key: cord-0006049-ys7an3rr authors: nan title: Book of Abstracts: 10th World Congress on Inflammation date: 2011-06-07 journal: Inflamm Res DOI: 10.1007/s00011-011-0341-6 sha: 4e744f58739095d750a561c2fa2364fb56cd6b9e doc_id: 6049 cord_uid: ys7an3rr nan The adaptive immune system with its antibodies, B and T cells arose around 500 million years ago in the first vertebrates and in all invertebrates, the defence mechanisms are purely innate. We have been working since 1985 on the innate immune system, using Drosophila as a model system. In Drosophila, an infection provokes the rapid synthesis of powerful antibiotic peptides. As an example, the basal level of expression of the antifungal peptide DROSOMYCIN is increased a thousand fold within 30 min of septic injury. The control of this expression involves at least two pathways that, for sake of simplicity, I refer to as the TOLL and the IMD pathways. The paramount role of the TOLL and IMD pathways in the host defence of Drosophila is illustrated by experiments in which mutant flies are challenged with fungi or bacteria. In TOLL-deficient mutants, survival to fungal, but not to bacterial infection, is severely compromised. By contrast, IMD mutants are markedly affected by bacterial infections but resist fungi. In vertebrates, recognition of microbes by the innate immune system takes place at the cellular level by a family of transmembrane receptors homolog to Drosophila Toll, namely the Toll like receptors or TLRs. In Drosophila however, these recognition events take place in the open circulatory system via soluble excreted recognition proteins like the peptidoglycan recognition proteins (PGRPs). In turn, these recognition steps must be conveyed onto Toll by extracellular proteolytic signalling pathways. Upstream of the IMD pathway, other PGRPs are recognizing Gram-negative microbial determinants. We are now interested in how these two pathways are activated and regulated. Although anatomy of the Drosophila immune system differs widely from the vertebrate immune system, we will discuss the possibility that the fruit fly, with its powerful genetics and simple organization, could become a new paradigm to study inflammation. Over the past three decades, there has been a growing appreciation of the physiological and pathophysiological importance of endogenous gaseous mediators, such as nitric oxide, carbon monoxide and hydrogen sulfide. All three gases were initially considered poisons, but their physiological importance is now unquestionable. H 2 S exerts a number of anti-inflammatory effects, including suppression of leukocyte adherence to the vascular endothelium, edema formation and NfKB activation. It also promotes resolution of inflammation and promotes wound healing. H 2 S may therefore be exploited in the design of more effective and/or less toxic anti-inflammatory compounds. In recent years, we have attempted to achieve this using hydrogen sulfidereleasing moieties, covalently linked to a number of different antiinflammatory drugs. For example, mesalamine is the first-line therapy for inflammatory bowel disease (Crohn's disease and ulcerative colitis), but it is a weak drug, with doses of up to 4 g per day required. An H2Sreleasing derivative of mesalamine (ATB-429) was shown to be significantly more potent in two models of colitis. In addition to reducing inflammation, ATB-429 suppressed expression of a number of proinflammatory cytokines (IL-1, TNF, IL-12) and markedly reduced granulocyte infiltration into the colonic tissue. Another class of therapeutics we have focused on is nonsteroidal anti-inflammatory drugs (NSAIDs). While very effective at reducing pain and inflammation, these drugs continue to be limited in their utility by significant toxicity in the gastrointestinal tract (the advent of selective COX-2 inhibitors has been only a modest advance in this regard). Several H 2 S-releasing NSAIDs have been developed and tested extensively. ATB-346 is an H 2 S-releasing derivative of naproxen. It exhibits comparable antiinflammatory and analgesic effects to naproxen, but does not produce any detectable GI injury in a variety of rodent models, including some in which gastrointestinal mucosal defence is severely impaired. Moreover, rather than impairing the healing of pre-existing ulcers, as is seen with naproxen and other NSAIDs, ATB-346 significantly enhanced healing. This approach could be taken to modify a wide range of drugs, as has been done with nitric oxide-releasing therapeutic agents. The incidence of chronic diseases is steadily rising over the past decades. Perturbations of gut microbial communities have been suggested in many of these. The main objective of the MetaHIT consortium is to explore associations of the bacterial genes, genomes or communities from the human gut with two chronic diseases, obesity and inflammatory bowel diseases. We developed a powerful new approach, Quantitative Metagenomics, to visualize the gut bacteria present in any individual. It is based on two main elements, the reference gene catalog of the gut bacterial genes and the high throughput sequencing of the total stool DNA. The reference gene catalog was established by massive Illumina sequencing of total fecal DNA from 124 individuals of European origin. It contains 3.3 million non-redundant genes, 150-fold more than encoded by our own genome. It captures over 85% of the genes from our cohort and includes about 80% of those found in previous studies of smaller scope, carried out in Japan and the US. This indicates that it represents well the human intestinal metagenome and has become known as our other genome (Qin et al. in Nature 464:59-65, 2010) . Two case/control studies, one of obese and lean individuals and the other of the ulcerative colitis patients and family matched controls will be presented. They reveal links between bacterial diversity and inflammation, which may be relevant to numerous pathologies. Furthermore, they unveil association of bacterial species to chronic disease. The species have a diagnostic/prognostic potential well above that of the alleles of our own genome, suggesting that personalized medicine should target, in preference, our other genome. KL-04 SENESCENCE AND INFLAMMATION: NEW LINKS, NEW TARGETS undergo a permanent arrest of cell proliferation (growth). Cellular senescence is also thought to cause or contribute to aging. How can the same process have such apparently incongruous effects (tumor suppression and aging)? The senescence growth arrest is accompanied by large-scale changes in gene expression. One outcome of these changes is a robust increase in the expression and secretion of numerous cytokines, growth factors and proteases, which we term the senescence-associated secretory phenotype (SASP). A striking feature of the SASP is that many SASP components are potent pro-inflammatory proteins. Inflammation, of course, drives virtually every major age-related disease, including, ironically, cancer. As an inflammatory response, cellular senescence also entails the expression of proteins that signal the immune system and targets senescent cells for clearance. Some senescent cells, however, escape immune clearance. Understanding how some senescent cells evade immune clearance provides novel targets for interventions aimed at facilitating the destruction of senescent cells by the immune system. Recent findings show that the SASP is primarily a DNA damage response (DDR) that may have evolved to allow damaged cells to communicate their compromised state to the surrounding tissue. The DDR engages a number of stress-responsive pathways that collaborate to stimulate the expression of SASP genes. These pathways include well-known transcriptional regulators of inflammation, such as NF-jB. They also include novel pathways that act at the level of translation, such as the mTOR pathway. These new data identify novel targets for suppressing the deleterious (pro-inflammatory) effects of cellular senescence, while preserving its beneficial (tumor suppressive) effects. Disclosure of interest: None declared. Interleukin 17 is a cytokine with central involvement in inflammatory immune responses. It is primarily produced by Th17 CD4 T cells, a subset of TCRcd T cells and some NKT cells. Over the past few years it has become clear that the production of IL-17 is regulated at multiple levels-both positively and negatively-to control its activity which when dysregulated can initiate pathogenic immunity and result in a variety of autoimmune syndromes. Cytokine production by Th17 effector cells is not a stable parameter, but instead appears to be modulated on several levels. As more states of CD4 T cell differentiation are uncovered, their flexibility is also beginning to be recognized. Components that control the plasticity of CD4 T cell populations include environmental influences at tissue sites, transcriptional circuitry and chromatin modifications. Understanding the rules that underlie adaptation and flexibility of T cell responses will be an essential part for future intervention strategies in human disease states. MicroRNAs (miRNAs) regulate gene expression at a post-transcriptional level by binding to complementary target mRNAs. They are involved into many aspects of immune system development, from hematopoiesis to activation of innate and adaptive immune response to infection. They also mediate complex multi-cellular regulations, for instance during regeneration of airway and skin epithelia. Epithelialmesenchymal interactions, which are critical during vertebrate embryo development, and for maintenance of homeostasis in adult tissues, are modulated by miRNAs. Thus, miR-155 is induced in human fibroblasts by inflammatory cytokines TNF-a and IL-1b while it is down-regulated by TGF-b. Keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, was established as a target in human lung fibroblasts. In vivo experiments using a mouse model of lung fibrosis have shown that miR-155 expression level is indeed correlated with the degree of lung fibrosis. miRNA dysregulation is also central to the development and pathophysiology of many cancers. We have recently highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor Hypoxia-Inducible Factor-1 (HIF-1) in lung cancer. Over-expression of miR-210 at late stages of Non-Small Cell Lung Cancer causes an alteration of cell viability, a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. Expression profiling of cells S6 Inflamm. Res. over-expressing miR-210 reveals a specific signature that includes several subunits of the Electron Transport Chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, a subunit of Succinate Dehydrogenase (SDH) is a validated target for miR-210, and SDHD knockdown mimics miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-offunction SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC components genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. We will discuss our current understanding of these general regulatory mechanisms, focusing on their involvement in inflammation. Triboulet et al. Science. 2007; 315:1579 -82. Pottier et al. PLoS One. 2009 4(8) The genetic component of Inflammatory Bowel Diseases (IBD) is among the best known for complex genetic disorders. The role of genes in Crohn's Disease (CD) appears to be stronger than in Ulcerative Colitis (UC) as demonstrated by the percentages of familial cases [8] [9] [10] vs. [6] [7] [8] ] and the concordance rates in monozygotic twins [20-50% (CD) vs. 2-6% (UC)]. Gene hunting was rarely fruitful in the past when using the functional candidate gene approach (e.g. HLA genes in UC), or linkage studies (e.g. NOD2 and SLC22A4/5 in CD). Conversely, the genome wide association studies (GWAS) have now provided more than 70 susceptibility genes for CD and more than 40 for UC in few years. These genes define some key biological functions like innate immunity and autophagy for CD and Th17 T-cell orientation for both diseases. However, despite of these important successes, GWAS failed to explain more than 25% of the heritability of IBD suggesting that the additional genetic variations, gene-gene or geneenvironment interaction and stochastic events may play a role in the predisposition to IBD. Nod1 and Nod2 are pattern recognition receptors of the innate immune system that detect peptidoglycan (PG) from the bacterial cell wall and trigger inflammation. Although both of these members of the Nod-like receptor (NLR) family respond to PG they respond to distinct substructures of this complex microbial-associated molecular pattern (MAMP). Indeed, Nod1 recognizes diaminopimelic acid-containing tripeptide fragments of PG, found mainly in Gram-negative bacteria, while Nod2 detects muramyl dipeptide, which is the minimal fragment of PG common to both Gram-negative and Gram-positive organisms. Detection of these muropeptides by Nod1 and Nod2 triggers a signal transduction cascade that culminates in the activation of NFjB and the production of pro-inflammatory mediators. Importantly, Nod1 and Nod2 have been implicated in inflammatory bowel disease (IBD), in particular Nod2 has been associated with Crohn's disease, yet a clear understanding of how dysfunctional Nod activation leads to aberrant inflammation is still lacking. While much of our previous work has focused on dissection of Nod signalling in in vitro and ex vivo models, our current interest is to understand how Nods function at the mucosal surface of the gastrointestinal tract and how they orchestrate inflammation and combat enteric infection. Specifically, we are interested in using bacterial colitis models, including the Citrobacter rodentium and streptomycin-treated Salmonella typhimurium models to probe Nod responses at the level of the intestinal mucosa. Our overall goal is to understand how Nod activation orchestrates mucosal responses and to delineate the functional importance of Nod triggering in order to gain a more complete understanding of their role in IBD. prausnitzii on resected ileal Crohn's mucosa was associated with a higher risk of endoscopic postoperative recurrence at 6 months. We thus hypothesized that F. prausnitzii could display anti-inflammatory effects. We thus analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro and in vivo models. In vitro PBMCs stimulation by F. prausnitzii led to significantly lower IL-12 and IFNc production levels and higher secretion of IL-10 [3] . Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis. These anti-inflammatory effects are most probably partly due to secreted metabolites able to block NF-jB activation and IL-8 production. We are now evaluating whether these properties are or not a general trait of F. prausnitzii species by isolating and testing other strains. In order to decipher the interactions between F. prausnitzii and the host, animal models with simplified microbiota are actually developed. We are also trying to identify the anti-inflammatory molecules in the supernatant of F. prausnitzii and the bacterial genes encoding these molecules. Finally, this knowledge might be useful for the application of probiotics or pharmacological molecules in CD for which the current treatments are poorly effective and merely aimed at maintenance of the disease. Counterbalancing dysbiosis using F. prausnitzii as a probiotic is thus a promising strategy in CD treatment. 1. Sokol et al. Inflamm Bowel Dis. 2006; 12:106-11. 2. Manichanh et al. Gut. 2006; 55:205-11. 3. Sokol et al. Proc Natl Acad Sci USA. 2008; 105(43) :16731-6. The increase in circulating inflammatory factors found in obese subjects and the discovery of macrophage accumulation in human adipose tissue has opened a new field of research in obesity. This allowed reconsidering aspects of obesity physiopathology. The so called ''low grade'' inflammatory state is characterized by the moderate but chronic systemic rise of an increasing panel of molecules. A small or even modest reduction of weight, known to ameliorate obesity associated risks, significantly reduces circulating inflammatory markers. However knowledge regarding the molecules linking obesity to its complications is still lacking. White adipose tissue contributes significantly to low-grade inflammation in obesity. This tissue is composed of mature adipocytes, precursors (preadipocytes), endothelial cells, macrophages, mast cells, blood vessels, nerves and lymphatic and connective tissue. The phenotype, amount and biology of adipose tissue components are altered profoundly in human obesity. The evaluation of transcriptomic interactions characterizing the obese adipose tissue demonstrated the strong relationship linking inflammatory processes to extra cellular matrix remodelling components. Our group showed that interstitial fibrosis accumulates in obese adipose tissue as in many organs affected by low-grade inflammation in chronic diseases (i.e. liver, lung, kidney pathologies). In addition, to structural and biological alterations of adipose tissue, there are perturbations of its dialogue with central (i.e. brain) and peripheral organs (i.e. muscle, intestine, liver, bone, vessels) via multiple signals including inflammatory mediators. The aim of this lecture is to discuss up-to-date current knowledge regarding the importance of inflammation and adipose tissue remodelling especially in human obesity. A clinical angle will be taken. Obesity is recognized as a low grade chronic inflammatory state. Several inflammatory factors are known to directly interact with insulin signaling pathway leading to insulin resistance. Such an effect is thought to contribute to the link between obesity and type 2 diabetes. Increasing evidences show that the adipose tissue (AT) itself is the site and the source of inflammation. Indeed, accumulation of innate and adaptive immune cells is observed in AT of various murine models of obesity (genetic and diet-induced obesity) as well as in human with increased adiposity index. Initial studies focused on adipose tissue macrophages (ATM). Characterization of human ATM (hATM) phenotypes and roles showed that hATM impact hAT remodeling, including adipocyte differentiation, angiogenesis and fibrosis. Through these local effects, ATMs may limit the AT capacity to buffer the toxic fatty acids into neutral triglycerides in adipocytes and thereby contribute to the ectopic lipid deposit into metabolic active tissues leading to insulin resistance. Kinetics studies of immune cells trafficking in AT clearly showed that T lymphocytes accumulate in mice AT before ATMs. The proportion of adipose tissue lymphocytes (ATL) subsets, including regulatory T lymphocytes, Th1 helper and cytotoxic T cells, are modulated with AT development. Analysis of the role of hATLs showed that hATL-derived secretions including interferon interfered with insulin-mediated up-regulation of the key lipogenic enzymes in adipocytes. Therefore, accumulation and activation of ATLs appears to be the primary event involved in obesityassociated inflammation and several recent evidences suggest that such an event might be related to cell-mediated immune response. The mammalian intestine provides a unique niche for a large community of bacterial symbionts that complements the host in digestive and anabolic pathways, as well as in protection from pathogens. Only a few bacterial phyla have adapted to this predominantly anaerobic environment, but hundreds of different species create an ecosystem that affects many facets of the host's physiology. Recent data show how particular symbionts are involved in the maturation of the immune system, in the intestine and beyond, and how dysbiosis, or alteration of that community, can deregulate immunity and lead to immunopathology. The extensive and dynamic interactions between the symbionts and the immune system are key to homeostasis and health, and require all the blends of so-called regulatory and proinflammatory immune reactions. Unfortunately, pro-inflammatory immunity leading to the generation of Th17 cells has been mainly associated with its role in immunopathology. Here I discuss the view that the immune system in general, and type 17 immunity in particular, develop to maintain the equilibrium of the host with its symbionts. A. Y. Rudensky 1, * 1 Immunology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Regulatory T lymphocytes (Treg cells) play a critical role in immune homeostasis by opposing immune-mediated inflammation in the context of autoimmunity, responses to infection, cancer, allergy, and interactions with commensal microbiota at the environmental interfaces. Transcription factor Foxp3 orchestrates differentiation and function of these cells. Our studies suggest that Foxp3 cooperates with other regulators of gene expression and co-factors to ensure the maintenance of its own expression and Treg cell suppressor function under basal and inflammatory conditions. In this regard, the ability of Treg cells to suppress Th17 mediated inflammatory responses in the intestines is dependent upon IL-10 receptor mediated activation of transcription factor Stat3 and its cooperation with Foxp3. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the brain and spinal cord leading to damage of nerve fibers resulting in neurological impairment and deficits. The cause of MS is still mostly unknown. Demyelination in the central nervous system in MS is thought to be mainly mediated by T helper 1 cells, although this dogma is currently heavily discussed since recent evidence suggests an active pathogenic role of T helper 17 and CD8 T cells in MS, which recapitulates earlier suggestions that virus may have pathogenic role in MS. The contribution of regulatory CD4 Foxp3+ T cells in the control of the disease is also controversial. Currently the unraveling of immune-mediated pathogenesis in MS relies mostly on animal models. So far there is not one animal model that reflects all aspects seen in MS, but still the many different models are very suitable for targeting specific aspects of this disease. By using rodent models we pinpointed several cellular and molecular pathways of potential relevance for human inflammatory diseases of the central nervous system. The lessons learned from these models will be discussed. Apoptosis is the most common form of physiological cell death and required for normal development and maintenance of tissue homeostasis, and its dysregulation is associated with various pathological conditions. In inflammation, for example, inhibition of apoptosis of inflammatory cells is a common phenomenon and essential to effectively fight against pathogens. On the other hand, specific tissue cells frequently undergo apoptosis in association with inflammatory responses that may cause or at least contribute to organ dysfunction. In contrast to apoptosis, autophagy is a cellular survival mechanism and usually induced when cells are exposed to stress. In infectious diseases, autophagy degrades intracellular pathogens and promotes cellular survival during pathogen invasion. Autophagy has also been shown to be involved in MHC class I and II antigen presentation, and may be required for the induction of self-tolerance. In addition, there is evidence that autophagy may provide negative feedback to dampen inflammatory signalling. Therefore, defects in autophagy may lead to autoimmunity. Moreover, in certain instances, induction of autophagy may promote cell death, including apoptosis, suggesting a cross-talk between apoptotic and autophagic pathways. Taken together, apoptosis and autophagy are critical to the overall fate of the cell and play important roles in immunity and inflammation. Physiological cell death, which occurs as a continuous byproduct of cellular turnover, is non-immunogenic or even tolerogenic, thereby avoiding autoimmunity. However, cancer cell death elicited by radiotherapy and some chemotherapeutic agents such as anthracyclines and oxaliplatin can be immunogenic. Immunogenic death involves changes in the composition of the cell surface, as well as the release of soluble immunogenic signals that occur in a defined temporal sequence. This 'key' then operates on a series of receptors expressed by dendritic cells (DC, the 'lock') to allow for the presentation of tumor antigens to T cells and for the initiation of a productive immune response. Immunogenic cell death is characterized by the early cell surface exposure of calreticulin, which determines the uptake of tumor antigens by DC. The late release of the protein high mobility group box 1 (HMGB1), which acts on toll-like receptor 4 (TLR4), is required for the presentation of antigens from dying tumor cells. In addition, the release of ATP from dying cells causes the P2RX7 purinergic receptor-dependent activation of the NLRP3 inflammasome in DC, thereby allowing them to release interleukin-1ß and to polarize tumor antigen-S10 Inflamm. Res. specific CD8 T cells towards a Tc1 cytokine pattern. We postulate that the immune system determines the long-term success of anticancer therapies, and that this immune response is dictated by immunogenic tumor cell death. Thus, therapeutic failure can result from failure to undergo immunogenic cell death (rather than cell death as such). Thus, agents that fail to induce immunogenic cell death cannot yield a long-term success in cancer therapy. Moreover, tumors that are intrinsically unable to undergo immunogenic cell death are incurable. Importantly, it appears that mitochondrial events determine whether cancer cells die or not in response to chemotherapy, while an endoplasmic reticulum stress (ER) response combined with autophagy determines whether this cell death is perceived as immunogenic. We suggest a series of strategies to restore the immunogenicity of cell death in the context of deficient ER stress and autophagy. Members of the Tumour Necrosis Factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains, assembled by a complex containing HOIL-1 and HOIP, were implicated in TNF signalling, yet their in vivo relevance remained uncertain. We identified Sharpin as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of the native TNF receptor signalling complex revealed RIP1 and NEMO to be linearly ubiquitinated. Mutation of the Sharpin gene causes chronic proliferative dermatitis (cpdm) in mice characterised by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1b was attenuated in cpdmderived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex Sharpin interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide in vivo evidence for the relevance of linear ubiquitination in preventing inflammation and regulating immune signalling. recruiting the required leukocyte subtypes necessary for the subsequent phases of response to infection. In addition, neutrophils were recently demonstrated to directly interact with immune and non-immune cell types, including dendritic cells (DC), NK cells, iNKT cells, polarized T cells and mesenchymal cells, a feature which greatly emphasizes their capacity to contribute as orchestrators of innate and adaptive immunity. All these novel aspects of the biology of neutrophils will be exhaustively covered, with particular emphasis on the more recent advances. Disclosure of interest: None declared. Human polymorphonuclear neutrophils (PMN) represent an essential cellular component of innate host defense against infection. Upon engagement with a suitably opsonized microbe, PMN ingest and confine the organism in a specialized compartment, the phagosome, where cytotoxins can be delivered. Concomitant with phagocytosis, there is coordinated assembly and activation of the NADPH oxidase, the source of oxidants to fuel microbial killing, and fusion of PMN granules, a source of a host of proteolytic enzymes and antimicrobial proteins. Prominent among the granule proteins delivered to the phagosome is myeloperoxidase (MPO), a hemeprotein uniquely able to generate hypochlorous acid (bleach), a potent antimicrobial agent, in the presence of H 2 O 2 . Although optimal PMN microbicidal activity relies on MPO-mediated production of HOCl and its capacity to modify microbial targets, efficient killing and degradation of ingested organisms reflects the coordinated activities of many bioactive species. The mechanisms underlying the generation and antimicrobial action in phagosomes will be highlighted, as will specific synergies between soluble circulating proteins and PMN responses that collaborate to eradicate invading microbes. Lastly, it is critical to recognize that many ingested microbes immediately sense and respond transcriptionally to the cytotoxins present in the phagosome. Successful microbial reactions allow ingested microbes such as staphylococci to survive and cause persistent infection. Only by understanding the molecular basis for the dynamic interactions between host and microbe will novel advances in antimicrobial therapy be made. Tissue macrophages can change their physiology in response to a variety of endogenous and exogenous signals generated in tissue. Many inflammatory signals can promote a classical macrophage activation state, characterized by the production of myriad inflammatory cytokines. These classically activated macrophages are an important component of host defense, but they are also responsible for host tissue damage during autoimmune diseases. We and others have identified signals that can 'reprogram' macrophages to assume physiologies that are quite distinct from classically activated macrophages. We have identified a so-called ''regulatory macrophage'' that produces high levels of the anti-inflammatory cytokine, IL-10. These cells are potent down-regulators of innate and adaptive immune responses and they can prevent or reverse autoimmune pathology. The 'reprogramming' step involves the activation of the Mitogen-Activated Protein Kinase, ERK, which leads to histone phosphorylation and chromatin remodeling at the IL-10 promoter. Regulatory macrophages secrete high levels of IL-10 but little to undetectable amounts of IL-12. Work is on-going to identify the various signals that can reprogram these cells to become regulatory macrophages and to discover biochemical markers that are specific to regulatory macrophages. The manipulation of the various macrophage subpopulations may lead to new approaches to diminish autoimmune pathologies, or conversely to develop more effective immune responses during vaccination. Neuronal control of inflammation and pain (SY06) behavioral illness including depression. We used oral antimicrobial therapy to induce dysbiosis in mice and assessed visceral pain responses and alterations in brain chemistry and behavior. Antibiotic treatment reduced lactobacilli and this was accompanied by a small increase in MPO activity but no structural damage in the gut. In addition, there was an increase in substance pain and in the visceromotor response to colorectal distension. These responses were attenuated in mice receiving either lactobacillus paracaseii or dexamethsone concomitantly with antibiotics. Oral antimicrobial therapy also increased Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus and an increase in anxiolytic behavior based on latency to step-down and light box-dark box preference testing. Antimicrobial-induced BDNF and behavioral changes were independent of vagus nerve integrity or changes in circulating pro-inflammatory cytokines and were not seen in germ free mice. The existence of a microbiome-brain communication was confirmed in adoptive transfer experiments where behavioral traits of one murine strain were induced in germ free recipients of another strain, following colonization with cecal bacteria. These results indicate that the intestinal microbiome communicates with the host enteric nervous system to alter visceral pain responses by an inflammation-dependent mechanism, and with the brain to alter behavior by an inflammationindependent mechanism. These findings may explain the frequent coexistence of pain and altered behavior in functional and inflammatory GI diseases. S. D. Brain Approximately 30% of arthritic patients consider that their medicines could be improved, especially in terms of pain relief. The work in this laboratory has concentrated on providing evidence for role of members of the transient receptor potential (TRP) family, especially TRPV1 and TRPA1 in murine models of inflammatory hyperalgesia. Our first study revealed a role for the TRPV1 receptor in attenuating thermal hyperalgesia and inflammatory swelling in a model of CFA-induced inflammation, in a situation where TNFa levels remained high. Interestingly, we also showed that capsaicin induced TRPV1-dependent vasoconstriction in the knee and the consequences of this are at present unknown. Further studies enabled a link between TNFa levels and TRPV1-dependent hyperalgesia to be made, a mechanism that is dependent on COX-2 and other cytokines, as well as TRPV1. More recently we have gained evidence that TRPA1 is also involved in mediating pain. In this case TRPA1 is important in mediating mechanical-induced hyperalgesia, an anti-hyperalgesic effect that appears independent of an effect on inflammation. Keeble et al. Arthritis Rheum. 2005 ;52:3248-56. Russell et al. Pain. 2009; 142:264-74. Fernandes et al. Arthritis Rheum. 2010 (in press Migraine is a highly prevalent chronic condition characterized by disabling attacks of pulsating head pain associated with nausea and vomiting, as well as other symptoms. The pathogenesis of migraine is still unknown, however, recent evidence supports the role of calcitonin gene-related peptide (CGRP) in the pathway which results in headache and the associated symptoms of migraine attack. The role of CGRP released from the central terminals of trigeminal neurons and other dorsal root ganglion neurons is still uncertain, whereas CGRP released from peripheral and perivascular endings of these neurons causes a marked and prolonged arterial vasodilatation. CGRP receptor antagonists have been found to ameliorate the migraine attack. It is possible that blockade of the hitherto not well defined central actions, or of the well known vasodilator effect of CGRP of the peptide, are key mechanisms for migraine treatment. A series of agents identified as triggers of migraine attacks have been found to release CGRP, and through this mechanism to produce meningeal vasodilatation. These include, among many other agents, ethanol, which acts via the sensitization of the transient receptor potential vanilloi-1 (TRPV1) channel, or ingredients of cigarette smoke via the TRPA (ankyrin)-1 channel. TRPA1 activation and CGRP release are also produced by umbellulone, a compound contained in Umbellularia californica, known also as the headache tree because, beginning first with the Native Americans, all the way through present times, the scent of its leaves has been recognized as a cause of headache. Sir Thomas Lewis in 1936 proposed that a proinflammatory substance was released from nocifensive neurons to produce vasodilatation and sensitization of these neurons to painful stimuli. Recent acquisition strongly suggests that this substance is CGRP, which via these, and possibly additional mechanisms, plays a major role in migraine mechanism. effector phase of various autoimmune inflammatory diseases. Our results indicate a surprising similarity between integrin and immunoreceptor signal transduction pathways in neutrophils and other cells. b 2 integrin-mediated neutrophil activation requires Src-family kinases, the ITAM-containing DAP12 and FcRc adapter protein, the Syk tyrosine kinase and phospholipase Cc2 (PLCc2). The same molecules also appear to participate in b 2 integrin-mediated activation of macrophages. Furthermore, DAP12/FcRc, Syk and PLCc2 are also required for osteoclast development and osteoclast-mediated bone resorption. Taken together, a signal transduction pathway reminiscent of that utilized by classical immunoreceptors mediates non-immunoreceptor signaling processes such as integrin signaling or osteoclast development and function. To test whether the above pathway also participates in pathological processes, we tested the development of K/BxN serum transfer arthritis, an autoantibodymediated arthritis model, in various genetically deficient mouse strains. Our results indicate that Src-family kinases, Syk and PLCc2 are all required for the autoantibody-mediated effector phase of this disease. Components of this pathway may prove to be suitable targets of the pharmacological therapy of autoantibody-mediated diseases such as certain subsets of rheumatoid arthritis. Anti-citrullinated protein antibodies (ACPA) are a very distinctive feature of rheumatoid arthritis (RA) patients. The presence of these antibodies is highly predictive for both the development of RA and the extent of associated joint destruction. Recent evidence indicates that well-known genetic risk factors for RA: the HLA-DRB1 shared epitope (SE) alleles and the PTPN22 T-allele are predominantly associated with anti-CCP positive RA. These reports, together with the finding that ACPA can exacerbate arthritis in mice, suggest that anti-peptidylcitrulline immunity plays an important role in the pathogenesis of the disease. Studies investigating at which point in time ACPA first appear, have revealed that these antibodies can often be detected several years before disease onset. The mere presence of ACPA therefore does not appear to be sufficient to precipitate disease. An explanation for this observation could be that the anti-citrullinated protein immune response first needs to mature more fully, in the course of which ACPA could acquire distinct characteristics which are instrumental in mediating tissue damage. Taking into consideration that ACPA can be detected before the clinical diagnosis of RA, and that the presence of ACPA is strongly associated with disease progression, we hypothesized that epitope spreading, avidity maturation and/or isotype usage of the ACPA response may play a role in the evolution of the disease. Our data reveal that broadening of isotypeusage and Epitope spreading with recognition of more citrullinated antigens occurs before onset of RA and that immunological differences in these ACPA are associated with the future disease course. S14 Inflamm. Res. Medical Faculty and University Hospitals, Geneva, Switzerland NOX enzymes are a gene family of reactive oxygen species (ROS)-generating NADPH oxidases. In mammals, 7 family members are known DUOX1, 2) . They transport electrons from cytosolic NADPH across the membrane and attach it to oxygen to form superoxide and down-stream ROS, including hydrogen peroxide. ROS are key players in inflammation, however in contrast with classical views, they have not only proinflammatory, but also antiinflammatory properties. I will discuss the two aspects. Concerning the proinflammatory properties of NOX-derived ROS and will discuss ROS-mediated neuroinflammation anddegeneration, commonly seen in a variety of diseases of the central nervous system, including Alzheimer's disease, ALS, stroke and epilepsy. Concerning the antiinflammatory properties of NOXderived ROS, I will discuss hyperinflammation and increased autoimmune disease in patients with chronic granulomatous disease (CGD, a deficiency in NOX2 or subunits thereof), as well as in mouse models of CGD. I will then discuss redox-sensitive signaling pathways which might account for the complex role of NOXderived ROS in inflammation. M. Pasparakis 1, * 1 Institute for Genetics, University of Cologne, Cologne, Germany Intracellular signal transduction pathways activated downstream of pattern recognition receptors and cytokine receptors play a critical role for the regulation of immune and inflammatory responses. The NF-jB pathway is one of the most important signalling cascades activated by cytokine and innate immune receptors. NF-jB regulates cellular responses to infection, injury and other stressful conditions requiring rapid reprogramming of gene expression. NF-jB activation has been implicated in the pathogenesis of many diseases, particularly chronic inflammatory conditions and cancer. NF-jB activation is mediated by the IjB kinase (IKK), which consists of two catalytic subunits, IKK1 (IKKa) and IKK2 (IKKb), and a regulatory subunit named NF-jB Essential Modulator (NEMO) or IKKc. In vivo studies in genetic mouse models provided experimental evidence for the fundamental functions of NF-jB signalling in inflammation. Using tissue specific ablation of IKK subunits we have studied the role of NF-jB for the maintenance of immune homeostasis in epithelial surfaces. Epidermal keratinocyte specific blockade of NF-jB signalling caused strong inflammatory skin lesions resembling human psoriasis. Moreover, epithelial cell specific blockade of canonical NF-jB activation caused severe chronic inflammatory colitis. These results revealed that NF-jB signalling in epithelial cells plays a critical role for the maintenance of immune homeostasis in epithelial tissues. Our current experiments investigate the mechanisms by which NF-jB signalling acts in epithelial cells to regulate tissue homeostasis and prevent the pathogenesis of chronic inflammation. The results of our most recent studies will be discussed. In the field of inflammation research, the most important advances in the past 10 years has been in the uncovering of multiple pathways involved in innate immunity. There are now 7 distinct receptor families that sense microbial products and in some cases products of inflamed tissues, and trigger the innate response, which includes induction of pro-inflammatory mediators as well as effector mechanisms in host defence. The best characterised are the Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs). Genetic variation in several of these components has been linked to inflammatory diseases, notably in the TLR system and in the NLR protein Nalp3 and associated proteins. Work on knockout mice and the use of inhibitors continues to validate some of these proteins in disease. From work on Nalp3 there has also been a resurgence of interest in the IL1 system as a key driver of inflammation in diseases such as gout and diabetes (both Type I and Type II). IL1 family members such as IL1beta, IL1alpha, IL18 and IL33 are providing important information on the role of Th17, Th1 and Th2 cells in disease. For investigators interested in signal transduction, the area has proved very fruitful in terms of the discovery of new signalling pathways and processes. We now have a good understanding of the major components activated by TLRs, notably the TIR domain-containing adapters that initiate signalling following recruitment to TIR domains within the TLRs themselves, the IRAK family of protein kinases that are then recruited, and a series of ubiquitination and phosphorylation reactions that ultimately lead to the activation of transcription factors such as NF-kappaB and IRF family members. Furthermore inhibitory mechanisms are also being revealed that are likely to important for resolution of inflammation, notably miRNAs such as miR21. As we continue to unravel the molecular details of these processes, new therapeutic options will present themselves. major processes of the resolution of inflammation, including inhibition of white blood cell recruitment and efferocytosis. In view of this 'duet' glucocorticoids and AnxA1/FPR2, we propose that detailed understanding of how the AnxA1/FPR2 pair impacts on experimental inflammation, and human cell biology, can provide novel indication for drug discovery programmes. Such a proposition is reinforced by the notion that potent anti-inflammatory lipids of the lipoxins and resolvins families can also signal via FPR2/ALX. Proof-of-concept studies in Fpr2 deficient mice reiterate the validity of this approach. Acute inflammation is characterized by the activation and recruitment of innate immune cells. The recruitment of neutrophils and monocytes to sites of inflammation is orchestrated by a range of inflammatory mediators including chemoattractant cytokines. Failure to properly resolve acute inflammation leads to chronic inflammation, the pathological process that underlies many important diseases including angina, arthritis and inflammatory bowel disease. Work in the Greaves Lab has shown that the macrophage chemoattractant cytokine chemerin (TIG2) acting through G protein coupled receptors (GPCRs) including ChemR23 (CMKLR1) can mediate macrophage chemotaxis and adhesion to the cell adhesion molecules VCAM-1 and ICAM-1 and the extracellular matrix molecule Fibronectin. While full-length chemerin is a potent mediator of macrophage chemotaxis we have shown that proteolytic digestion fragments of chemerin can act as potent anti-inflammatory mediators that enhance macrophage uptake of microbial debris and apoptotic cells in vitro and in vivo. Importantly we have shown that polyclonal antibodies that specifically block the chemoattractant effects of full length chemerin and the anti-inflammatory effects of chemerinderived C-terminal peptides lead to increased neutrophil and monocyte recruitment in a self resolving model of zymosan peritonitis. San Raffaele University and Research Institute, Milano, Italy HMGB1 is the prime example of a danger signal that originates from the damaged self rather than from invading pathogens. HMGB1 is passively released by cells that die traumatically, and actively secreted by severely distressed cells and by activated cells of the innate immunity system. HMGB1 is thus the archetypal Damage Associated Molecular Pattern molecule, or DAMP. Notably, HMGB1 signals via TLR4, the receptor for LPS, the archetypal Pathogen Associated Molecular Pattern molecule. In addition, HMGB1 forms highly inflammatory complexes with single-stranded DNA, LPS, IL-1b and nucleosomes, which interact with TLR9, TLR4, IL-1R and TLR2 receptors, respectively. The ability to act both alone and in combination with other inflammatory signals may be ideally designed to govern the choice between the sacrifice or reconstruction of tissues, as required by the presence or absence of pathogens. C-type lectin DNGR-1, also known as CLEC9A, which signals via Syk kinase. DNGR-1 is selectively expressed by CD8a + DC in mouse and their equivalents in human, a sub-group of DC that possesses specialised properties including a unique propensity to phagocytose dead cell debris and to crosspresent exogenous antigens to CD8 + T cells. DNGR-1 signalling via Syk in CD8a + mouse DC appears to regulate the retrieval and crosspresentation of dead cell-associated antigens and may also be involved in the activation of DC by dead cells. The study of DNGR-1 helps build a picture of the receptors and signalling pathways that regulate DC responses to self alterations and has applications in immunotherapy of cancer and infectious diseases. The molecular mechanisms by which lung injury triggers inflammation and fibrosis remain poorly understood. We reported that Nlrp3 inflammasome activation and IL-1R1 signaling were required for bleomycin-induced inflammation and fibrosis in mice. Now investigating endogenous molecules released after lung injury, we show uric acid and extracellular adenosine tri-phosphate (eATP) are danger signals activating the Nlrp3 inflammasome in lung inflammation and fibrosis. Uric acid and ATP are locally released upon bleomycininduced injury and involved in IL-1b production, lung inflammation and fibrosis. Fibrotic patients have elevated ATP content in BALF in comparison to control individuals. Since uric acid or silica crystals and soluble ATP are involved both in Nlrp3 activation and lung fibrotic diseases, we investigated whether ATP and purinergic signaling may be a corner stone of Nlrp3 inflammasome activation by particulate activators. We show that uric acid and silica crystals induce the active release of intracellular ATP by epithelial cells or macrophages in vitro. Moreover, IL-1b maturation and secretion by macrophages upon crystals depend on purinergic signaling through autocrine purinergic