for a better and more effective therapy. to me, it is absolutely incredible that in most countries, families do not have even package of c inhibitor at home for emergency cases, and that there are doctors who give androgens to children, even though they are still growing and the doctors should know how dangerous these androgens can be. and it is horrible that a lot of affected people are still misdiagnosed and therefore constantly living in danger. we must create the opportunity for every hae-affected person to get the right diagnosis and the same help, so that children can grow up as normally as possible, without missing school very often, and so that we all can go to work and enjoy life-just like the nonaffected, without horrible pains and horrible fears about the risk of suffocating to death or dying of pain. we are a minority-in every country-but together we are strong enough not to be stopped. in recent years, patient associations have had a fundamental role in the fight against rare diseases. many organizations now exist in various parts of the world, even for hae, to perform the important task of sharing information and maintaining a connection between patients and doctors. such associations perform indispensable functions, useful to both doctors and researchers. aims. aaee was founded as a nonprofit voluntary association in milan on march , , and has the following aims: d to spread knowledge of the disease and allow correct diagnosis d to make high-quality medical care and adequate treatment available to all patients d to issue an officially recognized health identification document describing the disease and its emergency therapy and containing the contact information of doctors who can be called for further information d to promote social and scientific meetings and congresses at a national and international level d to encourage meetings and exchanges among patients, between patients and doctors, and among doctors services. aaee represents approximately members and, in more than years of activity, has created a series of services to benefit patients: d in collaboration with the doctors of the diagnosis and cure center in milan, a -hour emergency telephone number was activated. d all patients have an emergency identification document indicating their diagnosis and its suggested treatment in more than language. d a direct link has been established with the pharmaceutical company that produces c -inh concentrate to locate this life-saving medicine at any time. d the minister of health and welfare has added hae to the list of rare diseases for which patients are exempted from paying for medicine. d a multilingual internet site (http://www.angioedemaereditario.org) offers medical and scientific information, useful addresses, and contact information for all hae associations worldwide. d regional representatives permit a more direct connection between patients and the association. d gynecologic and dental consultations are available. d to spread medical and scientific information as well as the association's activities, radio and television broadcasts have been organized. d every year, a national meeting is held in addition to regional meetings to exchange information between doctors and patients and between patients and the association. d some patients who have social roles in the community offer their experience and assistance to the association to deal correctly with various bureaucratic problems. d there are now other centers in addition to the in milan available to patients who need treatment, in rome, palermo, and civitanova marche. recent initiatives. recently, the association prepared a very simple form to collect data on the frequency of hae attacks in italian patients. information will be collected over the period of the next months as a brief trial. this will help aaee evaluate replies received from patients to make all necessary modifications and adapt the survey as much as possible to each involved country's needs and requirements. data will be collected in a proposed specific database. to make better use of the information, the aaee intends to put their internet site at everyone's disposal. in the last few weeks, the international pages have been modified, transforming them into a very simple but functional place where hae information and links throughout the world can easily be found. with the representatives of other associations, the members of aaee are working to create an international umbrella organization. after last year's short meeting on hae in palermo, where the first ideas were elaborated and after the budapest workshop, an international statute proposal now exists. during the last meeting, which took place in milan on october and , , an international committee and a medical panel of doctors and researchers were formed. hereditary angioedema and related disorders are characterized by acute episodes of unexplained swelling that are not responsive to antihistamines or corticosteroids. attacks may manifest in or more locations, most frequently the upper airways, face, hands, feet, genitalia, or intra-abdominal contents. untreated laryngeal swelling can result in death; angioedema of the small intestine can result in obstruction and mimic an acute abdomen. ultrasound may be useful to distinguish between abdominal pain attributable to hae and a true surgical emergency. occasionally, erythema marginatum or a similar, nonwhealing, nonurticarial rash may precede angioedema attacks; at other times, there may be no warning. episodes of hae may present as early as late infancy and may vary, even within the same individual, in frequency, severity, and location. attacks typically resolve within hours but may persist as long as days. aae, although much rarer than hae, often follows lymphoproliferative disorders or occasionally autoimmune, neoplastic, or infectious diseases. drug-induced angioedema may closely follow initiation of a drug regimen (often an antihypertensive agent) or appear many months later, whereas estrogendependent angioedema seems to follow initiation of pregnancy, ocs, or hrt more immediately. hereditary angioedema types i and ii are associated with various mutations to the c nh gene, located on chromosome . a frequently updated online database of these mutations is available to the public at http:// hae.biomembrane.hu; at the time of writing, different c nh mutations were known. the progression from low functional c -inh concentrations to attack genesis is still under debate; however, bradykinin has been identified as a mediator of angioedema attacks, and, unlike other proposed mediators, has been found to correlate with attack localization in early studies. nonetheless, this nonapeptide is unstable and cannot be measured via routine laboratory analyses. it is hypothesized that the local generation of bradykinin results in regional