receptor loops. Our results suggest that uric acid and silica crystals are competent to induce maturation and secretion of IL-1b through a process that involves as a first event the extracellular release of endogenous ATP and as a second event the signaling through several purinergic receptors that triggers the activation of the Nlrp3 inflammasome. In conclusion, we propose that blocking purinergic signaling could represent a novel strategy to reduce lung inflammation and fibrosis. Keywords: Uric acid crystals, ATP, Danger signal, Nlrp3 Inflammasome, Purinergic signaling. Giant cell arteritis (GCA) is an inflammatory blood vessel disease that primarily affects the aorta and its branches. Clinical manifestations include blindness, stroke, aortic aneurysm and failure-to-thrive. At a molecular level, GCA is characterized by the apparently unopposed stimulation of T cell immunity ordinarily mobilized to fight infection. The typical lesion is a granulomatous infiltrate that injures the blood vessel wall and leads to intimal hyperplasia and luminal occlusion. Early stages of the disease involve activation of wall-embedded dendritic cells which express a broad spectrum of pattern recognition receptors and respond to PAMPs and DAMPs. In humanized mice engrafted with human aorta and human T cells GCA can be elicited though initial stimulation with PAMPs. T cells recruited into the granulomatous lesions include Th1 and Th17 cells. The contribution of Th1 and Th17 cells to the disease process has been examined in patients undergoing two successive artery biopsies. Th17 cells are rapidly responsive to immunosuppressive therapy with corticosteroids whereas Th1 cells persist in the inflamed arteries despite continuous treatment. In treated patients vessel-wall inflammation represents a ''pure'' Th1 response. In untreated patients, antigen-presenting cells in the affected temporal arteries produce IL-12, IL-1b, IL-6 and IL-23. IL-12 production is resistant to corticosteroid therapy and promotes chronic vasculitis in the absence of Th17 responses. S18 Inflamm. Res. Conclusion: Multiple T cell lineages contribute to vasculitis with Th1 and Th17 cells being separable based on their susceptibility to steroidinduced immunosuppression. We conclude that at least two immuneactivation cascades are functional in GCA and propose that the disease instigators eliciting Th1 and Th17 immunity are distinct. awaited results, but also now some more specifically targeted monoclonal antibodies, like anti-TNF-alpha or anti-CD20 for ANCAassociated vasculitides, or anti-IL5 and anti-IgE for Churg-Strauss syndrome. However, the exact indications of these latter new agents, as well as their optimal dosage and duration, are not yet precisely defined. Therefore, at present, they are mostly prescribed for patients with disease refractory to conventional therapy, with promising results. Results of international ongoing trials will tell us in the few next years whether they may also have a place as first line treatments. The first results show that rituximab is as effective as cyclophosphamide (RAVE study, not published) to induce remission of Wegener's granulomatosis. Rituximab is now under evaluation for maintenance therapy. Other biotherapies have been evaluated. If etanercept does not seem effective, to maintain remission, infliximab is effective in severe and refractory ANCA-associated vasculitis. However relapses occur as soon the drug is stopped. It seems now reasonable to expect dramatic changes in the treatment of ANCA-associated vasculitis with a larger place for biotherapies. Osteoarthritis (SY12) OSTEOARTHRITIS, ANGIOGENESIS AND INFLAMMATION synovial inflammation in animal models of OA. Neovascularisation leads to ossification of articular cartilage and of chondro-osteophytes following growth factor expression by inflammatory cells within the subchondral bone. Inhibiting vascular growth or inflammation can each reduce structural progression in OA models. Inflammation contributes to the pain of OA, and angiogenesis may increase pain by exacerbating inflammation. Furthermore, angiogenesis leads to specific structural changes within the joint that may contribute to pain, including sensory nerve growth and reduced integrity of the osteochondral junction. In OA, fine, unmyelinated sensory nerve fibres grow alongside new blood vessels into structures that normally receive no innervation, including the non-calcified articular cartilage and the meniscal fibrocartilage. Determining specific molecular pathways of angiogenesis and inflammation in the OA joint should lead to targeted interventions with potential to improve symptoms and joint damage. Osteoarthritis is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors-such as advancing age, obesity, and trauma-that conspire to incite a cascade of pathophysiologic events within joint tissues. An important emerging theme in osteoarthritis is a broadening of focus from a disease of cartilage to one of the 'whole joint'. The synovium, bone, and cartilage are each involved in pathologic processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation, and the role played by genetics. In this review we summarize current scientific understanding of osteoarthritis and examine the pathobiologic mechanisms that contribute to progressive disease. Neutrophilic and eosinophilic granulocytes are key effector cells in host defence against invading bacteria and parasites; however overrecruitment, dysregulated activation and defective removal of these cells play a prominent role in the initiation and propagation of chronic inflammatory conditions such as asthma and COPD. Apoptosis of granulocytes occurs during the normal resolution process, renders these cells unresponsive to further stimulation and marks them for removal by phagocytes (e.g., macrophages) by mechanisms that switch the phenotype of macrophages from pro-to anti-inflammatory. Although there is little doubt that apoptosis plays a critical role in many physiological and pathological processes, its precise role in inflammatory diseases is only recently being unravelled. The findings that modulation of granulocyte apoptosis with pharmacological agents such as cyclin-dependent kinase (CDK) inhibitor drugs can precipitated inflammatory resolution by inducing inflammatory cell apoptosis and augmenting phagocytic removal of apoptotic cells will be discussed. We propose that by defining novel mechanisms that selectively accelerate neutrophil and eosinophil apoptosis together with those that promote phagocyte clearance of apoptotic granulocytes will provide the scientific foundation for innovative approaches to the therapy of human inflammatory diseases. University of Kentucky, Lexington, KY, United States PGE 2 is the dominant PG induced during the early phases of inflammation due to the dramatic increase in levels of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in response to pro-inflammatory cytokines and other stimuli to the inflammatory program. Pharmacologic inhibition of COX-2 enzyme activity either by non-specific or COX-2-selective non-steroidal anti-inflammatory drugs (NSAIDs) effectively blocks up-regulated production of PGE 2 . Blocking PGE 2 using NSAIDs also inhibits cytokine-stimulated upregulation of mPGES-1 expression, thereby enhancing their antiinflammatory effects. This mechanism proceeds via the interaction of PGE 2 and its EP2 and EP4 receptors and can be compensated by direct application of PGE 2 , EP2 or EP4 agonists, or by activating cAMP using forskolin. In addition to classical anti-inflammatory effects, PGE 2 acting via the EP4 receptor modulates the immune response promoting changes in the cytokine profile of antigen presenting cells to facilitate the IL-23/IL-17 axis important for autoimmune inflammatory diseases. In addition to inhibition of PGE 2 , NSAIDs also inhibit production of other PG, such as PGI 2 and PGD 2 . Since these PG, especially PGI 2 , contribute to inflammation and pain, NSAID therapeutic effectiveness could relate in part to this lack of specificity. However, lack of specificity towards pro-inflammatory cytokines undoubtedly contributes to adverse effects. Inhibition of PGI 2 , for example, may contribute to thrombophilia and atherogenesis as suggested by murine models. At present, it is unclear if specific inhibitors of mPGES-1 or EP4 receptors will be effective anti-inflammatory, analgesic, and antipyretic agents in humans. Development of drugs that inhibit mPGES-1 are hampered by species differences in activity leading to difficulties in implementation of murine models. However, experiments in PGES-1 null mice demonstrate that this strategy may yield promising results different from inhibition of COX-2. For example, mPGES-1 genetic deletion has important effects on neurons and microglia in a model of neuropathic pain that appear to be mediated via COX-1 rather than COX-2. Furthermore, mPGES-1 null mice appear protected from atherogenesis. Alternate strategies for inhibiting PGE 2 activity have now focused on EP4 receptor antagonists. In addition to showing promise in some animal models of arthritis and pain, EP4 receptor antagonists appear to inhibit platelet aggregation. Together, these properties suggest that EP4 receptor antagonism is another potential new therapeutic strategy for inflammation and pain. Sphingosine-1-phosphate (S1P) is a potent sphingolipid mediator formed inside cells by phosphorylation of sphingosine catalyzed by two sphingosine kinase isoenzymes, SphK1 and SphK2. SphK1 is activated by many pro-inflammatory cytokines leading to formation of S1P, which is exported by specific transporters to activate its own receptors in an autocrine and/or paracrine manner, a process, known as ''inside out signaling'' by S1P, is important for many of the immune cell functions known to be regulated by S1P and the diversity of these functions is explained by the repertoire of S1PR expression on various immune cells. We have recently also identified important roles for intracellular S1P during inflammation. We showed that activation of SphK1 by the pleiotropic cytokine TNF-alpha and production of S1P is necessary for Lys 63-linked polyubiquitination of RIP1, phosphorylation of IKK and IkBalpha, and IkBalpha degradation, leading to NF-kB activation, independently of S1P receptors. S1P specifically binds to TRAF2, a key component in NF-kB signaling triggered by TNF-alpha and stimulates its E3 ligase activity (Alvarez et al., Nature 465: 1084 . Thus, S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, suggesting a novel paradigm for regulation of Lys 63-linked polyubiquitination. This highlights the key role of SphK1 and its product S1P in TNFalpha signaling and the canonical NF-kB activation pathway and provides a mechanistic explanation for the numerous observations of the importance of SphK1 in inflammatory, anti-apoptotic, and immune processes. This work was supported by grants from the NIH R37GM043880, R01CA61774, RO1AI500941, and U19AI077435. Institut Pasteur, Paris, France Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. The viral replicative intermediate double-stranded RNA (dsRNA) is considered a potent activator of the innate anti-viral immune response. Bronchial epithelial cells are the primary target and the principal host for IAV. We previously used an in vitro approach to establish a role for the pattern-recognition Toll-like receptor (TLR)3 in the immune response of lung epithelial cells to IAV-derived dsRNA and we demonstrated that in vivo, IAV-TLR3 interaction critically contributes to viral pathology. However, additional cellular dsRNA-recognition proteins have been implicated as key sensors of viral infection. These include two caspase recruiting domain-containing RNA helicases, i.e. the retinoic acid-inducible gene I (RIG-I) protein and the melanoma differentiation-associated gene protein. In this context, we further demonstrated that the sensing of IAV by TLR3, RIG-I and MDA-5 clearly differs. Indeed, TLR3 activation appears critical for the induction of a pro-inflammatory response whereas RIG-I (but not MDA-5) activation is rather essential for the induction of a type I interferon-dependent antiviral response. Finally, we established that the innate immune response triggered by IAV is negatively regulated by the Suppressor of Cytokine Signaling (SOCS) 1 and SOCS3 proteins through a RIG-I/IFNAR-dependent pathway. T. Hussell 1, * 1 Imperial College London, National Heart and Lung Institute, Leukocyte Biology, London, United Kingdom The activity of innate immunity is not simply dictated by the presence of an antigen but also by the balance between negative regulatory and immune potentiator pathways. Even in the absence of an antigen, innate immunity can ''inflame'' if negative regulators are absent. This resting state is adaptable and dictated by environmental influences, host genetics and past infection history. A return to homeostasis post inflammation may therefore not leave the tissue in an identical state to that prior to the inflammatory event. We show that respiratory pathogens cause long term modification of the lung microenvironment by a de-sensitisation to bacterial products and an increase in the myeloid negative regulator CD200R. These two events prevent subsequent inflammatory damage while the lung is healing but also may predispose to bacterial colonisation of the lower respiratory tract. This modulation of lung homeostasis also occurs with prior non-infectious inflammatory events. In murine models exposed to the common allergen house dust mite (HDM), Streptococcus pneumoniae persists, becomes invasive and systemic and leads to mortality, whereas healthy mice clear the infection without untoward consequences. We identify the molecular mechanisms associated with bacterial susceptibility by showing the disruption of bacterial recognition by pattern recognition receptors due to sequestration or diversion of key receptors or downstream signalling components in the HDM-remodelled lung. A deeper understanding of the consequences arising from innate immune cell alteration during acute and chronic inflammation and the subsequent development of bacterial complications has important implications for future drug development. We harbor an enormous load of commensal bacteria in our intestines, which are physically excluded from entering the body by a single layer of epithelial cells and overlying mucus. The mucosal immune system is strategically situated in very close proximity to this massive load of bacteria, which can provide a wealth of stimuli for maturation and shaping of the immune system. Comparing germ-free mice, which lack all intestinal bacteria, to mice that are specifically colonized with a defined bacterial population clearly demonstrates that the presence of bacteria has strong immunomodulatory effects on both the mucosal and systemic immune systems. It is known that small numbers of live bacteria can penetrate past the epithelial cell barrier. The majority of bacteria that do penetrate are efficiently phagocytosed and killed by intestinal macrophages. In addition, dendritic cells can carry bacteria to the Peyer's patches and mesenteric lymph nodes where they stimulate B cells to switch to IgA. The numbers of IgA plasma cells in the lamina propria and the concentration of secretory IgA in the lumen of the gut both increase dramatically following colonization and some of the IgA that is induced is highly specific for the bacteria present in the flora. We have also found that intestinal CD4 + T cells also increase dramatically in response to the intestinal flora but overt inflammation, and the differentiation of T H 1 or T H 17 effector cells, is inhibited by commensaldependent activation and expansion of colonic lamina propria regulatory T cells. Furthermore, we have shown that impaired bacterial killing due to innate immune deficiencies leads to induction of a systemic antibody response, which can compensate for decreased innate responsiveness and protects clean mice from death. Importantly, this continuum between the innate and adaptive immune systems may also underlie the observed over reactivity to the intestinal microflora that is observed in inflammatory bowel disease. of alternatively activated macrophages (AMMs), that arise as a consequence of exposure to interleukins (IL)-4 and -13. AMMs have been implicated in wound repair and may also promote fibrosis. Using the dinitrobenzene sulphonic acid (DNBS) murine model of colitis we have found that peritoneum-derived macrophages differentiated in vitro and return to mice in a prophylactic or a treatment strategy significantly reduced the chemically-induced colitis: an event that was in part dependent on IL-10. Subsequent analyses has shown marked differences between these cells and bone marrow-derived AAMs, and intriguingly that a hyper-polarized AAM phenotype can be induced in vitro by exposure to specific pro-inflammatory and anti-inflammatory cytokines. Ongoing investigations in which he function and anti-colitic properties of AAMs are being investigated will be discussed. A.G. Buret 1, * 1 Inflammation Research Network, Dept. of Biological Sciences, University of Calgary, Calgary, AB, Canada Numerous reports have described how acute infections may trigger inflammatory responses. Conversely, recent discoveries highlighted the remarkable ability of some parasites to downregulate host immunity and minimize pathology, particularly with parasitic Helminthes. Less is known on how Protozoa may tone down immunity. Research into these mechanisms, and into the causative products, will be key to the development of new anti-infectives, but, perhaps even more importantly, novel interventions against autoimmune and inflammatory diseases. Complement lysis by extracellular Protozoa as well as modulation of the phagosomal compartment by intracellular parasites have been well described. In addition, cysteine proteases of various Protozoa, including Entamoeba histoloytica, are able to inhibit cellular immune responses in the intestine of the host, at least in part by modulating host prostaglandin synthesis and cleaving chemotactic cytokines. A variety of Protozoa, including Trypanosoma, Toxoplasma, and Leishmania. reduce pro-inflammatory cytokines. Trypanosoma reduces TNF-a and IL-12 in activated dendritic cells, making them producers of anti-inflammatory IL-10 and TGF-b. Similarly, upon activation by TLR's, dendritic cells exposed to Giardia produce more IL-10 and less IL-12, perhaps by modulating the release of lipoxins, as shown with Toxoplasma. Giardia is the most common cause of waterborne parasitic diarrhea worldwide. For reasons that remain obscure, the parasite does not trigger any overt inflammatory cell infiltration in the intestine. In addition to the effects on dentritic cells, ongoing research points to novel epithelial immuno-modulatory events. Indeed, Giardia is able to inhibit IL-1-induced CXCL8 production by intestinal epithelial cells via posttranslational mechanisms. Recent reports indicate that the production of diarrheal symptoms caused by Giardia infection may vary with between geographical areas of the World, and perhaps even protect against diarrhea. In view of the significance of community acquired diarrheagenic microbes, including E. coli pathotypes, as a cause of diarrhea in these endemic areas, interactions between Giardia, and pathogenic E. coli may represent an interesting avenue for future studies. Research needs to address whether and how enteric Protozoa such as Giardia may modulate host interactions with concurrent pathogens, as well as to assess its effects on the normal host microflora. Disclosure of interest: None Declared. Symposium sponsored by the SFAR (French Society of Anesthesiology and Critical Care Medicine) Biomarkers, which can be described as disease-associated molecular changes in body tissues and fluids, are tools for improving diagnosis and tailoring the treatment in the patients. Trauma patients are characterized by an easy to determine starting point, by comparison with sepsis. 1. Inflammatory markers in trauma patients. The immune response to trauma belongs to what has been described as the sterile inflammation. Trauma is responsible for an inflammatory response difficult to distinguish from inflammatory response to infection. Besides classical inflammatory markers such as CRP and procalcitonin, endogenous molecules termed damage associated molecular patterns (DAMPs) are involved, as these host derived nonmicrobial stimuli are released following tissue injury and have similar functions as pathogen associated molecular patterns (PAMPs) in terms of their ability to activate proinflammatory pathways. Among DAMPs, proteins such as S100 A8 and A9 have been recently studied in this context. 2. Other biomarkers utilization in trauma patients. Biomarkers of organ lesion/failure. All the organ can be involved in trauma and the list of biomarkers is non specific of trauma. Biomarkers such as troponin (cardiac muscle), isoform of CPK (muscles), lipase (pancreas), hepatic enzymes (liver contusion), biomarkers of axonal injury in head trauma indicate organ injury. -Biomarkers of infection have been studied in the early phase of trauma to detect an organ injury associated with dissemination of bacteria (such as hollow viscus perforation). Parameters such as simple white blood cell count, CRP or procalcitonin could indicate the presence of an infection, but Many protein-reactive chemicals can cause allergic contact dermatitis. This inflammatory skin disease is mediated by contact allergenspecific T cells. In order to trigger the adaptive immune response contact allergens must first activate the innate immune system to induce skin inflammation. Recent studies have revealed that contact allergen-mediated skin inflammation closely resembles the innate immune response to infection. Intriguingly, contact allergens activate similar innate immune mechanisms as microbial pathogens. Thus, metallic nickel ions can directly bind to human Toll-like receptor 4 (TLR4) and activate signal transduction. Some organic contact allergens indirectly activate TLR2 and TLR4 by causing the generation of endogenous ligands including breakdown products of the extracellular matrix component hyaluronic acid in the skin. Furthermore, by triggering the release of ATP from skin cells contact allergens activate the NLRP3 inflammasome via the ATP receptor P2X 7 . In addition, oxidative stress responses result from contact allergen encounter. The tight collaborative interplay of these innate immune and stress responses has important implications for the development of causative treatment strategies for allergic contact dermatitis and for the development of animal-free in vitro methods for the identification of contact allergens. The paradigm that pulmonary inflammation drives fibrogenesis has been challenged and it is proposed that, in some cases, the pathological process is mediated by alternative mechanisms in particle-related lung diseases. Our studies demonstrated that silica-induced lung fibrosis in mice results from the action of the immunosuppressive cytokines TGFb and IL-10 produced to limit the development of chronic inflammation. TGF-b and IL-10 induce lung collagen deposition when overexpressed in silica-treated mice. It is generally accepted that TGF-b and IL-10 are essential for regulatory T lymphocyte (T reg) expansion and immunosuppressive activity. We demonstrated that T regs are recruited to control inflammatory responses induced by silica particles. The persistent accumulation of immunosuppressive T regs in the lungs also contributes to pulmonary fibrosis by stimulating fibroblasts through the secretion of growth factors. In conclusion, immunosuppressive responses may also contribute to particle-induced lung fibrosis and represent an additional etiopathogenic pathway of lung fibrosis. R. Barouki 1, * 1 Universite´Paris Descartes, Inserm unit 747, Paris, France Persistent Organic Pollutants such as dioxin accumulate in adipose tissue and may exert long term effects in humans and animals. We have carried cellular experiments in order to determine novel mechanisms of toxicity of these pollutants and in vivo studies in an attempt to correlate the kinetics of these pollutants with biological and clinical parameters. The cellular response to the influx of potentially toxic xenobiotics is initiated by xenobiotics receptors. We have focused on the dioxin receptor (Arylhydrocarbon Receptor, AhR) and on its ligands. Functional genomics studies allowed us to identify a variety of genes that could mediate part of the AhR ligands toxicity. In both human liver cell lines and adipocyte cell lines, inflammatory genes were among the most potently induced by dioxin and certain PCBs. The upregulation of inflammatory genes by dioxin in adipose tissue was confirmed in vivo in mice. We have also studied target genes involved in focal adhesion, signal transduction by integrins and those mediating epithelial mesenchymal transition in a liver cell line. In another set of experiments we have assessed in human the effect of drastic weight loss in obese individuals on persistent organic pollutant release in the bloodstream and correlated this release with the regulation of gene expression in blood S24 Inflamm. Res. cells and in adipose tissue. We also correlated this release with a number of biological and clinical parameters and found significant effect on liver toxicity and metabolic parameters. Symposium sponsored by the JSIR (Japanese Society of Inflammation and Regeneration) SP-13 ANTI-OSTEOCLAST THERAPY H. Takayanagi 1, * 1 Tokyo Medical and Dental University, Tokyo, Japan Osteoclasts are bone-resorbing cells, which are involved in the pathogenesis of various bone and joint diseases including rheumatoid arthritis and osteoporosis. RANK lingad (RANKL) is a TNF family cytokine that is essential for the differentiation and function of osteoclasts. We have been studying the mechanism of osteoclast activation in the context of arthritis and found that aberrant expression of RANKL in synovium is responsible for the abnormal formation of osteoclasts. We identified IL-17-producing T helper cells (T H 17 cells) to be the exclusive T cell subset that has the ability to induce osteoclastogenesis and recently revealed IkBf to be a crucial regulator of Th17 development. These mechanisms provide a molecular basis for developing novel therapeutic strategies against bone destruction in arthritis. RANKL signals the cell through numerous immunomodulatory molecules such as TRAF6, NF-jB, c-Fos and nuclear factor of activated T cells (NFAT) c1. NFATc1 activation requires calcium signaling, which is mediated in osteoclasts by immunoreceptor tyrosine-based activation motif (ITAM) in dual membrane adaptors, Fc receptor (FcR) common c subunit (FcRc) and DNAX activating protein (DAP12). FcRc and DAP12 associate with multiple immunoreceptors such as OSCAR and TREM-2, which thus function as costimulatory receptors for RANK. Recent advances in the understanding of osteoclast regulation and osteoclast-targeted therapy will be discussed. Disrupted bone marrow (BM) hematopoiesis and delayed immune reconstitution are life-threatening complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, how GVHD impairs BM hematopoiesis has been largely unknown. Using several mouse models of MHC-mismatched allo-HSCT, we explored the effects of GVHD on the possible targets in BM, HSCs and hematopoietic niches. Lethally irradiated mice were transplanted with T cell depleted BM cells (TCD-BM) supplemented with T cells from allogeneic donor mice to induce GVHD. We first analyzed the hematopoietic reconstitution in MHC I-and MHC II-double mismatched GVHD model and found that the BM hematopoiesis, especially the B-lymphopoiesis, was severely impaired in the GVHD mice. The B-lymphopoiesis was also impaired in MHC II-single but not MHC I-single mismatched GVHD. This was further confirmed in a CD4 dominant minor mismatched model. Re-transplantation experiments revealed that GVHD-affected BM contains substantial number of HSCs that reconstitute all hematopoietic cell lineage. In contrast, hematopoietic niche in GVHD-affected BM failed to support the B-lymphopoiesis. Histological analysis of GVHD-affected BM revealed that osteoblasts were completely lost within 7 days after allo-HSCT. Accordingly, expression of mRNA for osteoblast markers, Runx2, Twist, Msx2, Osterix, Dlx5 and osteocalcin, was severely decreased in GVHD-affected BM. Moreover, the bone formation determined by bone histomorphometry was completely impaired in the GVHD mice. Administration of anti-CD4 antibody in the early time largely prevented BM-GVHD, while preserving GVL/ T effects. In conclusion, our study provides the first evidence for the destruction of osteoblastic niches mediated by allogeneic CD4 T cells during GVHD. The new concept of BM-GVHD would provide an explanation for disrupted hematopoiesis and delayed immune reconstitution after allo-HSCT and may bring novel preventive and therapeutic strategies for GVHD. Cancer and inflammation (SP05) Symposium sponsored by the IRA (Inflammation Research Society) melanoma treated with Ipilimumab in a randomized placebo controlled trial, the first drug of any type to do so, with more than 20% of patients alive for over 4 years after treatment. While this trial documents that CTLA-4 blockade can lead to durable responses in patients, there is clearly a need to increase the response rate. We have that administration of anti-CTLA-4 results in an increase of the frequency of CD4 T cells that express ICOS in both the tumor tissue and blood of bladder cancer patients. The ICOS + population contained effector CD4 T cells that made IFNc upon mstimulation with peptides from NY-ESO-1. We subsequently showed that survival of advanced melanoma patients treated with Ipilimumab had sustained elevation of ICOS + CD4 T cells ([twofold for 12 weeks) had significantly greater survival than those with no or transient increases. Together, these data suggested to us that ICOS might play an important role in the therapeutic effects of CTLA-4 blockade. To test this we used a mouse model of melanoma. We found that the efficacy of anti-CTLA-4 was markedly diminished in mice that were deficient in either ICOS or ICOS ligand (ICOSL), confirming that the ICOS/ICOSL pathway plays a critical role in anti-CTLA-4 therapy. These observations led us to test the possibility that engagement of ICOS could enhance the efficacy of anti-CTLA-4 therapy. To this end we transduced mouse B16F10 melanoma cells with a cDNA encoding ICOSL or a control construct. B16ICOSL + cells (Ivax) and control B16 cells were irradiated and used alone or in combination with anti-CTLA-4 to treat mice bearing established B16F10 tumors. We found that combination of the IVAX with anti-CTLA-4 was markedly more effective than the control vaccine plus anti-CTLA-4 or that of any single treatment alone. The increase in therapeutic efficacy was accompanied by a marked in increase in the density and functionality of CD4 and CD8 T cells within the tumor. These results suggest a novel strategy for manipulating the immune system to enhance anti-tumor responses: checkpoint blockade coupled with provision of agonist signals mediated by ICOS to enhance costimulation. Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged that cancer immunoediting would occur in three phases: Elimination (also known as cancer immunosurveillance, the host protective phase of the process), Equilibrium (the phase in which tumor cells that survive immune elimination remain under immunologic growth control resulting in a state of functional tumor dormancy) and Escape (the phase where clinically apparent tumors emerge because immune sculpting of the tumor cells has produced variants that display either reduced immunogenicity or enhanced immunosuppressive activity). Strong experimental data has now been obtained using mouse models of cancer to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process may also occur during the evolution of certain types of human cancer. Our efforts now focus on elucidating the molecular and cellular mechanisms that underlie each phase of cancer immunoediting and identifying the critical checkpoints that regulate progression from one phase of the process to the next. This approach has helped identify the nature of antigens seen by immunity in nascent developing cancers and has further shown that immunoselection is a major mechanism of immunoediting. Moreover, we have found that edited tumors can still be controlled by the immune system if natural mechanisms that prevent autoimmunity are suspended. As reported by others, we have confirmed that inhibition of CTLA-4 induces ejection of edited MCA sarcomas. However, we have also found that inhibition of PD-L1 does the same, although by perhaps different mechanisms. These differences will be discussed. F. Balkwill 1, * 1 Centre for Cancer and Inflammation, Barts and the London School of Medicine and Dentistry, London, United Kingdom A complex network of inflammatory cytokines and chemokines regulates communication between the malignant cells and supporting stroma in most experimental and human cancers. There is ample evidence from animal models that individual members of this cytokine network and their intracellular signalling pathways can make malignant cells resistant to apoptosis, can stimulate a tumor-promoting leukocyte infiltrate that may also suppress anti-cancer immune responses, and can regulate angiogenesis. Of all the individual cytokines and chemokines that have been implicated in tumor growth and spread, the most convincing and extensive animal model data relates to the cytokines TNF-a, IL-6 and the chemokine receptor CXCR4. These, and the CXCR4 ligand CXCL12, as well as the receptor Notch3 and its Jagged1 ligand, are co-expressed and coregulated in ovarian cancer cell lines and human ovarian cancer biopsies. We have named this co-regulation the TNF network. High levels of expression of TNF network gene pathways in cell lines and ovarian cancer biopsies is significantly associated with pathways and process important to cancer growth and spread including angiogenesis, cell adhesion, stromal development and the immune cell infiltrate. In ovarian cancer biopsies high levels of malignant cell staining for TNF network members related to poor prognosis. To translate these observations to clinical practice we have focused on IL-6, conducting a phase II trial of the anti-human IL-6 antibody CNTO328 in women with relapsed ovarian cancer. Our results showed that IL-6 may be a therapeutic target in ovarian cancer with multiple actions within the tumor microenvironment. Inflammation in severe sepsis: from bench to bedside (SP06) Symposium sponsored by the SRLF (Socie´te´de Re´animation de Langue Française) Among these receptors are the ''Toll-like receptors'' (TLRs), and associated molecules such as CD14 and MD-2, but also receptors important for the clearance of bacteria or bacterial products such as scavenger and mannose receptors. TLRs recognize bacterial endotoxin (TLR4/MD-2), bacterial lipopeptides and lipoteichoic acid (TLR2/1/6), viral RNA (TLR3), flagellin (TLR5), and CpG bacterial DNA (TLR9). Recognition of lipopolysaccharide (LPS, Gram-negative bacterial endotoxin). Ligation of microbial ligands by TLRs triggers intracellular signaling pathways. Upon ligand binding, many TLRs, in particular TLR2 and TLR4, activate inflammatory pathways via the so-called MyD88 pathway leading to NF-jB activation. TLR4, but also TLR3 activate also a non-MyD88 pathway, leading to the production of b-interferon. The step of recognition of (bacterial) PAMPs by TLRs is therefore essential to initiate an inflammatory response. TLRs have been also identified as key receptors in antigen presenting cells, boosting the adaptive immune system. Some TLRs are also expressed in non-professional immune cells such as endothelial and epithelial cells, and ligation of those TLRs can also initiate inflammatory responses in tissues. Most studies addressing TLR specificity have been performed using purified bacterial ligands and transfected cells. Only a few studies have investigated PAMPs and their receptors in mediating human cell activation using whole bacteria. Bacteria are essentially sensed by human immune cells as whole microorganisms. PAMPs can also stimulate immune cells in the form of microparticles and soluble mediators released from bacteria. The predominant PAMPs stimulating TLRs are largely unknown in the context of a whole bacterium. Using either TLR-transfected cells or human leukocytes, endothelial cells, or epithelial cells treated with specific anti-TLRs, -MD-2, and -CD14 monoclonal antibodies (mAbs), we have determined the contribution of cell-surface TLRs and their accessory proteins toward cellular responses to prototypic Gram-negative and Gram-positive bacterial strains responsible for human septic shock. We have shown that whole Gram-negative and Gram-positive bacteria activate human leukocytes, endothelial, and epithelial cells through TLRs and CD14. The blockade of these receptors with specific mAbs efficiently abrogated cell activation in response to their exposure to whole bacteria. TLR4 is the main TLR involved in cell activation by Gram-negative bacteria. In the absence of TLR4 or at high concentration of Gram-negative bacteria, TLR2 was also activated. Higher concentrations of Grampositive bacteria were needed to activate cells in a TLR2-dependent fashion. This activation appeared to be completely TLR4/MD-2 independent. These results largely confirm studies performed in genetically modified mice and demonstrate that antibody blockade of CD14 and TLRs can potentially decrease excessive and detrimental inflammation in response to bacteria, making it an attractive therapeutic avenue to explore in patients with severe sepsis and septic shock. Indeed, during the initial phase of shock, the blockade of TLR-dependent hyperactivation of cells without interfering with CD14-dependent internalization of bacteria and bacterial products might be beneficial. Interfering with the initial inflammatory response with TLR antagonists carries also the theoretical risk of bacterial overgrowth, a problem that should be minimized by an appropriate antibiotic therapy, particularly in patients with severe bacterial infection and septic shock. Work is underway to determine how leukocytes from premature babies recognize and get activated by bacteria, as well as immature neutrophils in adults, a prominent circulating cell type in patients with septic shock. These results will be presented. Elson G, Dunn-Siegrist I, Daubeuf The brain mediates via the autonomic nervous system and neurohormones the growth and proliferation of most if not all tissues involved in immunity, and all immune cells have membrane or cytosolic receptors for a number of neuromediators. The systemic inflammatory response to infection results in brain activation, which subsequently generates appropriate anti-inflammatory response. However, excess in pro-inflammatory mediators entering the brain can cause cerebral damage. In turn, dysfunction of the autonomic nervous and neuro-endocrine systems may alter immunity in a vicious circle resulting in metabolic derangements and organ failure. Two major pathways allow neuro-immune communications: the circumventricular organs (CVOs) located in the midline ventricular system, and the vagus nerve. CVOs are lacking a blood-brain barrier. Thus, they permit a direct communication between brain and blood stream. Some CVOs are located in the vicinity of neuroendocrine structures as and others are located close to brainstem autonomic centres. Thus, blood borne cytokines enter the brain through these areas, which express receptors for cytokines and bacterial fragments. In addition, specific carriers allow circulating cytokines to reach hypothalamic nuclei. Circulating IL-1 activates afferent vagal fibres terminating in the nucleus tractus solitarius, with subsequent stimulation of the hypothalamic-pituitary-adrenal axis. Efferent activity in the vagus nerve, termed 'cholinergic anti-inflammatory pathway', releases acetylcholine in the vicinity of macrophages within the reticuloendothelial system and leads to cellular deactivation and inhibition of cytokine release. The sympathetic and parasympathetic systems are thought to work hand-in-hand to modulate inflammatory responses. Diffuse endothelial activation induced by sepsis may result in bloodbrain barrier breakdown allowing circulating mediators to enter massively into the brain. They induce expression of CD40 and adhesion molecules on brain endothelial cells. NF-kappa-B induced over-expression of iNOS further increases blood-brain barrier permeability. Components of innate and adaptive immune systems are expressed in the brain during LPS challenge. They are first expressed within the CVOs, and then spread to deeper areas of the brain controlling neuroendocrine and autonomic functions. Toll-like receptors (TLR)4, TLR2 and TLR9 are expressed by glial cells, such as microglia, astrocytes and oligodendrocytes at rest and after exposure to LPS. Whether TLRs are expressed in neurones remains controversial. Endotoxin also up-regulates the iNOS in the brain, causing accumulation of NO which behaves as a neurotoxic effector. Prostaglandins and purines, such as ATP and adenosine, are further key mediators in brain response to sepsis, particularly inducing fever. A number of other mediators may be involved in the cerebral immune response, such as macrophage migrating inhibitory factor, macrophage inflammatory protein, endothelin-1, angiotensin II, plateletactivating factor, superoxide radicals and carbon monoxide. Exposure to LPS may cause apoptosis of brain cells via dysfunction of mitochondrial respiration, activation of mitogen-activated protein kinase and NF-kappa-B pathways, and release or accumulation of calcium and reactive oxygen species. In septic shock, post-mortem studies showed iNOS induced neurone and glial apoptosis, mainly within cardiovascular autonomic centres and the hippocampus. Cell culture experiments indicate an important role for microglial cells in immune surveillance, host defense and tissue repair. Indeed, they clear apoptotic cells and downregulate cytokines and encephalitogenic T-lymphocytes. During systemic inflammation, NO, cytokines and prostaglandins modulate brain neurotransmission, especially R-adrenergic system, GABAergic synapses, central muscarinic cholinergic regulation, corticotrophin releasing factor, ACTH, and vasopressin synthesis, and medullary autonomic centre output. Neurotoxic substances such as ammonium, or amino acids may also be involved. Prolonged LPS-exposure impaired synaptic transmission and neuronal excitability of pyramid neurons in the hippocampus, involved in behavioural and emotional systems. Finally, neurotransmitters and neurohormones modulate cerebral expression of inflammatory mediators. The final behavioural, neuroendocrine and autonomic responses are therefore variable because they depend on a complex and spatio-temporally organized process that involves both stimulatory and inhibitory pathways. J.P. Mira 1, * 1 Severe sepsis continues to be a major and increasing heath care burden worldwide. Despite aggressive organ support and optimal microbial therapy, sepsis remains the commonest cause of death in ICU with an overall mortality rate of 25-40%. The relative failure of conventional therapeutic strategies has stimulated a major interest in the development of new research axes, such as identification of genetic factors that influence sepsis susceptibility, therapeutic response, or outcome. Genetically-determined differences in host immune responses against pathogens might explain why some people get sick and die when they encounter a pathogen whereas others stay perfectly healthy. Explosion of knowledge both in human genomics and in host inflammatory response explains the increasing interest in immunogenetics over the last 15 years. Indeed, twin and adoptee studies have suggested more than 20 years ago, that host genetic factors are major determinants of susceptibility to infectious diseases in humans. Recently, candidate gene studies and human genome wide analysis have been used to identify infectious diseases susceptibility and resistance genes. Rarely, a single gene defect has been directly related to devastating consequences such as interferon-gamma receptor mutations leading to fatal infections with ubiquitous mycobacteria. For clinical practice, gene polymorphisms of specific immunological or physiological mediators appear to be of major importance. These genetic variants, which modify the regulation or function of either Pathogen Recognition Receptors or inflammatory mediators, have been associated with susceptibility and/or outcome of severe sepsis and septic shock. All steps of the host response to bacteria clearance have been shown to be potentially affected by genetic factors. However genetic studies in sepsis have produced contradictory results related in part to methodological faults. Improved adherence to published guidelines of good study design will help to ensure that genetic epidemiology contributes to a better classification of the heterogeneous septic population. The impact of these findings on the understanding of infectious disease pathogenesis and on the design of future preventive and therapeutic strategies should also be considerable. Symposium sponsored by the BIS (Brasilian Inflammation Society) Pulmonary fibrosis is a chronic respiratory disease with lethal consequences. Fibrosis may be triggered by a series of inflammatory conditions that usually precedes deposition of collagen in the lung parenchyma. Bleomycin-induced lung fibrosis in mice is the most commonly used model to study the pathogenesis of Pulmonary Fibrosis. Bleomycin instillation results in oxidative damage to the DNA of the alveolar epithelium and promotes cytokine release, which subsequently drives recruitment and activation of leukocytes in the airways. Leukocyte transmigration into the airways contributes to the chronification of the inflammatory process by inducing the production of cytokines, triggering fibroblast proliferation and excessive collagen deposition. Studies conducted by our group has shown that blockade of the chemokine receptor CXCR2 prevents development of fibrosis induced by bleomycin in mice. Importantly, blockade of CXCR2 was also protective even when started many days after induction of disease, suggesting it may be useful therapeutically in huams. The reduction of pulmonary fibrosis in those animals treated with CXCR2 inhibitors was associate with inhibition of angiogenesis. Similarly, preventive or therapeutic blockade of CCL3 with a chemokine binding protein-CCL3 (Evasin-1) decreased pulmonary fibrosis, an effect associate with decreased migration of leukocytes into the airways and reduced production of pro-fibrogenic cytokines. Chemokine receptors may signal via PI3 Kc. PI3 Kc deficiency led to attenuation of lung angiogenesis, reduction in leukocyte influx into the airways, decreased lung fibrosis and increased animal survival. Pharmacological inhibition of PI3 Kc caused functional changes in leukocytes, endothelial cells and fibroblasts in vitro, suggesting that PI3 Kc plays a role in multiple levels in the context of pulmonary fibrosis. More recently, we evaluated the role of Bradykinin receptors, B1 and B2, during the development of pulmonary fibrosis. B1R deficiency caused an increase in lung leukocyte influx, inflammatory cytokines and increased loss weight, and accelerated and increased mortality (100%) induced by bleomycin. However, B2R deficiency was associated with decreased production of inflammatory cytokines and chemokines and protected mice from lethality, weight loss, inflammation and fibrosis. These results unravel a novel role of B2 receptor in controlling inflammation and fibrosis after bleomycin challenge and suggest that B2 receptors may be interesting targets in the control of pulmonary fibrosis in humans. Inflamm. Res. Inflammasome activation with the production of IL-1b received substantial attention recently in inflammatory disease, but the role of inflammasome in the pathogenesis of asthma is not clear. Using an adjuvant-free model of allergic lung inflammation induced by ovalbumin, we investigated the role of NLRP3 inflammasome. We find that allergic airway inflammation depends on NLRP3-ASC complex producing IL-1b. Dendritic cell recruitment into lymph nodes, activation of Th2 lymphocytes in the lung and secretion of Th2 cytokines and chemokines including IL-33 and TSLP are reduced in the absence of NLRP3 and contribute to a dramatic reduction of allergic inflammation. Absence of NLRP3 and IL-1b is associated with reduced expression of other proinflammatory cytokines such as IL-13, IL-33, TSLP and IL-6. Mechanistically, we find that ATP which activates the inflammasome is released upon ovalbumin challenge. Furthermore, the critical role of IL-1R1 signaling in allergic inflammation is confirmed in IL-1R1, IL-1b and IL-1a deficient mice suggesting an additional role of IL-1a. By contrast t using the potent adjuvant alum during antigen immunization causes NLPR3 and IL-1R1 independent allergic asthma. In conclusion, NLRP3 inflammasome activation leading to IL-1 production is critical for the induction of a Th2 inflammatory allergic response, and the NLPR3 dependence of an allergic response may be determined by the adjuvant. Disclosure of interest: None declared. The importance of developing new animal models to assess the pathogenesis of glucocorticoid (GC)-resistant asthma has been stressed. Since following repeated allergen provocations, several features of asthma appeared less marked in many mice strains (Balb/ c, C57blk6 and others) as compared to A/J mice, we have investigated the sensitiveness of mice of strain A/J to allergic provocations in the presence or absence of dexamethasone (DEX) treatment. Animals were sensitized on days 0 and 14 by a mixture of Al(OH)3 and ovalbumin (OVA), and challenged for 2 or 4 consecutive days starting at day 19 post-sensitization. The animals were subjected to treatment with DEX (3 mg/kg, oral), or vehicle, 1 h before each provocation. Invasive methodology was employed for measurement of AHR. Peribronchial leukocyte infiltration and subepithelial airway fibrosis were analyzed by histomorphometry. Cytokines and chemokines were assessed by ELISA. All changes were done 24 h after the last challenge. Our findings show that A/J mice developed asthma-like pathological changes that become resistant to DEX treatment as they are exacerbated by successive provocations. Since, the expression of GATA-3 also appeared increased and less sensitive to DEX, it is not unlikely that an overexpression of GATA-3 may contribute to the state of GC refractoriness seen in this model. This study suggests the existence of a causative relationship between steroid resistance and continued allergic inflammation. Sexual dimorphism modulates Th1 and Th2 lymphocyte functions, and accordingly estradiol (E2) interferes with acute lung injury (ALI) due to intestinal I/R. Female rats are relatively resistant to end-organ injury caused by hemorrhagic shock and gut from female animals is more resistant than the males's to the deleterious effects of ischemic injury. Nitric oxide (NO) is a dual mediator of gut trauma-induced lung injury, but interaction of estradiol and NO is still not clear. Here we assessed the effect of E2 on the lung and intestinal inflammation after intestinal I/R and its interaction with the inducible NOS (iNOS) pathway. Anesthetized rats were subjected to occlusion of the superior mesenteric artery (SMA) for 45 min, followed by 2 h of reperfusion. Groups of rats were treated with E2 (17b estradiol, 280 ll/kg, s.c. 24 h before ischemia, or i.v. 30 min after induction of ischemia) and with the selective NOS inhibitor, aminoguanidine (50 mg/kg iv) or the NO-donor L-arginine (300 mg/kg, i.p.) 1 h before occlusion. Lung and intestinal vascular permeability (LVP and IVP) were assessed by Evans blue dye extravasation and neutrophil recruitment to the tissues by the myeloperoxidase (MPO) method. IL-10 was quantified by ELISA in cultured lung tissue. Female ovariectomized (OVX) rats had LVP, IVP and MPO increased after I/R whereas IL-10 levels were reduced. E2 reverted the increased LVP, but not IVP nor MPO. Aminoguanidine alone reduced LVP in OVX rats, but failed to do so when associated to E2. Aminoguanidine used in non-OVX rats markedly exacerbated LVP and IVP. Thus endogenous NO probably reduces the vascular effects of intestinal I/R. In addition, the NO-donor L-arginine imitated E2 since it reduced ALI after intestinal I/R. In confirmation, male rats treated with E2 were also protected. In conclusion, estradiol reduces lung inflammation after intestinal I/R, but the concomitant blockade of NOS suppresses protection. Supported by CNPq and FAPESP. Disclosure of interest: None declared. The auto-inflammatory diseases encompass a large group of inflammatory disorders defined as Mendelian genetic diseases involving the innate immune system, and especially interfering with the regulation of Interleukin 1 pathway. The core group of these diseases is represented by hereditary recurrent fever syndromes: Familial Mediterranean fever; Tumor necrosis factor receptor-associated periodic fever syndrome; the cryopyrinopathies (Cryopyrin-associated periodic syndrome or CAPS) which include familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and Chronic infantile neurological cutaneous and articular/Neonatal onset multisystemic inflammatory disease; the group of mevalonate kinase deficiency; nucleotide-binding oligomerisation domain, Leucine rich Repeat and Pyrin domain (NLRP12)-associated syndrome, and the deficiency of the interleukin 1-receptor antagonist. All these disorders are characterized by recurrent episodes of inflammatory attacks manifested by fever, local inflammatory symptoms due to serositis involving mainly the abdomen, the musculo-skeletal system, the thorax, and the skin, and an elevation of acute-phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA). Specificities characterize each of these entities. Other diseases sharing the Mendelian pattern of inheritance but not the intermittent character can be also considered as auto-inflammatory diseases such as pyogenic arthritis pyoderma gangrenosum and acne syndrome, Majeed syndrome, Ghosal syndrome. The concept of auto-inflammation is expanded to diseases in which innate immunity dysregulation is involved but without a Mendelian genetic basis such as atherosclerosis or gout; or disorders which share the implication of genes involved in Mendelian recurrent fever syndromes such as Crohn disease; or Inflammasomes are multimolecular platforms controlling caspase-1 activation and proinflammatory cytokine maturation like Interleukin-1beta (IL-1b). These platforms play major roles in the sensing of pathogens and stress signals. The best-characterized inflammasome is the NLRP3-inflammasome, which plays a critical role in protection against infections but also in auto-inflammatory disorders including Familial Periodic Fevers and gout disease. How the NLRP3-inflammasome activity is regulated is still not completely understood. Intracellular potassium levels as well as ROS production have been proposed to signal to NLRP3. New findings indicate that the NLRP3-inflammasome activity is controlled by autophagy. A growing number of illnesses, including Mendelian as well as multifactorial disorders, are recognized as autoinflammatory syndromes (AIS). So far, few studies have investigated susceptibility loci for complex AIS. However, major advances have been made in the characterization of monogenic AIS. Five genes have been involved in the first-recognized and most common AIS called hereditary recurrent fevers (HRF) and, depending on the definition of so-called AIS, 5-10 additional genes have been shown to be responsible for these disorders. Genetic testing allows confirmation of the diagnosis, avoids years of unsuccessful investigations, and permits targeted therapy, as well as genetic counselling. However, molecular analyses also show some limitations. (1) Most variations correspond to missense mutations whose deleterious effect remains often difficult to ascertain in sporadic cases due to the lack of appropriate functional assays. In addition, for several variations identified in HRF genes, it is not clear whether they are simple polymorphisms, confer a milder phenotype, or act as modifiers for other inflammatory loci. (2) Many patients with familial Mediterranean fever-FMF-(the most frequent monogenic AIS) carry only one mutation in the disease-causing gene (MEFV); this questions the recessive model of inheritance of FMF, although this observation depends on the clinical definition of the disease. (3) Notably, there is no molecular explanation in more than 70% of patients. Inflamm. Res. Several approaches can be used to identify new AIS genes: classical linkage studies, homozygosity mapping, as well as candidate gene approaches. The few recent novelties in the field include the involvement of NLRP12 in HRF, IL1RN in the DIRA syndrome, as well as IL10RA and IL10RB in early-onset enterocolitis. The discovery of anti TNF therapy, now routinely used in the treatment of rheumatoid arthritis and multiple diseases, was founded on several premises. First a hypothesis is that cytokines were important autoimmunity. Secondly the most accessible tissue to test was rheumatoid synovium. Thirdly by 1987/88 the molecular biology revolution had led to the cloning of multiple cytokine cDNAs, enabling the analysis of cytokine expression. Fourth, improved culture systems enabled the upregulated cytokine expression to be analysed and TNF emerged as a therapeutic target, with tested reproducibility. But the success is limited: there are no cures. Further work is need to address the reasons why this is so, and potential synergistic approaches may improve efficacy but at unknown risk. Importantly the cytokine analysis in diseased tissue approach has been successfully used by Claudia Monaco to show that upregulated cytokines are important in atherosclerosis, especially in active plaques. R.E. O'Hehir 1, * 1 Allergy, Immunology and Respiratory Medicine, Alfred Hospital, and Department of Immunology, Monash University, Melbourne, Australia Allergic diseases constitute major and growing health burdens worldwide. Th2-polarised immune responses to allergens drive the adverse reactions, notably IL-4 which induces IgE class switching and mast cell inflammatory mediator release and IL-5 which promotes local eosinophil activation. Allergen specific immunotherapy (SIT) can modify the natural course of allergic diseases. Conventionally this treatment is given subcutaneously but recently sublingual-swallow allergen-specific immunotherapy (SLIT) is gaining popularity, with meta-analyses confirming long lasting clinical efficacy with decreased adverse events. Data on the immunological mechanisms underlying effective SLIT are emerging. The most convincing studies to date indicate that SLIT induces decreased allergen-stimulated T-cell proliferation, immune deviation (decreased IL-4 and IL-5 and increased IFN-c), T cell anergy (allergen-specific non-responsiveness with enhanced IL-10 secretion), blocking antibodies (allergen-specific IgG 4 ), and induction of regulatory T cells, as have been established for subcutaneous injection immunotherapy. Peripheral T-cell tolerance to allergens is an important mechanism for maintaining homeostasis in healthy individuals and in clinically efficacious allergen-specific immunotherapy. It is likely that functional naturallyoccurring CD4 + CD25 + Foxp3 + CD127 lo T reg and peripherallyinduced IL-10-and/or TGF-b-secreting Treg are important in effective SLIT. Additionally, there is evidence that the oral mucosa presents distinct regulatory features with oral Langerhans-like DC playing a major role. The precise molecular delineation of the allergen-DC receptor interaction which favours tolerance induction and whether particular adjuvants specifically tailored for the sublingual route will allow improved allergen formulations and delivery strategies for greater efficacy of treatment remain to be determined. Epidemiological evidence from several independent groups suggests that the development of what eventually becomes chronic asthma is commonly initiated in early childhood, driven by a range of environmental factors which confer varying degrees of risk. Two factors stand out from the perspective of potential intervention targets. Firstly, the association between allergic sensitization to aeroallergens (as marked by IgE production) and asthma development has been recognised for many years, and in this context atopy can function as an independent risk factor. Secondly, early lower respiratory tract infection, particularly severe infection marked by inflammatory symptoms (wheeze and/or fever), also operates as an independent risk factor, and risk increases as a function of infection frequency. Moreover, maximum risk is encountered when these inflammatory pathways intersect, i.e., in children experiencing both sensitization and severe lower respiratory tract infections. This suggests direct interaction between these risk factors which results in inflammationmediated disturbance in the maturation of normal respiratory functions. These interactions also appear to operate when levels of IgE are relatively low, within the range commonly encountered transiently in up to 50% of the normal infant population. Ongoing studies in our lab on viral-induced severe asthma exacerbations provide a plausible explanation for these interactions, notably ''recruitment'' of underlying IgE-associated allergen-specific Th2 immunity into the inflammatory milieu of the infected airway via the innate immune response against the virus, which is mediated by upregulation of high affinity IgE-receptor expression on local airway mucosal Dendritic Cells. Additionally, it is becoming evident that mucosal dwelling bacteria also play a role in early asthma pathogenesis, and recent evidence on the nature of their participation will also be presented. Epigenetics, as the name implies, is used to describe the events that alter cell, organ whole animal phenotype that does not require changes in DNA sequence. It has been proposed to account for much of the missing heritability in many diseases including asthma, a chronic inflammatory disease of the airway. In addition to asthma, other airways diseases such as COPD and pulmonary fibrosis have been proposed to have epigenetic components. Changes in cell function involve co-ordinated post-translational modifications of histones and DNA including changes in DNA methylation and histone acetylation/methylation. These changes are under the control of several enzymes and enzyme complexes that write, read and erase these epigenetic marks. Epigenetic changes occur through life in response to environmental, physiological and pathological signals and may affect asthma susceptibility. For example, increasing the dietary content of methyl donors can increase asthma susceptibility and inflammatory components associated with airway hyperresponsiveness in animal models of asthma. Furthermore, changes in lung function and airway inflammation in response to environmental pollutants such as traffic/diesel pollution and cigarette smoking have been linked to changes in DNA methylation and histone acetylation. It is hoped that the development of novel drugs that target these writers, erasers and readers may produce novel therapeutic agents for chronic inflammatory diseases and at least cancer. Atherosclerosis is a chronic inflammatory disease of the arterial wall that is characterized by a disturbed equilibrium of immune responses and lipid accumulation, leading to the development of plaques. The atherogenic influx of mononuclear cells, namely monocytes and T-cells, and the interaction of antigen-presenting cells, T cells and monocytes is orchestrated by chemokines and their receptors. Studies using genedeficient mice and antagonists based on peptides and small molecules have generated insight into targeting chemokine-receptor axes for treating atherosclerosis, which might complement lipid-lowering strategies and risk factor modulation. Combined inhibition of multiple chemokine axes could interfere with the contributions of chemokines to disease progression at specific cells, stages or sites. In addition, the recently characterized heterophilic interactions of chemokines might present a novel target for the treatment and prevention of inflammatory diseases such as atherosclerosis. Macrophages play a pivotal role in the development of atherosclerosis. After recruitment in the sub-endothelial space, monocytes differentiate into macrophages, accumulate lipids thus forming foam cells and secrete pro-inflammatory and matrix-degrading factors, thus contributing to plaque development, inflammation and instability. Recently, a S32 Inflamm. Res. role for macrophages in adipose tissue dysfunction related to obesity and diabetes has also been demonstrated. Therefore, pharmacological modulation of macrophage functions represents an attractive strategy for the prevention and treatment of metabolic and cardiovascular diseases related to atherosclerosis. Macrophages are heterogeneous cells, which adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an ''alternative'' anti-inflammatory M2 macrophage phenotype. In recent years, the role of ligand-activated nuclear receptors, such as the peroxisome proliferator-activated receptors (PPAR) and Liver X receptors (LXR), in the modulation of macrophage polarization, lipid homeostasis and immune-inflammatory responses has been studied extensively. It has been shown that PPARc promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPARb/d in this process has been reported only in mice. Our findings show that, in contrast to PPARc, expression of PPARa and PPARbd overall do not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional protein that binds numerous ligands and controls multiple signaling pathways. LRP1 integrates TGFb and PDGF signaling pathways in vascular smooth muscle cells (vSMCs) and disruption of this signal integration results in increased TGFb signaling, Marfan-like changes of the vessel wall architecture, decreased cellular cholesterol export, massive foam cell accumulation and accelerated atherosclerosis. Recently, we found that the nuclear hormone receptor PPARc and the LDL receptor-related protein LRP1 functionally interact to control the metaplastic cartilaginous transformation of vSMCs. Loss of PPARc in vSMCs promotes cartilage formation in the medial muscular layer, but surprisingly this is prevented in the absence of LRP1. Wnt5a expression, and recapture and nuclear translocation of SPARC, both regulators of the cartilage inducers Sox9 and Cart1, require LRP1. By contrast, PPARc controls the expression of the Wnt5a antagonist sFRP2, and LRP1 and PPARc both suppress fibrosis-and calcification-promoting TGFb signals. Thus, LRP1 and PPARc jointly control the vascular wall architecture, by regulating cellular lipid and extracellular matrix homeostasis, as well as the differentiation state of vSMCs. Inflammatory processes are prominent in various types of human and experimental pulmonary hypertension (PH) and are increasingly recognized as major pathogenic components of pulmonary vascular remodeling. Macrophages, T and B lymphocytes, mast cells, and dendritic cells are present in the vascular lesions of PH, whether in idiopathic pulmonary arterial hypertension (PAH), or PAH related to more classical forms of inflammatory syndromes such as connective tissue diseases or human immunodeficiency virus. Similarly, the presence of circulating chemokines and cytokines and increased expression of growth (such as vascular endothelial growth factor and platelet-derived growth factor) and transcriptional (e.g., Nuclear Factor of Activated T cells or NFAT) factors in these patients are thought to contribute directly to further recruitment of inflammatory cells, and proliferation of smooth muscle and endothelial cells. Thus, the recognition of complex inflammatory disturbances in the vascular remodeling process offers potential specific targets for therapy and has recently lead to clinical trials investigating, e.g., the use of tyrosine kinase inhibitors. This presentation will provide an overview of specific inflammatory pathways involving cells, chemokines and cytokines, cellular dysfunctions, growth factors, highlighting their potential role in pulmonary vascular remodeling and the possibility of future targeted therapy. Introduction: PI3kc has recently been identified as a potential target for the treatment of inflammation, primarily due to its role in leukocyte chemotaxis. Moreover, this signalling molecule is vital for defence against invading microbes, and its loss could potentially result in persistent infection. We chose to examine the effects of genetic ablation of PI3K p110c on the development and resolution of acute murine colitis induced by the hapten trinitrobenzene sulfonic acid (TNBS). Methods: Colitis was induced in wild-type C57/Bl6 or PI3kc -/mice by intra-rectal administration of 4 mg TNBS in 30% ethanol. Colon tissue was collected on day 3 to examine severity of acute colitis, and on days 7 and 14 to observe the resolution of colitis. Indices of colitis assessed include: weight loss, gross inflammatory and histological score, and myeloperoxidase (MPO) activity. Results: While PI3kc -/mice exhibited significant protection from TNBS colitis by all measures during the early stages of colitis (i.e., 3 days post-TNBS), they also failed to properly resolve the inflammation by day 7 or 14. This was marked by a significant inability to gain weight following colitis ( Abnormalities of fat in the mesentery have been long recognized as characteristic features of Crohn's Disease (CD). The therapy with antibodies anti-TNF-a, i.e., infliximab, has been employed to treat CD patients. Although adipose tissue from CD patients can secretes a large number of hormones that act modulating immune responses and inflammation, there is no relate about infliximab action upon adipokine production by mesenteric adipose tissue (MAT) during intestinal inflammation. Thus, the aim of this work was to study infliximab effects upon adipokine production by MAT and intestinal inflammation in trinitrobenzenosulphonic acid (TNBS)-induced colitis in rats. Wistar rats with colitis induced by TNBS received infliximab (1 or 5 mg/kg/week). Colitis was characterized by histopathological analysis, MPO content and inflammatory mediators production in colon. Morphology, adipokines release and inflammatory markers were assessed in MAT. Infliximab treatment reduces MPO activity and improves all inflammatory parameters evaluated in colon. The TNF-aand IL-10 level in colon was not modified while iNOS expression was strongly inhibited in both infliximab groups. Leptin and adiponectin release by MAT was also inhibited in both infliximab groups, while resistin released was increased after infliximab treatment at 5 mg/kg dose. MAT also expressed level of PPAR-c and adipocyte size after infliximab (5 mg/kg) as healthy animals. TNF-a blockade in experimental colitis was able to reduce the inflammatory response in colon. In MAT, we could observe an inhibition in leptin production, an important pro-inflammatory adipokine. Interestingly, infliximab treatment restores adipocyte morphology and PPARc expression, which could act as an additional anti-inflammatory mediator, providing evidences that therapeutic intervention modifies not only intestinal inflammation but also MAT alterations induced by inflammation. Food allergy affects approximately 5% of children and 3% of the adult population in the western world. This disease is the manifestation of an abnormal immune response to antigens introduced into the organism orally and it is often mediated by IgE. Our group developed a mouse model for the study of food allergy induced to ovalbumin. One of the most remarkable alterations observed in the allergic mice is a loss of weight. However, the disturbances that trigger this loss of body weight are not clear. Thus, the aim of this study was to investigate the mechanisms involved in weight loss of mice with ovalbumin-induced food allergy. With this purpose, BALB/ c mice were subcutaneously sensitized with ovalbumin in aluminum hydroxide and challenged with the antigen containing diet for 7 days. The allergic mice showed significant weight loss with loss of epididymal adipose tissue, although it was not observed a reduction in food intake. These mice demonstrated adipose tissue inflammation characterized by increased leukocyte recruitment and infiltration of mast cells, macrophages and regulatory T cells in the stroma. Moreover, we demonstrated high concentrations of TNF-a, IL-6, IL-10 and the chemokine MCP-1/CCL-2 in this tissue. The metabolic changes in adipose tissue of allergic animals were represented by increased glucose uptake and lipolysis in adipocytes, resulting in atrophy of these cells. Changes were also seen in systemic metabolism characterized by decreased serum concentrations of glucose, triglycerides, total cholesterol and free fatty acids in allergic mice. Based on our results, we conclude that food allergy induce adipose tissue inflammation by producing mediators that lead to atrophy of this tissue. Activation of immune regulatory pathways is critically important for treatment of inflammation in autoimmunity, allergy, and transplantation. We discovered regulatory T cell epitopes (Tregitopes) located in IgG Fc and framework regions that induce regulatory T cells (Treg) in vitro and in vivo. We report efforts directed at using Tregitopes to suppress antigen (Ag)-specific inflammatory immune responses by inducing immunological tolerance. Studies in allergy (OVA model), transplantation (MLR, cardiac transplant model), protein therapeutics (OVA), gene therapy (AAV), and autoimmunity (EAE, NOD) have demonstrated that tolerance to a range of Ags can be induced by co-administration of Tregitopes with target Ags in vitro and in vivo. Tregitopes activate CD4 + CD25 + FoxP3 + natural Treg (nTregs). In vitro, co-incubation of Ag with Tregitopes leads to suppression of Teff responses (i.e. cytokine secretion, proliferation) and expansion of Ag-specific adaptive Tregs (aTregs). In vivo, co-administration of Tregitopes with a range of proteins (i.e. ovalbumin, and autoantigens) results in Treg expansion and functions, with suppression of Ag-specific T cell and antibody responses. The in vivo Tregitope effect is long-lasting; 100 days in transplant, and up to 30 weeks in the NOD mouse model. Tregitope treatment appears to be pleiotropic; in different model systems induction of IL-2, IL-10, suppression of gamma-interferon, induction of TGF-Beta and IL-9 are observed, perhaps due to the complex interplay of APC, nTregs, Teff, and aTregs. Stimulation of regulatory T cells to control a range of inflammation-mediated pathologies is promising. Moreover, the potential to stimulate Tregs in an Ag-specific manner may reduce side effects (i.e. infections) associated with more widely immunosuppressive treatments. Adaptation or incorporation of Tregitopes into established protein therapeutics also may have broad-reaching implications for the fields of allergy, autoimmunity and transplantation. We previously reported that NF-kB1-deficient (Nfkb1 -/-) mice had reduced CD4 T cell-dependent acute inflammatory arthritis (1) . We also previously reported that MoDC differentiated from monocyte precursors in the spleen during acute inflammatory peritonitis (2) . Here, we show that Nfkb1 -/mice are protected from acute inflammatory peritonitis and that protection in both of these models of acute inflammation is due to a requirement for the NF-kB1 subunit in CD4 T cells. Protection was not due to enhanced apoptosis or failure to upregulate cytokine receptors, but to a selective defect in the production of the cytokine GM-CSF by activated CD4 T cells. We also show that the in vivo differentiation of CD11c int CD11b hi Ly6C +/ hi MHCII hi MoDC, which could be identified in inflamed synovial tissue and draining lymphoid tissues, was dependent on the expression of NF-kB1 in CD4 T cells. These data provide in vivo evidence that NF-kB1-mediated production of GM-CSF by activated CD4 T cells is required for MoDC differentiation under inflammatory conditions. Abstract: Interleukin-4 mRNA exists naturally in two isoforms, one encoded by exons [1] [2] [3] [4] , and the other encoded by exons 1, 3, and 4 (IL-4d2). We have previously identified and named IL-4d2 mRNA. We and others studied IL-4d2 mRNA expression in pathology, and discovered changes in patients with scleroderma, asthma, tuberculosis, and other diseases. However, it is not known whether a corresponding IL-4d2 protein is also naturally produced and under what conditions. The in vivo effects of IL-4d2 protein have never been studied. We have developed an mAb against IL-4d2 with no cross-reactivity to IL-4. Freshly purified T cells from 10 patients with mild or moderate asthma and from 5 healthy controls were tested after up to 96 h activation with PMA/ionomycin. Levels of IL-4 and IL-4d2 mRNAs were measured by RT-Q-PCR, and secretion of IL-4 and IL-4d2 proteins was measured by ELISA. Basal expression of IL-4 and IL-4d2 mRNAs was detected in all samples. Stimulation caused a 5-12-fold increase in IL-4 mRNA, and a 20-70-fold increase in IL-4d2 mRNA, peaking at 12-24 h of activation. T cells from controls secreted IL-4 but not IL-4d2 protein, whereas T cells from asthmatics produced both IL-4 and IL-4d2 proteins in comparable amounts. We have recently described in mature neutrophils, a new survival pathway regulated by the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells. Notably, in neutrophils, which are non-proliferating cells, PCNA localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization occurring during myeloid differentiation. The first aim of the study was to elucidate the molecular basis of this PCNA cytoplasmic relocalization. We identified a leucine-rich nuclear export signal (NES) that can mediate an active CRM1dependent nuclear export. However, because this NES sequence was located at the inner face of the PCNA trimer, this NES was fully functional in PCNAY114A monomeric mutant, which accumulated within the cytoplasm. Using the DMF-differentiated PLB985 cell line, we next tested whether PCNA nuclear-to-cytoplasmic relocalization during differentiation would impact its anti-apoptotic activity in mature neutrophils. PLB985 cells expressing the monomeric and cytoplasmic PCNAY114A showed the same anti-apoptotic activity assessed by mitochondria depolarization as those expressing PCNA as compared to control PLB985. In contrast, no antiapoptotic activity was observed in PLB985 cells expressing the nuclear SV40NLSfused PCNA, thus strongly suggesting that nuclear-to-cytoplasmic relocalization but not trimerization was pivotal for PCNA survival activity in mature neutrophils. We concluded that (1) PCNA nuclearto-cytoplasmic relocalization is part of the specific features of the neutrophil differentiation programme since it is essential for its prosurvival activity and (2) Polymorphonuclear neutrophils (PMN) are key components of the innate response via several mechanisms, in particular the production of Neutrophil Extracellular Traps (NETs). NETs are composed of decondensed chromatin fibers associated with granule and cytoplasmic proteins and are released upon activation. NETs play an important role in antimicrobial defenses but seem also to contribute to some auto-immune and inflammatory diseases. Our aim was thus to investigate whether NETs could interact with dendritic cells and thus modulate key immune functions. We first developed a reproducible model to induce, isolate and quantify NETs from blood PMN. Calcium ionophore A23187 was chosen to induce NETs and the restriction enzyme Alu I allowed the recovery of heterogeneous-sized fragments of NETs, as observed by DNA electrophoresis. Their composition was assessed by Western blotting and the use of various antibodies against some proteins potentially involved in DC interactions; proteomic analysis is under investigation. The incubation of isolated NETs with moDC did not directly induce DC maturation as measured by phenotypic modifications (CD80, CD86, CD83, CD40, HLA-DR…). In contrast, NETs were able to modulate the effect of lipopolysaccharide (LPS) on moDC maturation. In particular, we could observe the decrease of LPS-induced HLA-DR up-regulation. Our preliminary results thus suggest that NET formation, that takes place during inflammation, could also constitute a regulatory mechanism by modulating some DC functions. The next steps of this study will be to better understand the mechanisms of NETs-DC interactions, in particular the signaling pathways involved and the consequences on T cell activation. These results add new arguments to document Aims: Bacterial recognition by mononuclear cells often initiates assembly of an inflammasome resulting in activation of caspase-1 and processing of IL-1b, a key pro-inflammatory cytokine. B. cepacia is one of the major opportunistic pathogens colonizing airways and inducing progressive respiratory inflammation in cystic fibrosis patients. However, the Burkholderia-sensing inflammasome remains uncharacterized. The purpose of this study was to investigate pyrin as a potential sensor of B. cepacia and its effect on caspase-1 activity. Methods: Human monocytes (Mo) and THP-1 cells, stably expressing either siRNA against pyrin or YFP-pyrin and universal inflammasome adaptor molecule ASC (YFP-ASC), were infected with B. cepacia and analyzed for the inflammasome activation. Results: B. cepacia efficiently activates the inflammasome and IL-1b release in Mo and THP-1 cells. Suppression of pyrin levels in Mo and THP-1 cells reduced caspase-1 activation and ability to process and release IL-1b by B. cepacia challenge. In contrast, over-expression of pyrin or ASC induced the robust IL-1b response to B. cepacia, which correlated with the enhanced host cell death. In initial efforts to identify the pathogen-associated molecular pattern (PAMP) responsible for B. cepacia recognition by pyrin, Mo were infected with Burkholderia mutants, defective in several types of secretion systems.Inflammasome activation and IL-1b release was abolished in cells infected with T4SS mutants of B. cepacia. Conclusions: For the first time we showed an association between pyrin levels and release of the major pro-inflammatory cytokine IL-1b by mononuclear cells infected with B. cepacia. The inflammatory reaction is likely induced by a yet uncharacterized PAMP of the T4SS. Pyrin in association with caspase-1 and ASC forms pyrin inflammasome and thus up-regulates mononuclear cell IL-1b processing and release in response to B. cepacia. CD36 receptor expressed on mononuclear phagocytes is a sensor of a broad range of microbial products that mediates phagocytosis and inflammation. CD36 is the main receptor mediating nonopsonic phagocytosis of P. falciparum parasitized erythrocytes (PfPEs). Accordingly, CD36 -/mice display a combined defect in parasite clearance and cytokine response leading to higher mortality rates. Interestingly, CD36 macrophage overexpression promotes PfPEs clearance and modulates parasite-induced TNFa secretion. CD36 is upregulated on macrophages following exposure to rosiglitazone and IL13, a Th2 cytokine, via the PPARc pathway. Nrf2 transcription factor has also been described to control CD36 expression on macrophages. Severe malaria is associated with an excessive production of proinflammatory cytokines such as TNFa. Here, we demonstrated that inflammatory processes generated through TLR2 engagement and TNFa negatively regulate CD36 expression both on human and murine macrophages, and hence decrease CD36-mediated PfPEs clearance, directly favoring the worsening of malaria infection in vivo. Our main objective was to determine whether PPARc or Nrf2 activators could restore CD36 expression and function on macrophages during inflammatory processes. In these conditions, we showed that PPARc ligands were ineffective to promote CD36 expression. This phenomenon was directly associated with a failure in the expression and activation of PPARc. Interestingly, pharmacological and molecular approaches using specific Nrf2 activators on PPARc-and Nrf2-deficient macrophages showed that CD36 expression and function were restored both on human and murine inflammatory macrophages via Nrf2, independently of PPARc. These results were validated in vivo where only Nrf2 and not PPARc activators improved the outcome of severe malaria highlighting that Nrf2 activators represent a novel class of host immunomodulatory drugs that might be useful for treatment of severe malaria. Disclosure of interest: None declared. Monocytes and macrophages endotoxin tolerance, and more widely Toll-like receptors (TLR) agonists tolerance is a phenomenon corresponding to a modification of functional response of these cells (particularly an alteration of proinflammatory cytokines) during iterative stimulation by theses agonists, notably the lipopolysaccharide (LPS). Tolerance is observed with monocytes and macrophages from many tissue (peritoneal cavity, spleen, bone marrow), but is not detected with alveolar macrophages (AM). We investigated the cytokine and cell specificities of lung microenvironment possibly involved in that singular behavior of AM. We confirmed the absence of tolerization of AM ex vivo in three different mice strains: BALB/c, C57BL/6 and 129SV. In vivo systemic inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) (produce in large amounts by type II pneumocytes, IIPC) led to a modification of the second response of AM to LPS. Similarly, AM obtained from interferon-c (IFN-c) receptor KO mice, or after in vivo inhibition of this cytokine, allowed the observation of tolerance. The responses of AM isolated from different S38 Inflamm. Res. KO mice (rag2-/-, rag2/cc-/-, cd3e-/-, l-/-, il-15-/-, Ja18-/-) lacking various leukocytes lineages led us to demonstrate the involvement of NK cells and B cells in the IFN-c production within the lung microenvironment. In summary, AM are resistant to tolerance to endotoxin. This functional singularity is underlined by the presence of GM-CSF (produced by IIPC) and of IFN-c of which production is depending of NK and B cells) within lung microenvironment. This subpopulation expresses phenotypic markers of iNKT cells and appears at day 14 post-infection concomitantly with an increase in TGF-b and IL-18 expression. This IL-17 producing iNKT population (iNKT-17), rather than IL-17 producing CD4 T cells (Th17), persist during the chronic phase in peripheral blood and in a large number of tissues, except colon. In addition, the levels of these cells were associated with disease progression to AIDS. Consistent with the role of TGF-b and IL-18 production, we demonstrated that, in vitro, TGFb and IL-18 induce the differentiation and expansion of iNKT-17 cells. The activity of IL-17 is classically defined by its ability to induce the expression of a variety of pro-inflammatory mediators, which ultimately leads to the recruitment and activation of phagocytes (polymorphonuclear neutrophils and monocytes Background: Recent studies suggest that prolonged alterations in innate immunity following a primary viral infection are orchestrated by up-regulation of negative regulators in innate immune effectors, primarily alveolar macrophages. The majority of negative regulators investigated in this study have not been explored following viral infection before and are therefore novel. Methods: Using a murine model, we tentatively assessed the involvement of various negative regulators [Glucocorticoid Receptor (GR) , TAM receptors, IL-10R, Ron-b, ST2L, EP2, LXR-a/b and PPAR-c] on alveolar macrophages following influenza. We also studied the antagonism of GR (by RU486 administration) following primary influenza infection and its effect on subsequent secondary bacterial challenge because of its therapeutic potential. Results: Following primary influenza, the expression profiles of the regulators altered dramatically. There was a biphasic expression pattern: early phase regulators, which peaked at day 7 (e.g. Axl, GR, PPAR-c, and GRK2) and late resolution phase regulators (e.g. MerTK, EP2, IL-10R, EP2 and LXR) which peaked around days 19-26. GR antagonism following primary influenza infection reduced the severity of subsequent secondary bacterial pneumonia. Discussion: The up-regulation of negative regulators in a biphasic manner is interesting and may represent any or all of the following: (a) apoptosis of alveolar macrophages leaving behind those that preferentially express a particular receptor, (b) maturation of macrophages at different times so that receptor expression is seen in waves, (c) recruitment of macrophages leaving a receptor at particular time points ( Introduction: The mechanisms underlying the pathogenesis of cerebral malaria (CM) remain unclear. The inflammatory infiltrate in the brain, with the recruitment of CD8+ T cells, and endothelium damage seems to be relevant in this process. Platelet-activating factor (PAF) is an inflammatory mediator involved in leukocyte recruitment and vascular permeability. Objective: To investigate the role of the PAFR in the outcome of Plasmodium berghei ANKA (PbA) infection and the relevance of this molecule for the neuroinflammatory process involved in CM. Method: C57Bl/6 wild-type (WT) and PAFR -/mice were infected with PbA (10 6 parasitized erythrocytes) by intraperitoneal injection. Parasitemia and survival parameters were monitored daily. Leukocyte rolling and adhesion were evaluated by intravital microscopy in the pial vessels. Recruitment and activation of CD8+ T cells of brainsequestered leukocytes were analyzed by FACS. The expression of cleaved caspase-3 in total brain extracts was analyzed through Western blot. Histopathological analyses were also performed. To further investigate the role of PAFR, WT mice were treated with PAFR specific antagonist 505) . Results: Infected PAFR -/mice resisted to the infection for a longer period (21 days) than WT (7 days). Perivascular inflammatory infiltrates and parenchymal hemorrhage were more intense in WT mice than PAFR -/mice. However, intravital microscopy showed similar levels of rolling and adhering leukocytes in WT and PAFR -/mice. The recruitment and activation of CD8+ T cells were reduced in PAFR-deficient mice, as the activation of caspase-3. In WT mice infected with PbA, treatment with UK74,505 was also associated with the reduction of infection severity. Objectives: Pseudomonas aeruginosa is often isolated with Candida albicans in critically ill patient airways. Lung sensitization with C. albicans in mice attenuated subsequent P. aeruginosa-induced lung injury and decreased bacterial burden. Interestingly, this protection persisted when direct interactions between the two pathogens were blocked. Our objective was therefore to determine the cellular mechanisms that may account for the Candida-triggered protective effect against P. aeruginosa infection. Methods: Strains were C. albicans CA SC5314 and P. aeruginosa PaO1. Balb/c Mice were colonized by endotracheal instillation of 10 5 CFU of C. albicans. P. aeruginosa pneumonia was induced 48 h later by endotracheal instillation of 10 7 CFU of P. aeruginosa. Lung immune response was assessed 24 h later in bronchoalveolar lavage (BAL) and lung homogenates from mice infected with PaO1 with or without C. albicans sensitization. BAL and lung cellularity were determined through flow cytometry (FACS). Results: Lung sensitization by a colonizing and persistent inoculum of C. albicans alone, led to significant alveolar macrophage cell recruitment and activation with a higher expression of MHC II in BAL (p \ 0.05 vs. control). In contrast, P. aeruginosa pneumonia alone (Pa) significantly increased both PMN and macrophages but decreased alveolar macrophage activation (p \ 0.05 vs. control). Importantly, lung sensitization by C. albicans significantly enhanced alveolar macrophage recruitment and activation with an overexpression of both CD86 and MHC II (p \ 0.05 vs. Pa). Consistently, lung total lymphoid cell populations were significantly decreased by P. aeruginosa pneumonia alone, but were significantly increased by C. albicans airway colonization alone and by Candida sensitization prior to P. aeruginosa pneumonia. Conclusion: C. albicans airway sensitization attenuates P. aeruginosa-induced lung injury through alveolar macrophage and possibly related lymphoid recruitment and activation. Disclosure of interest: None declared. MIF is a structurally unique inflammatory mediator that plays a critical role in several inflammatory diseases, including atherosclerosis. We recently discovered that MIF, contrary to its eponymous name, functions as a chemokine, promoting the recruitment of leukocytes to inflamed and atherogenic endothelium (Bernhagen et al., Nat. Med. 2007 ). This function is mainly based on non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, but MIF also binds to the MHC class II chaperone CD74. Blockade of Mif but not of the canonical ligands of Cxcr2 or Cxcr4, Cxl8 and Cxcl12, respectively, in mice with advanced atherosclerosis led to plaque regression. In an attempt to devise novel MIF/CXCR-based therapeutic approaches, we comprehensively mapped the MIF/CXCR interface and identified a pseudo-(E)LR and an N-likeloop motif in MIF as a critical structural basis for MIF's promiscuous interaction with CXCR2 (Weber et al., PNAS 2008; Kraemer et al., FASEB J. 2010) . Applying peptide spot array technology, here we fully characterize the MIF/CXCR4 interface and identify the motifs and residues on the receptor side of CXCR2 and CXCR4 responsible for MIF binding. Importantly, we will report on the identity, formation, and function of novel hetero-oligomeric MIF receptor complexes, which are involved in inflammatory monocyte and lymphocyte recruitment by MIF. Based on this previously unavailable structural information, we report on peptide-based anti-MIF strategies that may have future applicability in inflammatory disease and atherosclerosis. PIM kinases consist of three oncogenic serine/threonine kinases, whose activity is regulated at all levels of expression. PIM-1 and PIM-3 have been shown to be involved in T cell proliferation and survival following T cell receptor engagement and have reported roles in cytokine induced survival. These activities suggest that a PIM1/3 kinase inhibitor may be useful in targeting T cell mediated inflammatory conditions, such as Crohn's disease. Herein we report the ability of a small molecule PIM 1/3 kinase inhibitor to modulate disease progression and severity of the CD45RB adoptive transfer murine colitis model, which is a model where progression involves Th1 and Th17 cytokines. AR00472159 is a selective and potent inhibitor of PIM1 and 3 kinases in both the enzyme (160 pM PIM 1 and 300 pM PIM 3) and cellular setting (88 nM PIM 1 cell assay). When administered orally once daily at 10 and 100 mg/kg to CB17 SCID mice beginning 28 days after adoptive transfer of CD4 + CD45RBhi T cells (from Balb/c donors) and dosed for 21 days, AR00472159 significantly inhibited colon size (g/cm) by 68 and 69%, respectively. Colon pro-inflammatory cytokines IL-1b, IFNg and TNFa were also reduced with AR00472159 treatment. Furthermore, beneficial effects were seen by histopathology