increases in vascular permeability. the presence of other mechanisms, intermediaries, and moderating factors has not been discounted. case reports have shown that in some women, the occurrence or frequency of hae attacks appears to be influenced by estrogen. in these patients, puberty, pregnancy, or taking estrogen-containing ocs or hrt may initiate attacks. patients have also been described with normal concentrations of functional c -inh who nonetheless have angioedema attacks in response to increases in estrogen. bork et al found that approximately % of women with hae-i or hae type iii have a worsening of their attacks because of pregnancy or exogenous estrogen. laboratory diagnosis c -inh antigenic and functional assays are sufficient to diagnose hae-i, hae-ii, and aae. low c concentration is often a reliable confirmation but is not exclusive to c -inh deficiencies. currently, no routine laboratory methods can detect estrogen-dependent angioedema (formerly hae type iii). ideal prophylactic regimens are highly individualized and should consider the severity and frequency of the patient's attacks. -a alkylated androgens, antifibrinolytics, or c -inh concentrate have been used successfully for hae maintenance prophylaxis and/or short-term prophylaxis surrounding surgical or dental procedures. bork et al describe c -inh as an effective, if less frequently used, prophylactic agent. in some patients, antifibrinolytics may be sufficient to prevent attacks; however, attenuated androgens more often reduce the frequency and severity of attacks. patients receiving longterm androgens, especially pediatric patients, should be monitored for undesirable hepatic effects and other consequences such as virilization or delayed menarche; farkas et al suggest laboratory work-ups at intervals of to months. androgen overdosing is to be avoided; varga and other members of the workshop laboratory diagnosis panel note that, in hae-i, antigenic c -inh concentrations approaching % to % of normal are often sufficient to prevent attacks, and therefore, aiming steroid prophylaxis to achieve a % antigenic concentration subjects patients to an unnecessary risk. for the prophylaxis of aae, antifibrinolytic agents are recommended because patients with this condition are frequently resistant to attenuated androgens. danazol is a common prophylaxis for patients with hae who have frequent, severe angioedema attacks. despite known associated adverse effects such as virilization, weight gain, and decreases in hepatic function, the administration of danazol may result in an improvement of some conditions. contrary to findings in patients receiving high-dose danazol treatment for endometriosis, kollár et al reported that patients with hae receiving the minimum effective dose of danazol necessary to control their angioedema attacks did not have osteoporosis. indeed, their data suggest that such a danazol regimen may have a protective effect against bone loss. perricone et al noted a higher prevalence of polycystic or multifollicular ovaries in women with hae but found that danazol administered in hae-management doses, at least in a small sample of patients, improved ovarian condition. treatment varies depending on the location and severity of angioedema. peripheral edema often requires no treatment. for attacks involving the airways or severe abdominal manifestations, c -inh concentrate is the current acute attack therapy of choice for patients with hae. in countries where this is not available, personal importation of c -inh concentrate may be an option; alternatively, the administration of ffp or an antifibrinolytic, although less effective, may be of some benefit in emergencies. supportive measures such as rehydration, antiemetics, and pain control may be necessary depending on the site and severity of the attack. new uses for existing therapy, such as prophylactic or home administration of c -inh concentrate, have been studied on a small scale and proven successful. new therapies, including versions of recombinant c -inh, a kallikrein inhibitor, and a bradykinin receptor- antagonist, are in development. (marco cicardi, md,* milan, italy) since the first comprehensive clinical description of hae in , knowledge of the disease has been expanding. as have many other genetic defects, hae has benefited in the last years from the spread of molecular biology, which brought a tremendous advance in the understanding of the molecular basis of the disease. however, what appears remarkably unique to hae is that within the last years, this bulk of information has been directly transferred into therapeutic approaches. the dream of research from the bench to the bedside is becoming real in hae, and the rule that there is no interest in developing drugs for treating a small number of patients seems to have vanished. revolutionary new techniques for identifying and producing drugs, such as peptide selection from phage display libraries and transgenic animals as source of recombinant proteins, have been implemented to obtain agents active in hae. currently, completely new compounds, a kallikrein inhibitor, a bradykinin receptor antagonist, and a recombinant c -inh, are under clinical evaluation for the treatment of hae. this disease is facing the new pharmacology. chemicals antagonizing biologic systems are being substituted with disease-tailored agents, and in hae, this does not necessarily mean gene therapy. this close relationship between a pharmacologic agent and a disease results in the need for a close relationship between patients and industry. in , the first c esterase inhibitor deficiency workshop was held in hungary with the idea of having patients, scientists, physicians, and industries contributing with equal authority. since that time, a real hae community has been created, and patient support groups are actively involved in designing and carrying clinical studies and are supporting initiatives, such as the hae register, aimed to improve the understanding of the disease and its treatment. aside from the new scientific knowledge or the new therapeutic compounds, recent years' frank and open discussion between different parties is a major achievement for the advancement of the hae cause. many sections of this supplement have repeatedly highlighted how disabling and risky it has been for most patients living with hae, but patients can now have great hopes for the future. the major concern remains the difficulty that many patients still have in reaching correct diagnosis and treatment. deaths caused by hae and similar angioedematous disorders still occur in developed countries because the majority of physicians, ignoring the existence of hae, still label these patients as allergic. we hope that this supplement will help bring more hae, aae, and nonallergic edema patients to the correct diagnosis. institute in the