with up to 85% inhibition of mucosal thickening, 64% inhibition of colon inflammation and 65% inhibition of colon gland loss. These data support an important role of PIM kinases in a Th1/Th17 driven model of colitis The engineering of a latent cytokine using the latency associated peptide (LAP) of TGF-b1 fused via an MMP cleavage site to interferon (IFN)-b prolongs in vivo half-life and the MMP cleavage site permits IFN-b release specifically at a site of inflammation (1) . We aimed to assess whether small peptides, such as VIP, aMSH and c 3 MSH can be similarly delivered and modulate inflammation and arthritis. Latent forms of VIP, aMSH and c 3 MSH were generated by fusion to the LAP of TGF-b through an MMP cleavage site, purified, and ability to be hydrolyzed by MMP1 confirmed. The biological activity of LAP-c 3 MSH was compared to protein alone in vivo in murine monosodium urate crystal (MSU) peritonitis (16 h Gr1 + cell influx) and LAP-MMP (control), LAP-MMP-VIP or LAP-MMP-aMSH were delivered by gene delivery via encoding plasmid DNA i.m. in collagen-induced arthritis (CIA). The recruitment of MSU induced PMN (GR1+) infiltration was reduced by 35% with c 3 MSH at 1 nmol whereas administration of purified latent LAP-MMP-c 3 MSH at 0.03 nmol attenuated leucocyte influx by 50%. Gene delivery of plasmids coding LAP-MMP-VIP and LAP-MMP-aMSH at disease onset reduced the development of CIA compared to empty LAP-MMP. A significantly lower degree of paw inflammation, inflammatory cell influx, and the erosion of cartilage and bone was seen with LAP-MMP-VIP and LAP-MMP-aMSH. Collagen type II autoantibody and serum inflammatory cytokine production were also reduced in these two groups when compared to LAP-MMP. The short half lives of VIP and aMSH limit their therapeutic use. Incorporation of these small anti-inflammatory peptides into the LAP shell and delivered either locally or through gene therapy can control inflammatory and rheumatic disease. This concept can be developed to control human arthritides and other autoimmune diseases. Adams The Weizmann Institute of Science, Rehovot, Israel Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS, associated with complex anti-myelin autoimmunity. Among all approaches proposed for MS therapy, an approach that neutralizes only the pathogenic T cells reacting against myelin, while leaving the innocent immune cells intact, is the ultimate goal in the immune-specific therapy for MS. The multiplicity of primary target antigens, along side the dynamic nature of autoimmunity in MS, whereby the specificity of anti-myelin pathogenic autoreactivities may shift or expand in the same patient with disease progression, impose major difficulties in devising immune-specific therapy to MS. To overcome this multiplicity and the potential complexity of pathogenic autoreactivities in MS, we have put forward the concept of concomitant multi-antigen/multi-epitope targeting as, a conceivably more effective approach to immunotherapy of MS. We constructed an EAE/MS-related synthetic human Target Multiple sclerosis (MS) is an inflammatory, autoimmune disease characterized by massive demyelination associated with axonal damage and neuronal loss, typically accompanied with episodes of relapses/remissions. Both in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), the development of central nervous system (CNS) inflammation was shown to be associated with robust upregulation of the CXCL12 expressed in the CNS. However, the dynamic of CXCL12 at the different stages of EAE and in particular at recovery and indeed, its expression by cells of neural lineages under such circumstances remains unclear. We noted that while CXCL12 expression in the CNS is increased in correlation with the development of severe pathological and clinical EAE, interestingly, the level of CXCL12 remains high during spontaneous clinical recovery associated with reduction of CNS inflammation. We show that not only astrocytes, but also newly generated neuronal and oligodendrocyte progenitors express CXCL12 in the CNS of mice at the peak of EAE and during recovery. CXCL12 immunoreactivity in neuronal and oligodendrocyte progenitor cells was further verified on in vitro differentiated adult neural stem/progenitor cells (aNPCs). In vitro studies further show that addition of CXCL12 enhances the differentiation of aNPCs towards neuronal and oligodendrocyte progenitors. Moreover, the addition of AMD3100, a CXCR4 antagonist, markedly inhibited the effect of both the exogenous and endogenous CXCL12 in promoting the differentiation of neuronal and oligodendrocyte progenitors, and their mature counterparts. Our data highlight the postinflammatory steady expression of CXCL12 by neuronal and oligodendrocyte progenitors and demonstrate its involvement in differentiation/maturation of aNPCs, implicating a role for CXCL12 in neural repair. Disclosure of interest: None declared. IL-6 is implicated in the pathogenesis of various immunoinflammatory disorders of the CNS. IL-6 signals via binding to either the membrane bound IL-6Ra (classical signaling) or soluble (s)IL-6Ra (trans-signaling) that then form a complex with gp130 to activate the JAK/STAT and SHP2/MAPK pathways. Here we determined the relative importance of classical-versus trans-signaling in mediating IL-6 actions in the CNS. Biogenic mice were generated with CNS-restricted, astrocyte-targeted production of IL-6 and co-production of the specific inhibitor of IL-6 trans-signaling, sgp130-Fc (termed GFAP-IL6/sgp130 mice). Transgene-encoded IL-6 mRNA levels were similar in the brain of GFAP-IL6 and GFAP-IL6/sgp130 mice. However, GFAP-IL6/sgp130 mice had decreased pY-STAT3 in the brain due to a marked reduction in the total number of pY-STAT3-positive cells and the absence of detectable pY-STAT3 in specific cell types. These changes in pY-STAT3 were associated with a disproportionate reduction in the expression of various inflammatory markers as well as reduced vascular pathology and bloodbrain barrier leakage. Degenerative changes in the cerebellum characteristic of GFAP-IL6 mice were almost absent in GFAP-IL6/sgp130 mice. The findings suggest that in the CNS: (1) The medial prefrontal cortex (mPFC) and the basolateral nucleus of the amygdala (BLA) are anatomically and functionally interconnected cooperating for the integration of emotionally salient sensory input. Specifically, the unpleasantness and affective component of chronic pain are driven by prelimbic and infralimbic (PL/IL) divisions of the mPFC and amygdala reciprocal connections. These regions can be profoundly affected by persistent pain generated by peripheral nerve injury or chronic inflammation in rodents. We show in this study that nociceptive responding BLA ? mPFC pyramidal neurons undergo deactivation following carrageenan-induced inflammation together with downregulation of the endovanilloid/glutamate transmission (while GABAergic tone was enhanced) that can participate for impairing pain behaviour S44 Inflamm. Res. mediated by BLA-mPFC circuits. In particular, it has been found here that pyramidal cells are inhibited by intra-plantar injection of carrageenan. We show unprecedented evidence of a neural mechanism by which selective metabotropic glutamate receptor subtype 1 (mGluR1) up-regulation in the BLA may impair mPFC function in inflammatory pain conditions. These results appear opposite to those ones found in neuropathic pain conditions (excitatory effect on pyramidal neurons). Thus amygdala can lead to differential changes in functionally distinct subsets of mPFC neurons in inflammatory versus neuropathic pain. In conclusion, we believe that the present data will help the understanding of the cross-talk between amygdala and mPFC, and the role of mGluR1 in the BLA for the functional changes of amygdalo-cortical interactions within the framework of the pain-related neuronal affective mechanisms. Disclosure of interest: None declared. Necdin has been shown to promote neuronal survival, differentiation and restricts proliferation in hematopoietic stem cells. We tested the hypotheses that necdin is expressed in inflammatory cells and modulates inflammation in stroke. Necdin was specifically upregulated in cells derived from the ischemic hemispheres which was CD11b + sorted (60 min of middle cerebral artery occlusion). To corroborate, necdin + / Iba1 + double positive cells were observed and assumed the activated 'ameboid' morphology in brain sections. To test the role of necdin in growth arrest of inflammatory cells, we generated lentiviral particles (LVPs) to downregulate necdin at protein levels via RNA interference (RNAi). In concurrent with MCAO, LVPs were intra-arterially injected at the ipsilateral side. Cell counts show significantly higher amounts of BrdU + , Iba1 + , BrdU + /Iba1 + double positive and BrdU + /Iba1 + /LVP-EGFP + triple positive cells in the ischemic cortex in comparison to the control. Analysis of EGFP expressing bone marrow chimeric mice showed that necdin + cells were not derived from the peripheral macrophages invading the brain after MCAO. In summary, necdin is expressed in the hyperactivated subpopulation of microglia and restricts their proliferation. The aim of the present study was to evaluate the contribution of Peroxisome Proliferator-Activated receptors (PPARs) and Liver X receptor (LXR) in an experimental model of spinal cord injury (SCI) in mice. To this purpose we used: (1) a high affinity ligand for PPAR-c (15d-PGJ2); (2) a high affinity PPAR-b/d agonist (GW0742); and (3) a potent LXR receptor ligand (T0901317). Moreover, to evaluate the contribution of PPAR-a, we used mice with a targeted disruption of the PPAR-a gene (PPAR-aKO). Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. 15d-PGJ2, GW0742, and T0901317 treatments, 1 and 6 h after the SCI, significantly reduced: (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, (4) pro-inflammmaory cytokines expression, (5) NF-jB activation, (6) iNOS expression and (6) Tumor M2-PK is known to be present in high concentrations in tumor tissue, plasma and other body fluids. Plasma determination of Tumor M2-PK has been shown to discriminate between benign/premalignant and malignant conditions. Tumor M2-PK has also been reported to be increased in some inflammatory conditions. The present study explores the ability of Tumor M2-PK expression to differentiate malignancy from chronic inflammatory condition (GERD) and assess it as a noninvasive biomarker in early diagnosis of esophageal cancer. The Tumor M2-PK concentration was quantitatively measured in EDTA-plasma of 218 individuals by a sandwich ELISA method (Schebo Ò Tech, Germany The myeloid derived suppressor cell (Gr1 + CD11b + , MDSC) is a recently described protumorigenic cell type with various immunosuppressive and proangiogenic affects. 5-Fluorouracil (5-FU), a pyrimidine analog chemotherapeutic drug has been shown to reduce MDSC recruitment in vivo in a xenograft model of lymphoma and to selectively kill MDSC in vitro. We have previously shown that MDSC are upregulated and correlate with neoplastic changes in the colon in the interleukin-10 deficient mouse (IL-10 -/-). The aim of this study was to test whether targeting MDSC in vivo with 5-FU affects cancer development in vivo in the IL-10 -/model of colitis associated cancer. 6-week-old IL-10 -/mice were given 6 weekly intraperitoneal injections of 50 mg/Kg 5-FU or saline control (n = 5). Animals were sacrificed at 12 weeks and colons were harvested. Inflammatory changes, polyps and dysplasia scores were evaluated macroscopically and histologically using previously published scoring systems. Gr1 + CD11b + cells per unit area of mucosa (cells/10 6 mm 2 ) were determined via immunohistochemical analysis of colon sections with anti-mouse antibodies. Ischemia induce a hypoxia situation starting a sequence of events that are amplified after reperfusion leading to severe organ injury and dysfunction by production of reactive oxygen species, leukocyte infiltrate and protein extravasation. Inflammation has been considered the most important cause of injury in organs subjected to ischemia-reperfusion (I/R). The aim of this study was evaluate the endogenous mechanisms responsible for regulation of local and systemic inflammation after intestinal I/R in mice genetically selected for maximal (AIR max ) or minimal (AIR min ) local Acute Inflammatory Response. After 45 min of intestinal ischemia, mice were submitted to reperfusion (0, 1, 4 and 24 h). The extent of lung injury was evaluated by myeloperoxidase (MPO) activity and cellular infiltration in the lung parenchyma. Hypoxic genes and inflammatory cytokine expressions were evaluated in the lung by Real Time PCR. It was observed that AIR max I/R mice had a progressive neutrophilia starting after ischemia and peaking at 4 h of reperfusion (2.7 ± 0.3 9 10 6 cell/ml). On the other hand, AIR min I/R mice had a low infiltrate with equivalent levels compared to the control groups (1.2 ± 0.2 9 10 6 cell/ml). Corroborating these results the MPO activity was higher in the lungs of AIRmax I/R than AIRmin I/R mice (p \ 0.01). A high expression of Hif 1-a, Vhl, Il-1b and Il-6 was observed in AIRmax I/R mice. Conversely, AIR min I/R mice presented low expression of these genes. The positive correlation found in AIR mice between the inflammatory reactivity and hypoxic genes expression suggest that the genes responsible for regulating the acute inflammatory response, fixed by selective breeding, can modulate the expression of genes involved in situations of hypoxia, and consequently modify the lung injury intensity hypoxia-dependent. Tumor microenvironment components contribute to metastases of different carcinomas through TLR2-mediated stimulation of production of pro-tumorigenic cytokines by inflammatory myeloid cells. One of subsets within the inflammatory myeloid cells compartment is immature and includes myeloid-derived suppressor cells (MDSC), accumulating in tumor-bearing hosts in response to various inflammatory mediators. We supposed that TLR2 might become more readily available on the surface of MDSC in response to component of complement C5a, one of the most active inflammatory mediators, concentration of which is increased in a tumor microenvironment. In our experiments, C57BL/6.hMUC1 transgenic mice with growing subcutaneous pancreatic tumors PancO2.hMUC1 exhibited a significant accumulation of myeloid-derived suppressor cells (MDSC) with a phenotype CD11b + Gr1 + versus non-tumor-bearing control mice. The majority of MDSC ([70%) expressed inflammatory markers S100A9 and CD88 (C5a receptor). At a maximum dose of 100 ng/ml recombinant C5a increased the proportion of monocytic S100A9 high , TLR2 high cells within the CD11b + , Gr1 + MDSC more than twofold during 6-h incubation (P \ 0.05, three repeats with cells harvested from separate animals). Increased expression of TLR2 was associated with decreasing the expression of CD88 (presumably due to ligand-receptor internalization). We suggest that rapid C5a-induced increase in availability of TLR2 on the MDSC surface may promote metastazing through activation of MDSC known to tolerize anti-tumor T cells. Hypothetically, MDSC activation may be supported by TLR2 ligands hyaluronan and versican commonly overaccumulated within the tumor microenvironment. We also hypothesize that co-accumulation of TLR2 high MDSC and versican/hyaluronan ligands within the same compartments may serve as candidate biomarker pattern for propensity of a tumor to metastasize. Polycyclic Aromatic Hydrocarbons (PAHs), such as DMBA, induce a decrease in bone marrow cells (BMC) numbers and hematological alterations resulting in an immunosuppressive state. DMBA metabolism depends on the activation of the aryl hydrocarbon receptor (AhR). Mice genetically selected for high (AIR max ) or low (AIR min ) acute inflammatory response to s.c. injection of Biogel P100 presented a complete segregation of Ahr alleles endowed with low (Ahr d ) or high (Ahr b1 ) affinity to PAHs, respectively. Accordingly, AIR max are more resistant than AIR min to DMBA induced skin and lung carcinogenesis. We investigated the effect of DMBA treatment on BMC of AIR selected mice and its possible impact on acute inflammatory response. AIR max and AIR min mice were treated with a single i.p. dose of 50 mg/kg DMBA in olive oil. Flow cytometric analysis was used to determine hematopoietic stem cells (HSC) (Lin -/Sca-1 + /c-Kit + ) and neutrophils (Gr-1 hi / CD11b hi ). Proliferation index of BMC was determined in response to GM-CSF stimulus. Acute inflammation response was also evaluated after 24 h of Biogel P100 subcutaneous (s.c.) injection. DMBA treatment resulted in a significant (p \ 0.01) decrease in neutrophil population and increase of HSC on bone marrow in AIRmin mice only. Blast cells from DMBA-treated AIRmin presented displastic nucleus, which is one of the distinguishing features of preleukemia, and myeloid cells showed low proliferation capacity after in vitro GM-CSF stimulation. These effects on myeloid BMC reflect on an impaired cellular migration to the inflammatory site 24 h after Biogel injection. This investigation demonstrates that AIR max mice are protected and AIRmin are prone to acute bone marrow cytotoxic and presumable preleukemic effects of DMBA. The complete segregation of alleles at the Ahr locus found in AIR mice might contribute to their differential inflammatory responses and to the phenotypes of susceptibility and resistance of BMC to DMBA-induced effects. At birth, immune system switches from a predominant Th2 profile to a balanced Th1/Th2 profile. This switch may be modulated by food factors which may participate in epigenetic regulation of immune orientation key genes expression. This study aims at identifying the incidence of daily ingestion of iron by juvenile mice on the orientation of immune response following the induction of an experimental colitis or food allergy. Six groups of male juvenile Balb/c mice were treated during 6 weeks with vehicle or iron solution (150 mg/kg/day-100 ll/day po). In parallel, half of the animals were submitted to sham or ovalbumin sensitization (1 mg/ml-100 ll/day po) and challenge (100 mg/ml-100 ll po). In the other half, we induced a sham or experimental TNBS colitis (40 mg/kg in 50% Ethanol 1 week followup) after the 6 weeks of iron treatment. Spleen expression of T-bet and GATA3 were evaluated by RT PCR. Their epigenetic regulations were assayed by the analysis of cytosine methylation levels of their respective promoter sequence. In Th1 model, T-bet/GATA3 ratio increase is mainly linked to T-bet promoter gene hypomethylation and thus gene overexpression. The Th2 model revealed upregulation of GATA3/T-bet ratio consecutive to GATA3 expression increase and T-bet expression inhibition. However, no modification of promoter gene methylation profile was detected. Iron prevented T-bet/GATA3 ratio upregulation and modulation of T-bet promoter gene methylation in the Th1 model. On the other hand, it prevented GATA3/T-bet ratio increase in the Th2 model but could not modulate methylation S48 Inflamm. Res. profile. Chronic daily intake of iron in juvenile individuals interfere with T-bet and GATA3 gene expression reestablishing in both models the initial Th1/Th2 balance. In Th1 mediated immune response, it is linked to an epigenetic regulation of the promoter region methylation profile while the Th2 model revealed to be more complex and possibly, the resulting orientation will be associated to other epigenetic phenomenon. Hydrogen sulphide (H 2 S), a gaseous mediator synthesized in several mammalian tissues by two main enzymes CBS and CSE, increases under inflammatory conditions or sepsis. Since H 2 S and H 2 S-releasing molecules afford potent inhibitory properties on the process of leukocyte trafficking, we tested whether endogenous Annexin A1 (AnxA1) could display intermediary functions. Treatment of human PMNs the H 2 S donor NaHS (10-100 lM) provoked prompted and intense mobilization ([50%) of AnxA1 from the cytosolic pool to the cell surface, supporting the inhibitory effects of the gas in the flow chamber assay. Such in vitro actions could be translated in analyses of the inflamed microcirculation, where NaHS (100 lmol/kg s.c., -1 h) afforded marked inhibition of IL-1-induced cell adhesion and emigration in the mesenteric vessels of wild type, but not AnxA1 -/-, mice. Next, we investigated whether endogenous AnxA1 could modulate H 2 S synthesis, indicating existence of a positive circuit. To this end, a marked increase in CBS and/or CSE expression in a variety of tissues (aorta, kidney, spleen) tested from AnxA1 -/mice, as compared to wild type animals, was quantified by qPCR. Moreover, NaHS was able to counteract the increase in expression in iNOS and COX2 (fourfold and sevenfold reduction, respectively) upon LPSstimulation of bone marrow derived macrophages (BMDM), though it was totally inactive in cells prepared from AnxA1 -/mice. Taken together, these data strongly suggest-for the first time-the existence of a positive interlink between AnxA1 and the H 2 S pathway, providing a novel mechanistic explanation for the exquisite properties of H 2 S in the control of experimental inflammation. These finding may be relevant to innovative discovery programmes aiming at harnessing the biological properties of H 2 S. Supported by the Wellcome Trust (programme grant 086867/Z/08/Z) Disclosure of interest: None declared. Tissue infiltration of macrophages (Mph), critical for innate immunity, is also involved in pathologies, such as chronic inflammation and cancer. In vivo, Mphs migrate mostly in a constrained three-dimensional (3D) environment. We have recently shown that, depending on the extracellular matrix (ECM) architecture, human Mphs are able to migrate in 3D using two distinct migration modes: they use the protease-independent amoeboid migration mode (like other leukocytes) in porous ECM and the mesenchymal migration mode in non-porous ECM. In addition, we have identified the phagocyte specific tyrosine kinase Hck as an effector of Mph mesenchymal migration. Now we show that the mesenchymal migration of macrophages in ECM is matrix metalloproteinase (MMP)-independent, whereas it is MMPdependent inside tumour cell spheroids. ECM degradation is the result of several cellular processes: (1) formation of 3D podosomes with proteolytic activity, (2) matrix compaction, and (3) matrix internalization. By using composite matrices, we show that a single Mph is able to switch from the amoeboid to the mesenchymal migration mode and vice versa. To identify molecular determinants of MDM 3D migration, we have undertaken a transcriptomic analysis to compare gene expression profiles between 2D-and 3D-migrations and between 3D amoeboid versus 3D-mesenchymal migration. Analysis of transcriptomic signatures are currently under analysis. Characterization of Mph migration effectors will provide pharmacological targets to control deleterious Mph tissue infiltration. Disclosure of interest: None declared. Mice with a naturally occurring Csfm(op)/Csfm(op) (op/op) gene mutation lack functional macrophage-colony stimulating factor (M-CSF) and are deficient of M-CSF-derived macrophages. They are severely monocytopenic and their remaining M-CSF-independent macrophages were shown to differ in differentiation and distinct functions when compared with phenotypically normal mice of the same background. Macrophages from op/op mice are able to sufficiently perform effector functions in vitro, such as phagocytosis and nitric oxide (NO) production. There are quantitative differences when peritoneal elicited macrophages were tested. Those from op/op mice showed a stronger response than those from control mice with regard to NO or IL-12 release. Interestingly, these same characteristics have been described to B-1 cell derived phagocytes. In this study, the peritoneal cell subpopulations of op/op mice and their littermates were analyzed. The aim was to investigate whether op/op peritoneal ''macrophages'' were B-1 cell derived phagocytes. The deficiency of cfms gene in op/op mice did not affect peritoneal B-1 cell population. Further, these cells are able to differentiate into phagocytes under LPS stimulus. The origin of these phagocytes was related to B-1 cells considering the VH11 Ig gene rearrangement detected in these cells. It also could be postulate that B-1 cell differentiation into phagocyte is independent of M-CSF. We also speculate that the MCSF-independent macrophage described in literature could be derived from B-1 cells. Investigations are being performed to provide more definitive evidence for the existence of B-1 cell derived phagocytes in vivo. Proteinase activated receptor-2 (PAR 2 ) is a G protein-coupled receptor that is activated by proteolytic cleavage. Previously, PAR 2 activation has been shown to cause pain in the joint (Helyes et al. 2010 . Eur J Pain); however, the mechanisms responsible for this are unclear. To elucidate neuronal PAR 2 expression in joints, 10 ll of a 2% Fluoro-gold (FG) solution was injected into the knee joints of 4 naïve male Wistar rats to retrogradely label knee joint afferent neurones. Four days later, dorsal root ganglia (DRGs) from L3, L4 and L5 were removed, sectioned and stained with an anti-PAR 2 antibody. Cells positive for FG and for PAR 2 were counted and neuronal diameters measured. To measure joint nociception objectively, electrophysiological recordings were made from single unit afferent nerve fibres in the knee joints of male Wistar rats during both normal and noxious rotation of the joint. Afferent fibre firing rate was recorded during 10 s rotations made before and over a period of 15 min after close intra-arterial injection of the PAR 2 activating peptide, 2-furoyl-LIGRL-NH 2 (100 nmol/100 ll). Rats were either naïve (n = 20) or pretreated with the TRPV1 antagonist, SB366791 (500 lg/ kg, i.p. 30 min prior to nerve recordings; n = 6) It was observed that 59 ± 5% of the FG positive cells in the DRGs were also positive for PAR 2 , and PAR 2 was broadly expressed across all sizes of DRG neurones innervating the knee joint. 2-furoyl-LIGRL-NH 2 caused a significant increase in firing rate in joint afferent neurones during both normal and noxious rotation over a time period of 15 min. This effect could be blocked by pre-treatment with SB366791 (p \ 0.05 compared to 2-furoyl-LIGRL-NH 2 alone, using two way ANOVA). In conclusion, PAR 2 is expressed in joint sensory neurones and activation of PAR 2 causes sensitization of these afferents via a TRPV1-dependent mechanism. Disclosure of interest: None declared. Central nucleus of amygdala (CeA), ''nociceptive amygdala'', is a crucial area controlling the reciprocal interactions between chronic pain and its affective consequences. It is under glutamate-induced plasticity occurring after prolonged pain stimuli which leads to neuron hypersensitivity. CeA pain control pass through rostral ventromedial medulla (RVM) modulation. Metabotropic glutamate receptor subtype 7 and 8 (mGluR7 and mGluR8), which can be now selectively stimulated/inhibited with highly specific ligands, may ''gently'' affect glutamate transmission. AMN082 and (S)-3,4-DCPG, selective agonists for mGluR7 and mGluR8 receptors respectively, have been locally administered within CeA and behavioural and electrophysiological changes have been investigated in models of inflammatory chronic pain. mGluR7 and mGluR8 receptors play different, even opposite, actions on pain-control and induced changes in RVM. Moreover, mGluR7 shows to function only under normal conditions whereas mGluR8 requires a chronic pain-induced plasticity to play as analgesic. Having mGluR7 facilitated pain transmission, a selective mGluR7 antagonist, MPPIP has been tested. Inflamm. Res. It played an antinociceptive action, as expected. Painful sensation is a hallmark of the inflammatory response induced by pathogens or tissue damage. A large spectrum of molecules released within the inflamed tissue such as neuropeptides, prostaglandins or proteases induces pain by stimulating primary afferent neurons in situ. Activity of primary sensitive fibers can be counteracted by peripheral endogenous regulatory mechanisms involving local opioid release by leukocytes infiltrating the inflammatory site. Since pain is an inherent component of inflammation often accompanying immune response, we investigated the endogenous regulation of CFA-induced inflammatory pain in the context of adaptive T cell immune response. The nociceptive response to mechanical stimuli was studied using von Frey filaments in mice immunized with OVA in CFA. The nociceptive response of nude versus wild type mice was dramatically increased, demonstrating T cell deficiency associated with increased pain sensitivity. Based on adoptive transfer experiments of OVA-specific CD4 + T lymphocytes into nude mice, we show that antigen-specific activated but not resting T lymphocytes are responsible for the spontaneous relief of inflammation-induced pain following antigen challenge. The analgesia was dependent on opioid release by antigen-primed CD4 + T lymphocytes at the inflammatory site. Indeed, T cell-mediated analgesia was inhibited by local injection of an opioid receptor antagonist, unable to cross the blood-brain barrier. Notably, we found opioid precursor mRNA to be more than sevenfold increased in antigen-specific activated CD4 + T lymphocytes, as compared to resting T lymphocytes in vivo. Taken together, our results show that CD4 + T lymphocytes acquire anti-nociceptive effector properties when specifically primed by antigen and point out analgesia as a property linked to the effector phase of adaptive T cell response. Disclosure of interest: None declared. Objective: Recent findings based on basic and clinical research implicate important molecules and pathogenesis in rheumatoid arthritis (RA). However, there are still many unknown facts, due to the complexity of the disease and the numerous molecular players involved, and the integrated systems approach is needed to understand this disease. Methods: A systems approach was performed to characterize the RAassociated lists of pathways, diagnostic markers, and therapeutic targets. Twelve global gene expression datasets obtained from the joint tissues or cell lines of patients with RA and osteoarthritis were analyzed through data integration with orthogonal non-negative matrix factorization, functional enrichment, and network modeling process. Results: By the integrative analysis, we identified core RA-associated genes (RAGs). By mapping RAGs into functional pathways and diseases, we categorized key RA cellular processes, common pathological phenotypes, and their related diseases. Then, we reconstructed networks describing key RA processes and their inter-relationships based on their protein-protein and protein-DNA interaction. From these network models, we established cell type dominant signature, anti-TNF-alpha therapy perturbed domain, and putative regulatory mechanisms. Finally, we predicted essential molecules that can control or indicate such key processes. The list of potential markers and targets included 20 genes, which were selected based on the networks from RAGs identified from the integration of secretion data and cell/ tissue specificity data. Conclusion: We introduce lists of diagnostic markers and therapeutic targets identified using a systems approach. Also, suggested network models and panel of essential molecules can shed novel insights into RA pathogenesis and also serve as an important resource for discovery of therapeutic targets and diagnostic markers. Background: IL-23 is a pro-inflammatory cytokine known to be essential for the differentiation of the Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. Its subunit, IL-23 p19, is specific of this cytokine. We had previously demonstrated for IL-1beta and TNF-alpha, that active immunization against these cytokines could be protective in animal models of arthritis. Objectives: The aim of this study was to evaluate the effect of two vaccines targeting the IL-23 p19 subunit, IL23-K1 and IL23-K2, on collagen-induced-arthritis (CIA), a model of rheumatoid arthritis. Methods: Using bioinformatics, we defined two peptides in IL-23 p19. Each peptide was coupled with keyhole limpet hemocyanin (KLH). Anti-murine IL-23 immunization was performed by injecting intramuscularly IL23-K1 or IL23-K2 formulated in incomplete Freund adjuvant (IFA), four times (D0, 7, 28, 49) We identified the novel target kinase MAPKAPK5 as a mediator of matrix metalloprotease 1 (MMP1) production in synovial fibroblasts from rheumatoid arthritis (RA) patients (RASFs) [1] . GLPG0259 was selected as a clinical candidate out of a series of potent, small molecule MAPKAPK5 inhibitors. Here we characterize the effect of GLPG0259 on pro-inflammatory cytokine and MMP production in vitro and ex vivo and evaluate GLPG0259's effect on disease progression in a collagen-induced arthritis (CIA) mouse model of RA. GLPG0259 and reference kinase inhibitors were assessed in human RASF cultures and in an ex vivo assay by measuring IL-6 and MMPs in synovial biopsies from CIA rats. GLPG0259 was thoroughly tested in the mouse CIA model (clinical score, histology, immunohistochemistry, X-ray radiography and lCT and in vivo imaging of protease activity). GLPG0259 concentrationdependently inhibited the release of IL-6 and MMPs in RASFs and synovial tissue, to levels better than or comparable to inhibitors of p38, JAK and SYK kinases. In contrast to p38 inhibitors, GLPG0259 inhibited MMP3 expression in synovial tissue. GLPG0259 did not block phosphorylation of JNK, ERK and p38 MAP kinases, indicating cytokine/MMP release is mediated by a downstream kinase. In the mouse CIA model, GLPG0259 reduced paw inflammation and bone destruction at doses of 1 mg/kg and higher. At 30 mg/kg, a well tolerated dose, GLPG0259 completely blocked disease progression, similar to the TNFa blocker Enbrel Ò . Immunohistochemistry and in vivo imaging showed that GLPG0259 reduced the expression and activity of MMPs within affected joints. GLPG0259, an orally bioavailable inhibitor of MAPKAPK5, has anti-inflammatory and bone protecting activity similar to or better than other therapeutically relevant inhibitors, thereby showing potential as a first-in-class candidate drug for the treatment of RA. GLPG0259 is currently being evaluated in a Phase II trial in RA patients. [1] Objective: Our objective was to investigate putative epigenetic mechanisms regulating 5-LO expression in human mast cells (HMC-1), macrophages, neutrophils, and RA-affected synovial fibroblasts (RASF). Methods: 5-LO gene expression/activity in cultured cells were assessed by Western blotting, quantitative RT-PCR, and by lipid metabolite assays (ELISA). DNA/promoter methylation was analyzed by nested PCR/ genomic DNA bisulfite sequencing. Histone (e.g., H3) methylation and acetylation patterns at promoter, exonic, and intronic sequences of the 5-LO gene were quantitated by chromatin immunoprecipitation (ChIP) assays. RT2 miRNA PCR Arrays were used to screen for 5-LO mRNA 3 0 UTR specific miRNA expression under study conditions. Results: In untreated mast, macrophage and neutrophil-like cells, the degree of DNA CpG island methylation in the 5-LO gene proximal promoter correlated inversely with 5-LO expression. In contrast, RASF were 5-LO negative but both distal and proximal promoter regions were hypomethylated; ChIP analysis resolved this inconsistency when it was revealed that histone H3 is hypermethylated/ hypoacetylated at Lys-9 and -27 residues in all promoter, exonic, and intronic sequences tested, favouring promoter silencing. Dexamethasone (DEX) treatment of HMC-1 cells increased basal expression levels of 5-LO, a process associated with reduced methylation but increased acetylation of histone H3 on lysines 9 and 27. These results were confirmed with cell-permeable histone deacetylase and methyltransferase inhibitors. RT2 miRNA PCR arrays revealed little correlation between the levels of 5-LO mRNA and protein expression and 32 miRNAs in RASF and HMC-1 mast cells. Conclusion: DNA/histone modifications represent mechanisms governing both basal and dysregulated 5-LO gene expression. However, epigenetic mechanisms function in a cell specific manner making disease control targeting DNA/histone modification enzymes with specific pharmacotherapy challenging. Disclosure of interest: None declared. Inflamm. Res. The systemic inflammatory response induced by sepsis is the major cause of acute lung injury (ALI). Our previous work showed that in ALI induced by instillation of LPS, the NFkB nuclear translocation and cytokines production was reduced in diabetics. In the present study we investigated if the same occurs in ALI secondary to sepsis. Methods: Diabetes was induced in male Wistar rats by alloxan and sepsis by cecal ligation and puncture surgery (CLP). After 6 h, bronchoalveolar lavage (BAL) was performed to measure cytokines by ELISA. Lung tissue was prepared for evaluation of edema, cell infiltration (HE staining), Transforming growth factor (TGF) b, asmooth muscle actin (SMA) (immunohistochemistry), COX-2 (western blot) and for MyD88 and SOCS1 mRNA (RT-PCR). Results: Morphological analysis of the lung after 6 h of CLP showed edema and mononuclear cell infiltrate in the BAL and parenchyma. Remodeling was evaluated by the increased number of cells expressing TGF-b and a-SMA. These parameters were lower in diabetics. Comparing the CLP with the sham operated group we found that non-diabetics exhibited an anti inflammatory profile (high IL10, TGFb and COX-2 and low IL-1b and TNFa). In diabetics this polarization was not observed and the concentration of mediators was much lower than in non-diabetics. Alveolar macrophages from diabetics with sepsis compared to non diabetics show lower expression of MyD88 mRNA and overexpression of SOCs1 mRNA. Conclusion: The initial phase of ALI induced by CLP is characterized by edema, mononuclear cell infiltrate and signs of early remodeling. Concomitantly, the lung exhibited an anti inflammatory profile. These characteristics were less intense in diabetics. The impaired expression of MyD88 in macrophages from diabetics with sepsis can explain the milder lung inflammation observed in diabetics. Financial support: FAPESP and CNPq Disclosure of interest: None declared. Increased production of NO and prostaglandins associated with a decrease in the formation of a vasoconstrictor arachidonic acid product, 20-HETE, contributes to endotoxin (ET)-induced hypotension, vascular hyporeactivity, multiple organ failure, and high mortality rate. Therefore, we hypothesized that use of stable analogues of 20-HETE may provide an alternative strategy for the treatment of septic shock. To test this hypothesis, we investigated the effects of a synthetic analog of ,14(Z)-dienoyl]glycine (5, , on the ET-induced changes in iNOS-hsp90 complex formation, PKG and NADPH oxidase activity, peroxynitrite production, and COX-1/COX-2 and CYP4A1 protein expression in rats and mortality in mice. Blood pressure fell by 28 mmHg and heart rate rose by 47 beats/min in male Wistar rats treated with sublethal dose of ET (10 mg/kg, i.p.). ET-induced fall in blood pressure was associated with an increase in iNOS-hsp90 association, phosphorylated VASP, gp91 phox , p47 phox , nitrotyrosine and COX-2 protein expression, a decrease in COX-1 and CYP4A1 protein expression in heart, thoracic aorta, kidney and superior mesenteric artery, and elevated serum nitrotyrosine levels. All of the ET-induced changes, except for COX-1 protein expression, were prevented by 5,14-HEDGE given 1 h after ET injection. Treatment of male Balb/c mice with 5,14-HEDGE 1 h after injection of ET raised the survival rate from 84 to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, prevented the effects of 5,14-HEDGE on the ET-induced changes, except for iNOS-hsp90 association. These data suggest that 5,14-HEDGE reverses hypotension and improves survival during endotoxemia, and may be regarded as a potential therapeutic agent in the treatment of septic shock. Systemic infections leading to sepsis and septic shock are a major cause of mortality in intensive health care units. Gram-negative sepsis is accompanied by an overactivated innate immune response as well as excessive coagulation mainly induced by endoxins released from the bacteria. Due to rising antibiotic resistance and current lack of effective treatments there is an urgent need for new therapies. The aim of the present study was to explore the immunomodulatory effects and therapeutic potential of the thrombin-derived C-terminal peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) and the related HVF18 (HVFRLKKWIQKVIDQFGE) in regard to endotoxinmediated shock and Pseudomonas aeruginosa sepsis. Both peptides abrogate endotoxin effects in vitro and in vivo. In human blood they interfere with coagulation by modulating tissue factor-mediated clotting. Furthermore, in a mouse model of Pseudomonas aeruginosa sepsis, treatment with GKY25 leads to reduction of the inflammatory response, as well as decreased organ damage and reduced mortality. Taken together, the ability of these peptides to modulate both inflammation as well as coagulation, makes them attractive therapeutic candidates in the development of novel treatments for sepsis. Anesthesiology and Intensive Care Unit, Oita University, Oita, Japan Background: Autophagy is the process cells recycle their own nonessential, redundant, or damaged organelles and macromolecular components. It is an adaptive response to sublethal stress, such as nutrient deprivation, that supplies the cell with metabolites it can use for fuel. The purpose of this study was to determine whether unbridled autophagy is related to sepsis induced organ dysfunction, and autophagy induced organ dysfunction can be compensated by nutritional intervention in mice cecal ligation and puncture model. Methods: All experiments were conducted in accordance with the Institutional Care and Use Committee. Male C57BL/6 mice (20-23 g) were randomly allocated into four groups: Group C (sham operated), and Group S (food restriction for 12 h), Group CLP (cecal ligation and puncuture model), and Group CLP + 5%G (cecal ligation and puncture model treated with 5% glucose subdermal injection). Electron microscopic examination of liver were done for autophagosome and autolysosome detection. Immunoblotting of liver samples were done for detection of LC3. Blood samples were also examined such as ALT, AST, blood glucose, NEFA, and HMGB1. Statistical differences were determined by ANOVA and repeated measured ANOVA, followed by Dennett's test. Result: AST, ALT and HMGB1 levels were highest in the Group CLP among the other groups; however, the Group CLP + 5%G was significantly lower than the Group CLP. On the other hand, autophagic levels detected by microscope and Immunoblotting was significantly increased in both Group S and Group CLP than other groups. Conclusion: The results of our study show that autophagy was induced with cecal ligation and puncture model like as food restriction, and excessive autophagy may cause sepsis induced liver damage. 5% glucose administration can restore excess autophagy and liver damage. Nutritional intervention may improve autophagy associated organ dysfunction in several critical states. Disclosure of interest: None declared. Carbohydrate-containing molecules are distributed on external layers of all living organisms, being involved in many functions related to induction of the immune system. This work was focused on the effect of galactofuranosyl epitopes (carbohydrates usually observed in pathogenic microorganisms) on the phagocytic activity of peritoneal macrophages and also on the survival rate of mice with polymicrobial sepsis induced by cecal ligation and puncture (CLP). To synthesize galactofuranoside derivatives, galactose was converted to methyl and n-Octyl band agalactofuranosides (GFB-Me, GFA-Me, GFB-O and GFA-O). The methyl galactofuranosides were converted to 5,6-O-isopropylidene derivatives . NMR and ESI-MS analysis confirmed the authenticity of all derivatives. GFB-Me and GFB-O had an immune stimulant effect at 5 lg/mL on macrophage phagocytic activity, with an enhancement of 35.12 and 11.90% respectively, but GFA-Me and GFA-O gave a low response. GFA-5,6-IPP and GFB-5,6-IPP had negative values relative to the control group -4.17 and -34.22%, respectively. These results showed that the free HO-5 and HO-6 and b-configuration are essential for the galactofuranosyl units to have phagocytic activity, which also stimulated peroxide, superoxide anion and nitric oxide production. GFB-Me and GFB-Me-5,6-IPP demonstrated the most significant effects on isolated macrophages and they have been selected to in vivo tests on septic mice. GFB-Me and GFB-Me-5,6-IPP increased significantly the mortality rate of septic mice. However, GFB-Me started killing earlier than GFB-Me-5,6-IPP. The treatment with GFB-Me elevated significantly the levels of all cytokines evaluated (TNF-a, IL-1b, IL-6 and IL-10). Both molecules were able to exacerbate mortality by sepsis in mice by a mechanism that depends on an increased levels of cytokines. These results reinforce the relevance of galactofuranosyl epitopes as components of pathogens and its virulence. Disclosure of interest: None declared. Autoimmunity, connective tissue diseases (OC06) Interestingly and in contrast to the IL-23p19 knockout mice, the IL-17RA deficient showed a Th2-like phenotype in splenic CD4 + T cells at day 69. No difference was noted for FoxP3 expression in the splenic CD4 + T cells between the three different mouse groups. Moreover, the CII-specific IgG2a levels in the sera of IL-17RA-/was significantly lower compared to the control group at day 20 and lower although not statistically significant at day 69. At this latter time point, CII-specific IgG1 levels in the sera of IL-17RA-/-was increased although not statistically significant compared to the control. In conclusion, these data revealed a critical role for the IL-17RA signaling in the development of autoimmune inflammation of the joint. Moreover, these data show a Th2-like phenotype in IL-17RA-/-mice immunized with CII, suggesting that IL-17 receptor signaling is involved in the suppression of Th2 cytokines in autoimmune collagen arthritis. T regulatory (Treg) cells are specialized T lymphocyte subsets playing a role in sustaining peripheral tolerance. Treg cells produce a number of cytokines, such as IL-10 and TGF-b, and can be characterized by various markers including FOXP3, CD25 and cytotoxic T lymphocyte antigen-4 (CTLA-4). Moreover, glucocorticoid-induced TNF receptor family-related (GITR) has been indicated as a molecule involved in Treg cell activity regulation in mice (Nocentini et al. 2007 ). Aim of our study was to investigate GITR expression in human CD4 + T lymphocytes and its possible role in Treg cell function. Results indicate that a subset of CD4 + T cells, characterized by GITR expression and low levels of CD25 expression (CD4 + CD25 low-GITR + ), is present in peripheral blood representing a low percentage of the entire CD4 + T cell population ( It is generally assumed that autoimmune inner ear disease (AIED) is caused by cochlear inflammation and glucocorticoids restore hearing by immunosuppression. However, little attention has been given to the susceptibility of cochlear ion and water transport pathways in AIED and steroid control over their recovery. Therefore, MRL/lpr autoimmune disease mice were evaluated to determine how systemic disease and glucocorticoid treatment affect the ion homeostasis mechanisms in the ear. Mice were tested for hearing at either 2 months of age (prior to disease, N = 8) or 6 months of age (after hearing loss onset, N = 7). Additional 6 month old mice (N = 8) were given oral prednisolone for 2 weeks. Inner ears from all were collected for qRT-PCR of 24 genes responsible for endolymph homeostasis, including channels for transport of sodium, potassium, chloride, and water, as well as tight junctions and gap junctions. Mice developed hearing loss at 6 months and 22 of the 24 cochlear ion and water transport genes were significantly downregulated. Inflammatory cytokine genes were actually suppressed in the ear, not elevated. Steroid treatment significantly increased expression of several homeostasis genes, including the potassium channel Kcne1, the epithelial sodium channel, several gap junction proteins (Gja1, Gjb2, Gjb6), and aquaporin 3, all of which are involved in K + transport and endolymph production. Steroid treatment had no significant effect on inflammatory gene expression in the cochlea. It was concluded that circulating autoantibodies and immune complexes may cause hearing loss by disrupting the blood labyrinth barrier and genes required for endolymph homeostasis. Steroid treatment appeared to improve hearing by restoring these homeostatic genes without impacting inflammation. This provides new insight into one potential mechanism of AIED and how glucocorticoid therapies may help to restore collagen synthesis whereas IL-13 promotes fibrosis by stimulating TGF-b production. The aim of the present study was to determine the effect of TGF-b on IL-13 expression by peripheral blood lymphocytes (PBLs) from SSc patients (n = 8) compared to healthy donors (n = 10). Jurkat cell line was used as a Th2 model. Cells were treated or not with TGF-b 5 ng/ml for 2-48 h in the presence or not of inhibitors for Smad and MAPkinase pathways. Quantification of IL-13 mRNA levels and mRNA stability were studied by real-time PCR. Jurkat cells were also transiently transfected with IL-13 reporter gene. Smad and MAPkinase pathways as well as GATA-3 expression were assessed by immunoblot. GATA-3 nuclear transactivation was analyzed by electrophoretic mobility shift assay and ChIP assays. Our results showed that TGF-b inhibited IL-13 mRNA steady state levels by 20-40% without affecting mRNA stability in Jurkat cells and PBLs form healthy donors. Inhibition of IL-13 production involved both Smad-and p38-dependent pathways and repression of GATA-3 transcription factor binding capacity to IL-13 promoter. In contrast to the inhibition detected in Jurkat cells and healthy PBLs, TGF-b increased IL-13 mRNA and protein expression by 35-43% in PBLs from SSc patients as compared to PBLs from healthy donors. This study demonstrates that TGF-b downregulates IL-13 production via GATA-3 as a consequence of signaling through both Smad and p38 pathways in physiological situation whereas it becomes a potent activator in SSc pathological situation. Inhibition of GATA-3 expression or its IL-13 promoter-DNA binding ability might provide a new approach for SSc treatment. Even though there are clinical trials assessing granulocyte-macrophage colony-stimulating factor (GM-CSF) blockade in rheumatoid arthritis (RA), questions remain as to how GM-CSF acts as a proinflammatory cytokine. For the regulation of arthritis progression by GM-CSF the aims of this study were to determine (1) whether there are systemic effects, (2) the changes in synovial tissue leukocyte populations and (3) the arthritis model dependence on GM-CSF. The collagen-induced arthritis (CIA) and K/BxN serum transfer arthritis models were tested in GM-CSF-/-mice and/or wild-type mice using an anti-GM-CSF mAb. Cell populations were assessed by differential staining and flow cytometry. GM-CSF blockade, while ameliorating CIA development, was found to have systemic effects, limiting the increase in circulating Ly6C hi monocytes and neutrophils. GM-CSF blockade also led to fewer synovial macrophages (both Ly6C hi and Ly6C lo ), neutrophils and lymphocytes. In the absence of GM-CSF, K/BxN serum transfer arthritis initially developed normally; however, the numbers of Ly6C hi monocytes and synovial macrophages (both Ly6C hi and Ly6C lo ) were again reduced, along with the peak disease severity and maintenance. Thus GM-CSF is a key player in two arthritis models, participating in interactions between hemopoietic cells, locally and even systemically, to control myeloid cell number as well as presumably to ''activate'' them. These results could be useful for the analysis of the current clinical trials targeting GM-CSF in RA. Disclosure of interest: None declared. Fac. Medicine, Dept. Pediatrics, Div. Immunology, Universite´De Sherbrooke, Sherbrooke, 2 Immunology, Univ. Toronto, Toronto, Canada The cellular and molecular events preceding the initial activation of autoreactive CD8 T cells in several autoimmune diseases remain unclear. Recent reports have implicated IL-6, IL-21 and IL-7 in the triggering of autoreactive CD8 T cells in mouse models of autoimmune diseases. We have shown that IL-21 and IL-6 synergize with IL-7 or IL-15 to induce antigen-independent proliferation of naive CD8 T cells, with a concomitant increase in antigen responsiveness. We postulated that the cytokine-induced augmentation of TCR sensitivity (referred herein as 'cytokine priming') might enable stimulation of potentially autoreactive CD8 T cells by low affinity self antigens. Here, we show that cytokine priming enables naive TCR transgenic CD8 T cells to respond to low affinity TCR ligands, resulting in proliferation and display of effector functions. We have used a transgenic mouse model of autoimmune type 1 diabetes to show that cytokine-primed autoreactive CD8 T cells acquire the capacity to cause disease following stimulation with weak TCR agonists, and that the diabetogenic potential of these cells is dependent on the continuous availability of IL-15. These findings demonstrate a novel mechanism by which inflammatory cytokines could contribute to the triggering of autoreactive CD8 T cells and thus have important implications for autoimmune diseases associated with microbial infections and chronic inflammation. Disclosure of interest: None declared. Monocytes can be subdivided into discrete populations using surface markers, and in humans these fall into two subsets, the CD14 + CD16 -(*90%) and CD14 + CD16 + (*10%) cells. We have previously demonstrated that in co-culture a mixed population of monocytes can activate endothelial cells, leading to the recruitment of flowing neutrophils. Here we extend this work to study the inflammatory activity of purified subsets. CD14 + CD16and CD14 + CD16 + monocytes were cultured on 0.4 lM pore inserts with endothelial cells for 24 h. The ability of the endothelial cells to recruit neutrophils was then tested in a flow based adhesion assay. In some experiments, monocyte conditioned medium, anti-IL-6 antibody or human recombinant IL-6 were added to cocultures. Cytokine secretion in cocultures was measured by luminex (Millipore). Mixed monocytes and the CD14 + CD16population both induced endothelial cell activation resulting in moderate levels of neutrophil recruitment from flow, while CD14 + CD16 + monocytes induced significantly higher levels of neutrophil recruitment. Mixed monocytes and CD14 + CD16both generated high levels of IL-6 in co-culture, whereas CD14 + CD16 + generated significantly less IL-6. Addition of IL-6 rich supernatant from CD14 + CD16coculture to CD14 + CD16 + cocultures significantly reduced the neutrophil recruitment driven by CD14 + CD16 + cells. Addition of recombinant IL-6 to CD14+ CD16+ cocultures also reduced the level of neutrophil recruitment. These observations suggest that IL-6 plays a role in crosstalk between monocyte subsets and is capable of modulating the inflammatory response of endothelial cells. Compromised epithelial barrier function and tight junction alterations are hallmarks of Inflammatory Bowel Disease (IBD). Several studies have identified a role for proinflammatory cytokines such as TNFa and INFc in epithelial barrier defects observed in the context of IBD. Interleukin (IL)-18 is the latest member of the IL-1 family of cytokines to be identified. Elevated levels of IL-18 have been reported in mucosal tissue from IBD patients. Furthermore, neutralization of IL-18 was shown to reduce disease severity in several murine models of colitis, suggesting a role for this cytokine in the pathogenesis of IBD. Nevertheless, the role of IL-18 in intestinal epithelial barrier function remains unclear. The major aims of our study were to determine the effect of IL-18 on epithelial tight junction structure and function, and to investigate the mechanism involved in this process. Colonic epithelial Caco-2 monolayers were challenged with IL-18 (50 ng/mL) for 24 h and samples were processed for immunocytochemistry, immunoblotting, and permeability measurements. Our findings demonstrated that treatment with IL-18 selectively disrupts tight junctional occludin, without affecting the distribution of claudin-4 or -5. This effect is accompanied by a significant increase in the expression and activity level of myosin light chain kinase (MLCK Lnk (SH2B3) belongs to a family of SH2-containing proteins and is a signaling adaptor molecule that plays regulatory functions in the homeostasis and proliferation of immune cells. We previously showed that Lnk is expressed in endothelial cells (EC) and phosphorylated in response to TNF (1, 2) . We further demonstrated that, in EC, Lnk is a negative regulator of TNF signaling that inhibits E-selectin and VCAM-1 expression in response to TNF (3) . Mechanistically, Lnk activates PI3kinase through Akt phosphorylation. Sustained Lnk-mediated activation of PI3-kinase in TNFa-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, while phosphorylation of p38 and JNK MAPKs was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFa-treated ECs. In the present study we show that integrin ligation, using anti-CD29 Abs, also promotes Lnk activation and regulation in EC whereas integrin-mediated signaling (Akt and GSK3b phosphorylation) consecutive to CD29 ligation is abrogated in EC when Lnk is not expressed. We found that Lnk binds to ILK and activates FAK and paxillin. Overall, Lnk causes F-actin stress fibers, cytoskeleton reorganization and increases both the number and density of focal adhesions. Functionally, Lnk expression dramatically increases EC adhesion, slows down EC migration and inhibits apoptosis induced by anoïkis. Transfection of mutants and SiRNA identified alpha-parvin as a molecular partner of Lnk mediating the negative regulation of EC migration. Thus, the adaptor Lnk is an effective key regulator of inflammatory-and integrin-signaling controlling EC activation, migration and death, all critical in vascular remodeling and regeneration. Our current work in animal models also proposes to modulate Lnk in graft EC to provide a cytoprotective signal in organ transplantation. (1) The accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that BiP, an ER chaperone, was highly expressed in RA synovium and synoviocytes. The proinflammatory cytokines, such as TNF-a and IL-1b, but not IL-10, increased the BiP expression in synoviocytes. Thapsigargin and tunicamycin, ER stressors, increased the BiP and Bcl-2 expression in synoviocytes, which was greater in RA synoviocytes than in OA synoviocytes. Downregulation of BiP transcripts using siRNA increased apoptosis of RA synoviocytes, while abolishing the TNF-a-and TGF-b-induced synoviocyte proliferation and cyclin D1 up-regulation. Moreover, BiP siRNA inhibited VEGF 165 -stimulated proliferation, capillary tube formation, wounding migration, chemotaxis of endothelial cells in vitro. Downregulation of BiP expression in RA synoviocytes that engrafted into immunodeficient mice also significantly reduced synoviocyte proliferation and angiogenesis. BiP siRNA also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants that engrafted into immunodeficient mice. Conversely, a selective BiP-inducer, BIX, prevented synoviocyte apoptosis induced by thapsigargin, and increased synoviocyte proliferation in mice with experimental osteoarthritis when injected intra-articularly. In summary, BiP is crucial to Phospholipase Ce (PLCe) is an effector of Ras and Rap small GTPases. We have for the first time shown by using PLCe-deficient mice that PLCe plays a critical role in the induction of cytokine production in non-immune cells in a variety of inflammatory reactions, such as those involved in tumor promotion. The role of PLCe in tumorigenesis is suggested also in humans by recent genome-wide studies that have identified SNPs in the PLCe gene (PLCE1) as a risk factor for gastric adenocarcinoma and esophageal squamous cell carcinoma. Here, to further investigate its role in inflammation, we create transgenic mice overexpressing PLCe in epidermal keratinocytes. The transgenic mice spontaneously develop chronic skin inflammation sharing the features of human psoriasis, such as adherent silvery scale formation and aberrant infiltration of IL-22-producing CD4 + T cells. The keratinocytes in the PLCe transgenic mice highly produce IL-23, which the skin symptom development depends on as demonstrated by the reversion of the skin phenotype upon its neutralization. Importantly, overproduction of IL-23 is induced even in wild-type keratinocytes in vitro by stimulating with the conditioned medium from those overexpressing PLCe. These results suggest that increased IL-23 expression in the PLCe transgenic mouse skin is mediated by certain autocrine/paracrine factors secreted by PLCe-overexpressing keratinocytes. Disclosure of interest: None declared. OC-077 TLR5 ACTIVATION/IL-1B SIGNALING: A NEW PATHWAY FOR ALVEOLAR MACROPHAGE PHAGOCYTOSIS AND BACTERIAL KILLING OF PSEUDOMONAS AERUGINOSA chronic lung infections. Although Toll-like receptor (TLR) 4 and TLR5 are respectively implicated in the recognition of LPS and flagellin of P.a and in the control of lung infections, very little is known about their roles in the early activation of alveolar macrophages (AMs). Here, we show that flagellin/flagellum-TLR5 interaction is essential to induce P.a clearance by AMs. In contrast to WT PAK (a wild-type P.a strain), unflagelled PAK strain or a strain expressing a flagellin mutated into TLR5-recognition site were not killed by either MH-S cells or primary AMs. We showed reduced uptake of unflagelled or flagellin-mutated PAK in MH-S cells and primary AMs, when compared to WT PAK. Furthermore, primary AMs from TLR5-/-mice were unable to kill PAK, when compared to primary AMs from WT mice. A deficit in PAK phagocytosis by primary TLR5-/-AMs was also observed. The importance of recognition by TLR5 for AM-mediated clearance of PAK is recapitulated in vivo in mice. We showed that TLR5-mediated PAK recognition and uptake by AMs were associated with high IL-1b synthesis, which was reduced with PAK mutants or with primary TLR5-/-AMs. We demonstrated that IL-1b release was associated with the ability to kill PAK since adding recombinant IL-1RA inhibited significantly the early clearance of PAK by AMs. In conclusion, we demonstrated for the first time that TLR5 plays a major role in the early clearance of P.a by AMs and that the flagellum-TLR5 interaction is required for effective phagocytosis of P.a and for the induction of IL-1b synthesis. Our data described a role of IL-1b in the control of P.a killing by AMs, a mechanism that we are currently further dissecting. Disclosure of interest: None declared. Lipid mediators and metabolic diseases (OC08) Background: We recently reported that endocannabinoid 2-arachidonoyl-glycerol (2-AG) activates human neutrophils independently of CB receptor activation. We found the effects of 2-AG were mediated by an autocrine activation loop involving arachidonic acid (AA) and its subsequent metabolism to leukotriene (LT) B 4 . In the present study, we tested whether 2-AG and AA could also stimulate neutrophil functions linked to host defense, more specifically the release of microbicidal peptides as well as their bacterial and virucidal activities. Results: We initially tested if AA and 2-AG were triggering the release of the microbicidal peptides a-defensins and LL-37. We found that both 2-AG and AA stimulated the release of a large amount (lg) of a-defensins as well as a limited amount (ng) of LL-37. This release occurred rapidly and paralleled the biosynthesis of LTB 4 . Importantly, the supernatants of neutrophils activated with nanomolar concentration of AA, 2-AG or LTB 4 almost completely inhibited the infectivity of herpes simplex virus-1 (HSV-1). Additionally, the supernatants of 2-AG-and AA-stimulated neutrophils strongly attenuated the growth Escherichia coli and Staphylococcus aureus. Importantly, all the effects of AA and 2-AG mentioned above were Introduction: Toll-like receptors (TLRs) are pattern recognition receptors which recognize molecules in microorganisms. Leukotrienes (LTs), lipid mediators derived from 5-lipoxygenase (5-LO) pathway, has been recognized as modulators of FcgR-dependent phagocytosis. Taking this information into account, our aim was to evaluate the participation of TLRs ligands on FcgR-mediated phagocytosis. Methods: Rat alveolar macrophages (AMs) or mouse (SV129 or 5-LO -/-) peritoneal macrophages (PMs) were treated with TLR ligands and challenged with IgG-sRBCs. After 90 min, phagocytosis was evaluated by a colorimetric assay. Lipid mediators (LTB 4 and cys LTs) were measured by EIA. Western blotting was also used. Results: Except for CpG, all TLR agonists tested (TLR2, TLR3, and TLR4) were able to amplify phagocytosis of IgG-sRBC by AMs in a concentration-dependent manner. Phagocytosis amplification induced by TLR3 ligand was a time-dependent, fact not observed for TLR2 and TLR4 ligands. AMs treatment with zileuton also impaired TLR4 ligand-induced phagocytosis amplification, suggesting the participation of 5-LO products in this phenomenon. In order to confirm LTs involvement in TLRsinduced FcgR-mediated phagocytosis amplification, we observed that PMs obtained from 5-LO -/mice did not presented any difference on phagocytosis when stimulated with TLR2, 3, and 4 ligands. LTB 4 production induced by IgG engagement was amplified by TLR2 and TLR4 ligands, but not by TLR3. Similar results were obtained when cys-LTs were quantified, whose production was increased by TLR2, TLR3, and TLR4. ERK1/2 and p38, mitogen-activated proteins kinases ( Secreted phospholipases A2 (sPLA2) are a family of enzymes which catalyze the hydrolysis of glycerophospholipids liberating lysophospholipids and fatty acids. Among sPLA2 s, the human group X (hGX)-sPLA2 has the highest catalytic activity towards phosphatidylcholine (PC) the major phospholipid of cell membranes and blood lipoproteins. Increased sPLA2 activity has been documented in several inflammatory disorders and the expression of the sPLA2 groups IIA, III, V and X has been detected in human atherosclerotic lesions, indicating that sPLA2s are an important link between lipids and inflammation, both involved in atherosclerosis. This pathology linked to chronic inflammation of the arterial wall is characterized by the accumulation of Low-Density Lipoproteins (LDL) and foamy cells within the subendothelial space. Recently the presence of dentritic cells (DC), the most potent antigen presenting cells in atherosclerotic lesions raised the question about their role in disease progression. We have previously shown that modified lipoproteins can modulate DC function, interfering with their ability to stimulate T cells. In this study, we show that hGX-sPLA2-treated LDL induced DC maturation resulting in a characteristic mature DC phenotype and in enhanced DC ability to activate IFNc secretion from T cells. hGX-sPLA2 phospholipolysis of LDL produced high levels of lipid mediators, such as lysophosphatidylcholine and free fatty acids, which also modulated DC maturation. Our results indicate that hGX-sPLA2 can induce DC maturation, both directly by hydrolyzing PC from cell membranes and indirectly by LDL phospholipolysis, though resulting in different cytokine secretion profiles, pointing to differential modulation of DC function. In conclusion, the secretion of hGX-sPLA2 in inflamed tissues may contribute to the local activation of DC through the production of lipid mediators from LDL and/or cell plasma membranes. Introduction: Obesity, a chronic inflammatory status, is associated with a cellular remodeling of white adipose tissue (WAT) with the presence of inflammatory cells [1] . Visceral WAT which hypertrophy is associated with metabolic complications, is more vascularized and inflammatory than subcutaneous WAT [2] . We hypothesized that endothelial cells from the visceral WAT through their inflammatory secretions could alter adipocyte biological functions. Methods: To reproduce tissue architecture, we developed a new experimental 3D model where human mature adipocytes are included in a peptidic hydrogel. In this 3D gel, the adipocytes maintained their metabolic functions including adipokine secretion (leptin and adiponectin) for 1 week. We performed co-culture of these cells both isolated from visceral WAT of obese subjects in our 3D experimental design. Results: Endothelial cells provoke a decrease of adipokine secretions (-50% for leptin and -20% for adiponectin, p \ 0.01). An increased secretion of inflammatory cytokines including IL-6, CXCL-8, CCL-5 and G-CSF was observed in co-culture adipocytes/endothelial cells. By studying the secretory profile of angiogenesis-related molecules, we also highlighted the increased secretion of IGFBP-3 in these experimental conditions. Conclusion: Increased secretion of cytokines and chemokines as IL-6 and CCL-5, with conversely decreased secretion of the anti-inflammatory adiponectin could perpetuate a vicious cycle in hypertrophied and inflammatory WAT. Inflammation and angiogenesis being interrelated processes, G-CSF, CXCL-8 and IGFBP-3, all involved in endothelial precursor recruitment, could promote neovascularization contributing to tissue repair. Inflammation is frequently associated with metabolic syndrome (MetS). Analytes such as adiponectin, C-peptide, CRP, cystatin C, ferritin, IL-1a, IL-6, insulin, leptin, PAI-1, resistin, TNFa, take part in physiological processes such as insulin secretion, glucose regulation, fatty acid metabolism, inflammation, iron homeostasis, energy homeostasis/ endocrine regulation, fibrin regulation. Other molecules involved in the inflammation response are the adhesion molecules. Biochip array technology enables the determination of multiple analytes from a single sample and this approach offers more information than single analyte determination as provides a patient's profile at a single point in time. This is illustrated here with examples of analytical parameters of biochip arrays for the determination of analytes related to the MetS and inflammation. Simultaneous chemiluminescent immunoassays are employed for biomarker detection and the microarrays of capture ligands define the discrete test areas for each of the analytes in each biochip (9 9 9 mm). The immunoassays were applied to the Evidence Investigator TM analyser. The MetS Array I enables the multiplex determination of C-peptide, ferritin, IL-1a, IL-6, insulin, leptin, PAI-1, resistin, TNFa and the MetS Array II measures simultaneously adiponectin, CRP and cystatin C with sensitivity values ranging from\3.5 pg/ml (IL-1a) to\1 mg/ l (CRP). For all the assays the intra-assay and inter-assay precision, expressed as %CV, were typically\15%. The adhesion molecules array allows simultaneous measurement of the soluble forms of L-, P-, E-selectins, VCAM-1 and ICAM-1 with analytical sensitivity values ranging from 0. Mast cells, basophils and eosinophils represent the first line of host defence in allergic inflammation reaction. We investigated the mechanism by which the anti-allergic drugs di-sodium cromoglycate and sodium nedocromil inhibit histamine and eicosanoid release from human mast cells (HMCs) as well as peripheral blood basophils (PBBs) using biochemical techniques. Cord derived HMCs were cultured in vitro and PBBs obtained from human donors or cardiac puncture in mice. Subsequent antigen challenged led to a prompt release of histamine and prostaglandin D 2 (PGD 2 ), which was inhibited by cromoglycate, nedocromil, dexamethasone and human recombinant Annexin-A1 (Anx-A1). Treatment of HMCs with cromoglycate or nedocromil induced a rapid phosphorylation on the Ser 27 residue of Anx-A1, hence its secretion, secondary to inhibition of phosphatase activity and activation of PKC. Treatment of the HMCs or PBBs with a specific neutralising anti-Anx-A1 (but not irrelevant) monoclonal antibodies reversed the inhibitory effect of these drugs on histamine and PGD 2 -released. To highlight the crucial importance of Anx-A1 in this rapid effect of these drugs we tested the effect of nedocromil in Anx-A1 null mice. Cromones did not inhibit the release of histamine nor prostaglandin D2 from basophils from Anx-A1 null mice. Background: The engulfment of apoptotic leukocytes (efferocytosis) by macrophages during the resolution of inflammation is essential for homeostasis and results in macrophage reprogramming/immunesilencing. Materials and methods: To characterize pro-resolving properties of CD11b low macrophages and their modulation by pro-resolving agents, zymosan A-initiated peritonitis was used, followed by immunostaining and FACS sorting to distinct macrophage subpopulations. Sorted CD11b high and CD11b low macrophages were analyzed for protein markers by Western blot, efferocytic scores using fluorescent microscopy, and secretion of cytokines ex vivo using ELISA. Results: Here, we show CD11b high macrophages convert to CD11b low ones and stop efferocytosing apoptotic PMN after reaching an engulfment threshold in vivo. In addition, CD11b low macrophages are distinct from either M1 or M2 in their protein expression signature and express proresolving properties, such as diminished responses to different TLR ligands ex vivo and propensity to emigrate from resolving inflammation sites to lymphoid organs. Of interest, we found the pro-resolving lipid mediators resolvin (Rv) E1 and RvD1, as well as the glucocorticoid dexamethasone (Dex), to enhance satiated-efferocytosis and consequently CD11b low macrophage conversion from their CD11b high counterparts, whereas genetic deficiency in the chemokine scavenging receptor D6 resulted in delayed satiation and reduced immune-silencing of macrophages, and inhibits their departure from resolving inflammation sites. Conclusions: We suggest satiated-efferocytosis is a novel phagocyte property displayed by CD11b low macrophages and regulated by proresolving mediators. Moreover, satiated-efferocytosis is required for CD11b low macrophage emigration from resolving inflammation sites and the return of tissue homeostasis. Thus, satiated-efferocytosis is essential for the completion of timely-and spatially-coordinated resolution of acute inflammation. Disclosure of interest: None declared. Enterohemorrhagic Escherichia coli (EHEC) induces elevated levels of IL-1beta in infected patients. However, the molecular mechanisms involved in the activation of IL-1beta by EHEC are unknown. Inflammasomes are multiprotein complexes in the cytosol that process proIL-1beta to bioactive IL-1beta. To identify the inflammasomes involved in IL-1beta induction by EHEC, we infected macrophages derived from wild type and mice deficient in inflammasome components with EHEC and analyzed the processing and release of mature IL-1beta. EHEC-induced IL-1beta production was dependent on caspase-1 and the adaptor ASC. Notably, both NLRP3, a cytosolic sensor for microbial and endogenous products, and AIM2, a cytosolic sensor for DNA, were absolutely essential for EHEC-induced IL-1beta production. Though EHEC is a non-cytosolic pathogen, bacterial products such as DNA were observed in the cytosol of infected cells, providing a potential means by which cytosolic inflammasomes might be activated. This also indicated that phagosomal degradation of bacteria is a prerequisite for inflammasome activation by EHEC. Supporting this hypothesis, blocking lysosomal acidification and type I interferon signaling that are essential for bacterial lysis in phagosome, led to a marked decrease in EHEC-induced IL-1beta secretion. Thus, our results demonstrate that EHEC potently activates multiple inflammasomes as a result of release of bacterial products from phagosomes into the cytosol. Disclosure of interest: None declared. Dengue virus (DENV) infection is the most important mosquito-borne disease on Earth. In the past few years, several studies have shown the importance of inflammasome complex activation during infectious processes. Then, we evaluated the role played by inflammasome activation during host response to primary DENV infection. To this end a mouse experimental model, utilizing a DENV-2 strain that causes a severe disease in mice was used. Wild-type (WT) infected mice showed increased levels of cleaved Caspase-1 in spleen. However, Casp-1 cleavage in spleen of DENV-2-infected ASC -/mice was reduced. These animals, as well as Casp-1 -/mice, presented a more severe disease manifestation after DENV infection. In addition, viral loads in spleen of ASC -/and Casp-1 -/--infected mice were markedly elevated and lethality rates after infection were strikingly higher. Of note, both groups showed reduced IL-1bproduction after DENV infection, when compared to WT-infected mice. Hence, IL-1ra-treated mice showed marked susceptibility to DENV-2 infection when compared with infected-WT mice, showing similar alterations to that seen in ASC -/and Casp-1 -/--infected mice. Of note, there was marked increase in reactive oxygen species (ROS) after DENV infection in WT mice, and inhibition of NADPH oxidase by Apocynin (APO) treatment or infection of gp91phox -/mice resulted in reduced ROS production and in inhibition of IL-1bproduction after DENV infection. In a similar way to the response seen in mice deficient for inflammasome components and in IL-1ra treated mice, APO-treated and gp91phox-/-mice showed more marked susceptibility to DENV infection. These data suggest that ROSmediated inflammasome activation is essential for host ability to deal with DENV infection, in part through promotion of IL-1b production and control of viral replication. Acute gout is an intensely painful form of inflammatory arthritis caused by the formation of monosodium urate (MSU) crystals in the joint and characterised by the infiltration of leukocytes including monocytes. Currently, the role of MSU-crystal recruited monocytes in the progression of the inflammatory response is unclear with monocyte differentiation into macrophages being linked with either antiinflammatory or pro-inflammatory responses to MSU crystals in vitro. Using a murine model of acute MSU crystal-induced inflammation we profiled the differentiation pathway of MSU crystal-recruited monocytes in vivo. Our findings showed that newly recruited monocytes exhibited poor phagocytic capacity, expressed low levels of pro-IL-1b and failed to produce pro-inflammatory cytokines alone, or in response to MSU crystal restimulation ex vivo. However, over time MSU crystal-recruited monocytes exhibited increased phagocytic capacity and expression of CD14, and the accumulation of intracellular pro-IL-1b, active caspase-1 and intracellular IL-1b, indicative of differentiation into an M1-like macrophage. Consistent with this, MSU crystal restimulation triggered the production of pro-inflammatory cytokines by these cells ex vivo, and initiated IL-1b production and leukocyte recruitment in vivo. MSU crystal-recruited monocytes differentiate into pro-inflammatory M1-like macrophages in vivo. These data show for the first time that differentiation of MSU crystal-recruited monocytes into a pro-inflammatory M1-like macrophage in vivo, combined with ongoing MSU crystal deposition, may play a significant role in abrogating resolution and perpetuating inflammation in gout. Disclosure of interest: None declared. Wegener's granulomatosis (WG) is an autoimmune disease characterized by granulomatous inflammation of the respiratory tract and systemic vasculitis. Vasculitis involves small-to medium-size vessels and mostly affects lungs and kidneys. Given the current lack of a mouse model for this human disease, it is important to conceive alternatives to investigate the pathogenesis of this syndrome. We have previously described [1] a peculiar NK-like CD4 T cell subset probably involved in WG vascular damage. We here investigated the role of the endothelium, target of the vascular injury. We analyzed the phenotype and functional features of human microvascular endothelial cells (MECs) derived from renal glomerula, lung and skin. We observed consistent differences between the three tissues in the basal expression of activation and adhesion markers (ICAM-1; CX3CL1; MHC-I) along with NK cell receptor (NCR) ligands (MICA/B-; ULBPs; PVR). Moreover the MECs stimulation with different factors induced distinct changes in phenotype and cytokine production (IL-8; IL-6; IL-15; CX3CL1). Finally, we showed that MECs derived from the three tissues have different ability to stimulate NK cells. Of note, renal-and lung-derived MECs elicited NK cell response more efficiently than the skin-derived. Our findings demonstrate that endothelial cells depending on their tissue origin can differently stimulate cells of the immune system. These data could contribute to explain why vasculitis may selectively affect specific organs, as for Wegener's disease, in which lung and kidney vessels are particularly concerned. Dysregulation of the antiviral immune response may contribute to autoimmune diseases. Here, we hypothesized that altered expression or function of MAVS, a key molecule downstream of the viral sensors RIG-I and MDA-5, may impair antiviral cell signalling and thereby influence the risk for systemic lupus erythematosus (SLE), the prototype autoimmune disease. We used molecular techniques to screen nonsynonymous single nucleotide polymorphisms (SNPs) in the MAVS gene for functional significance in human cell lines and identified one critical loss-of-function variant (C79F, rs11905552). This SNP substantially reduced expression of type I interferon (IFN) and other proinflammatory mediators and was found almost exclusively in the African-American population. Importantly, in African-American SLE patients, the C79F allele was associated with low type I IFN production and absence of anti-RNA-binding protein autoantibodies. These serologic associations were not related to a distinct, functionally neutral, MAVS SNP Q198K. Hence, this is the first demonstration that an uncommon genetic variant in the MAVS gene has a functional impact upon the anti-viral IFN pathway in vivo in humans and is associated with a novel sub-phenotype in SLE. Disclosure of interest: None declared. CO-releasing molecules (CORM) can reproduce the effects of CO derived from heme oxygenase-1 (HO-1) activity and show antiinflammatory properties in a range of diseases with an inflammatory component. We have demonstrated previously the protective actions of CORM-2 in osteoarthritic (OA) cartilage. However, little is known about the effects of these molecules in OA synovitis. In this study we explored the effects of CORM-2 in OA synoviocytes stimulated with interleukin (IL)-1b. Synovial tissue samples were obtained from 15 OA patients undergoing total knee joint replacement. Synoviocytes were obtained by digestion with collagenase and cultured until third passage. Cells were stimulated with IL-1b (10 ng/ml) in the presence or absence of CORM-2 at various concentrations (50-200 lM) or RuCl 3 (200 lM) as negative control. CORM-2 markedly decreased cell proliferation induced by IL-1b and it did not modify HO-1 expression, thus discarding the participation of this protein in the effects of CORM-2. This agent was able to reduce both protein and mRNA levels of IL-8, CCL2 and CCL20, effects that were accompanied by a marked reduction in the migration promoted by IL-1b. At the highest concentration employed, this treatment also showed beneficial effects by decreasing reactive oxygen species generation. Interestingly, CORM-2 also reduced, in a concentration-dependent manner, the activity and expression of degenerative mediators such as MMP-1 and MMP-3. Further analyses revealed that CORM-2 significantly counteracted the activation of mitogen-activated protein kinases and nuclear factor jB signalling pathways, which could be responsible, at least in part, for the protective effects elicited by CORM-2 in this cellular system. In conclusion, CORM-2 exerts anti-inflammatory and antidegenerative effects on OA synoviocytes. CO-releasing molecules represent new and interesting strategies to counteract synovial inflammation during osteoarthritis. Disclosure of interest: None declared. Due to their genetically-determined capacity to respond to proinflammatory stimuli, keratinocytes have a role in the pathogenesis of psoriasis. Upon IFN-c and TNF-a exposure, psoriatic keratinocytes express exaggerated levels of inflammatory mediators, and show aberrant hyperproliferation and terminal differentiation. However, the thickening of psoriasic skin also results from a resistance of keratinocytes to cytokine-induced apoptosis. Recently, we found that SOCS1 and SOCS3 counteract IFN-c-induced effects in keratinocytes and are very highly expressed by psoriatic cell strains. Due to the well-known anti-apoptotic effects of SOCS1 and SOCS3, we investigated on their possible involvement in protecting psoriatic cells from IFN-c/TNF-a-induced apoptosis through the PI3 K/AKT signaling, which has definite anti-apoptotic functions in keratinocytes. Other than expressing increased SOCS1 and SOCS3, psoriatic keratinocytes showed high levels of phosphorylated AKT in response to IFN-c and TNF-a. Moreover, the expression of the anti-apoptotic Bcl-X L and Bcl-2 genes could not be down-regulated by IFN-c/TNF-a in psoriatic keratinocytes, differently to what observed in healthy cells. Keratinocytes stably transfected with SOCS3 were more resistant to IFN-c/ TNF-a-induced apoptosis compared to control clones, and showed high levels of Bcl-X L, Bcl-2 and phosphorylated AKT. Vice versa, SOCS3 silencing resulted in enhanced IFN-c/TNF-a-induced apoptosis, correlating with impaired AKT phosphorylation, and reduced Bcl-X L and Bcl-2 levels. SOCS1 manipulation in keratinocytes had effects similar to that of SOCS3 even though less significant. Our data indicate that SOCS3 and SOCS1 can contribute to the anti-apoptotic programs occurring in IFN-c/TNF-a-activated psoriatic keratinocytes by sustaining AKT activation and Bcl-X L and Bcl-2 expression. We are currently investigating whether SOCS3 and AKT inhibition in psoriatic keratinocytes renders these cells sensitive to apoptosis. Disclosure of interest: None declared. TNF plays a major inflammatory role in rheumatoid arthritis (RA), and anti-TNF targeted treatments are highly effective in this disease. In the present study, we aimed to elucidate the interplay between TNF and CD4 + CD25 + FoxP3 + regulatory T cells (Treg), and the impact of anti-TNF therapies on those cells. We used a TNF-driven disease, specifically human TNF transgenic (TTg) mouse model of arthritis. In the first part of our study, we observed that, in 7 weeks old-TTg mice, hTNF overexpression induced lower Treg proportions in secondary lymphoid organs, as compared to age-matched wild type (wt) mice. However, the frequency of Treg cells increased with time in TTg mice, reaching the same proportion than wt mice at 24 weeks. TNFR2 expression by Treg, also increased with time in TTg mice. In the second part of our study, TTg mice were treated with either passive (infliximab) or active (TNF-K: vaccination against TNF) anti-TNF. Compared to untreated mice, hTNF blockade with either infliximab or TNF-K resulted in an increased Treg frequency. In lymph nodes (LN), our study showed that hTNF blockade induced an expansion of induced Treg cells defined by the CD4 + CD25 + FoxP3 + CD62Lphenotype, an up-regulation of CTLA-4 expression by Treg cells, accompanied by an increased Treg suppressive activity on effector T cell proliferation. In conclusion, in a strictly hTNF dependent inflammation model, we show for the first time that TNF can have different effects on Treg, depending on the duration of exposure and on disease phase. Our work also shows that TNF blockade either by TNF-K or infliximab could depend not only on TNF neutralization but also on Treg upregulation. Disclosure of interest: J. Osteoarthritis (OA) is characterized by chondrocyte premature aging involving altogether an hypertrophic phenotype, a DNA damage accumulation and presence of senescence-mediated secretory factors such as Il-1b, Il-8 and MMPs. Molecular mechanisms involved in OA are still under investigation. Here we identified by genome wide miRarray analysis that p16INK4A-regulatory microRNA, hsa-miR-24 is one target of three ex vivo osteoarthritic mimicking models namely Il-1b, IL-8 and oxidative stress-treated chondrocytes. hsa-miR-24 expression is repressed upon IL-1 treatment while p16INK4A accumulates. Furthermore, we show by RT-qPCR that hsa-miR-24 expression increases during early steps of ex vivo chondrogenic differentiation of mesenchymal stem cells and decreases while p16INK4A increases correlating with an hypertrophic stage. Finally, preliminary results based on specific antagomir approach suggest that hsa-miR-24 down regulation is sufficient to trigger p16INK4A-dependent premature aging on chondrocyte and expression of hypertrophic markers such as MMP13. Altogether, we proposed that hsa-miR-24 is one of the first microRNA in human having an central anti-aging role by preventing the onset of one age-related disease. Disclosure of interest: None declared. We have previously demonstrated that Transient Receptor Potential Vanilloid 4 (TRPV4) activation in intestinal epithelial cells caused intracellular calcium mobilization and chemokine release. Further, TRPV4 activation in mouse intestine induced colitis, suggesting that this channel could participate to intestinal inflammation. Many mediators are released at inflammatory sites, including serine proteases known to activate Protease-Activated Receptors (PAR), but also endogenous gas, such as hydrogen sulfide (H 2 S). We hypothesized that such mediators are able to regulate TRPV4 functions in intestinal epithelial cells Methods: TRPV4 response was evaluated by measuring calcium flux with a Fluo-8 probe in Normal derived Colon Mucosa (NCM460) cells. Cells were exposed to PAR 1 -(TFLLRN-NH 2 ), PAR 2 -(SLI-GRL-NH 2 ), or PAR 4 -activating peptide (AYPGFK-NH 2 ), Sodium Hydrogen Sulfide (NaHS) a H 2 S donor, or their vehicle, 3 min before a second stimulation with the specific agonist of TRPV4, 4aPDD (4aphorbol-12,13-didenoate) or its vehicle. Results: TRPV4 agonist induced a dose-dependent calcium flux in NCM460 cells. The response to 10 lM of 4aPDD was significantly increased when cells were pre-incubated with PAR 1 -(100 lM) PAR 2 -(10 lM), but not PAR 4 -activating peptide (200 lM). Intestinal epithelial cells did not respond to low concentration (1 lM) of 4aPDD. However, if cells were pre-treated with the PAR 1 -(100 lM) or the PAR 2 -(10 lM), but not the PAR 4 agonist, they became responsive to 1 lM of the TRPV4 agonist. Incubation of NCM460 cells with NaHS (200-1,000 lM) dose-dependently decreased the calcium response to pro-inflammatory dose of TRPV4 agonist (50 lM). Conclusions: Activation of PAR 1 , PAR 2 but not PAR 4 potentiated the cellular response of TRPV4 but also sensitized the receptor, potentially enhancing its pro-inflammatory effects. However, NaHS exposure decreased cellular response to TRPV4 agonist, thereby potentially exerting anti-inflammatory effects in the gut. Disclosure of interest: None declared. Secreted phospholipases A2 (sPLA2), are now considered attractive therapeutic targets and potential biomarkers, as they contribute in inflammation through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2 is expressed in human atherosclerotic lesions and that the phospholipolysis of LDL by hGX sPLA2 results in a proinflammatory LDL particle (LDL-X), which perturbs endothelial function through the induction of endoplasmic reticulum (ER) stress. Alteration in ER homeostasis triggers the unfolded protein response (UPR) which regulates the balance between survival and apoptosis. In the present study we aimed to identify the molecular pathways directing LDL-X-stress responses to survival or apoptosis in endothelial cells. Endothelial cells (HUVEC), activated with LDL-X induced all three branches of the UPR response with concomitant activation of apoptotic and survival pathways (BCL2 family of proteins). WB and RT-QPCR analysis showed a significant decrease in cyclinD1 expression, suggesting that the regulation of cyclinD1 in response to LDL-X induced-ER stress is a checkpoint control, which prevents cell-cycle progression until homeostasis is restored. Furthermore, immunofluorescence analysis of the mitochondria in the LDL-X treated cells showed altered morphology and shrinkage indicating mitochondrial dysfunction. In parallel, we observed upregulation of antioxidant/detoxifying enzymes, with concomitant activation of NRF2, whose main role is to protect cells from oxidative stress. As LDL-X is not oxidized, we postulated that in addition to the modification of LDL, the lipid mediators produced from LDL hydrolysis, arachidonic acid and Lyso-PC, activate the antioxidant response and this was further confirmed by WB analysis, RT-QPCR and immunofluorescence experiments of HUVEC cells stimulated with the lipid mediators. Conclusion: LDL-X induces inflammation in endothelial cells through a lipotoxic stress which implicates mitochondria. Disclosure of interest: None declared. Deposition of uric acid crystals in joints causes gout disease. Prolonged airway exposure to silica crystals leads to silicosis development. Aluminum hydroxide (alum) salts are frequently used as vaccine adjuvant. These micrometer size particles activate innate immunity through the Nlrp3 inflammasome resulting in IL-1b production. The inflammasome is activated not only by particles derived from endogenous aggregates or environmental pollutants but also by a broad spectrum of situations, from metabolic stress to pathogenrelated molecules. Particles-mediated inflammasome activation has been linked to reactive oxygen species involvement and lysosome destabilization. In an other hand, ATP is present in all living cells and extracellular ATP is a well known soluble danger signal able to activate the Nlrp3 inflammasome through P2X7 receptor and pannexin-1 pore formation. Here we establish a link between ATP and particles signaling. First, we show that uric acid, silica and alum particles trigger endogenous ATP release depending on functional P2X7 receptor and pannexin-1 hemichannel proteins. Then, we observe that maturation and secretion of IL-1b were dramatically impaired in presence of purinergic receptors or pannexin-1 pore antagonists. This demonstrates that crystals are competent to induce maturation and secretion of IL-1b through a process that involves as a first event the extracellular release of endogenous ATP. Moreover, we show that not only ATP but also ADP and UTP are able to stimulate IL-1b production through multiple purinergic receptors signaling, rendering even more interesting to better decipher purinergic-mediated inflammatory processes. These data demonstrate that nucleotides are endogenous danger signal generated in response to particle exposures and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome and trigger IL-1b maturation and secretion. Disclosure of interest: None declared. IL-27 and sOx40L are produced mainly by antigen presenting cells and are powerful immunomodulatory molecules. For instance, IL-27 enhances B cell proliferation and OX40L reduces Th2 response. To date platelets