netherlands, and has consulting agreements with genmab and lev pharmaceuticals. m. juers is an employee of aventis behring gmbh, a zlb behring company, and has aventis stock options. h. longhurst has received support/funding from dyax corp., pharming, aventis behring, and jerini. j. nuijens is employed by pharming group nv, a biotechnology company developing recombinant c nh. c. o'grady has received support/funding from dyax corp., pharming, aventis behring, jerini, and baxter. p. späth is a collaborator of zlb behring. k. williams is an independent writing contractor paid for this and other projects by dyax corp.; her husband is a senior-level employee of dyax corp. and owns shares of dyax corp. stock. all other authors-none. hereditary angioneurotic edema: report of a large kindred with defect in c' esterase inhibitor and review of the literature the house of the seven gables on giant urticaria Ü ber akutes umschriebenes hautö dem hereditary angio-neurotic oedema a biochemical abnormality in hereditary angioneurotic edema: absence of serum inhibitor of c' -esterase hereditary and acquired c -inhibitor deficiency: biological and clinical characteristics in patients clinical, biochemical, and genetic characterization of a novel estrogen-dependent inherited form of angioedema increased vascular permeability in c inhibitor-deficient mice mediated by the bradykinin type receptor complement abnormalities with lower extremities discomfort in vietnamese immigrants role of the second component of complement (c ) and plasmin in kinin release in hereditary angioneurotic edema kinin formation in hereditary angioedema plasma: evidence against kinin derivation from c and in support of ''spontaneous'' formation of bradykinin bradykinin-mediated angioedema plasma bradykinin in angio-oedema the pathogenesis of hereditary angioedema bradykinin and the pathophysiology of angioedema activation of factor xii and cleavage of high molecular weight kininogen during acute attacks in hereditary and acquired c -inhibitor deficiencies endothelial cells contribute to vascular leakage in patients with c inhibitor deficiency and angioedema heat shock protein catalyzes activation of the prekallikrein-kininogen complex in the absence of factor xii activation of the bradykinin-forming cascade on endothelial cells: a role for heat shock protein exogenous oestrogen as an alternative to food allergy in the aetiology of angioneurotic oedema hormonally exacerbated hereditary angioedema effect of sex hormones on the complement-related clinical disorder of hereditary angioedema hereditary angio-oedema: new clinical observations and autoimmune screening, complement and kallikrein-kinin analyses danazol: endocrine pharmacology and therapeutic applications hereditary angio-oedema: a review with particular reference to pathogenesis and treatment local bradykinin generation in hereditary angioedema role of the p residue of complement inhibitor (ala ) in determination of target protease specificity: inhibition of complement and contact system proteases post-translational mechanisms of endothelial nitric oxide synthase regulation by bradykinin pathways for bradykinin formation and inflammatory disease hereditary angioedema: the clinical syndrome and its management type i hereditary angio-oedema: variability of clinical presentation and course within two large kindreds pathogenetic and clinical aspects of c inhibitor deficiency hypovolemic shock caused by ascites in hereditary angioedema hereditary angioneurotic edema with severe hypovolemic shock hereditary angioedema: an infrequent cause of abdominal pain with ascites sonographic appearances of the abdominal manifestations of hereditary angioedema changes in splenoportal axis calibre and flow in a patient affected by hereditary angioedema sonographic findings in abdominal hereditary angioedema ct of angioedema of the small bowel image of the month: hereditary angioedema (hae) hereditary angioedema with gastrointestinal involvement: endoscopic appearance abdominal attack of hereditary angioedema associated with marked leucocytosis: a case report hereditary or acquired angioedema caused by functional deficiency of c inhibitor-a still unfamiliar disease picture asphyxiation by laryngeal edema in patients with hereditary angioedema treatment of episodes of laryngeal edema with c inhibitor concentrate in patients with hereditary angioedema erythema marginatum preceding an acute oedematous attack of hereditary angioneurotic oedema bladder involvement in hereditary angioedema neurological manifestations of angioedema: report of two cases and review of the literature hereditary angioneurotic edema, i: case reports and review of the literature hemodynamic effects of bradykinin on systemic and pulmonary circulation in healthy and hypertensive humans analysis of an exon polymorphism of the b bradykinin receptor gene and its transcript in normal subjects and patients with c inhibitor deficiency eradication of helicobacter pylori and improvement of hereditary angioneurotic oedema contributions to the study of the favouring role of chronic urinary infections in inducing and starting drug-allergic-type reactions acquired angioedema associated with sinusitis helicobacter pylori infection and skin diseases hereditary angioedema associated with subacute cutaneous lupus erythematosus rheumatoid arthritis and hereditary angioedema immunoregulatory disorders associated with hereditary angioedema, i: clinical manifestations of autoimmune disease immunoregulatory disorders associated with hereditary angioedema, ii: serologic and cellular abnormalities lupus erythematosus associated with c inhibitor deficiency c inhibitor deficiency in a patient with rheumatoid arthritis-increased risk of adverse effects of penicillamine? glomerulonephritis and hereditary angioedema: report of cases a family with hereditary angioedema and multiple immunologic disorders association of systemic lupus erythematosus and sle-like syndromes with hereditary and acquired complement deficiency states long-term follow-up of non-systemic lupus erythematosus glomerulonephritis in patients with hereditary angioedema: report of four cases a case of hereditary angioneurotic edema associated with systemic lupus erythematosus inherited deficiencies of complement components in man epidemiology and estimated population burden of selected autoimmune diseases in the united states hereditary angioneurotic edema and charcot-marie-tooth disease in the same family association of hereditary angioedema and hereditary breast cancer association of celiac disease and hereditary angioneurotic edema coincidence of hereditary angioedema (hae) with crohn's disease reduction in transmission of hepatitis c after the introduction of a heat-treatment step in the production of c -inhibitor concentrate ultrasonography in the diagnosis of hereditary