were not reported to be able to release neither IL-27 nor sOX40L. We sought to examine the levels of IL-27 and sOx40L in supernatants of apheresis platelet concentrate preparations (PCs) during storage and in left over products involved in transfusions that induced acute transfusion reactions (ATR). Samples from PCs were taken for analysis each day during their 7-day storage. We recorded 4 ATR episodes for which PC left over products could be analyzed. Two fractions (supernatant and lysates of platelets) from each ATR-associated PC (and 10 controls) were prepared. A proteomic approach was used to determine inflammatory factors involved in ATR and ELISA was used for quantify these factors. After a 1-day storage of PCs we found elevated levels of IL-27 and sOx40L (respectively at 18,020 ± 5,844 and 374 ± 50 pg/mL/ 10 9 unit) that increased respectively by 2.2 and 2.25-fold at storage day 7 versus day 0. Moreover, supernatants of ATR-associated PCs contained higher and corresponding platelet lysates contained lower, IL-27 and sOx40L levels, compared to control PCs (P \ 0.05). We exposed B and T cells to supernatants and cell lysates of ATR-associated PCs and observed an increase of CD86 and CD69 expression only for PCs supernatants. This over-expression of activation markers was abolished when B and T cells were preincubated with IL-27 and sOx40L blocking antibodies. We demonstrated that platelets contain and release IL-27 and sOx40L that are implicated in ATR. These data emphasize the importance of investigating, in a multi-center collaborative study, larger series of ATR-associated PCs to determine specific reagents or processes to reduce IL-27 and sOx40L production during collection and storage of platelet components. Disclosure of interest: None declared. ). This work examines whether aberrant presentation of MPO could attenuate autoimmunity and anti-MPO glomerulonephritis. Autoimmunity was induced by immunizing mice (with MPO or pep52) then disease was triggered with low dose anti-GBM globulin which induced glomerular neutrophil influx and MPO deposition. Aberrant presentation of MPO was performed by intranasal insufflation of pep52 or immunization with MPO-pulsed bone marrow-derived CD40-/-dendritic cells (DCs). Nasal insufflation of pep52 over 3 consecutive days protected mice from the development of autoimmunity when subsequently immunized with 200 lg pep52, compared with control mice given nasal insufflation with an irrelevant peptide. The frequency of CD4+ cells responding to MPO by producing IFNc was 457 ± 107 (per 10 6 , control mice) versus 289 ± 90 (tolerized mice) and IL-17A-producing cells was 231 ± 154 versus 49 ± 32; p \ 0.001. Anti-MPO IgG titres were also reduced. When pep52 immunized mice were challenged with anti-GBM globulin, GN was reduced in tolerized mice with reduced glomerular segmental necrosis, glomerular neutrophils, macrophages, CD4+ cells and proteinuria. Immunization of C57BL/6 mice with MPO-pulsed CD40+/+ DCs induced strong anti-MPO autoimmunity which induced GN when triggered with anti-GBM globulin. However, immunization with CD40-/-DCs protected from the development of autoimmunity and GN. Draining lymph node cells isolated from CD40-/-DC-immunized mice showed higher levels of IL-10 suggesting the induction of Tr1 regulatory T cells. Aberrant presentation of immunodominant peptide or MPO autoantigen tolerizes and protects from autoimmune anti-MPO GN. Disclosure of interest: None declared. In IgA nephropathy (IgAN), the commonest primary glomerulonephritis worldwide, mesangial proliferation and renal inflammation are key pathological features for disease progression. Our group has identified the transferrin receptor 1 (TfR1 or CD71) as a mesangial IgA1 receptor and we had shown that in cultured mesangial cells TfR1 activation by polymeric IgA1 (pIgA1) induces IL-6 production and cell proliferation. However the mechanism underlying this cytokine production and cellular proliferation is still unclear. Herein, we developed a new inducible mouse model of IgAN, in which patients' IgA induced mesangial cell proliferation, IL-6 and TNF-a production, monocytes/macrophage renal infiltration and renal dysfunction. These responses were associated with PI3K/Akt/m-TOR pathway activation, once sustained PI3K/Akt pathway activation in PTEN± mice amplified patients' IgA1 effects, whereas blocking of this pathway by rapamycin was inhibitory. Concomitant with PI3K/ Akt/m-TOR pathway activation we observed an increased TfR1 expression and platelet-derived growth factor receptor-beta (PDGFRß) expression and phosphorylation. Interestingly, PDGFR inhibition abrogated mesangial cell proliferation but not the inflammatory cytokine production. In vitro overexpression of TfR1 increased cell sensitivity to PDGF-BB suggesting that mesangial cell proliferation is due to a convergence of TfR1 and PDGFR signaling. We conclude that PI3 K/Akt/m-TOR pathway is pivotal to IgAN inflammation by controlling renal inflammatory cytokines production and is also important to mesangial cell proliferation via a modulation of PDGFR signaling threshold. Disclosure of interest: None declared. Renal tubulointerstitial fibrosis is the end result of chronic inflammation and it is regulated by a complex network of cytokines, including the pro-fibrotic factor TGF-b1 and the two anti-fibrotic cytokines BMP-7 and HGF. One of the molecules that might orchestrate the balance between TGF-b1 and HGF/BMP-7 is CD44, which is a surface glycoprotein involved in inflammation and whose expression is up-regulated on tubular epithelial cells upon kidney injury. All CD44 isoforms differ in the extracellular domain and are therefore interacting with different molecules. The CD44 standard (CD44s) isoform can facilitate TGF-b1 signaling, while the CD44variant 3 (CD44v3) is capable of binding HGF and BMP-7 due to its attached heparan sulfate moieties. Using transgenic mice overexpressing either CD44s or CD44v3, specifically on proximal tubular epithelial cells (TECs), we studied the progression of obstructive nephropathy (UUO), as a model of chronic kidney diseases. Wt and transgenic mice (n = 8/group) were subjected to UUO, and sacrificed at 1, 3, 7, and 14 days. Primary TECs were isolated from Wt and transgenic mice (n = 3/group), and stimulated with r-TGF-b1. Statistics were done with the Mann-Whitney test. At day 1 upon UUO the CD44 s transgenic mice showed significantly more tubular damage compared to the Wt and the CD44v3 mice, while no differences were found at later time-point. At day 1, the CD44v3 UUO kidneys revealed significantly less myofibroblasts accumulation together with less TGF-b1 signaling and increased BMP-7 signaling compared to the Wt and CD44 s kidneys. In vitro stimulations of primary TECs with TGF-b1 showed that, in response to TGF-b1, CD44v3-TECs synthesize less a-SMA, CTGF, TGF-b1 and collagen type1 mRNAs then the Wt-, and CD44 s-TECs. In conclusion, these results suggest that CD44v3 decreases tubular injury and myofibroblast accumulation in early stage of chronic obstructive nephropathy through downregulation of TGF-b1 pro-fibrotic signaling. Introduction: Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN). CC chemokine receptor (CCR)2 regulates monocyte/macrophage migration into injured tissues. However, the direct role of CCR2 in the progression of DN remains unknown. We hypothesize that deficiency or blockade of CCR2 confers kidney protection in DN. Methods: Experiments were conducted in Ins2 Akita mice with or without the selective CCR2 antagonist, RS504393 (2 mg/kg/day via osmotic minipump) for 12 weeks or in CCR2 deficient mice (CCR2 -/-) and their wild type littermate (CCR2 +/+ ) following STZ induced diabetes for 6 weeks. Results: Blocking CCR2 using RS504393 in Ins2 Akita mice significantly attenuated albuminuria (p \ 0.005), histological changes and kidney macrophage recruitment (p \ 0.005) compared to Ins2 Akita + vehicle. Furthermore, mice lacking CCR2 (CCR2 -/-) mimicked CCR2 blockade by reducing albuminuria to normal range, diabetes-mediated histological changes, kidney fibronectin mRNA expression, and inflammatory cytokine production (TNF-a and IFN-g) compared to CCR2 +/+ mice. Bone-marrow derived monocytes from CCR2 +/+ mice adoptively transferred into CCR2 -/mice reversed the renal tissue protective effect in diabetic CCR2 -/mice. In vitro, podocytes grown on high glucose (HG) media are associated with significant increase in macrophage migration from those grown in normal glucose (NG) media using transwell migration assay and this effect was blocked with the addition of anti-MCP-1 antibody. In addition, M1 macrophages induced podocyte permeability; but not M2 macrophages. Inflammatory processes play a crucial role in all stages of atherogenesis: from early endothelial activation to the eventual rupture of the atherosclerotic plaque. Activation of the canonical NF-kB signal trans-duction pathway, mediated by phosphorylation and degradation of IkBa by the IkB-kinase (IKK) complex, is a crucial step in proinflammatory target gene expression such as that of chemokines or adhesion molecules, which have been implicated in atherosclerosis. The role of the COP9 signalosome (CSN), a multi-functional protein complex with functions in NF-kB and b2-integrin signaling, in atherosclerosis has been unknown. Here, super-complex formation between IKK and the CSN was indicated for by IKK2/CSN5, IKK2/ CSN3 and NEMO/CSN2 coimmunoprecipitation in human umbilical vein endothelial cells (HUVECs). The CSN interacts with IKKs and attenuates IKK phosphorylation as observed by siRNA knockdown (KD) of CSN5 or CSN2 under pro-atherogenic conditions. Next, a TNFa-dependent degradation of IkBa was markedly enhanced (52%) by CSN5-or CSN2-siRNA treatment (KD efficiency: *40%) and a concomitant increase of NF-kB activity between 60 and 70%, as readout by reporter gene and binding activity assay, respectively in HU-VECs. Inversely, transient overexpression of CSN5 using CTAP-CSN5 fusion construct led to an increase in IkBa levels and a reduction in NF-kB activity. Furthermore, CSN5-depleted cells stimulated with TNFa showed an increase in MCP1, VCAM1, E-selectin and also ICAM1 expression when compared to control cells. This was translated into increase monocytes arrest on the surface of CSN5 siRNA-treated HUVEC monolayers. Finally, doubleimmunostaining for CSN5 and CD31 in human atherosclerotic plaque showed the presence of CSN5 in endothelial cells. Taken together, the CSN controls the activity of NF-kB in atherogenically stimulated endothelial cells and might have anti-inflammatory and anti-atherogenic functions. Disclosure of interest: None declared. Hydrogen sulfide (H 2 S) has been implicated in cardioprotection during ischemia/reperfusion (I/R) injury. Autophagy is the primary pathway for degrading and recycling long-lived proteins, and its activation has recently been shown to mediate myocardial injury in response to I/R injury. In this study we investigated the ability of H 2 S preventing autophagy as a potential mechanism underlying its cardioprotective effects against I/R injury. Male rats were subjected to myocardial ischemia for 45 min followed by 2 h reperfusion. NaHS, a H 2 S donor, at doses of 10, 30, 100 lmol/kg was injected i.p. at 30 min before ischemia. The autophage related genes beclin 1 and ATG5 mRNA were elevated in the area at risk (AAR) of hearts subjected to I/R. NaHS reduced myocardial infarct size and attenuated the increase of beclin 1 and ATG5 expressions. In vitro, cultured neonatal rat cardiomyocytes were subjected to 24 h hypoxia followed by 2 h reoxygenation, and NaHS at doses of 10, 30, 50, 100 lmol/L were added 30 min prior to hypoxia. Detection of microtubule-associated protein 1 light chain 3 (LC3) conversion, i.e., LC3-I to LC3-II, and monodansylcadaverine (MDC) staining were also used for the determination of autophagy activity in cardiomyocytes. Inflamm. Res. Cardiac hypertrophy, accompanied by progressive inflammation, and consequent heart failure continue to burden Western society. In the current study, we show that miR-155, an inflammatory cell-expressed microRNA, stimulates the secretion of pro-hypertrophic andinflammatory factors by macrophages and thereby causes adverse inflammation and heart failure. We subjected miR-155 KO and WT mice to 1 and 4 weeks of angiotensin II (AngII) to induce cardiac hypertrophy, inflammation and failure. Whereas AngII significantly increased both cardiac mass and hypertrophic signalling in WT mice, the absence of miR-155 inhibited this hypertrophic response. Moreover, absence of miR-155 prevented inflammatory cell influx in the heart following AngII, more specifically the influx of macrophages. These data were independently confirmed using antagomiRs against miR-155 in AngII-treated mice. In situ hybridization predominantly identified cardiac miR-155 in macrophages and very little was detected in cardiac myocytes. Therefore, we hypothesized that macrophage miR-155 mediates pro-hypertrophic signalling towards cardiac myocytes. Indeed, both bone marrow transplantation with miR-155 KO and WT mice and in vitro experiments independently confirmed that miR-155 in the macrophage mediates cardiac hypertrophy. We found that miR-155 represses its direct target Suppressor of Cytokine Signalling-1 in the macrophage, leading to increased IL-6 secretion and consequent STAT3 activity, implicated in cardiac inflammation and hypertrophy. These data reveal miR-155 expression in the macrophage to be mandatory to allow cardiac hypertrophy, inflammation and failure, by permitting cytokine signalling, identifying miR-155 as a crucial inflammatory regulator of cardiac hypertrophy. Disclosure of interest: None declared. Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle for 1 h, after that, the kidney was reperfused immediately. Four hours after the surgery, 2 9 10 5 ASCs were intraperitoneally administered, and animals were followed for 24 h post-treatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 9 10 5 ASC at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis already is present. At 24 h after reperfusion, ASCtreated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4, IL-10 and HO-1 expression increased despite a lack of ASC in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition and decreased staining for FSP-1, type I collagen and hypoxyprobe. The renal protection seen in ASCtreated animals was followed by reduced serum levels of TNF-a, KC, RANTES and IL-1a. Surprisingly, treatment with ASC at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition. Disclosure of interest: None declared. Acute lung injury (ALI), a devastating disorder characterized by hypoxemia and overwhelming pulmonary inflammation, remains a primary factor of morbidity and mortality in critically ill patients. Extracellular adenosine has been implicated as central signalling molecule during conditions of limited oxygen availability (hypoxia), important in attenuating tissue damage, acute inflammation and the preservation of pulmonary barrier function. During periods of acute inflammation extracellular levels of adenosine are increased in the affected tissues, yet the molecular mechanisms are presently unknown. The Equilibrative Nucleoside Transporters (ENTs) have significant impact on regulation of the levels of extracellular adenosine. During periods of hypoxia, the expression levels of ENT1 and -2 are significantly reduced, leading to the attenuation of adenosine uptake and resulting in an increased extracellular adenosine concentration. Cellular hypoxia is part of the pathophysiological correlate of an acute inflammatory process, yet in addition a variety of cytokines are released upon the stimulation of the immune system. Given this, we investigated consequences of pulmonary acute inflammation on the expression of ENTs. Our initial studies with endothelia and pulmonary epithelia demonstrate attenuation of adenosine uptake as result of diminished expression of ENTs during acute inflammation in vitro. Studies with siRNA confirmed the major contribution of ENT2 as main adenosine transporter in lung. Furthermore, examination of the ENT2 promoter suggests nuclear factor-kappa B in ENT2 repression. Additional in vivo studies using a murine model for ALI showed that pharmacological inhibition of ENTs results in improved pulmonary barrier function, attenuated neutrophil accumulation and reduced release of pro-inflammatory cytokines during ALI. Taken together, these findings reveal transcriptional repression of ENTs as an innate protective response during acute pulmonary inflammation. NF-kB plays a central role in immunity, inflammation, development, cell survival and has been indicated under a number of pathological conditions of lung disease, including asthma, chronic bronchitis, and chronic obstructive pulmonary disease (COPD). In this study, we assessed the in vivo activation of NF-kB signaling in lung tissue using a bioluminescence imaging system (IVIS) to monitor activation of an NF-kB promoter in response to lipopolysaccharide (LPS) stimulation. A plasmid contained responsive elements (RE) of NF-kB and luciferase as a reporter gene has been delivered intravenously in nude mice at the concentration of 40 ug per mouse using in vivo-jetPEI TM from Polyplus as a transfectant agent. One week after DNA delivery the transient transgenic mice had been imaged in order to check the baseline activation of the NF-kB pathway. The day after, the mice have been treated with LPS 15 ug per mouse intratracheally and the lungs imaged using bioluminescence (BLI) at 2, 4, 7 and 24 h. The ability of the IKK2 inhibitor MLN120B orally administered at the dose of 300 mg/kg to counteract NF-kB activation has been evaluated. The maximum peak of NF-kB activation was reached at 4 h with seven-tenfolds of induction in comparison to the saline group and at 24 h the signal dropped down at basal level. In the group treated with MLN120B was observed a 50% inhibition of LPS-induced NF-kB stimulation, an effect that was in good agreement with the inhibition of p65 nuclear translocation evaluated ex vivo in lung homogenates. In this experiment we showed that is feasible to monitor NF-kB activation in vivo in lung tissue in a non-invasive way by BLI and create a new in vivo tool for drug discovery process. Introduction: Inosine has been suggested to exert anti-inflammatory effects in a wide range of inflammatory conditions, including acute lung inflammation. Objective: This study intended to investigate the possible involvement of adenosine receptors in inosine anti-inflammatory effects in a murine model of ovalbumin-induced asthma. Methods: Female balb/c mice, sensitized (day 0) and boosted (day 7) subcutaneously with OVA (10 lg) were challenged at day 14 and 15 with aerosolized OVA (1%) and treated intraperitoneally 30 min prior with inosine (0.001-10 mg/kg) or with the selective adenosine receptors antagonists A 1 (DPCPX), A 2A (ZM241385), A 2B (alloxazine) or non-selective (caffeine), 30 min before inosine injection (10 mg/kg). After 24 h, the bronchoalveolar lavage (BAL) was obtained to perform the cell counting. Lungs fragments were obtained and cultured-24 h (explant) for determination of cytokines Results: Inosine treatment reduced the number of total leukocytes (85 ± 2%) with ID 50 of 0.094 (0.023-0.37) mg/kg, macrophages (89 ± 11%), lymphocytes (92 ± 3%) and eosinophils (97 ± 2%) recovered in the BAL at 10 mg/kg, when compared with the allergic control group. Pre-treatment with ZM241385 and caffeine, reverted 70 ± 5 and 97.6 ± 8.1% the inosine effect on the total cell count, and 86.5 ± 7.9% and 96.2 ± 4.1% the macrophage count, respectively. Also, caffeine treatment reverted the lymphocyte cell count in 68.0 ± 14.5%. DPCPX or alloxazine treatment did not cause any effect. Inosine also reduced the levels of IL-4 (67 ± 8%) and IL-5 (55 ± 8%) in explants, an effect reverted in 96. Background: Massive recruitment of neutrophils and extracellular release of their azurophil granules (AG) leads to chronic proteolytic and oxidative stresses in cystic fibrosis (CF) airways. Previously, we demonstrated that neutrophils are reprogrammed upon entry into CF airway (via cAMP and mTOR pathways), and then go to release AG actively. Better understanding of these reprogrammed neutrophils is essential to develop efficient treatments for fatal CF airway disease. Methods: We used novel retroviral envelope probes for FACS analysis of surface metabolite transporters on CF airway neutrophils (glucose transporter Glut1, inorganic phosphate transporters Pit1 and Pit2, and neutral amino acid -AA-transporter ASCT2), as well as mass spectrometry to characterize AA metabolites (assumingly, derived from proteolysis) in CF airway fluid. Results: We found increased expression of Glut1 (+81%) and PiT1 (+77%) on CF airway neutrophils, compared to blood neutrophils. Moreover, CF airway neutrophils with high AG release compared to those with low AG release (as identified in our previous studies), displayed major increases in Glut1 (+29%), PiT1 (+315%), ASCT2 (+145%) and PiT2 (+259%), consistent with the idea that AG release and metabolic reprogramming occur concomitantly. Next, we found that CF airway fluid is highly enriched in AA metabolites, starting in newborns and infants. CF airway fluid contained 300 + AA metabolites, with[95% of them increased tenfold or more compared to airway fluid from healthy controls. AA metabolites correlated highly with neutrophil count and anabolic AA, some of which are transported by ASCT2, were very high in CF airway fluid. Conclusions: Our results suggest that neutrophils not only proteolyze the airway tissue and thereby condition the extracellular fluid towards high AA content, but that they also use these AA, and other nutrients, to drive their reprogramming. This new pathway may be amenable to therapeutic modulation. Disclosure of interest: None declared. Reactive oxygen species (ROS) have an established role in inflammation and host defense. However, how they contribute to the pathogenesis of autoinflammatory diseases is not clear. Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a familial autosomal dominant periodic fever syndrome characterized by prolonged febrile episodes, serositis, dermal inflammation and an elevated risk of amyloidosis. Mutations in the extracellular domain of TNFR1 associated with TRAPS cause misfolding and accumulation of mutant TNFR1 protein in the endoplasmic reticulum. PBMC from TRAPS patients and myeloid cells from mice heterozygous for TRAPS-associated TNFR1 mutations exhibit enhanced basal MAPK activity and enhanced production of TNF, IL-1 and IL-6 in response to LPS that is dependent on p38 and JNK activity. Production of TNF and IL-6 in response to LPS is independent of the NALP3 inflammasome, IL-1 or Caspase-1. TNFR1 mutant heterozygous mice do not develop spontaneous fevers but are hypersensitive to LPS-induced endotoxic shock. We hypothesized that ROS might sustain MAPK activation and consequent inflammatory responses in cells from TRAPS patients. Indeed, we found that a variety of antioxidants could significantly reduce inflammatory cytokine production by cells from TRAPS patients and knock-in mice. Rather than NADPH oxidases, we find that mitochondrial respiration provides the ROS necessary for enhanced cytokine production in TRAPS. TNFR1 mutant cells exhibit enhanced mitochondrial oxidative capacity and ROS generation. Pharmacological agents reduce inflammatory cytokine production after LPS stimulation in both cells from TRAPS patients and healthy controls in proportion to their ability to block mitochondrial ROS production but not simply respiration. These findings suggest that mitochondrial ROS are required for optimal inflammatory responses and may be a novel therapeutic target in TRAPS and other autoinflammatory diseases. Disclosure of interest: None declared. Dengue is one of the most important mosquito-borne viral diseases. There are no treatments or vaccines available and mechanisms involved in host response to infection are poorly understood. However, there are several reports documenting the generation of free radicals after viral infections. Then, we evaluated the role played by reactive oxygen species (ROS) and nitric oxide (NO) generation during host response to primary DENV infection. To this end a mouse experimental model, utilizing a DENV-3 strain that causes a severe disease in mice was used. Wild-type (WT) infected mice showed increased levels of ROS, NO and peroxynitrite (ONOO -) in spleen. Inhibition of NADPH oxidase by Apocynin (APO) treatment or infection of gp91phox -/mice and inhibition of iNOS by infection of iNOS -/mice resulted in reduced ROS and NO production followed by ONOOdecreased generation. These animals presented a more severe disease manifestation after DENV infection, showing increased thrombocytopenia, marked hemoconcentration, elevated plasmatic transaminases activity, enhanced neutrophil arrest and tissue injury in liver and elevated systemic production of TNF-a and IL-6. In addition, viral loads in spleen of all knockout and treated mice were markedly elevated and lethality rates after infection were strikingly higher. Of note, APO treated, gp91phox -/and iNOS -/mice showed reduced ONOOproduction after DENV infection, when compared to WT-infected mice, suggesting this specie is of great importance during host response to DENV infection. Hence, FeTPPs (a Peroxynitrite scavenger) treated mice showed marked susceptibility to DENV-3 infection when compared with infected-WT mice. These data suggest that ROS and NO generation are essential for host ability to deal with DENV infection, in part through promotion ONOOproduction and control of viral replication. Increasing evidence showed that proteases and proteinase-activated receptor (PARs) activation are involved in several intestinal inflammatory conditions. We hypothesized that serine proteases could modulate the injury induced by acute mesenteric ischemia/ reperfusion. Methods: C57/Bl6 mice were subjected to 90 min of intestinal ischemia followed by 0 or 2 h of reperfusion (I/R). Mice were treated with FUT-175, a potent serine-protease inhibitor or with the PAR 1 antagonist SCH-79797. PAR 2 -/mice were subjected to I/R and compared to PAR 2 +/+ . Serine-protease activity was determined in plasma and intestinal tissues. Myeloperoxidase (MPO) activity (an index of granulocyte recruitment), chemokine expression and western blot were performed on intestinal tissues. Results: After the ischemic period, plasma and tissue serine protease activity levels were increased compared to the activity measures in tissues from sham, and this increase was maintained for 2 h after reperfusion. Trypsin 25 kDa band was detected in I/R tissue. Mouse treatment with FUT-175 increased survival after I/R, inhibited tissue protease activity and also significantly decreased MPO activity, KC and MCP-1 intestinal expression. We investigated whether serine proteases modulate granulocyte recruitment by a mechanism dependent on PARs activation. MPO activity levels were significantly reduced in I/R group treated with the PAR1 antagonist SCH-79797 and in I/R PAR 2 -/compared respectively to vehicle group and PAR 2 +/+ mice. Conclusion: This study showed that increased proteolytic activity and PAR activation play an important role in intestinal I/R injury. Inhibition of PAR-activating serine proteases could be beneficial to reduce post-ischemic tissue inflammation. Disclosure of interest: None declared. Aims: Activation of protease-activated receptor 2 (PAR 2 ) has been shown to increase proliferation of colonic epithelial cells and inhibit apoptosis in airway cells, actions that are consistent with the healing of damaged tissue. We hypothesized that PAR 2 was important in resolving colitis by modulating the survival of colonic epithelial cells. Methods: (1) Colitis was induced in wild-type (WT) and PAR 2 -/-C57Bl/6 mice by the oral administration of 2.5% dextran sodium sulfate (DSS) for 1 wk. Mice were either killed at the end of DSS treatment or maintained on regular drinking water for 4 more weeks. Colonic MPO activity and levels of PGE 2 and PGD 2 were determined for mice killed at the end of DSS treatment. (2) HT-29 colonic epithelial cells were treated with PAR 2 activating peptide 2-furoyl-LIGRLO (2fLI; 5 lM) followed by IFNc and TNFa to induce apoptosis. Western blotting for cleaved caspase-3 and PARP, as well as Annexin-V/PI flow cytometry, were used to quantify apoptosis. Results: (1) PAR 2 -/mice lost weight earlier than WT after DSS treatment compared to controls and had significantly higher MPO levels than WT at the earlier time point. Colonic PGE 2 was increased in both strains after DSS treatment, while PGD 2 was only increased in WT. PAR 2 -/mice showed increased levels of cellular infiltration and loss of architecture at the later time point compared to WT. (2) Pretreatment with 2fLI, but not the reverse peptide, was able to significantly reduce levels of IFN-c + TNF-a-induced apoptosis by all methods of detection. 2fLI decreased levels of cleaved caspase-8 and but did not change levels of Bax, Bcl-2 or Bcl-xL as determined by Western blot. Conclusions: PAR 2 promotes the resolution of inflammation by stimulating the production of the pro-resolution eicosanoid PGD 2 and by reducing cytokine-induced epithelial apoptosis. Our findings may represent important physiological mechanisms whereby proteases facilitate colonic healing after inflammation. Disclosure of interest: None declared. Imbalance between proteases and their endogenous inhibitors such as Elafin or Secretory Leukocyte Protease Inhibitor (SLPI) has been identified in colonic tissues from inflammatory bowel disease (IBD) patients. We tested here whether the administration of recombinant food-grade lactic acid bacteria (LAB) expressing these protease inhibitors could restore the proteolytic balance and thus decrease inflammation in a mouse model of IBD. Methods: Colitis was induced in mice with DSS (5% in drinking water) for 7 days. During DSS exposure, mice were orally treated daily with either recombinant LAB (either Lactococcus lactis or Lactobacillus casei) expressing Elafin, SLPI or IL-10, or with wildtype (wt) LAB as a control. A L. lactis strain inactivated for the extracellular housekeeping protease HtrA was also used to express Elafin and tested in the same model, to potentially increase the levels and quality of secreted proteins and thus activity of recombinant Elafin. At day 7, colonic tissues were analyzed for macroscopic damage scores, cytokines secretion and myeloperoxydase activity. Proteolytic activity (trypsin-like and elastase) was also assessed in colon washes. Results: Mice were significantly protected against colitis by recombinant LAB strains producing protease inhibitors, while wt LAB failed to modify any parameters of inflammation. All LAB strains producing Elafin and SLPI were more efficient at decreasing intestinal inflammation induced by DSS treatment than the ones producing IL-10. The protective effects of L. lactis expressing Elafin were enhanced when htrA gene was inactivated. Conclusion: Oral treatments with recombinant LAB producing Elafin or SLPI restored the proteolytic balance and protected mice from colitis. Food-grade LAB could be used as carriers of beneficial proteins such as protease inhibitors (Elafin and SLPI) for their delivery in the gut. Such recombinant bacteria could be considered as effective treatments to reduce inflammation in IBD. Disclosure of interest: None declared. Inflamm. Res. Nakajo-Nishimura syndrome (NNS) (MIM 256040) is a disorder that segregates as an autosomal recessive fashion and shows autoinflammatory symptoms including periodic fever, partial lipodystrophy, contracture of joints, skin rash and calcification of basal ganglia. We detected the gene locus using the SNP microarray-based homozygosity mapping following to determine the affected gene by direct sequencing. A mutation was found in a gene encoding the proteasome subunit in NNS patients. As predicted by structural modeling, fractioned analysis revealed assembly defect of proteasome and decrease of all 3 different peptidase activities (chymotrypsin-like, trypsin-like, and caspase-like activities). Accumulation of ubiquitinated proteins were detected in the cells and skin biopsy sections from patients with NNS by western blotting and immunohistochemistry. The productions of inflammatory cytokines were increased in cultured NNS cells compared with controls, as increased concentrations in sera from patients with NNS. Our findings reveal that decrease of proteasome activity is associated with a novel mutation of the proteasome subunit in NNS. The Ca 2+ -binding proteins S100A9 and S100A8 are predominantly expressed by neutrophils and monocytes/macrophages. Once secreted, these proteins act as damage-associated molecular patterns (DAMPs) by triggering inflammatory responses. Indeed, previous studies showed that S100A9 induces leukocyte activation and promotes their migration toward inflammatory sites. Interestingly, high levels of S100A8 and S100A9 are found in the serums and at inflammatory sites of patients with chronic inflammatory diseases like rheumatoid arthritis, and these levels correlate with disease activity. In this study, neutralizing antibodies were used in the murine collagen-induced arthritis model to investigate the importance of S100A8 and S100A9 in rheumatoid arthritis. Anti-S100A8 or anti-S100A9 mAbs were injected twice a week starting 1 day before arthritis induction. Anti-S100A9 injections led to a 50% reduction of disease intensity while anti-S100A8 injection caused a worsening of the disease. The anti-S100A8-associated results are consistent with the anti-inflammatory properties of S100A8 protein after its oxidation. Lower bone destruction was observed in anti-S100A9-treated animals compared to the isotype control group. Cytokine production was also decreased as exemplified by a marked reduction of IL-6 in serum of anti-S100A9-treated animals. Considering that human S100A9 induces the production and secretion of various cytokines like IL-6, MCP-1, MIP-1a, IL-1b, and TNF-a by human peripheral blood mononuclear cells, we suggest that S100A9 amplify the immune response through the production of pro-inflammatory soluble factors. Treatment with monoclonal anti-S100A9 may breach the amplification of the immune response by reducing pro-inflammatory cytokine secretion and leukocyte migration, leading to less tissue destruction. Our results show that S100A9 is a potential therapeutic target in inflammatory diseases unlike S100A8 which seems to be relevant in the resolution of inflammation. Background and Aim: SRT2104 is a highly selective small molecule activator of the silent information regulator transcript (SIRT1) that has broad anti-inflammatory effects in cell cultures and rodents. The aim of the current study was to determine the effect of SRT2104 on the inflammatory and coagulation responses in normal healthy male subjects after exposure to LPS. Methods: This double-blind, placebo-controlled study consisted of three treatment arms (N = 8 per arm): (1) Oral SRT2104 (2 g/day) for seven consecutive days; (2) placebo on days 1-6 and SRT2104 (2 g) on day 7; (3) placebo for seven consecutive days; on day 7, all subjects received intravenous LPS 3 h after dosing with SRT2104/ placebo. Results: SRT2104 significantly attenuated LPS-induced release of IL-6 (p = 0.0777 and 0.0318 for groups 1 and 2 vs. group 3, respectively) and IL-8 (p = 0.0470 and 0.0249, respectively). There was a trend toward lower TNFa and IL-10 levels. SRT2104 attenuated coagulation activation reflected by significantly lowered plasma levels of the prothrombin fragment F1 + 2 concentration (p = 0.0384 and 0.0368, respectively). SRT2104 did not impact LPS-induced changes in gene expression profiles of blood leukocytes as determined by whole genome arrays performed before and 4 h after LPS. CRP levels measured 24 h post-LPS were significantly lower in the SRT2104 group 2 versus placebo (p = 0.0179). Clinical signs associated with LPS tended to be reduced after SRT2104 administration. AEs were primarily those expected following LPS. Conclusion: This is the first clinical study to demonstrate biological responses consistent with the activation of SIRT1 by a small molecule. SRT2104 was shown to significantly attenuate LPS-induced IL-6 and IL-8 release and activation of coagulation. The effects on coagulation are differentiated from those previously observed for steroid agents in a similar setting and suggest a unique potential as a therapeutic strategy for inflammatory disease. Disclosure of interest: None declared. Anticalins are a new class of therapeutic proteins based on human lipocalins. We reasoned that given the diversity of compounds that these molecules can naturally bind, it should be possible to engineer and identify Anticalins with specificity for IL-4Ra, a receptor known to play a key role in allergy and asthma pathogenesis. Methods and results: Phage display technology was used to isolate a human tear lipocalin-derived Anticalin to specifically recognize human IL-4Ra with low picomolar potency. The protein can be expressed at high yield using a scalable E.coli process and exhibits drug-like properties (Tm of 64°C + refolding after thermal denaturation) and resistance to multiple stress conditions. A dissociation constant of Kd = 14 pM was measured for the interaction with human IL-4Ra by surface plasmon resonance (SPR). The crossreactivity profile, competitive antagonistic properties and potency towards both the IL-4Ra/gamma and the IL4Ra/IL13Ra receptor complexes were confirmed in FACS-based binding studies using transfected cells and multiple disease relevant cell-based assays. Furthermore, Anticalins can also be effectively delivered by inhalation to the lung, due in part to their relative small size of 17 kDa and robust biophysical properties, and exhibit an extended pulmonary half-life in rodents. Conclusions: Our data describe the discovery and characterization of an inhalable IL-4Ra Anticalin (PRS-060), a potential therapeutic for the treatment of allergic diseases, including asthma. PRS-060 displays numerous differentiating features posited to be relevant for the proposed therapeutic concept, including, but not limited to inhibition of IL-4 and IL-13-dependent pathways with high potency, local administration and action in the lung via the inhaled route, favourable safety and tolerability (human scaffold, lack of immune effector cell interactions due to the absence of an Fc domain), robustness (dry powder formulation) and low production costs. The aim was to scrutinize gene activation following neutrophil in vivo extravasation and IL-1 in vitro stimulation in healthy study subjects and to address the question if neutrophils render a potential to tune the composition of inflammatory mediators. In vivo extravasated neutrophils were collected by the skin chamber method and compared to circulating neutrophils following a density separation. Gene expression was assessed by gene array and confirmed by quantitative PCR. Protein expression of selected markers was assessed by flow cytometry, immunoelectron microscopy and ELISA. Expression of the IL-1 receptor type 1 (IL-1R1) increased following extravasation at both gene and protein level. Flow cytometry indicated an increased surface expression that was further induced following stimulation. Immunoelectron microscopy confirmed an increased pool of IL-1R1 both at the cell surface as well as intracellular, following extravasation. Furthermore, NFkB1 and several chemokines regulated by NFkB such as CCL3, CCL4, CCL20 and CXCL2 were up-regulated following extravasation, and the corresponding chemokines were locally produced at high concentrations at the inflammatory site. In addition, IL-1 in vitro activation of purified granulocytes induced an increased gene expression of NFkB, CCL3, CCL4, CCL20 and CXCL2, similar to gene activation following extravasation. Furthermore, a release of CCL3 and CCL4 was detected following 5 h of in vitro IL-1 stimulation. The present results indicate that neutrophils could tune the proinflammatory milieu following extravasation and IL-1 stimulation by the production of chemokines. This renders the neutrophil a potential to bridge the consecutive accumulation of mononuclear leukocytes. Disclosure of interest: None declared. Crotoxin, the main toxin of Crotalus durissus terrificus rattlesnake venom presents long-lasting anti-inflammatory properties. A single dose of crotoxin inhibits paw edema, leukocyte-endothelium interaction and consequently cell migration induced by carrageenan. Leukocyte-endothelium interaction induced by carrageenan involves adhesion molecules expression, such as P-Selectin and ICAM-1. The aim of this study was to demonstrate histologically the inhibitory effect of crotoxin on cell migration to the subcutaneous tissue of footpads injected with carrageenan, and investigate the effect of this toxin on ICAM-1 and P-Selectin expression at this tissue. A single dose of crotoxin (0.89 lg/50 lL s.c.) or saline (control) was administered 1 h before carrageenan (300 lg/50 lL) or saline intraplantar injection in mice. For histological analysis, the footpad was removed 6 h after carrageenan. The tissue was processed and sections were stained with H/E. To evaluate P-Selectin and ICAM-1 expression, the footpad of the treated animals with crotoxin was removed 30 min or 1 h after carrageenan and analyzed by immunohistochemical. Tissues were incubated with antibody against these adhesion molecules and incubated with secondary antibody peroxidase conjugate. Histological analyses showed that the treatment with crotoxin (1 h) reduced edema and cellular infiltration when compared to control. The same treatment decreased the expression of P-Selectin and ICAM-1 when compared to control. These results suggest that the inhibitory effect of crotoxin on cell migration involves the action of this toxin on the expression of these adhesion molecules plays a key role in inflammation. These results contribute to characterization of anti-inflammatory properties of the crotoxin and reinforce that this toxin is a potential natural product in controlling inflammatory diseases. Supported by: CAPES, FAPESP and INCCTOX. Disclosure of interest: None declared. Introduction: The fundamental role of serine proteases and their inhibitors on pathophysiological processes has been evidenced. The mechanisms of action of Amblyomin-X, a Kunitz-type serine protease inhibitor designed from the cDNA library of the Amblyomma cajennense tick, were here characterized in the vascular endothelial growth factor (VEGF)-induced angiogenesis. Methods: In vivo intravital microscopy assay was carried out in the dorsal skin chamber in male Swiss mice and optical microscopy evaluation was performed in the chick embryo chorioallantoic membrane (CAM). t-End cell lineage was simultaneously treated with amblyomin-X and VEGF and employed to quantify cell migration and tube formation by optical microscopy; cell adherence to matrigel by colorimetric assay; apoptosis, necrosis, proliferation, cell cycle and adhesion molecules expression by flow cytometry. Results: Topical application of Amblyomin-X, simultaneously to VEGF, inhibited the VEGF-induced angiogenesis in the mice dorsal subcutaneous microvasculature and in the CAM. Treatment of t-End with Amblyomin-X delayed the cell cycle by maintaining them in G0/ G1 phase; inhibited cell migration, adherence, proliferation, ICAM-1, VCAM-1 and PECAM-1 protein expressions. Conclusion: Amblyomin-X blocks the in vivo VEGF-induced angiogenesis by affecting endothelial cells functions involved in different steps of the process. Besides contributing to the comprehension of pathophysiological mechanisms of Kunitz type-serine protease inhibitors, data herein have shown support further studies focusing on Amblyomin-X as a scientific tool to the design of therapeutic molecules. Financial support:FAPESP (08-57850-8; 08/56072-1). Disclosure of interest: None declared. Circulating polymorphonuclear leucocytes are recruited in the wound by soluble mediators, causing cell adherence, transcapillary migration and chemotaxis. In a later stage T cells play a modulatory role in wound healing, although the full range of their effects is incompletely understood. There is no evidence that B lymphocytes play a significant role in wound healing. Aim. The question arises which and when immune cells and their specific subsets accumulate in the fast and delayed healing wounds. This applies particularly to lower limb non-healing ulcers. Methods. In order to define which blood immune cells reveal predilection for wound cells or matrix, an in vitro adherence test was worked out. Briefly, cryopreserved tissue sections were covered with blood leukocyte suspension at 4°C, incubated for 30 min, the non-adherent cells were washed out. This test allowed to show (a) adhesion to fibroblasts, keratinocytes, endothelial cells, matrix, (b) phenotypes of adhering cells stained with monoclonal antibodies. Venous ulcer edge biopsy specimens obtained from 15 patients and skin fragments of five healthy undergoing various vein surgery were studied. Results: Ulcers. Neutrophils adhered to granulation tissue around capillaries and to matrix, forming clusters. Few adhered to fibroblasts. They also stuck to epidermis. CD68+ve and elastase+ve monocytes were evenly distributed close to capillary lumen. Few scattered irregularly distributed CD3 T cells were seen on granulation tissue but none on epidermis. On normal skin section adherence of neutrophils, monocytes and lymphocytes was almost nil. Interestingly, all cell types strongly adhered to glass. Conclusions. Preponderance of neutrophils and monocytes over lymphocytes on granulation tissue sections suggests presence of a signal for their accumulation as in the scavenging phase of wound healing. The signal might be microbes and autoimmune tissue-antigen-specific cohorts of granulocytes and lymphocytes. Disclosure of interest: None declared. CD1d-restricted invariant natural killer T (iNKT) cells play an important role in the regulation of diverse immune responses, including allergy, autoimmunity, and cancer. MicroRNAs (miRNAs), a class of 21-25 nt single-stranded non-coding small RNAs, are increasingly being recognized as important regulators of gene expression through the inhibition of effective mRNA translation. We recently reported that miRNAs expressed in iNKT cell lineage are potent regulators of iNKT cell development, maturation, and function. However, the role of specific miRNAs in the iNKT cells has not been addressed yet. Using miRNA arrays, we found that the expression of miR-150 was significantly upregulated in mature iNKT cells compared to immature iNKT cells in the thymus. Interestingly, mice with miR-150 deficiency have a defect in iNKT cell development in the thymus and iNKT cell maturation was blocked at developmental stage 1 and 2, while the peripheral iNKT cells maturated normally. Furthermore, miR-150-deficient peripheral iNKT cells produce more cytokine interferon-gamma compared to that of wild type after PMA/ionomycin stimulation. Thus, our results suggest that miR-150 controls iNKT cell thymic maturation and regulates iNKT cell function. Disclosure of interest: None declared. Pharmacology, Tabriz University 0f Medical Science, Tabriz, 2 Immunology, 3 Pharmacology, Tehran University of Medical Science, Tehran, Islamic Republic of Iran Introduction: Most pharmacological effects of Metformin, the antidiabetic drug is mediated through activation of Adenosine Monophosphate-activated Protein Kinase (AMPK). AMPK is a key sensor of cellular AMP/ATP ratio. AMPK activation by Metformin has a number of other biological effects including inhibiting of mammalian Target of Rapamycin (mTOR) as a key signaling process in cells that regulates cell growth, cell proliferation, cell motility, protein S84 Inflamm. Res. synthesis, and transcription. Recent epidemiological studies demonstrate that Metformin lowers the risk for several types of cancer in diabetic patients. Concerning the critical role of angiogenesis in the incidence and progression of tumors, we investigated the effect of Metformin on Human Umbilical Vein Endothelial Cells (HUVECs) proliferation and migration and its possible mechanism by inhibiting AMPK signaling using Compound C. Method: We studied the effect of Metformin on morphology and viability of HUVECs using MTT and LDH assays, proliferation by MTT and cell numbering, and migration by wound repair method. Moreover, the effect of Metformin on VEGFR2 expression was evaluated by flow cytometry. Metformin at concentrations of 0.5-3 mM effectively reduced the number of endothelial cells by 5.5-55%, without being cytotoxic to these cells. Similarly, the cell proliferation and migration were markedly (P \ 0.001) inhibited by Metformin. The inhibitory effects of Metformin on the endothelial cells number and migration were reversed partially by Compound C (P \ 0.01). Interestingly, compound C (10 mM) alone showed a slight but significant inhibitory effect on the endothelial cells number (16%) and migration (16.5%). Metformin at the studied concentrations had no significant effect on VEGFR2 expression. This study indicates that Metformin exerts potent anti-angiogenic effects on HU-VECs and AMPK activity, at least in part, is required for this effect. Ficolins are a family of pattern-recognition molecules, which interact with microorganisms surface and trigger complement activation. M-ficolin has been identified in lung epithelial cells, in monocytes and in easily mobilizable granules of neutrophils. A peculiarity of M-ficolin is its ability to bind sialylated ligands on autologous neutrophils or monocytes, but the physiological function of this binding is unknown. Our aim was to analyze the responses induced by M-ficolin interaction with neutrophils and to evaluate whether leukosialin (CD43), a membrane sialoglycoprotein related to cell adhesion, aggregation and migration, is a potential target receptor for the M-ficolin. We confirmed that biotinylated rM-ficolin binds to neutrophils via a calcium-dependent interaction inhibited by N-acetylglucosamine (GlcNAc), thus involving its capacity to bind acetylated ligands including sialic acid. Interestingly, binding of rM-ficolin inhibited neutrophils labeling with anti-CD43 mAb, but not with antibodies to another membrane glycoprotein, CD66. The effect of rM-ficolin on CD43 immunolabeling was abolished by GlcNAc but not by lactose, thus confirming the steric hindrance of CD43 by exogenous M-ficolin. Fluorescence microscopy showed an even distribution of biotinylated-rM-ficolin on resting neutrophil surface at 4°C but a polar redistribution at 37°C, similar to the antibody-induced capping of CD43. Exposure to recombinant M-ficolin triggered neutrophil functions, such as aggregation, adhesion and respiratory burst in a dose-dependent manner, effects already described for CD43 antibody cross-linking. The Y271F mutant of M-ficolin, unable to bind sialic acid, was unable to enhance neutrophil activation and did not prevent access of anti-CD43 mAb to its antigen. These results strongly suggest that CD43 is a receptor for M-ficolin and show that M-ficolin interaction with the cell surface, possibly via the cross-linking of CD43, induces potent neutrophil activation. Disclosure of interest: None declared. Knock-out mice from experimental inflammatory diseases shed new light on an important participation of the complement alternative pathway in neutrophil-mediated diseases such as rheumatoid arthritis, membranoproliferative glomerulonephritis or vasculitis with antineutrophil cytoplasmic autoantibodies (ANCA). We have recently described an activation of the complement alternative pathway by TNF-stimulated neutrophils (PMN). We now analyzed the consequences of this activation. PMN, stimulated or not with TNF, were incubated with normal human serum or sera immunodepleted in C5, C2 or factor B, to distinguish classical and alternative pathways involvement. PMN activation was assessed by flow cytometry measurement of CD11b expression or oxidative burst. Complement activation on the surface of PMN, stimulated by coagulation-derived products or by TNF, further activated PMN, enhancing the degranulation and oxidative burst. Complement-induced PMN activation was inhibited by a C5aR-antagonist and in C5-depleted serum, emphasizing the role of C5a and possibly C5b-9 complexes, detected on TNF and serum-activated PMN. Anti-MPO or -PR3 ANCA amplified complement activation exclusively on TNF-stimulated adherent PMN a condition known to allow the access of ANCAs to their antigens. This activation involved the classical pathway but amplification by the alternative pathway was required. Microparticles, released by PMN in vitro and highly present in plasma of acute vasculitis patients, efficiently activated the complement alternative pathway. In conclusion, PMN activate the complement alternative pathway, which, in turn, amplifies PMN pro-inflammatory responses, thus representing a new amplifying loop in the inflammation process. This mechanism probably explains the complement involvement in PMN-mediated diseases such as ANCA-associated vasculitis. Disclosure of interest: None declared. Snake venom metalloproteinases (SVMP) are classified as P1-P3, according to their domains composition and are involved in local inflammatory reactions observed in envenomed victims. Using three SVMPs, we evaluated the participation of different domains in alterations of the leukocyte-endothelial interactions induced by Bothrops venoms in the microcirculation of the cremaster of mice. We also evaluated, by immunofluorescence and ELISA, the expression of ICAM-1 (CD54) and PECAM-1 (CD31), molecules responsible by leukocytes adhesion and migration, respectively. The toxins used were: Jararhagin (JAR), a P3 SVMP with a strong hemorrhagic activity, which has catalytic, disintegrin-like, and cysteinerich domains; JAR-C, a degraded form of JAR devoid only of the catalytic domain, with no hemorrhagic activity; and BnP1, a weakly hemorrhagic P1 SVMP, which has only the catalytic domain. Toxins (0.5 lg) or PBS (100 lL) were injected into the scrotal bag of mice. Adhered and emigrated leukocytes were counted in a post-capillary venule by intravital microscopy 2, 4 or 24 h after the injections. The number of adhered and emigrated cells increased in all times studied. Adhered cells diminished after 24 h of the toxins injection when compared to 2 h, but emigrated cells significantly increased in this period. There was a significant increase in the expression of CD54 after 2 and 4 h, followed by a decrease 24 h after the injection. We Introduction: Translocator protein (TSPO) is found in peripheral cells, as immune and endothelial cells, and may be related to the benzodiazepine actions on immune system. This worked investigated TSPO-binding drugs effects on leukocyte-endothelial interactions and on adhesion molecules expressions. Methods: Intravital microscopy assays were performed in the mesentery microcirculation of male Wistar rats after topical application of TSPO ligands (PK 11195 or Ro 5-4864; 100 nM) and fMLP (10 -8 M; topical application). In other set of assays, circulating leukocytes were in vitro incubated with TSPO ligands (100 nM) and 1 h after incubated with fMLP (10 -8 M) to quantify adhesion molecules expressions by flow cytometry. Cells were also pre-treated with nifedipine (100 nM) to investigate the role of intracellular calcium on TSPO ligands effects on L-selectin expression. All procedures were performed according to protocols approved by the Brazilian Society of Science of Laboratory Animals for proper care and use of experimental animals. Results: fMLP stimulation decreased rolling cells and increased adherent cells, and Ro5-4864 pre-treatment abolished these effects. fMLP in vitro stimulation reduced and enhanced L-selectin and b2integrin expression on neutrophils, respectively, and Ro5-4864 treatment inhibited the reduction on L-selectin expression caused by fMLP. Pre-treatment with nifedipine reversed Ro5-4864 effects on L-selectin expression after fMLP stimulation. Conclusions: Results herein presented show that TSPO ligand alters in vivo leukocyte-endothelial interactions evoked by fMLP. And more, Ro5-4864, but not PK11195, blocks the fMLP effect on neutrophil L-selectin expression depending on intracellular calcium levels. Introduction: Translocator protein (TSPO) involvement on benzodiazepine's actions on immune system seems to be dependent on endogenous glucocorticoids actions. This work evaluated the effects of TSPO-binding agents and the participation of the cytosolic glucocorticoid receptor (CGR) on leukocyte-endothelial interactions. Methods: RU 38486 (10 mg/kg; i.p.; 18 and 1 h before experiments; RU) or vehicle-treated (VT) adult male Wistar rats were anaesthetized and intravital microscopy assays were performed in the mesentery microcirculatory network after topical application of RPMI or TSPO ligands PK 11195 (100 nM) or . In other set of assays, circulating leukocytes were obtained from VT or RU-treated animals, and in vitro incubated with TSPO ligands (100 nM) to quantify adhesion molecules expressions by flow cytometry. All procedures were performed according to protocols approved by the Brazilian Society of Science of Laboratory Animals for proper care and use of experimental animals. Results: RU treatment enhanced the number of rolling and adhered cells, and Ro5-4864 treatment reduced only the number of rolling cells. RU treatment also enhanced L-selectin and decreased b2-integrin expressions in circulating neutrophils, and only L-selectin enhancement was reversed by both TSPO ligands. RU treatment did not affect adhesion molecules expression on circulating lymphocytes, but additional incubation with in vitro Ro5-4864 decreased L-selectin and b2-integrin expressions. Conclusions: TSPO ligands interfere with in vivo leukocyte-endothelium interactions evoked by RU treatment, which may be dependent on alterations on adhesion molecules expression. Therefore, an interaction of CGR and TSPO may be proposed on leukocyte-endothelial interactions. Financial support:Fapesp 09/52245-1. Disclosure of interest: None declared. Inflamm. Res. The development of CD4 + memory T lymphocytes during immune responses is not completely understood. In this study, to define the molecular signature of CD4 + memory T cells, we investigated the gene expression profile and methylome of antigen specific memory CD4 + T cells generated using T cells from DO11.10 OVA specific TCR transgenic mice. More than 12 million 5 0 -end tags were sequenced from naïve effector and memory T cell libraries with a next generation sequencer, corresponding to approximately 12,000-14,000 different protein-coding genes in this single cell type. Several novel up-and down-regulated genes were found in memory CD4 + T cells, which are possible candidate genes related to development and maintenance of the memory phase. In addition, when the genome-wide DNA methylation profile was investigated in memory CD4 T cells, 466 DMRs (differentially methylated regions) in 438 genes were identified. Interestingly, most DMRs in naïve and memory T cells were demethylated during T cell differentiation. Moreover, the majority of the DMRs were localized to intronic sites and not to CpG islands. Among these DMRs, only 5% were correlated with expressed genes that related to ''signal transduction'', ''cell communication'' and ''immune response''. Moreover, these DMR were different between various T cell subsets, indicating that the methylation status of T cell subsets reflects T cell conditions. Our findings indicate that the genes associated with DMR are key genes related to memory phenotype that undergo variable changes in DNA methylation during CD4 + T cell differentiation. Disclosure of interest: None declared. The present study examined the diverse role of mediators from C sensory neurons and mast cells (MC) in paw inflammation induced by intraplantar (i.pl.) injection of Concanavalin A (Con A) in Dark Agouti (DA) male rats. Depletion of C sensory neurons was performed by neonatal administration of capsaicin, while systemic depletion of MC was performed by successive injections of increasing doses of compound 48/80 in adult rats. The result showed that neonatal depletion of C sensory neurons diminished Con A-induced increase in paw diameter and plasma extravasation, while MC depletion increased both parameters. Although opposing effects on paw diameter and plasma extravasation were recorded, both treatments similarly reduced the influx of immune cells to inflamed paws. However, both treatments led to the decrease in proportion of T cells and increase in proportion of resident macrophages in draining lymph nodes (DLN). According to results, neuropeptides secreted from activated C sensory neurons have pivotal role in acquiring local inflammatory response, influencing both vascular and cellular components. On the other hand, mediators released from MC are not indispensable for inflammatory edema formation, but are critical for cell recruitment to the inflammatory focus. Mediators secreted either from C sensory neurons or MC also affect microenvironment in DLN during Con A-induced inflammation. Diverse evidence supports the hypothesis that in hemorrhagic and hemolytic episodes free heme might be responsible for the inflammatory response in the lung. Starting to the premise that the alveolar macrophages (AM) is the main defender of lung environment, we investigated the effects of free heme (ferriprotoporphyrin IX) in alveolar macrophage activation. Heme stimulation is able to increases AM responsiveness, enhances IL-1b, IL-6 and IL-10 release, induces HO-1 expression and stimulates ROS production by rat AM. In addition, heme stimulation induces p47 phox phosphorylation, 5-LO and FLAP expression, as well as NF-kB nuclear translocation, suggesting that these proteins can be related to pro-inflammatory effects by heme in AM. All these events are directly involved in microbicidal functions of macrophages and prompted us to investigate the role of heme in these functions. In this study, we observed that heme induces phagocytosis and bacterial killing, key events responsible by pathogens removal. Taken together, these results suggest a role for heme as a proinflammatory agent able to induce antimicrobicidal activity in alveolar macrophage. The understanding of the effect of heme in pulmonary inflammatory processes can lead to the establishment of new strategies to ameliorate tissue damage associated with hemorrhagic episodes and severe hemolysis. Galectins are structurally related proteins characterised by their carbohydrate recognition domains and an affinity for b-galactosides. Galectin-9 (Gal-9) was initially characterized as a potent eosinophil chemoattractant but has subsequently been found to have profound immunosuppressive effects in models of collagen-induced arthritis, acute graft versus host disease and carrageenan paw oedema. Its effects on neutrophil recruitment and behaviour during inflammation have not been thoroughly investigated. The objectives of the present study were therefore to investigate whether Gal-9 modulates neutrophil recruitment during the acute inflammatory response. To determine the effect of Gal-9 on leukocyte trafficking in vivo a model of zymosan peritonitis was performed. Mice were pre-injected i.p. with Gal-9 (1-30 lg) followed by 0.2 mg zymosan. Peritoneal lavages were collected and leukocyte influx assessed. To pinpoint whether Gal-9 affects a specific step of the leukocyte recruitment cascade flow chamber assays were performed. Freshly isolated neutrophils were incubated with Gal-9 (3-30 nM) and perfused over endothelial monolayers. The number of interacting neutrophils were quantified in six random fields. The effect of Gal-9 on neutrophil annexin V expression, ERK phosphorylation and adhesion molecule expression was also assessed. Pre-treatment with Gal-9 resulted in a significant increase in the numbers of neutrophils and monocyte/ macrophages recruited into the peritoneal cavity. The flow chamber experiments identified a specific effect of Gal-9 on neutrophil transmigration with a concomitant reduction in neutrophil rolling, significant at 10 nM. In contrast to other galectins, Gal-9 was found to induce Annexin V exposure in the absence of other activating stimuli and also induced ERK phosphorylation. These data indicate a role for Gal-9 in neutrophil recruitment during acute inflammation. Disclosure of interest: None declared. , on the other. We found that there were 4,206 genes (20%) different between BMM and GM-BMM, but only 17% of these differentially expressed genes were identical across the species. By gene ontology (GO) classification, 34 and 41% of differentially expressed GO categories in the mouse and human gene lists, respectively, were common across the species; however, 84% of the most significant 50 differentially expressed GO categories were common. By molecular pathway analysis using the Pathway Interaction Database (http://pid.nci.nih.gov/), 57 (77%) NCI-Nature and 37 (82%) BioCarta differentially expressed pathways were common across the species. RT-PCR was used to confirm these patterns. In conclusion, BMM and GM-BMM would appear to be reasonable models for human monocyte-derived macrophage populations when relative gene expression in response to M-CSF and GM-CSF is analyzed at the ''pathway'' level rather than at the level of individual genes. Disclosure of interest: None declared. Objective: Visfatin is a novel proinflammatory adipokine secreted from adipocytes, which has been linked to atherosclerotic plaque formation, recently. However, controversial results exist about the role of visfatin in the pathogenesis of atherosclerosis. Therefore, we investigated whether visfatin could regulate monocyte/macrophage differentiation and expression of scavenger receptors (SRs), containing CD36, SR-A, and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1), responsible for foam cell formation in macrophages, a critical step in atherogenesis. Methods and Results: The THP-1 monocytic cells were differentiated into a macrophage-like phenotype by incubation with phorbol-12myristate-13-acetate (PMA) in the presence or absence of visfatin. In this study, we show that visfatin enhanced the oxLDL uptake by inducing CD36 and LOX-1 expression, but not SR-A, and led to the production of inflammatory cytokines such as IL-1b, IL-6, and TNF-a. We also found that PMA-induced THP-1 cells became more adherent with a concomitant increase of ICAM-1 protein in the presence of visfatin. Moreover, immunofluorescence microscopy revealed that visfatin induces peripheral membrane ruffling, spreading and actin polymerization, suggesting that visfatin alters morphology and cytoskeletal organization. Visfatin-induced effects were involved with JNK pathway as determined by inhibition with JNK inhibitor, SP600125 and we also observed activation of JNK by visfatin. Conclusions: Taken together, these data suggest that visfatin promote PMA-induced THP-1 cell differentiation into trapping macrophage phenotype and contribute to macrophage foam cell formation by increasing CD36 and LOX-1 expression and inflammatory cytokine production, consequently leading to the progression of atherosclerosis. Disclosure of interest: None declared. regulation of the leukocyte migration in response to inflammatory stimuli has not been fully elucidated. We assessed the effects of nicotinamide on neutrophil recruitment in the pleural cavity of mice, and the microcirculation of the murine cremaster muscle using intravital microscopy. BALB/c mice were treated with nicotinamide per os (Nic, 1,000 mg kg -1 ) 30 min before and 1 h after challenge with an intrapleural injection of carrageenan (Cg, 200 lg/cavity), zymosan (Zy, 200 lg/cavity), or leukotriene B 4 (LTB 4 , 500 ng/cavity). Neutrophil migration was assessed 4 h after the challenge through pleural wash. To perform intravital microscopy studies, BALB/c mice were pretreated with Nic (1,000 mg kg -1 ) 30 min before and 1 h after challenge with Cg (100 lg directly added to cremaster muscle). Rodamine 6G (0.1 mg/ml; i.v.) was injected prior to light fluorescence microscopy. The number of rolling and adherent leukocytes was determined during video analysis. Nic (1,000 mg kg -1 ) inhibited the neutrophil recruitment induced by Cg (PBS, 1.9 ± 0.6; Cg, 27.1 ± 5.6; **Cg + Nic, 5.4 ± 0.6), Zy (PBS, 0.04 ± 0.01; Zy, 113.7 ± 22.9; ***Zy + Nic, 0.18 ± 2.7), or LTB 4 (PBS, 0.08 ± 0.02; LTB 4 , 6.4 ± 1.5; ***Nic + LTB 4 Exposure to cadmium is associated with the development of pulmonary damage such as emphysema and lung cancer. This metal is also a powerful inducer of different proinflammatory and cell cycle regulatory proteins in many biologic models. Previously we showed that prolonged exposure of low concentration of cadmium resulted in up regulation of proinflammatory cytokines and cell cycle regulatory molecules in mice lung cell. The present study was undertaken to determine molecular mechanism of inflammation and its relation to cell proliferation in a transformed human lung adenocarcinoma epithelial cell line (A549) in response to cadmium chloride. In comparative studies, we examine that short-duration exposure to lower doses of cadmium significantly increase the growth of A549 cells, whereas, higher doses are toxic and cause cell death. We also observed that cadmium induced elevated expression of epidermal growth factor receptor (EGFR) along with different proinflammatory cytokines like IL-1b, TNF-a, IL-6. The possible occurrence of cell proliferation events was evaluated via analysis of the physical state of the DNA and the expression of Ki67 and PCNA. We also checked the pattern of expression of different cell cycle regulatory molecules involved in the onset of cell proliferation. Our results indicate that cadmium treatment appears to induce inflammatory and growth responses in transformed A549 cell line by activating epidermal growth factor receptor and its downstream modulators. These results may contribute to better understand the toxic mechanism of cadmium moreover; the expression profile of cadmium induced regulatory molecules could provide potential biomarkers for cadmium exposure. Disclosure of interest: None declared. Chronic allograft nephropathy (CAN) is a major obstacle to the establishment of survival of the transplanted kidney. The trait most associated with the decline of renal function in this pathology is the interstitial tubule fibrosis (FTI) and tubular atrophy (TA). Recent studies suggest that certain interstitial myofibroblasts derive from renal tubular cells that have undergone the process of epithelialmesenchymal transdifferentiation (EMT). In this study, the objective was to induce the transdifferentiation of epithelial cells derived from human proximal tubular epithelial (HK-2), using as inducer hydrogen peroxide. Methods: To this end, epithelial cells were incubated with Keratinocyte-SFM medium (GIBCO) with 5% fetal calf serum (GIBCO), deprived of serum during the experiment. Cells were treated with concentrations of H 2 O 2 (0.10, 0.25, 0.50, 1.0 and 2.0 mM/mL) and evaluated in the time of incubation for 24, 48 and 72 h for induction of EMT by oxidative stress. TGF-b was used in concentration (10 ng/mL) as positive control. Cells without treatment were used with negative control. Evidence was given by EMT microscopy, cell viability, cell motility and immunocytochemistry of molecules related to the process, such as cytokeratin, E-cadherin, vimentin and a-SMA. Results: We observed the cell viability assay that the better dose to sublethal condition was 0.25 mM/mL at 24, 48 and 72 h in relationship to control, but when tested with cell motility, the doses of 0.25 and 0.50 mM H 2 O 2 , when done for 48 and 72 h, provided the best results of migration, supported by immunocytochemistry analysis, in which cells are subjected to oxidative stress by altering the expression of surface proteins of epithelial cells begin to express proteins mesenchymal cells. Studies from our laboratory have identified the macrophage F-actinassociated and tyrosine phosphorylated protein (MAYP), also known as proline, serine, threonine, phosphatase-interacting protein 2 (PSTPIP2), as a regulator of actin bundling, filopodia formation and directed motility in macrophages (Chitu et al. 2005) . Two missense mutations, I282N(Lupo) and L98P(cmo), in the MAYP/PSTPIP2 gene lead to autoinflammatory disease in mice characterized by skin inflammation, cartilage and bone destruction (Grosse et al. 2006; Chitu et al. 2009 ). This phenotype closely resembles human chronic recurrent multifocal osteomyelitis (Ferguson et al. 2006) . PSTPIP2 is expressed predominantly in macrophages and osteoclasts (Chitu and Stanley 2007) and regulates their differentiation and activation in vitro and in vivo. Absence of PSTPIP2 in cmo mice leads to the expansion of myeloid progenitors thus priming macrophage and osteoclast differentiation. In addition, compared to wt macrophages, cmo macrophages produce more osteoclastogenic cytokines in response to LPS challenge. Structure-function studies reveal that interactions of PSTPIP2 with PTP-PEST and membrane phospholipids are critical for its ability to control the development and activation of macrophages and osteoclasts. Disclosure of interest: None declared. Introduction: TLRs expressed in endothelial cells and leukocytes are pattern recognition receptors that detect invading microorganisms and sterile molecules to trigger immune and inflammatory responses. TLR activity is closely linked to the risk of many inflammatory diseases and immune disorders. Combined proteomic and transcriptomic approaches applied to analyze Thalassophryne nattereri venom complexity revealed the identity of the major toxins as a family of new proteins displaying kininogenase activity, the Natterins. Objective: In this study, we investigated the modulation of TLR activity by Natterins and the underlying mechanism. Methods and results: Interaction of leukocytes with cremaster microvessels was monitored by using an intravital microscope in Swiss, C57BL/6 (WT) and TLR2 KO and MyD88 KO and C3H/HePas (WT) and C3H/HeJ (TLR4 mutant) mice. LPS (0.02 lg/mL), KC (0.01 lg/mL) or PAR4 agonist (0.02 lg/mL) in 20 lL were administered topically in cremaster muscle after 6 h of treatment with Natterins (0.02 lg/mL) injected by intraescrotal route. When rolling behavior was determined after topical application of LPS or KC in mice pre-treated with Natterins, the proportions of rolling leukocytes in venules were almost the same in control-mice. In contrast, no inhibitory effect was observed in Natterinstreated mice challenged with topical application of PAR4 agonist. The anti-inflammatory effect of Natterins was not modified by metyrapone or ZnPP IX. Finally, the rolling of leukocytes induced by LPS or KC was not impaired in Natterins-treated TLR2 KO, MyD88 KO or TLR4 mutant mice, indicating that TLR2-or TLR4-MyD88-mediated signals are required for the anti-inflammatory effect induced by Natterins. Conclusion: These data provide a molecular level explanation for the anti-inflammatory function of Natterins in LPS-or KC-induced inflammation that is dependent on TLR2-and TLR4-MyD88 signaling. Support: FAPESP and CNPq. Disclosure of interest: None declared. Corneal neovascularization is a side effect of numerous inflammatory and non-inflammatory ocular surface disorders. During corneal injury, angiogenic factors are released from both corneal cells and infiltrating inflammatory immune cells. The angiogenic factors induce endothelial cells proliferation, migration, and tubulogenesis. Angiomotin, an angiostatin binding protein, is involved in endothelial migration during angiogenesis. Angiomotin protein is a key factor for endothelial migration. Therefore, blockade of Angiomotin function will leads to inhibition of angiogenesis. In this study we evaluated the expression of Angiomotin in alkali induced inflammatory corneal neovascularization in mice. A total of 20 BALB/c mice divided into two groups for this study. First, animals were anesthetized by administration of ketamine and xylazine combination intraperitoneally. Further topical anesthesia induced by 0.5% proparacaine hydrochloride on animals right cornea. Alkali burn corneal neovascularization induced by a silver nitrate cauterization technique. Mice were sacrificed 3 and 6 days post injury and eyes were enucleated for analysis. Corneas were removed and RNA was extracted using TRIzol reagent. The first-strand cDNA was synthesized from 1 lg of total RNA. Real-time PCR was performed using TaqMan gene expression master mix and specific primers and probes of Beta-actin and Angiomotin genes. The results showed that Angiomotin expression increases during corneal neovascularization. Angiomotin expression increased in both groups of animals and its expression was higher in day 6 in comparison to day 3. The results of this study confirm the over-expression of Angiomotin during inflammatory induced corneal neovascularization. Based on these results, we here propose this idea that Angiomotin can be a new target for designing new therapeutic option for corneal neovascularization. Disclosure of interest: None declared. K. J. Roberts 1,* , S. W. Edwards 1 , R. J. Moots 2 Exogenously added Nampt (100 ng/mL) delayed both constitutive neutrophil apoptosis, and the turnover of the anti-apoptotic protein Mcl-1. However, it did not have any significant effect on functions such as chemotaxis and the respiratory burst. Inhibition of the enzymatic activity of Nampt (using the inhibitor FK866/APO866 at concentrations from 1 to 100 nM) inhibited the activity of the respiratory burst in a time and dose-dependent manner. Inhibition of Nampt also significantly inhibited transcription of inflammatory cytokines such as TNFa. These data confirm the importance of Nampt in regulating neutrophil apoptosis, but reveal a new function for Nampt in controlling functions that would contribute to the inflammatory process. Therapeutic inhibition of Nampt in inflammatory diseases would be predicted to decrease the release of tissue-damaging reactive oxygen species and the release of pro-inflammatory cytokines from activated neutrophils. Disclosure of interest: None declared. Bothrops jararaca snake venom (BjV) is a complex mixture of biologically active proteins, such as proteinases, phospholipase A2, cysteine-rich secretory protein, lectin-like protein, C-type lectin and L-amino acid oxidase. Besides them, it have been identified a variety of pharmacologically active peptides in the low molecular weight fraction (Bj-LMWF) from BjV, such as Bradykinin-potentiating peptides, natriuretics peptides and disintegrins. It has been shown that BjV induces a significant leukocytes accumulation, manly neutrophils, at the local tissue damage. In the present study we investigate the contribution of Bj-LMWF to induce lung leukocyte influx after gastrocnemius intramuscular injection. The Bj-LMWF was obtained through a Millipore centrifuge filter device with a molecular weight cut-off 10KDa. To confirm the absence of proteolytic enzymes or others proteins ([10 kDa) of the venom, the filtrate contained the LMWF was analysed by SDS-PAGE silver stained, gelatinolytic activity and MALDI-TOF mass spectrometry. The lung leukocyte influx was induced in male Swiss mice by injection of BjV (1.2 mg/ kg) or Bj-LMWF (0.24 mg/kg), in gastrocnemius muscle. Cell influx was evaluated by bronchoalveolar lavage at 15 min, 1, 6 and 24 h after venom or Bj-LMWF injection. Results showed that BjV and Bj-LMWF evoked a significant cell accumulation in the lung from 6 up to 24 h following the injection. Differential cell counts from BjV mice demonstrated that neutrophils were the predominant cells accumulating at 6 h and mononuclear cells at 24 h. Bj-LMWF significantly increased the number of neutrophils at 6 h as well as mononuclear cells at 6 and 24 h. In conclusion, both BjV and Bj-LMWF have a similar activity as pro-inflammatory response to lung leukocyte migration. The fact that Bj-LMWF induces inflammation suggests that non-enzymatic peptides elicit inflammatory events perhaps by interacting with specific cell membrane acceptors. Lab Pathophysiology, 2 Lab Immunophatology, 3 Lab Immunochemistry, Butantan Institute, Sa˜o Paulo, Brazil Severe local inflammatory symptoms are common in envenoming induced by Bothrops jararaca (Bj) snakebites. It is known that metalloproteases is the main class of toxins involved in the inflammatory activity of this venom. A protein related to mammals high molecular mass kininogen (HK), purified from the plasma of Bj snakes was tested on inflammatory activity of Bj venom in vivo, using intravital microscopy. Bj venom (1 lg) or Jararhagin (JAR) (0.5 lg), a hemorrhagic metalloproteinase isolated from Bj venom, incubated or not with BjHK (2 lg) were injected (100 lL) into the scrotal bag of mice. The microcirculation of cremaster muscle was analyzed 2 or 24 h after the injections. We analyzed a segment of a post-capillary venule during 5 min and counted adhered and emigrated leukocytes, comparing with the observed in control group injected with sterile buffered saline. Results showed that, in groups injected with Bj venom or JAR, adhered and emigrated leukocytes were significantly increased in all times studied. Adhered cells were diminished after 24 h of the toxins injection, when compared to 2 h, but emigrated cells were significantly increased. When incubated with BjHK, the Bj venom or JAR did not induce alterations of leukocyte-endothelial interactions in the microcirculation. This inhibition was similar to the observed when Bj venom and JAR were treated with o-phenanthroline, despite of the mechanism of action of BjHK on the catalytic activity of JAR not be like a chelating agent. We can conclude that BjHK inhibits the inflammatory activity of the Bj venom by inhibiting the activity of their metalloproteinases. In human envenoming by South American rattlesnakes there are no inflammatory reactions at the site of the bite. Experimentally, the venom of the Crotalus durissus terrificus (CdtV) shows a significant anti-inflammatory action, inhibiting the acute inflammatory reaction induced by thioglycolate or carrageenan. Still, the CdtV alters some functions of macrophages, cells that in chronic inflammatory processes are fused to form multinucleated giant cells (MGC). This process depends on the participation of actin filaments (F-actin). Our objectives were to evaluate the effect of the CdtV on the dynamic of F-actin rearrangement on the formation of MGC in mice subjected to a chronic inflammatory stimulus and to assess the fraction of the venom responsible for the inhibitory effect on the formation of MGC. After an ion exchange chromatography of CdtV, three different fractions are obtained: PI, PII (corresponding to the pure crotoxin-CTX, the main toxin of this venom) and PIII. We implanted round glass cover slips on the s.c of mice pretreated with a non-toxic doses CdtV or fractions. After 4, 7, 14 and 21 days of the implantation, cover slips were removed, stained with hematoxylin/eosin in order to count the formed MGC or processed for immunohistochemical identification of F-actin. The formation of MGC was significantly inhibited in cover slips removed after 4, 7 and 14 days of the group treated with the crude venom as well as in the group treated with crotoxin, when compared to saline treated groups. The immunostainings for F-actin were also significantly inhibited in all times studied when compared to control groups. The inhibition of F-actin expression was positively correlated with the inhibition of the giant cells formation observed previously in cover slips implanted in the same period and stained with h/e. The compiled data shows a significant inhibitory action of the CdtV on the progression of the chronic inflammatory response. Galectin-3, an endogenous beta-galactoside-binding lectin with both intracellular and extracellular localizations, is expressed and secreted by various inflammatory cells. In vivo experiments with galectin-3 -/mice emphasized the critical role of this lectin in inflammatory responses but did not allow distinguishing extracellular and intracellular effects. Modulations of polymorphonuclear neutrophils (PMN) responses by galectin-3 have been described, but with contradictory results regarding the need of neutrophil priming. Objective: The aim of this study was to analyze galectin-3 extracellular effects on human PMN functions. Results: Although galectin-3 is highly expressed intracellularly in neutrophils, as shown by western blot analysis of cell lysates, we could not detect galectin-3 on the surface of resting or activated neutrophils. Exogenous recombinant galectin-3 was able to bind to PMN via its lectin site (inhibited by lactose) and this binding was significantly increased (60%) when PMN were activated with fMLP. A homogeneous binding of biotinylated galectin 3 was observed by immunofluorescence on PMN at 4°C. Transfer to 37°C resulted in a capping of cell-bound galectin 3, which was co-localized with a known galectin 3 ligand, CD66 and with another major membrane glycoprotein, leukosialin CD43, but not with the CD11b/CD18 integrin. Galectin-3 (0.8 mM) triggered PMN degranulation, as shown by CD11b upregulation. Moreover, it was able to further enhance the CD11b up-regulation triggered by suboptimal TNF-a concentrations (1 ng/ml). Finally, galectin-3 induced the adhesion of PMN to gelatin-coated plates in a dose-dependent manner. Gal 3 preincubation with polymyxin B did not modify its ability to activate neutrophils, excluding an effect of contaminating LPS. Conclusion: These results show that exogenous galectin-3 activates both naive and primed PMN, possibly via an interaction with CD66 and CD43, thus confirming its pro-inflammatory function. Disclosure of interest: None declared. Plants from Anacardiaceae family occur from Brazil. Its leaves and stem bark are used in folk medicine against general inflammations. The aim of this study was to evaluate the anti-inflammatory action of the ethanol extract and substances obtained from Anacardiaceae leaves (EFl). Pleurisy was induced by an intra-thoracic injection of zymosan (Zym 100 lg/cav.) or carrageen (Car 300 lg/cav.) in mice oral pre-treated with EFl (12.5-400 mg/kg), and purified substances: gallate acid (GA), methyl gallate (Mg) and penta-galloylglucose (Pg). Pleural wash was used to evaluate leukocyte migration, protein extravasation, cytokines KC and IL-6 production and LTB4. Mice received an intra-articular (i.a.) injection of Zym. (500 mg/cav.) and the knee-joint swelling was evaluated by measurement of left knee joints using a digital caliper. Knee synovial cavities were washed to analyze leukocyte migration. The experiments were realized under approval of Committee on Ethical Use of Laboratory Animals of Fundação Oswaldo Cruz (licence n. L0052/08. The statistically analyzed was realized by one-way ANOVA and Student-Newman-Keuls post-test (p \ 0.05). The pre-treatment with EFl reduced in doseresponse way the total leukocyte influx (100-400 mg/kg) in pleurisy induced by Zym. The pre-treatment with EFl was also able to inhibit the neutrophil influx and protein extravasation in the pleurisy induced by Car. The EFl, GA, Mg and Pg showed an anti-inflammatory effect on pleurisy induced by Zym reducing the LTB4, KC and IL-6 production. EFl also inhibited neutrophil influx and the oedema in Zym. induced arthritis. The results describe the anti-inflammatory activity to EFl suggesting a putative new phytomedicine. Disclosure of interest: None declared. Although proteinase-activated receptor (PAR)-4 has been implicated in inflammation, it's role in regulating the neutrophil recruitment in response to chemoattractants has not been demonstrated. To investigate the contribution of serine-proteinases and PAR-4 activation to the neutrophil migration in response to carrageenan or trypsin, we examined the effects of aprotinin or PAR-4 antagonist tcY-NH 2 on the neutrophil migration induced by these chemoattractants. BALB/c mice were treated with intrapleural (i.pl.) injections of trypsin (Tryp, 0.1-10 lg), and the number of infiltrating neutrophils evaluated 4 h after trypsin injection through pleural wash. Mice were also treated with aprotinin i.pl. (Aprot, 0.01-0.1 lg) or tcY-NH 2 (0.1-10 ng) 1 h before the i.pl. injection of carrageenan (Cg, 30 lg) or trypsin (10 lg). Neutrophil migration was assessed 4 h after the challenge through pleural wash. Trypsin induced a dose-dependent neutrophil recruitment (PBS, 0.02 ± 0.01; Tryp 0.1 lg, 0.06 ± 0.01; Tryp 1.0 lg, 0.3 ± 0.05; **Tryp 10 lg, 1.2 ± 0.4 9 10 4 neutrophils/ cavity). This recruitment was inhibited by tcY-NH 2 pretreatment (PBS, 3.0 ± 0.2; Tryp, 11.2 ± 0.3; ***tcY-NH 2 + Tryp, 4.6 ± 0.7 9 10 4 neutrophils/cavity). tcY-NH 2 or Aprotinin treatment inhibited the neutrophil recruitment induced by Cg in a dose-dependent manner (PBS, 1.6 ± 0.2; Cg, 7.9 ± 0.4; ***tcY-NH2 0,1 lg + Cg, 2.6 ± 0.4, ***tcY-NH 2 0.3 lg + Cg, 4.2 ± 0.3, ***tcY-NH 2 1 lg/cavity + Cg. 1.9 ± 0.3.10 4 neutrophils/cavity), (PBS + PBS, 1.9 ± 0.3; PBS + Cg, 11.2 ± 1.1; *Aprot 0.01 lg/ cavity + Cg, 7.6 ± 1.0, *Aprot 0.03 lg/cavity + Cg, 8.3 ± 0.9, ***Aprot 0.1 lg/cavity + Cg, 5.2 ± 0.8 9 10 4 neutrophils/cavity). ANOVA followed by Tukey-test, (*p \ 0.05, **p \ 0.01, ***p \ 0.001). These data demonstrate that trypsin is an important component to mediating the neutrophil migration in carrageenaninduced pleurisy, and that the activation of PAR-4 plays an important role in regulating this migration. Disclosure of interest: None declared. The Lnk adaptor protein is mainly expressed in the haematopoietic system. It possesses a potential dimerization (DD), a PH and SH2 functional domains and a conserved C-terminal tyrosine phosphorylation site that allow its interaction with different signalling effectors. Mice deficient for this protein have demonstrated its role as a negative regulator of signalling pathways controlling the proliferation of haematopoietic stem cells (HSC), myeloid and B-lymphoid progenitors. This phenotype is partly due to hypersensibility to several cytokines and growth factors, notably Stem Cell Factor (SCF). Our previous studies have implicated for the first time Lnk as an important inhibitor of SCFdependent migration of primary mast cells. However, the molecular mechanism underlying this regulation has not been identified. To analyze the functional contribution of the different Lnk domains to Kitdependent cell migration, we used Lnk-deficient bone marrow-derived mast cells (BMMCs) expressing wild-type or Lnk mutated forms as our cellular system. First, we examined the effect of Lnk on the actin reorganization and cell spreading by immunofluorescence. Our results showed that the Lnk SH2 domain is important for inhibiting SCFmediated actin polymerization and cell spreading of BMMC. In order to dissect at the molecular level the mechanism by which Lnk negatively regulates Kit-dependent cytoskeleton reorganization and cell migration, we examined the activation of two signalling effectors involved in these processes, the SHP-2 phosphatase and the Vav1 protein. Our results showed that Lnk down-regulates SCF-mediated activation of both molecules. Interestingly, our preliminary data suggest that Vav1 and Lnk interact in a SCF-independent fashion; we are currently confirming this association. Altogether, our findings suggest that Lnk down-regulates specific SCF-dependent signalling pathways, implicated in cytoskeleton rearrangement and migration of primary mast cells. Disclosure of interest: None declared. HCV is a highly immunogenic infectious agent leading to the development of an intense humoral and cellular response. The HCV infection of cells rapidly triggers intracellular signaling events leading to cytokine production and changes in the pattern of expression of cell adhesion molecules, providing a barrier to replication and viral spread. The aim of this study was to characterize the expression of adhesion molecules and cytokine profile in peripheral blood from blood donors of the Foundation HEMOAM HCV positive. We analyzed blood samples from 50 individuals that were reactive in serological screening test for HCV, and 53 samples negative of healthy blood donors. The expression of cell adhesion molecules in leukocytes was analyzed by flow cytometry and serum cytokine was performed by ELISA. The prevalence of HCV infection in the population of blood donors of the Foundation HEMOAM living in Manaus, from August/2009 to July/2010, was 0.1% and was greater prevalent in the group of first-time blood donors, compared to repeat blood donors. It was observed that the expression of cell adhesion molecules on leukocytes in peripheral blood is influenced by HCV infection, resulting in changes in the pattern of leukocyte recruitment to the liver and thus alters the course of the disease. There is evidence that the immune response against HCV in the population of blood donors, provide a toward Th1 profile, characterized by a high concentration of IL-12 and IL-10 in peripheral blood. It was found that the concentration of cytokines in peripheral blood from donors infected with HCV correlated with the expression pattern of cell adhesion molecules of leukocytes. However, our results reinforce the importance of the relationship between cell adhesion molecules, cytokine production and HCV infection. It is well documented that irritant inoculation into either the peritoneal cavity (PC) or other sites of mice generate a factor/s that stimulate bone marrow production of monocytes. We hypothesized that cells from the peritoneal cavity might also respond to remote inflammatory stimuli. Searching for data to support this hypothesis, LPS, BCG or Ehrlich ascitic tumor were inoculated in the footpad of Balb/c mice and B-1a and B-1b cells, macrophages and B-2 cells were analyzed by flow cytometry after different time intervals of irritant inoculation. Results show that the peritoneal macrophage population increased in the PC with a simple needle puncture in the foot pad after 24 and 48 h. When tumor was inoculated, macrophage population also increased persisting for 72 h. The number of B-1 cells increased after 48 h of tumor inoculation and B-2 cell population declined in the three periods studied. Therefore, we may suggest that alteration in PC cells is remote-stimulus dependent since these modifications were not observed after BCG and LPS inoculation. Only B-1 cell population increased 24 h after BCG stimulus. Also, animals were inoculated with Ehrlich tumor in the paw and 24 h after, CFSE solution was inoculated in the foot pad or in PC. 16 h after, the presence of CFSE + cells in spleen, bone marrow, popliteal lymph node and PC was evaluated. Results show that cells in the paw migrate to the popliteal lymph node and PC. Yet, cells in the CP migrate to the same lymph node. CFSE + cells were not detected in bone marrow or spleen. Data obtained so far show that the peritoneal cavity is not physiologically limited to itself but interacts with pathological manifestations that occur remote to its limits by mechanisms under investigation in our laboratory. Heart rate variability (HRV) is reduced in several clinical settings associated with systemic inflammation. The underlying mechanism of decreased HRV during systemic inflammation is unknown. It appears that the inflammatory cytokines might play a role, since epidemiologic studies has shown that circulating levels of interleukin-6 (IL-6) correlate significantly with indices