angioneurotic oedema ultrasonography for early diagnosis of hereditary angioneurotic oedema ultrasonography in the diagnosis and monitoring of ascites in acute abdominal attacks of hereditary angioneurotic oedema clinical management of hereditary angio-oedema in children acute abdominal attack of hereditary angioneurotic oedema associated with ultrasound abnormalities suggestive of acute hepatitis angioedema: manifestations and management hereditary angioedema: a broad review for clinicians detection of c inhibitor mutations in patients with hereditary angioedema a review of the reported defects in the human c esterase inhibitor gene producing hereditary angioedema including four new mutations hereditary angioedema with normal c -inhibitor activity in women hereditary angioedema type iii: an additional french pedigree with autosomal dominant transmission hereditary angioneurotic edema: two genetic variants c inhibitor and hereditary angioneurotic edema behavior in vivo of normal and dysfunctional c inhibitor in normal subjects and patients with hereditary angioneurotic edema crucial residues in the carboxy-terminal end of c inhibitor revealed by pathogenic mutants impaired in secretion or function estrogen-dependent inherited angioedema serum interspecies differences in metabolic pathways of bradykinin and [des-arg ]bk: influence of enalaprilat handbook of immunopharmacology: the kinin system des-arg -bradykinin metabolism in patients who presented hypersensitivity reactions during hemodialysis: role of serum ace and aminopeptidase p aminopeptidase p in individuals with a history of angio-oedema on ace inhibitors possible contraindication of angiotensin converting enzyme inhibitors in patients with hereditary angioedema exacerbation of angioedema by an angiotensin converting enzyme inhibitor in a patient with variant form hereditary angioedema angioedema due to angiotensin-converting enzyme inhibitors hereditary angioedema first apparent in the ninth decade during treatment with ace inhibitor reply (hereditary angioedema type iii: an additional french pedigree with autosomal dominant transmission) inherited and acquired deficiencies of c esterase inhibitor in man hereditary angioedema and normal c -inhibitor activity in women methyltestosterone therapy for hereditary episodic edema (hereditary angioneurotic edema) acquired c esterase inhibitor deficiency and angioedema: a review hereditary angioedema: a decade of management with stanozolol rapid fibrinolysis, augmented hageman factor (factor xii) titers, and decreased c esterase inhibitor titers in women taking oral contraceptives c inactivator level in pregnancy c -esterase inhibitor in uncomplicated pregnancy and mild and moderate preeclampsia c esterase inhibitor in pregnancy acquired c inhibitor deficiency in lymphosarcoma the metabolism of c inhibitor and c q in patients with acquired c -inhibitor deficiency acquired angioedema and anti-c -inhibitor autoantibody autoimmune c inhibitor deficiency: report of eight patients relevance of lymphoproliferative disorders and of anti-c inhibitor autoantibodies in acquired angio-oedema acquired angioedema with lymphoproliferative disorder: association of c inhibitor deficiency with cellular abnormality acquired c inhibitor deficiency in a case of lymphosarcoma of the spleen: reversal of complement abnormalities after splenectomy acquired c inhibitor deficiency with angioedema symptoms in a patient infected with echinococcus granulosus acquired angioedema associated with chronic hepatitis c angioedema due to acquired c -esterase inhibitor deficiency in a patient with helicobacter pylori infection angiooedema due to acquired deficiency of c -esterase inhibitor associated with leucocytoclastic vasculitis acquired deficiency of the inhibitor of the first component of complement: report of five additional cases with commentary on the syndrome recurrent febrile panniculitis and hepatitis in two patients with acquired complement deficiency and paraproteinemia acquired c inhibitor deficiency associated with systemic lupus erythematosus affecting the central nervous system acquired c esterase inhibitor deficiency in two patients presenting with a lupus-like syndrome and anticardiolipin antibodies angioedema with acquired deficiency of the c inhibitor: a constellation of syndromes an igg autoantibody which inactivates c -inhibitor autoantibody facilitated cleavage of c -inhibitor in autoimmune angioedema acquired c inhibitor (c -inh) deficiency type ii: replacement therapy with c -inh and analysis of patients' c -inh and anti-c -inh autoantibodies autoimmune c -inhibitor deficiency acquired angioedema with c inhibitor deficiency: is the distinction between type i and type ii still relevant? prevalence of monoclonal gammopathy in patients presenting with acquired angioedema type clinical and biological distinctions between type i and type ii acquired angioedema autoantibodies and lymphoproliferative diseases in acquired c -inhibitor deficiencies idiopathic nonhistaminergic angioedema drug-induced angioedema without urticaria valsartan-induced angioedema quantification of c -inhibitor functional activities by immunodiffusion assay in plasma of patients with hereditary angioedema-evidence of a functionally critical level of c -inhibitor concentration interaction of i-labelled complement subcomponents c- r and c- s with protease inhibitors in plasma inactivation of factor xii active fragment in normal plasma: predominant role of c- -inhibitor distribution of plasma kallikrein between c- inactivator and alpha -macroglobulin in plasma utilizing a new assay for alpha -macroglobulin-kallikrein complexes on the role of c -inhibitor as inhibitor of tissue-type plasminogen activator in human plasma inactivation of factor xia in human plasma assessed by measuring factor xia-protease inhibitor complexes: major role for c -inhibitor proteolytic activities of two types of mannose-binding lectin-associated serine protease the serpin superfamily of proteinase inhibitors: structure, function, and regulation regional assignment of the human c -inhibitor gene to q -q detailed physical map of human chromosomal region q - shows high meiotic recombination rate around the men locus complete nucleotide sequence of the gene for human c inhibitor with an unusually high density of alu elements recombinations between alu repeat sequences that result in partial deletions within the c inhibitor gene recombinational biases in the rearranged c -inhibitor genes of hereditary angioedema patients contiguous deletion and duplication mutations resulting in type hereditary angioneurotic edema cpg mutations in the reactive site of human c inhibitor molecular genetics of c inhibitor frequent de novo mutations and exon deletions in the c inhibitor gene of patients with angioedema cooh-terminal substitutions in the serpin c inhibitor that cause loop overinsertion and subsequent multimerization transinhibition of c inhibitor synthesis in type i hereditary angioneurotic edema the catabolism of c (-)-inhibitor and the pathogenesis of hereditary angio-edema c inhibitor. functional analysis of naturally-occurring mutant proteins c inhibitor hinge region mutations produce dysfunction by different mechanisms unique c inhibitor dysfunction in a kindred without angioedema, ii: identification of an ala -val substitution and functional analysis of the recombinant mutant protein a hinge region mutation in c -inhibitor (ala -thr) results in nonsubstrate-like behavior and in polymerization of the molecule structural and functional aspects of c -inhibitor c inhibitor gene sequence facilitates frameshift mutations c inhibitor gene and hereditary angioedema the rapid detection of unknown mutations in nucleic acids enzymatic methods for mutation scanning reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations chemical cleavage of mismatch: a new look at an established method c inhibitor mutations which affect intracellular transport and secretion in type i hereditary angioedema current methods of mutation detection efficient detection of point mutations on color-coded strands of target dna exhaustive mutation scanning by fluorescence-assisted mismatch analysis discloses new genotype-phenotype correlations in angiodema denaturing high-performance liquid chromatography: a review mutation detection by denaturing gradient gel electrophoresis (dgge) five novel mutations in the c inhibitor gene (c nh) leading to a premature stop codon in patients with type i hereditary angioedema single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection molecular defects in hereditary angioneurotic edema detection of c inhibitor (serping /c nh) mutations in exon in patients with hereditary angioedema: evidence for novel mutations molecular basis for the deficiency of complement inhibitor in type i hereditary angioneurotic edema altered c inhibitor genes in type i hereditary angioedema restriction fragment length polymorphism analysis of the c -inhibitor gene in hereditary c -inhibitor deficiency rapid detection by fluorescent multiplex pcr of exon deletions and duplications in the c inhibitor gene of hereditary angioedema patients the effect of sequence variations within the coding region of the c inhibitor gene on disease expression and protein function in families with hereditary angio-oedema the promoter of the c inhibitor gene contains a polypurine.polypyrimidine segment that enhances transcriptional activity mutation screening of the c inhibitor gene among hungarian patients with hereditary angioedema hereditary angioedema with a de novo mutation of exon in the c inhibitor gene showing recurrent edema of the hands around the peripheral joints: importance for the differential diagnosis of joint swelling type i c inhibitor deficiency with a small messenger rna resulting from deletion of one exon synthesis of c inhibitor in fibroblasts from patients with type i and type ii hereditary angioneurotic edema impaired production of both normal and mutant c inhibitor proteins in type i hereditary angioedema with a duplication in exon expression of functional human c inhibitor in cos cells characterization of recombinant c inhibitor p variants characterization of c inhibitor-ta: a dysfunctional c inh with deletion of lysine c inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition a surprising new protein superfamily containing ovalbumin, antithrombin-iii, and alpha -proteinase inhibitor primary structure of the reactive site of human c -inhibitor molecular cloning of human c inhibitor: sequence homologies with alpha -antitrypsin and other members of the serpins superfamily the serpins are an expanding superfamily of structurally similar but functionally diverse proteins: evolution, mechanism of inhibition, novel functions, and a revised nomenclature c -esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema in vitro interaction of c -inhibitor with thrombin c inactivator inhibition by plasmin plasminogen activator in normal subjects after exercise and venous occlusion: t-pa circulates as complexes with c -inhibitor and pai- potentiation of c inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma modulation of contact system proteases by glycosaminoglycans: selective enhancement of the inhibition of factor xia the potentiation of human c -inhibitor by dextran sulphate is transient in vivo: studies in a rat model human c inhibitor: primary structure, cdna cloning, and chromosomal localization the functional integrity of the serpin domain of c -inhibitor depends on the unique n-terminal domain, as revealed by a pathological mutant simultaneous turnover of normal and dysfunctional c inhibitor as a probe of in vivo activation of c and contact activatable proteases regulation of c inhibitor synthesis the role of sialic acid in the functional activity and the hepatic clearance of c -inh clearance of human native, proteinase-complexed, and proteolytically inactivated c -inhibitor in rats a randomized, controlled trial to study the efficacy and safety of c inhibitor concentrate in treating hereditary angioedema a new role for c- -inhibitor in homeostasis: control of activation of the first component of human complement activation of the complement, coagulation, fibrinolytic and kallikrein-kinin systems during attacks of hereditary angioedema activation of the fourth component of complement (c ): assessment by rocket immunoelectrophoresis and correlation with the metabolism of c i-pla( ) activation during apoptosis promotes the exposure of membrane lysophosphatidylcholine leading to binding by natural immunoglobulin m antibodies and complement activation c-reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an antiinflammatory innate immune response: implications for systemic autoimmunity c q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited chromatin-independent binding of serum amyloid p component to apoptotic cells surface-mediated defense reactions: the plasma contact activation system quantification of plasma factor xiia-c -inhibitor and kallikrein-c -inhibitor complexes in sepsis prekallikrein activation and high-molecular-weight kininogen consumption in hereditary angioedema activation of the coagulation cascade in c -inhibitor deficiencies generation of plasmin during acute attacks of hereditary angioedema inactivation of factor xia in vivo: studies in chimpanzees and in humans reduction of contact activation related fibrinolytic activity in factor xii deficient patients: further evidence for the role of the contact system in fibrinolysis in vivo the role of factor xii in contact system activation effect of bradykinin-receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects roles for the kallikrein-kinin system in inflammatory exudation and pain: lessons from studies on kininogen-deficient rats angioedema induced by a peptide derived from complement component c microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms binding and activation properties of human factor xii, prekallikrein, and derived peptides with acidic lipid vesicles dextran sulphate inhibits phospholipid and sulphatide mediated autoactivation of factor xii inhibition of the activation of hageman factor (factor xii) by platelet factor inhibition of the activation of hageman factor (factor xii) by beta -glycoprotein i angioneurotic edema of the upper airways and antihypertensive therapy vasopeptidase inhibition: a double-edged sword? hereditary angioedema: genealogic studies and clinical considerations on genetic forms in a case study of patients late angioedema caused by ace inhibitors underestimated angioedema due to ace inhibitors: common and inadequately diagnosed increased sensitivity to bradykinin among african americans angioedema and cough in nigerian patients receiving ace inhibitors angioedema due to ace inhibitors: increased risk in patients of african origin angiotensin ii receptor blocker-associated angioedema: on the heels of ace inhibitor angioedema angiotensin ii receptor blockers in patients with ace inhibitor-induced angioedema angioedema associated with angiotensin ii receptor antagonists: challenging our knowledge of angioedema and its etiology mechanisms of angiotensin ii-induced expression of b kinin receptors omapatrilat: bristol-myers squibb omapatrilat and enalapril in patients with hypertension: the omapatrilat cardiovascular treatment vs. enalapril (octave) trial omapatrilat-the story of overture and octave vasopeptidase inhibitors-concepts and evidence biochemical basis of angioedema associated with recombinant tissue plasminogen activator treatment: an in vitro experimental approach bradykinin and des-arg( )-bradykinin metabolic pathways and kinetics of activation of human plasma bioregulation of kinins: kallikreins, kininogens, and kininases bradykinin receptors effects of hormone replacement therapy on serum angiotensin-converting enzyme activity and plasma bradykinin in postmenopausal women according to angiotensin-converting enzyme-genotype hormone replacement therapy and serum angiotensin-convertingenzyme activity in postmenopausal women decreased synthesis of serum carboxypeptidase n (scpn) in familial scpn deficiency familial carboxypeptidase n deficiency factor xii does not initiate prekallikrein activation on endothelial cells activation of the plasma kallikrein/kinin system on endothelial cell membranes the plasma kallikrein-kinin system counterbalances the renin-angiotensin system the physiologic basis of assembly and activation of the plasma kallikrein/kinin system mechanism of enhanced kinin release from high molecular weight kininogen by plasma kallikrein after its exposure to plasmin detection of active kallikrein in induced blister fluids of hereditary angioedema patients dose-dependent effects of postmenopausal estrogen and progestin on antithrombin iii and factor xii increased euglobulin fibrinolytic potential in women on oral contraceptives low in oestrogen-levels of extrinsic and intrinsic plasminogen activators, prekallikrein, factor xii, and c -inactivator molecular basis of estrogen regulation of hageman factor xii gene expression contact factors in plasma from women on oral contraception-significance of factor xi for the measured activity of factor xii biochemical and molecular pharmacology of kinin receptors transcription factor nuclear factor kappab regulates the inducible expression of the human b receptor gene in inflammation receptors for kinins: from classical pharmacology to molecular biology mechanisms regulating the expression, self-maintenance, and signaling-function of the bradykinin b and b receptors regulation of bradykinin b -receptor expression by oestrogen coexistence of hereditary angioedema and turner's syndrome hereditary angioedema precipitated by estrogen replacement therapy in a menopausal woman recurrent episodes of skin angioedema and severe attacks of abdominal pain induced by oral contraceptives or hormone replacement therapy recurrent angioedema: familial and oestrogen-induced angioedema and oral contraception recurrent angioedema associated with hypogonadism or anti-androgen therapy estrogen induction and contact phase activation of human factor xii enhanced expression of factor xii (hageman factor) in isolated livers of estrogenand prolactin-treated rats the influence of estrogen and prolactin on hageman factor (factor xii) titer in ovariectomized and hypophysectomized rats differential regulation of kininogen gene expression by estrogen and progesterone in vivo the effect of oestrogen dose and progestogen type on haemostatic changes in women taking low dose oral contraceptives effects of a very low-estrogen oral contraceptive on clotting factors, carbohydrate metabolism and plasma lipids and lipoproteins contact activation factors in plasma from women using oral contraceptives-increased levels of factor xii, kinin-free high molecular weight kininogen and acetone-activated kallikrein estrogen-associated thromboembolism contact factor mediated fibrinolysis is increased by the combined oral contraceptive pill prospective randomized study of effects of unopposed estrogen replacement therapy on markers of coagulation and inflammation in postmenopausal women positive impact of hormone replacement therapy on the fibrinolytic system: a long-term randomized controlled study in healthy postmenopausal women postmenopausal hormone replacement therapy increases coagulation activity and fibrinolysis effect of long-term hormone replacement therapy on angiotensin-converting enzyme activity and bradykinin in postmenopausal women with essential hypertension and normotensive postmenopausal women estrogen regulation of angiotensin-converting enzyme mrna sex hormones in hereditary angioneurotic oedema the complete drug reference bethesda: american society of health-system pharmacists drugdexÒ system hereditary angioedema and oral contraception effects of methylamine and heparin on a rapid chromogenic assay of c -esterase inhibitor in plasma effect of time, temperature and additives on a functional assay of c inhibitor effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women management and diagnostic guidelines for urticaria and angio-oedema hereditary angioneurotic oedema in three families: symptomatic heterogeneity, complement analysis and therapeutic trials hereditary angioneurotic oedema in finland: clinical, immunological and genealogical studies hereditary angioedema due to deficit of c esterase inhibitor hereditary angioneurotic edema: a clinical survey c -inh defect as an example of deficiency disease hereditary angioedema: danazol therapy in a -year-old child hereditary angioneurotic oedema: current management in pregnancy a multicentre evaluation of the diagnostic efficiency of serological investigations for c inhibitor deficiency inherited and acquired deficiencies of c esterase inhibitor in humans c is activated in hereditary angioedema, and c /c -inhibitor complexes rise during physical stress in untreated patients contact system in healthy term newborns: reference values in cord blood application of a monoclonal antibody against a neoepitope on activated c in an elisa for the quantification of complement activation via the classical pathway c inhibitor and diagnosis of hereditary angioedema in newborns complement components in newborns and their mothers determined by electroimmunoassay angioedema: a review on the acquired, allergic or non-allergic, and the hereditary forms serum complement levels in infancy: age related changes influence of age and sex on serum complement components in children development of the human coagulation system in the full-term infant plasma protease inhibitors in premature infants: influence of gestational age, postnatal age, and health status serum complement profiles in infants and children misdiagnosis of hereditary angio-oedema type and type acute consumption of c inhibitor in a patient with acquired c -inhibitor deficiency syndrome normal complement c values do not exclude hereditary angioedema hereditary angioneurotic oedema treatment of hereditary angioedema with a vapor-heated c inhibitor concentrate replacement therapy in hereditary angioedema: successful treatment of acute episodes of angioedema with partly purified c inhibitor c inh concentrate in the therapy of hereditary angioedema c -esterase inhibitor transfusions in patients with hereditary angioedema replacement therapy in hereditary angioedema: successful treatment of two patients with fresh frozen plasma c -inhibitor deficiency and angioedema treatment of hereditary angioedema letter: anaphylactic reaction to aprotinin dx- and hae: a developmental perspective the synthetic kunitz domain protein dx- to treat angioedema in patients with hereditary angioedema role of kinins in seasonal allergic rhinitis: icatibant, a bradykinin b receptor antagonist, abolishes the hyperresponsiveness and nasal eosinophilia induced by antigen efficacy and tolerability of icatibant (hoe ) in patients with moderately severe chronic bronchial asthma jerini announces positive phase ii study results with icatibant for the treatment of hereditary angioedema a phase i study of recombinant human c -inh in asymptomatic patients with hereditary angioedema-hae pharming features clinical results of c inhibitor at investigator meeting [press release treatment of hereditary angioedema with danazol: reversal of clinical and biochemical abnormalities morphologic evaluation of the liver in hereditary angioedema patients on long-term treatment with androgen derivatives side effects of long-term prophylaxis with attenuated androgens in hereditary angioedema: comparison of treated and untreated patients the long-term safety of danazol in women with hereditary angioedema long-term treatment of hereditary angioedema with attenuated androgens: a survey of a -year experience danazol therapy: an unusual aetiology of hepatocellular carcinoma hepatocellular adenomas in patients taking danazol for hereditary angio-oedema danazol-induced hepatocellular adenoma in patients with hereditary angio-oedema a case of hereditary angioneurotic oedema, successfully treated with epsilonaminocaproic acid: studies on c' esterase inhibitor, c' activation, plasminogen level and histamine metabolism long-term prophylaxis with c -inhibitor (c inh) concentrate in patients with recurrent angioedema caused by hereditary and acquired c -inhibitor deficiency pharmacokinetic parameters of c -inhibitor concentrate in patients with hereditary angio-oedema (hae)-a prospective study acute airway obstruction following tooth extraction in hereditary angioedema oral manifestations and dental management of patients with hereditary angioedema hereditary angioedema: report of case prophylactic use of epsilon aminocaproic acid for oral surgery in a patient with hereditary angioneurotic edema tranexamic acid: preoperative prophylactic therapy for patients with hereditary angioneurotic edema intravenous tranexamic acid in the management of hereditary angio-oedema potentially fatal hereditary angioedema: a review and case report the efficacy of short-term danazol prophylaxis in hereditary angioedema patients undergoing maxillofacial and dental procedures successful off-pump coronary artery bypass graft surgery in a patient with hereditary angioedema preoperative prophylaxis for c esterase-inhibitor deficiency in patients undergoing oral surgery: a report of three cases hereditary angioedema: uncomplicated maxillofacial surgery using short-term c inhibitor replacement therapy c -esterase inhibitor concentrate prevents upper airway obstruction in hereditary angio-oedema acquired angioedema as the presenting feature of lymphoproliferative disorders of mature b-lymphocytes spontaneous regression of acquired c esterase inhibitor deficiency associated with splenic marginal zone lymphoma presenting with recurrent angio-oedema acquired angioedema associated with rectal carcinoma and its response to danazol therapy: acquired angioedema treated with danazol activation of the contact system and fibrinolysis in autoimmune acquired angioedema: a rationale for prophylactic use of tranexamic acid angioneurotic edema with acquired c -inhibitor deficiency and autoantibody to c -inhibitor: response to plasmapheresis and cytotoxic therapy purity, activity, and virus safety of a pasteurized antithrombin concentrate a factor viii concentrate, highly purified and heated in solution virus-safe plasma proteins: elimination of viruses of risk by the manufacturing procedure inactivation of hepatitis a virus by pasteurization and elimination of picornaviruses during manufacture of factor viii concentrate absence of anti-human immunodeficiency virus types and seroconversion after the treatment of hemophilia a or von willebrand's disease with pasteurized factor viii concentrate absence of hepatitis after treatment with a pasteurized factor viii concentrate in patients with hemophilia and no previous transfusions effectiveness of alternative treatments for reducing potential viral contaminants from plasma-derived products inactivation of parvovirus b during pasteurization of human serum albumin a novel coronavirus associated with severe acute respiratory syndrome virus safety of pasteurized clotting factor concentrates: an eleven year follow up hepatitis c and pasteurized c -inhibitor concentrate prospective virus safety follow-up after therapy with a pasteurized c -inhibitor concentrate prophylactic treatment with pasteurised c inhibitor in herditary angioedema (hae)-a prospective months follow up prospective follow up of clinical manifestation and therapy in children with hereditary angioedema (hae) high-titer screening pcr: a successful strategy for reducing the parvovirus b load in plasma pools for fractionation prospective audit of adverse reactions occurring in primary antibody-deficient patients receiving intravenous immunoglobulin state of care for hemophilia in pediatric patients hereditary angioedema treatment of acute attacks of hereditary angioedema with c -inhibitor concentrate clinical studies of sudden upper airway obstruction in patients with hereditary angioedema due to c esterase inhibitor deficiency proteolytic inactivation of plasma c -inhibitor in sepsis expression of active human c inhibitor serpin domain in escherichia coli n-and o-glycans of recombinant human c inhibitor expressed in the milk of transgenic rabbits production of complex human glycoproteins in yeast recombinant human c -inhibitor produced in pichia pastoris has the same inhibitory capacity as plasma c -inhibitor recombinant human c -inhibitor produced in pichia pastoris has the same inhibitory capacity as plasma c -inhibitor recombinant c inhibitor p /p variants display resistance to catalytic inactivation by stimulated neutrophils the effect of danazol in the treatment of chronic cystic mastitis danazol-induced pseudomenopause in the management of endometriosis effects of danazol on pulsatile gonadotropin patterns and on serum estradiol levels in normally cycling women cortical and trabecular bone mineral content in women with endometriosis: effect of gonadotropin-releasing hormone agonist and danazol a comparison of the skeletal effects of goserelin and danazol in premenopausal women with endometriosis evidence of similar increases in bone turnover during nafarelin and danazol use in women with endometriosis the effects of nafarelin and danazol on vertebral trabecular bone mass in patients with endometriosis zoladex (goserelin acetate implant) in the treatment of endometriosis: a randomized comparison with danazol. the zoladex endometriosis study group bone mass in endometriosis patients treated with gnrh agonist implant or danazol excretion of urinary n-telopeptides reflects changes in bone turnover during ovarian suppression and indicates individually variable estradiol threshold for bone loss mechanism of mammalian ovulation complement, complement activation and anaphylatoxins in human ovarian follicular fluid cystic ovaries in women affected with hereditary angioedema increased plasma beta-endorphin levels in hereditary angioedema elevated levels of plasma beta-endorphin and gamma -melanocyte stimulating hormone in the polycystic ovary syndrome impaired human ovarian follicular fluid complement function in hereditary angioedema functionally active complement is present in human ovarian follicular fluid and can be activated by seminal plasma inherited and acquired deficiency of c esterase inhibitor in humans transmission of hepatitis g virus in patients with angioedema treated with steam-heated plasma concentrates of c inhibitor loss of danazol responsiveness in angioedema with c inhibitor deficiency complement and complement fixation immunochemical quantitation of the third, fourth and fifth components of human complement: concentrations in the serum of healthy adults recurrent abdominal pain as the sole manifestation of hereditary angioedema in multiple family members danazol therapy for hereditary angio-oedema in children epsilon aminocaproic acid therapy of hereditary angioneurotic edema: a double-blind study danazol for children with immune thrombocytopenic purpura hereditary angio-oedema in children tonsillectomy in a patient with hereditary angioedema after prophylaxis with c inhibitor concentrate a cluster of mutations within a short triplet repeat in the c inhibitor gene dysfunctional c inhibitor ta: deletion of lys- results in acquisition of an n-glycosylation site characterisation of nucleotide sequence variants and disease-specific mutations involving the # end of the c -inhibitor gene in hereditary angio-oedema an rna splice site mutation in the c -inhibitor gene causes type i hereditary angio-oedema a point mutation in exon of the c -inhibitor gene causing type i hereditary angioedema a novel donor splice site mutation in the c inhibitor gene of a patient with type i hereditary angioneurotic edema nonsense mutations affect c inhibitor messenger rna levels in patients with type i hereditary angioneurotic edema identification of a novel mutation of c inhibitor gene in a chinese family with hereditary angioedema a single base deletion from the c -inhibitor gene causes type i hereditary angio-oedema c -inhibitor gene nucleotide insertion causes type ii hereditary angio-oedema substrate properties of c inhibitor ma (alanine -glutamic acid): genetic and structural evidence suggesting that the p -region contains critical determinants of serine protease inhibitor/substrate status identification of type i hereditary angio-oedema (hae) mutations type ii hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine- in the c inhibitor gene a dysfunctional c inhibitor protein with a new reactive center mutation type ii hereditary angio-oedema associated with two mutations in one allele of the c -inhibitor gene around the reactive-site coding region c -inhibitors and their genes: an update identification of a c/t mutation in the reactive-site coding region of the c -inhibitor gene and its detection by an improved mutation-specific polymerase chain reaction method identification of a new p residue mutation ( arg-ser) in a dysfunctional c inhibitor protein contained in a type ii hereditary angioedema plasma a mutation unique in serine protease inhibitors (serpins) identified in a family with type ii hereditary angioneurotic edema a de novo deletion in the c inhibitor gene in a case of sporadic hereditary angioneurotic edema a point mutation in the c -inhibitor gene causes type i hereditary angiooedema clusters of intragenic alu repeats predispose the human c inhibitor locus to deleterious rearrangements structural and functional aspects of c -inhibitor n, none; r, rare (< attack/mo); f, frequent (> attack/mo). *e-aminocaproic acid is not shown because it had no effect or caused intolerance, and therefore, it was replaced by tranexamic acid.cover. carbon backbone of a -dimensional model of c -inhibitor, with amino acids involved in hae. this model is made by homology modeling and is based on the crystal structure of other serpins. the a-helices are depicted in red, the b-strands in light blue. amino acids mutated in hae patients are depicted in yellow ball-and-stick. the reactive site, p -arg, is depicted in magenta ball-and-stick. ineke bos. adapted with permission from bos et al.