of depressed HRV in various clinical conditions. Since there is no report to show a cause-effect relationship between IL-6 and decreased HRV, the present investigation was carried out to study the peripheral and central effects of IL-6 on heart rate dynamic in mice. Adult male BALB/c mice were used in the study. RT-PCR was performed to study the expression of IL-6 receptor in mouse atrial and the results showed that gp130 mRNA was detectable in the atrium. The effect of IL-6 was also studies on chronotropic responsiveness of isolated atria to adrenergic and cholinergic stimulations. Incubation of isolated atria with 10 ng/ ml of IL-6 was associated with a significant hypo-responsiveness to cholinergic stimulation (log IC 50 of carbacholine changed from -6.26 ± 0.1 in controls to -5.59 ± 0.19 following incubation with IL-6, P \ 0.05). The chronotropic responsiveness to adrenergic stimulation was identical with or without incubation with IL-6. To examine the systemic effects of IL-6 on HRV, studies were performed in two separate experiments. Intraperitoneal injection of IL-6 (200 ng/ mouse) was associated with a significant decrease in HRV parameters (SDNN, SD1, and SD2). While intracerebroventricular injection of IL-6 (50 ng/mouse) had no significant effect on HRV parameters. These data are in line with a peripheral role for IL-6 in the genesis of decreased HRV during systemic inflammation. Understanding the interaction between cytokines and cardiovascular regulatory system could potentially lead to development of strategies for treatment and management of patients with inflammatory diseases. Disclosure of interest: None declared. Objective: To investigate the prevalence and characteristics of patients with adult-onset Still's disease (AOSD), serum levels of cytokines/chemokines were measured and examined the association of systemic manifestations, especially hemophagocytic syndrome (HPS) in complicated with AOSD, Methods: Seventeen patients diagnosed as having AOSD and 20 healthy controls, with informed consents, were enrolled. We analyzed clinical and laboratory findings retrospectively. Serial serum samples were obtained from patients with active and inactive AOSD and controls. Cytokines/chemokines, including IL-18, CXCL8, CXCL10, CCL2 and CX3CL1 were determined by ELISA. Multivariate analysis was used to evaluate the correlation between serum chemokine levels and disease activity and clinical features of AOSD. Results: Significantly higher serum levels of all cytokines/chemokines (IL-18, CXCL8, CXCL10, CCL2 and CX3CL1) were observed in patients with active AOSD compared with healthy controls. The elevated CX3CL1 levels seen in AOSD patients correlated positively with clinical activity, as well as with CRP and ferritin levels, although there were no significant correlation between serum levels of other chemokines and either CRP or ferritin levels. In 17 patients with AOSD, 4 patients were complicated with HPS. Levels of serum CX3CL1 and ferritin were all significantly higher in AOSD patients with HPS than in those without HPS. Notably, serum CX3CL1 levels were significantly diminished following successful treatment and clinical improvement. Conclusion: The serum CX3CL1 level may be used as a clinical marker to assess disease activity of AOSD. We suggest that high In a limited number of patients with active RA, we undertook a pilot study to characterize the phenotype and function of effector and Treg cells obtained from the synovial fluid of affected joints and compared them with that of the autologous peripheral blood. Multi-color flow cytometry is employed for this purpose. Our preliminary data reveals significant enrichment of both Th17 (IL-17A+) and Treg cells (FoxP3+) in the local disease site of RA patients (Synovium) over their peripheral blood. The most interesting observation we made was the significantly reduced IL-10 production by CD4+FoxP3+ Treg cells derived from the local disease site(affected articular joints).However, under same in vitro condition higher percentage of Treg cells(CD4+CD25hi, FoxP3+) from the peripheral blood of the same patients could produce IL-10. Our results suggest (1) intact homing of Treg cells (in parallel with Th17 cell enrichment) to the diseased synovial fluid and (2) there is definitive functional impairment of the locally recruited Treg cells, at least in terms of IL-10 production. We also studied the expression of chemokine receptors such as CCR4, CCR6, etc. on the Treg cells which may be involved in their selective recruitment to the pathologic site of RA. We feel that these preliminary findings are quite interesting and require further studies with more elaborate experiments to understand the possible mechanism(s) contributing to the languidness of locally recruited Treg cells in terms of suppressive cytokine production (IL-10). Identifying the complex immune interplay between T effector and T reg cells might help in restoration of locally recruited Treg cell's function for clinical application in rheumatoid arthritis. Disclosure of interest: None declared. Pharmacology, Chiesi Farmaceutici S.P.A., Parma, Italy CXCL8/Interleukin-8 is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined here the acute effect of aqueous cigarette smoke extract (CSE) on CXCL8 expression in primary cultures of normal human airway smooth muscle cells (ASMC). In parallel, CXCL8 released in the culture medium was measured by ELISA. We found that CXCL8 mRNA levels markedly increased (up to 100-fold) upon CSE exposure in a concentration and time-dependent manner and such effect was accompanied by the accumulation of the CXCL8 protein in the culture medium. CSE-evoked elevation of CXCL8 mRNA was mimicked by the smoke component acrolein and was fully prevented by the a,b-unsaturated aldehydes scavenger N-acetylcysteine (NAC). Inhibition of protein synthesis with cycloheximide (CHX) per se caused a super-induction of CXCL8 mRNA levels, indicating that gene products maintaining basal CXCL8 mRNA at low levels are constitutively expressed in ASMC. Upregulation of CXCL8 mRNA levels evoked by CSE and acrolein, but not by TNF-alpha, was negatively modulated by CHX indicating the involvement of secondary response genes. By western blot analysis we observed that CSE-evoked upregulation of CXCL8 mRNA was preceded and accompanied by phosphorylation of p38 MAPK. In cells simulated with CSE, and subsequently treated with actinomycin D to block mRNA neosynthesis, pharmacological inhibition of p38 MAPK strongly accelerated the decay of CXCL8 mRNA levels, indicating that p38 MAPK signalling enhances the stability of CXCL8 mRNA. In sum, p38 MAPK-mediated stabilization of mRNA underlies upregulation of CXCL8 elicited by CSE. Given the pivotal role of CXCL8 in neutrophil chemotaxis and activation, this study sheds light on the mechanisms through which cigarette smoke can initiate inflammatory responses in the lung. Disclosure of interest: None declared. S. Sobhanian 1,* , A. Sotoodeh Jahromi 1 , P. Zareian 1 1 Jahrom University of Medical Sciences, Jahrom, Islamic Republic of Iran Background and objective: Pregnancy is related to glucose metabolism disorders and insulin resistance Insulin Resistance May Facilitate Supply of Appropriate nutrients particularly of glucose to fetus for fetal growth and metabolism. The mechanism responsible for insulin resistance has not been clearly stated. The purpose of this study was to evaluate the role of resistin and in insulin resistance during pregnancy. Approach: Serum resistin and TNF-a concentrations were measured by ELISA in 86 healthy pregnant women (26, 23 and 37 of them in the 1st, 2nd and 3rd trimesters, respectively) and in 21 healthy non pregnant women in a cross sectional study. Results: Resistin concentration was significantly higher in the third trimester (9.5 ± 3.3 ng mL -1 ) as compared with non pregnant women (7 ± 3.3 ng mL -1 ). Serum TNF-a level were also significantly increase in pregnant women (2.6 ± 1.9 pg mL -1 ) as compared with maternal healthy controls (0.8 ± 0.7 pg mL -1 ). There were significant correlation between gestational age and BMI (r = 0.28, p = 0.01), resistin (r = 0.36, p = 0.002) and TNF-a (r = -0.44, p \ 0.0001). There was not significant correlation between gestational age and insulin resistance (IR). We also did not found correlation between IR and resistin as well as between IR and TNF-a in pregnant women. Conclusion: TNF-a and resistin do not appear to contribute greatly to pregnancy induced insulin resistance in healthy pregnancy. Disclosure of interest: None declared. Objective: Tissue fluid/lymph (TF) contains cytokines, chemokines, growth factors originating from blood, parenchymatous and infiltrating cells. These proteins regulate immune processes but also influence cellular events in lymph nodes draining inflammatory tissues. Aim: To measure concentration of pro-and anti-inflammatory cytokines and chemokines in human lower limb skin tissue fluid/lymph in normal subjects, patients with rheumatoid arthritis, obstructive lymphedema without and with bacterial dermatitis. Methods: TF was collected from lymphatics in lower leg and cytokines measured with ELISA. Results: (1) pro-and anti-inflammatory (IL1b, TNFa, IL1Ra, MIP1a, MCP1, IL6, IL12, TGF b), (2) regulating epidermal and dermal cellular (KGF, MMP9, TIMP 1 and 2, PDGF BB) and (3) Objective: This study was performed to identify the effect of vitamin D on the pathogenic behavior of primary Th17 cells on synovial fibroblast (RASF) activation both from patients with early RA. Here, we show in Th17-RASF co-culture experiments an increase of IL-6, IL-8, and MMP-1 and MMP-3. Vitamin D significantly suppressed the production of IL-6 and MMP-3 in these co cultures. Interestingly, the specific enhanced autocrine production of IL-17 due to this Th17-RASF interaction was significantly inhibited by vitamin D. In addition, markedly suppressed expression of RORgammat and significantly enhanced expression of GATA3 was noted in the presence of vitamin D. No effect of vitamin D was observed on T-bet and FoxP3 expression. In addition, lower IL-22 and enhanced IL-10 production was found in the presence of vitamin D. The regulatory effects of vitamin D on IL-17 production in the Th17-RASF co culture was comparable to neutralizing IL-17 and on IL-6 production comparable to neutralizing TNF or IL-17. In conclusion, these data show that vitamin D modulates the pathogenic behavior of primary Th17 cells in their activation of synovial fibroblasts. In addition, vitamin D suppresses the proinflammatory IL-17 loop between Th17 and RASF cells. These data suggest that the activation of the vitamin D pathway may have therapeutic potential for the treatment or even prevention of persistent arthritis. Disclosure of interest: None declared. Objectives: Orthodontically-induced inflammatory root resorption (OIIRR) is an unavoidable pathologic consequence of orthodontic tooth movement. OIIRR is a sterile inflammatory process that is extremely complex and involves various disparate components, including mechanical forces and certain known biologic messengers. Interleukin (IL)-8, a member of the CXC chemokine family of cytokines, is potent chemoattractants for neutrophils. Recent studies demonstrated that IL-8 and RANKL contributed to alveolar bone remodeling during orthodontic tooth movement. However, little is known about the relationship between OIIRR and IL-8/RANKL. The aim of this study was to investigate how IL-8 and RANKL contribute to root resorption during orthodontic tooth movement. Materials and methods: Forty male 6-week-old Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. We determined the expressions of CINC-1/CXCR2 (cytokine-induced neutrophil chemoattractant; CINC-1/CINC-1 receptor) and RANKL proteins in root resorption area using immunohistochemistry. Furthermore, we investigated the effects of compression forces (CF) on IL-8, and IL-8 on RANKL production by human periodontal ligament (hPDL) cells for 48 h. We observed an effect of chemokine treatment on rat odonto/osteoclasts in dentin slices that recapitulated root resorption. Result: The immunoreactivity for CINC-1/CXCR2 and RANKL was detected in odontoclasts and PDL fibroblasts by the orthodontic force of 50 g on day 7. CF increased the secretion and the expression of mRNA of IL-8 from hPDL cells in a magnitude-dependent manner. Addition of IL-8 increased the expression of mRNA of RANKL from hPDL cells. Moreover, CINC-1 stimulated osteoclastogenesis from rat osteoclast precursor cells. Conclusion: IL-8/RANKL may facilitate the process of root resorption due to excessive orthodontic force. Disclosure of interest: None declared. Materials and methods: In vivo study, 10 g of orthodontic force were applied to the molars to induce experimental tooth movement in rats. Immunohistochemical staining with HSP70 was performed. In vitro study, hPDL cells were subjected to 1.0, 2.0, or 4.0 g/cm 2 of CF for 24 h, and were treated with recombinant human HSP70 for 12 h. The mRNA expression of HSP70, TNF-a, and RANKL from hPDL cells subjected to CF were determined by real-time PCR. Effects of HSP70 on the CF-induced TNF-a and RANKL expression were determined. Results: In vivo study, on 1 day after tooth movement, the immunoreactivity of HSP70 was weakly. On 2, 3 and 4 days, HSP70 positive reaction in fibroblasts was recognized more than on 1 day. Further, HSP70 positive reaction was increased on 7 and 14 days. In vitro study, the mRNA expression (CF 4.0 g/cm 2 ) of HSP70 markedly increased to 12 h. In particular, the increase from 9 to 12 h was significantly higher than other duration. In contrast, the mRNA expression (CF 4.0 g/cm 2 ) of TNF-a and RANKL significantly increased after the application and peaked at 9 h. However, the mRNA expression of TNF-a and RANKL gradually decreased with the increasing level of HSP70 at 12 h after the application. Furthermore, exogenous HSP70 partially inhibited the CF-induced TNF-a and RANKL expression in a dose-dependent manner at 6 and 12 h. Conclusions: These results indicated that HSP70 inhibited the expression of TNF-a and RANKL in hPDL cells in response to CF. In conclusion, HSP70 may function as a homeostatic mechanism to compensate for PDL cell changes occurring during orthodontic tooth movement. Disclosure of interest: None declared. Background: Although is known that surgery and anaesthesia can influence the release of mediators in patients, the role of volatile and venous anaesthetics on the modulation of cytokines in patients submitted to minor elective surgeries is not well elucidated. We evaluated and compared pro-and anti-inflammatory cytokines in patients submitted to non-invasive elective surgeries under isoflurane anaesthesia or total intravenous anaesthesia (TIVA) with propofol. Methods: ASA I adult patients submitted to otorhinolaryngology surgery were randomly allocated to receive anaesthesia with isoflurane 1 MAC (minimum alveolar concentration) (n = 16) or propofol 3-5 mg mL -1 (n = 18). Venous blood samples were drawn from each patient at baseline (T1), 120 min after anaesthesia induction (T2) and on the first postoperative day (T3). Plasma concentration of interleukins IL-1b, IL-6, IL-8, IL-10 and IL-12 and tumor necrosis factor (TNF-a) were measured by Cytometric Bead Array (CBA) using flow cytometry. Results: The pro-inflammatory IL-6 concentration significantly increased in isoflurane group at T2 (4.55 pg mL -1 ) and T3 (8.10 pg mL -1 ) versus baseline (2.10 pg mL -1 ) whereas in propofol group this increasing occurred only at T3 (12.80 pg mL -1 ) versus baseline (2.10 pg mL -1 ). Besides, in TIVA group IL-8 had also raised just on the first postoperative day T3 (8.20 pg mL -1 ) versus baseline (5.65 pg mL -1 ). There were no significant differences of IL-1b, IL-10, IL-12 and TNF-a within or between groups. Conclusions: ASA I patients submitted to non-invasive surgeries isoflurane anaesthesia promotes prompt and progressive IL-6 release while TIVA with propofol promotes late IL-6 and IL-8 release. Patients submitted to surgical procedure have inflammatory response because of the trauma of the surgery and possibly due to the anesthetics. The role of volatile anesthetics on the modulation of cytokines in patients submitted to non-invasive surgey is not well elucidated. Sevoflurane is widely used for inhalation anesthesia. This study aimed to evaluate the cytokines (pro-inflammatory: IL-1beta, IL-6, IL-8, IL-12 and TNF-alpha and anti-inflammatory IL-10) in plasma from ASA I patients scheduled for otorhinological surgery under inhalation anesthesia with sevoflurane at Botucatu Medical School Hospital, UNESP. Patients with a disease, smokers, alcoholics, obeses, those who were occupationally exposed, those who had any kind of infection or inflammation within the previous 30 days, those who were under medication or who had received radiation were excluded. After approval of The Ethical Committee for Human Research, the study was conducted in 15 patients of both genders, aged from 18 to 44, who were submitted to surgery with 1.0-1.5 minimum alveolar concentration of sevoflurane. Blood samples were drawn at four time points: before anesthesia induction (T1-baseline), before the beginning of the surgery (T2), 120 min after anesthesia induction (T3) and on the first post-operative day (T4). Cytokines were evaluated using the kit BD Human Inflammation Cytometric Bead Array by flow cytometry. Friedman test was used to compare cytokine profile among four sampling times. There were no significant differences in the concentrations of all cytokines among the four time points evaluated (P [ 0.05), with exception of IL-6, that increased on the first postoperative day (T4) in relation to baseline (T1) and T2 (P \ 0.001). In conclusion, sevoflurane anesthesia does not change concentrations of anti-or pro-inflammatory cytokines during surgical procedure, but promotes late IL-6 release after otorhinological surgery in adult ASA I patients. Objective: Orthodontically induced inflammatory root resorption (OIIRR) is an unavoidable pathological consequence of orthodontic tooth movement. Cause of OIIRR has not yet been conducted. IL-17 from rheumatoid arthritis (RA) patient enhances local inflammation and increases the production of inflammatory cytokines, which further promote RANKL expression and activity. We reported that compressed periodontal ligament cells obtained from tissues with severe external apical root resorption produced a large amount of IL-17 and RANKL in vitro. Therefore, in the present study, the expressions of RANK, RANKL, IL-17, IL-17 receptor, IL-23 receptor and Cathepsin-K were investigated using immunohistochemical analysis, and presence of Th17 cells was confirmed using immunofluorescence confocal analysis in RA model mice in root resorption during experimental tooth movement. Materials and methods: 8-week-old male BALB/cAJcL mice (control) and SKG/Jcl mice (RA) were subjected to an orthodontic force of 10 g with a closed coil spring ligated to the maxillary first molar cleat by a stainless steel ligature wire to induce a mesial tipping movement of the maxillary first molars for 7 days. Each sample was sliced into Objective: Orthodontically induced root resorption (OIRR) is one of side-effects in orthodontic treatment, and this resorbed root cannot be recovered again. The occurrence of severe root resorption is higher in patients with allergies or asthma, therefore the immune factors may be involved with the occurrence of root resorption. Further, we also reported that orthodontic patients with severe root resorption have high level proinflammatory cytokines such as receptor activator of NF-jB ligand (RANKL) and interleukin (IL)-17 in gingival crevicular fluid (GCF).Recently, it has been revealed that IL-17 plays an important role in bone destruction of rheumatoid arthritis by inducing IL-6 production in fibroblast-like synoviocytes. However, the relationship among IL-17, IL-6 and OIRR is not clear. Therefore, we investigated the effects of IL-17 on IL-6 production from human periodontal ligament (hPDL) cells. Furthermore, the expression of IL-17 and IL-6 in root resorptive lacunaes were observed by immunohistochemical staining. Inflamm. Res. Materials and methods: To induce experimental tooth movement in rats, 50 g of orthodontic force was applied for 7 days. Immunohistochemical staining with TRAP, IL-17, IL-17R, and IL-6 was performed. In vitro study, hPDL cells were stimulated by IL-17 (0-100 ng/ml) for up to 72 h. Real time PCR and ELISA were estimated the expression of IL-6. In addition, the effect of IL-17 on osteoclastogenesis was assessed by TRAP staining and activity of actin ring. Result: In immunohistochemical staining, TRAP, IL-17, IL-17R and IL-6 positive cells were significantly increased in the root resorption area at 7 days. In vitro study, IL-17 induced IL-6 production from hPDL cells in a dose-dependent manner. Furthermore, the number of TRAP positive cells and osteoclasts with actin ring significantly increased. Objective: Orthodontically induced inflammatory root resorption (OIIRR) is an unavoidable pathological consequence of orthodontic treatment. Orthodontic forces are known to produce mechanical damage and inflammatory reactions in the dental pulp and periodontal ligament. Interleukin (IL)-17 is an inflammatory cytokine which T helper (Th) 17 cell derived. However, the relationship between IL-17 and osteoclast development is not fully understood. Our previous study found that the level of IL-17 in gingival crevicular fluid (GCF) obtained from orthodontic patients with severe root resorption increased. We examined that the effects of IL-17 on the expression of receptor activator of nuclear factor-B ligand (RANKL) by human dental pulp (hDP) cells in vitro, and the expression of IL-17, IL-17 receptor, IL-6 and RANKL in root resorption area during experimental tooth movement in rats. Materials and methods: hDP cells were stimulated by IL-17 (0-100 ng/ml) for up to 48 h. The expression of RANKL and OPG mRNA was determined by using real-time PCR. In animal study, forty 6-week-old male Wistar rats were subjected to an orthodontic force of 10 or 50 g with a closed coil spring (wire size: 0.005 in., diameter: 1/12 in.). Experimental tooth movement was undertaken for 7 days. Each sample was prepared for haematoxylin and eosin (H and E) and immunohistochemistry staining for tartrate-resistant acid phosphatase (TRAP), IL-17, IL-17R, IL-6 and RANKL in root resorption area. Results: Stimulation with IL-17 increased the levels of RANKL mRNA expression from hDP cells in a dose-dependent manner. On days 7, immunoreactivity for IL-17, IL-17R, IL-6 and RANKL was detected in odontoclasts with an orthodontic force of 50 g, but not 10 g. Conclusions: These results indicate that IL-17 may modulate the mRNA expression of RANKL in hDP cells.IL-17 might be involved in the progress of inflammation in pulp tissue and the incidence of severe root resorption during orthodontic treatment. Disclosure of interest: None declared. The interleukin-6 is released in response to inflammation and cancer can function to promote growth and facilitate invasion of several forms of tumours including that of the prostate. The PSMA and PSA are implicated in the development and progression of prostate cancer. The increased secretion of IL-6 and co-expression of PSMA-PSA in prostate tumorigenesis may be correlated. The aim of this study was to relate the expression of IL-6 and their receptors with PSMA-PSA profiles in pathologic prostate tissues to investigate their role in prostate tumorigenesis. The study was carried out in 9 benign prostatic hyperplastic (BPH) and 17 cancerous human prostates (PC). Immunohistochemical analysis was performed. Our results showed that in BPH, (PSMA+, PSA+) was positive in 4/9 (44.5%), (PSMA+, PSA-) was present in 3/9 (33.3%), (PSMA-, PSA+) was absent, and (PSMA-, PSA-) was present in 2/9 (22.2%) of patients. While, in PC, (PSMA+, PSA+) was present in 12/17 (70.58%), (PSMA+, PSA-) was present in 4/17 (23.52%), (PSMA-, PSA+) was present in 1/17 (5.88%) of patients and (PSMA-, PSA-) was absent. Concerning (PSMA+, PSA+) profile, IL-6 and it's receptor IL-6Ra was expressed in all the patients with BPH and in the most of PC patients (75 and 91.66%, respectively), whereas it's second receptor gp130 was present in 50% of BPH and in 83.3% of PC. Alternatively, IL-6 and it's receptors were expressed in the most of patients in the other profiles in BPH and also in PC. Among (PSMA+, PSA+, IL-6) profile, PSA is more expressed than PSMA (16.25 ± 2.49 and 31.28 ± 5.3, respectively) in BPH. Inversely, PSMA is more expressed than PSA in PC patients within the same profile (32.69 ± 7.63 and 20.45 ± 1.31, respectively).There was an association between the high expression of IL-6 and PSMA-PSA co-expression in BPH and PC. A better understanding of biologic mechanism of this association connecting with the role of several signaling pathways within PSMA-PSA profiles will be interesting. Disclosure of interest: None declared. Dipeptidyl peptidase IV (DPPIV) is a protease preferentially cleaving N-terminal dipeptides from peptides with Pro or Ala in the penultimate position. It regulates the activities of physiologically important compounds: chemokines, neuropeptides, hormones and growth factors, and is expressed on the surface of various cell types and is found also in soluble form. To gain new insight into the physiological role of DPPIV in experimental model of inflammation, we explored blood plasma DPPIV activity during cyclophosphamide (CPA)-induced cystitis in rats. We compared the DPPIV activity in blood plasma of CPA injected rats with and without treatment by proline rich peptide (PRP), a cytokine from neurosecretory granules of neurohypophysis. PRP had been demonstrated as a unique regulator of activity of neurons, strong antibacterial agent, and mediator of the hypothalamus-neurohypophysis-bone marrow-thymus axis. In the present work, the Wistar line white rats (130-160 g) were divided on three groups. Two experimental groups were intraperitoneally administrated CPA (25 mg/animal), the control group received saline. On the next day, one of experimental groups was injected with PRP (20 lg/animal). At 3rd, 5th, 7th, 9th, 11th, 13th days after CPA administration, the animals were anaesthetized, per 1 ml of blood were taken by cardiopuncture, and plasma was separated from the erythrocytes by centrifugation during 5 min at 2,000 rpm. In CPA injected animals, along with the cystitis development, we observed the time dependent inhibition of DPPIV activity in blood plasma peaked at 60% drop on the 7th day and the recovery of this activity after 11th day. In the PRP treated animals the inhibition of DPPIV activity peaked at 40% drop, and the occurrence of cystitis was milder. In conclusion, it seams the using of PRP along with CPA as a chemotherapeutic agent diminished the side effects of CPA as cystitis and on the other hand, protected the activity of important protease, DPPIV. Disclosure of interest: None declared. Colitis induced by the hapten trinitrobenzene sulfonic acid (TNBS) with reactivation is a good experimental model for studying inflammatory bowel disease pathogenesis and appropriate therapeutics, mainly Crohn's disease (CD). TNBS administration repeatedly provides induction of colitis relapse and remission periods and the establishment of chronic disease features, such as the mesenteric adipose tissue (MAT) alterations observed in CD. Lymph node activation and the role of perinodal adipose tissue (PAT) have been poorly studied in this model. Thus, the aim of this work was study the interactions of lymph nodes and PAT during reactivated TNBS-colitis in Wistar rats. The alterations of PAT evaluated were fatty acid profile and adipokine production by gas chromatography and ELISA, respectively. Lymph node cells were cultured and cytokines production evaluated by ELISA. Fatty acid analysis of PAT reveals an increase of x-6 polyunsaturated fatty acids during colitis, such as linoleic acid, gamma-linolenic acid and arachidonic acid, resulting in a ratio x6/x3 different from healthy controls. x-6 arachidonic acid level and ratio x6/x3 were not modified in lymph node cells or serum. PAT also produces elevated levels of pro-and anti-inflammatory adipokines during colitis. Lymph node cells release high levels of IFN-c and TNF-a but not IL-10, characterizing the predominant Th-1 response associated with this disease. Additionally, T cells collected from lymph nodes from animals with colitis demonstrated increased IFN-c production via a COX-2-dependent pathway after supplementation with x-6 arachidonic acid. Our results suggesting that PAT modification could contribute to the lymph node cell activation observed during colitis, increasing the Th-1 response. In mammals the chemokine system modulates leukocyte migration during inflammatory process. Neutrophil recruitment depends mainly of the CXC chemokine family, such as CXCL8 and its receptors CXCR1 and CXCR2. The zebrafish (zf) inflammatory process also involves leukocyte recruitment. Sequence analysis studies have identified several members of CC and CXC chemokine families. Additionally, recent studies have shown that teleost have two distinct lineages of CXCL8, with distinct expression profiles at early inflammatory stages: the CXCL8-Lineage 1 (CXCL8-L1) and the CXCL8-Lineage 2 (CXCL8-L2). Both chemokine lineages seem to have a powerful chemotactic activity in carp. Here we address the role of zfCXCL8-L1 and L2, in neutrophil recruitment, under inflammatory conditions. We have been employing splice morpholinos (MOs) designed to block zfCXCL8 expression in the Tg(mpx:GFP)i114 transgenic line. To monitor neutrophil recruitment two different stimuli were used: tail transection and injection of fluorescent bacteria. Briefly, one-cell stage Tg(mpx:GFP)i114 embryos were microinjected with the zfCXCL8 MOs or with MO standard control. Next, tg larvae with 3 days post-fertilization (dpf) were tail transected or injected with fluorescent bacteria. Larvae were mounted in 1% of agarose low-melting point and time-lapse images were taken at different time points to address the total neutrophil number recruited to the injuries. Furthermore, a gene expression study of cxcl8-l1, cxcl8-l2, cxcr1 and cxcr2 was made by qPCR using extracted RNA from 3dpf larvae in different times of inflammation. Our data show that cxcl8-l1 and l2 genes are over-expressed under inflammatory conditions. Additionally, in zfCXCL8 morphants the in vivo time-lapse images have shown a significant decrease in the neutrophil number recruited to the inflamed area. These results support the idea that also in zebrafish CXCL8 has an important role in modulation of neutrophil recruitment under inflammatory conditions. Disclosure of interest: None declared. Introduction: Hydroxyapatite (HA) is widely used for prosthesis coating and is known for its generation of wear particles. This S100 Inflamm. Res. material release mounts an inflammatory response which could result in implant loss. To improve the bioactivity of the implant, electrodeposited biphasic ceramic containing 50% HA and 50% tricalcium phosphate (TCP) was elaborated. In this work, we have studied the regulation of inflammatory mediators production induced by the biphasic biomaterial compared to standard HA coating or titanium alloys (Ti6Al4V). Material and methods: Freshly elutriated human monocytes were exposed to the materials for 4 and 24 h. IL-1b, -6, -8, -10, TNF-a, TIMP-1 and -2 concentrations were measured with ELISA. Gelatinolytic activities of conditioned media were examined by gelatin zymography. Total mRNAs were extracted and variations of mRNA production were measured by RT-qPCR. Results and discussion: Pro-inflammatory mediators production study had highlighted an up-regulation of IL-1b and TNF-a production, and an increased IL-6 mRNA level by HA-stimulated cells. IL-8 was more produced by HA/TCP-stimulated cells and HA coating induced an increased IL-10 concentration. Moreover we had stressed that HAinduced MMP-2 mRNA production and activity were decreased by comparison to HA/TCP condition whereas MMP-9 did not exhibit any variations at either mRNA or enzymatic activity levels. Tissular inhibitors of MMPs (TIMP)-1 and -2, did not exhibit any variations between the two studied coatings. Our data have highlighted that HA/ TCP had a lesser inflammatory potential than HA coating leading to a decreased in inflammatory cells activation. Therefore, this regulation of inflammation process was counterbalanced with an increased in ECM turnover. Conclusion: These results suggest that HA/TCP might be used as a novel biomaterial for prosthesis coatings to improve implant integration and lifespan. Introduction: During obesity, a chronic inflammatory state, white adipose tissue (WAT) undergoes profound cellular remodeling with accumulation of immune cells like macrophages and lymphocytes. Visceral WAT which development is associated with obesity-related complications, is more inflammatory than subcutaneous WAT [1] . Using cDNA microarray analysis, we identified CXCL2 among the most over-expressed genes in human inflammatory preadipocytes. We aimed at precising the role of CXCL2 that we characterized for the first time in human WAT. Results: The circulating levels of CXCL2 measured by Elisa assay were significantly higher in obese than in lean age-matched subjects (p \ 0.05). CXCL2 mRNA levels determined by RT-PCR, were also increased in WAT of obese subjects (p \ 0.05). CXCL2 secretion was significantly higher in visceral compared to subcutaneous WAT (156 vs. 24 ng/ml, p \ 0.001). In visceral WAT, CXCL2 expression was positively correlated to M1 (TNF and IL-6) while not to M2 (CD206 and AMAC-1) macrophage markers. Adhesion/transmigration of human monocytes to/through endothelial cells from visceral WAT performed as described in [2] was enhanced by CXCL2. Immunohistological analysis showed a higher number of activated neutrophils adherent to the endothelium of visceral WAT from obese compared to lean subjects. In vitro adhesion of neutrophils to endothelial cells from visceral WAT was also increased by CXCL2. The same stimulatory effect was observed with conditioned media from visceral WAT whereas neutralizing antibody of CXCL2 abolished it. Conclusion: CXCL2 could be considered a new adipokine upregulated in WAT during human obesity. CXCL2 could participate in the inflammation of visceral WAT by recruiting blood monocytes and neutrophils. Introduction and objectives: Scorpion envenomation is a common medical problem in many countries, and it is an important cause of morbidity and mortality. Animal venom is able to induce systemic alterations similar to those observed in acute-phase inflammatory responses. There are many studies regarding the venom actions, however little is known about the interactions the fractions IX (Ts2) or X (Ts6) with the immune system. The aim of this study was to investigate in vivo, the ability of Ts2 and Ts6 induce cytokines and nitric oxide release, as well as lipid bodies (LB) formation in peritoneal cavity of mice. Methodology and results: Ts2 or Ts6 (250 lg/kg) or PBS were intraperitoneally injected in the peritoneal cavity of 129sv mice and after 4, 24, 48 and 96 h the lavage was collected. The cytokines were measured by ELISA and nitric oxide (NO) by Greiss method. Ts2 or Ts6 toxins were not able to stimulate NO, however they stimulated the release of inflammatory cytokines, such as, IL-6, TNF-a, TGF-b, IL-1b and IFN-c in different manner depending of the time analyzed. To evaluate LB, cells were stained by osmium tetroxide and counted in optical microscope. LB formation was increased after 24 h of the injection of toxins. Neutrophils were increased in peritoneal cavity between hours 4 and 96 after injection of toxins. Conclusion: Our results show that Ts2 and Ts6 induced recruitment of inflammatory cells (neutrophils) and inflammatory mediators. Ts2 and Ts6 induced the formation of LB, which could be related with eicosanoids production. Disclosure of interest: None declared. Purpose: Corneal infections are the main cause for worldwide preventable blindness. Corneal cells respond to infections by triggering an inflammatory response through toll-like receptors (TLRs). Vitamin D (1,25(OH) 2 D 3 ) modulates immune responses in certain cells. We hypothesise 1,25(OH) 2 D 3 plays an immunomodulatory role and there exists a coordinated cross-talk between vitamin D and TLR signalling to reduce inflammation and remove infection. Methods: Three different cell types-primary corneal epithelial cells (PHCEC), corneal fibroblasts (HKF) and scleral fibroblasts (HSF) were generated from each donor corneal-scleral button remnants from transplant surgery. The mRNA and protein expression of VDR, vitamin D metabolizing enzymes 1a-OHase and 24-OHase and their regulation with TLR activation was investigated by RT-PCR, immunohistochemistry, real-time PCR and superarray. The culture supernatants were analysed for the production of cytokines using ELISA and multiplex bead immunoassays. Results: Immunolocalisation studies identified VDR, 1a-OHase and 24-OHase as cytoplasmic in the epithelial cells and keratocytes. Only the PHCEC converted the inactive (25(OH)D 3 ) to active 1,25(OH) 2 D 3 . Following TLR activation (TLR3 and 4), the mRNA and protein expression of pro-inflammatory cytokines and chemokines and anti-inflammatory cytokine (IL-10) were up-regulated in corneal cells. Under these conditions, the mRNAs for 1a-OHase and 24-OHase were also up-regulated. 1,25(OH) 2 D 3 inhibited the cytokine production upon TLR activation. Only PHCEC showed stimulation in anti-microbial peptide-cathelicidin expression with TLR stimulation which was enhanced by vitamin D. Conclusions: In the cornea vitamin D acts as a 'double-edged sword' where on one hand it offers immuno-suppression by up-regulating anti-inflammatory cytokines and down-regulating pro-inflammatory cytokines and on the other hand it up-regulates anti-microbial peptides in defence against microbial infections. Disclosure of interest: None declared. Background: Although measurements of inflammatory factor concentrations are thought to be affected by testing procedures, little is known about the effect of sample type. This study compared (a) the extraction of selected inflammatory factors from human plasma and serum samples, and (b) the effect of thaw-freeze cycles on the concentrations of these factors in plasma and serum. Methods: Participants enrolled in a study examining the role of acute inflammation in the development of Complex Regional Pain Syndrome following a wrist fracture, consented to an assessment that included a blood test. Serum and plasma samples were collected from the uninjured arm within 7-14 days of fracture and stored at -80°C. Alternate samples were exposed to 0, 1 or 2 prolonged thaw-freeze cycles. A Bio-Rad multiplex-17 bead array cytokine assay and Luminex technology were used to test for the cytokines IL-1b, IL-6, IL-8, TNF-a, IFN-c, IL-10, IL-12p70 and the chemokines MCP-1 and MIP-1b. Out of range low values were replaced with the established minimal detectable value for the test. Results: Samples were collected from 25 participants (11 female and 14 male, mean age 43.96 ± 17.26). Concentrations were below the detectable limit of the test for many of the inflammatory markers. Concentrations of IL-1b, IL-6, IL-8, TNF-a, IFN-c, IL-10, MCP-1 and MIP-1b were higher in serum than in plasma; IL-12p70 was measured as higher in plasma than in serum. However, paired sample t tests revealed no significant group differences between concentrations obtained from plasma and from serum (p \ 0.05). Inflammatory factors were not affected by freeze-thaw cycles, except for IL-8 (p \ 0.05). Conclusions: Inflammatory factor concentrations measured by multiplex bead array are not affected by the sample medium (plasma or serum) or by one or two thaw-freeze cycles (except IL-8). Other factors, such as handling requirements, should be considered when selecting a sample medium for testing of inflammatory factors. Disclosure of interest: None declared. The pathogenesis of development of nasal polyposis is incompletely understood. The aim of this study was the investigation of the role of inflammatory cells, adhesion molecules, intermediate filaments and chemokine receptors in the development of nasal polyposis. Totally, 35 patients were enrolled (Group 1, 10 patients with Samter syndrome; Group 2, 10 patients with diffuse polyposis without signs of Samter syndrome; Group 3, 5 patients with solitary nasal polyps; Group 4, 10 controls). Expression of CD105, CD106, CD62E, CD4, CD8, CXCR4, CD147, CD90, CD104, BF45, vimentin, pancytokeratin and muscle specific actin (MSA)from all patients' specimens were determined. Expression of CD4, CD8 and CD106 were similar between the groups. Ratio of patients expressing CD4 in Group 1, Group 2 and Group 3 were higher than the controls. Ratio of patients expressing CD8 antigen were significantly higher in all three groups than the control group. Expression of CD147 in Group 3 and Group 4 was significantly higher than in Group 1 and Group 2. CD98 expression was higher in Group 1, Group 2 and Group 3 than in Group 4. The ratio of patients expressing vimentin in Group 1, Group 2 and Group 3 were significantly higher than in Group 4. Immunostaining for pancytokeratin was positive in all S102 Inflamm. Res. patients. In conclusion, inflammatory cell, adhesion molecule, intermediate filament and chemokine receptor profiles in nasals polyps differ among both different patient groups and control subjects. Electro-magnetic fields (EMFs) interact with inflammatory mechanisms. To investigate the activity of EMFs on the release of TNF-a we did dose in a monocyte culture isolated from the blood of eight healthy donors. We studied ''in vitro'' the effects of TAMMEF System (Therapeutic Application of a Musically Modulated Electromagnetic Field), Electromagnetic Low Frequency ELF or untreated cells on LPS induced TNF-a release. ELF apparatus modulate the frequency of 100 Hz, sinusoidal waveform; TAMMEF System produces a field whose parameters are modified in time and varying within the respective range, so that in a single application all possible codes can occur. We used a pair of electromagnetic plates controlled by an audio tape player, recorded with a classical excerpt. The human lymphomonocytes culture were positioned between the polar expansions and exposed at ELF and TAMMEF electromagnetic field for an hour each 24 h 4 days before LPS stimulation. Sham group was positioned away from magnetic fields. TNF-a determination was made by Elisa test; the data were analyzed by Student's ''t'' test and ANOVA. The baseline release of TNF-a did not differ between the three study groups (intergroup difference, +34.1, +33.1, +38.3% for Sham, ELF and TAMMEF treated groups, respectively). The increase of TNF-a was greater after ELF $$treatment (+440, +387, +370, +407% at 24th 48th 72th and 96th hours) versus TAMMEF groups (+159, +278, +298, +256% in the same times) in comparison to the Control group (+247, +356, +216, +311%). There was a statistical significant difference (p = 0.01) between ELF and TAMMEF whereas the difference between control vs ELF and control vs TAMMEF were not significant (F = 0.2 and 0.1, respectively). LPS induces a stronger TNF-a release in culture undergone to ELF in comparison to TAMMEF, in each time of the experimental protocol, and this seems to indicate that TAMMEF is able to induce a complex modulation of LPS induced TNF-a release. Disclosure of interest: None declared. Airway epithelial cells are a major source of proinflammatory cytokines and chemokines and have a key role in the pathogenesis of respiratory diseases such asthma and COPD. The severity of these diseases is associated with increased levels of IFNc and TNFa and with a reduced sensitivity to steroids. Recently, it has been demonstrated that combination of TNFa and IFNc impairs the ability of steroids to inhibit the expression of pro-asthmatic genes, such as IL6 and IL8, in airway smooth muscle cells. It has not been studied whether a similar phenomenon occurs in the airway epithelial cells. The aim of this study was therefore to investigate whether IFNc, in combination with TNFa, reduces steroid sensitivity in A549 human lung epithelial cells and human primary bronchial epithelial cells (NHBE cells). IFNc synergistically enhanced the expression of TNFa-induced inflammatory mediators such as IL6 and Rantes, but did not affect TNFa-induced IL8 expression in A549 cells. The ability of the steroid fluticasone propionate (FP) to inhibit the secretion of these cytokines was similar in response to TNFa alone or in response to TNFa in combination with IFNc, suggesting that IFNc doesn't affect the steroid trans-repression activity. Moreover, in A549 cells stably transfected with a glucocorticoid response element (GRE)-driven reporter gene, increasing concentrations of IFNc, up to 200 ng/ml, did not alter steroid trans-activation activity. However, combination of TNFa and IFNc produced high expression of IP10 and ICAM1 that is completely steroid insensitive. Same trend of steroid sensitivity for TNFa-and IFNc -induced inflammatory genes has been observed in primary NHBE cells from different donors. We conclude that the reduction of steroid responsiveness by combination of TNFa and IFNc is cell type specific and that there is a set of IFNc-induced genes intrinsically steroid insensitive. Disclosure of interest: B. Maschera employee of: GSK and participate in share scheme, K. Simpson employee of: GSK and participate in share scheme. Objectives: Exercise has been introduced as a model of the stress response hence having some impact on immune system reactions. To obtain information about lymphocyte turnover in cells recruited during exercise, we recently analyzed the interactions between exercise stress and the immune system to provide an opportunity to link basic and clinical physiology and to evaluate the role of stress like activities and immune related sub population of t-cells. Methods: The study was performed using sera samples of 34 exercisers as the case group and sera samples of 15 non-exercisers as the control group. Two blood samples were taken before and after exercise of which serum was extracted. Cell blood count (CBC), CD3, CD4 and CD8 were measured for all participants. Data were analysed using ANOVA and Spearman analysis tests. Results: The mean ± SD of CD3, CD4, CD8 and CD4/CD8 were 64.7 ± 6.3, 41.25 ± 5.07, 21.75 ± 5.49 and 2.08 ± 0.63 in case group while 64.33 ± 4.56, 42.86 ± 5.44, 20.33 ± 4.93 and 2.34 ± 0.76 in control group, respectively. There were a significant correlation between CD3 (r = 0.424, p = 0.01) and CD8 (r = 0.661, p = 0.000) with WBC count in case group, while this correlation was not found for control group. The mean CD8 level was significantly higher in $$women (24.66) compared to men (19.50) (95% CI -8.82-1.50, P = 0.008). The CD4/CD8 ratio was increased significantly in control than case group but this relationship was not significant. Conclusion: The mean OD value of CD3 and CD8 and the CD4/CD8 ratio were all significantly different among exercisers compared to non-exercisers indicating that immune system can be affected by exercise depending on the nature and quality of exercise. Objective: The objective of this study was to examine the effects of proteasome inhibitor MG132 on cyclooxygenase 2 (COX-2) induction and IL-8 secretion induced by IL-1beta in human osteoarthritic meniscus. Methods: Human meniscal chondrocytes were enzymatically isolated from osteoarthritic knee cartilage. The cultured meniscus cells were characterized by immunocytochemistry. Western blotting was used to assess the induction of COX-2 by IL-1beta stimulation (10 ng/ml), in the presence or absence of MG132 (35 lM) or NF-kB SN50 peptide (100 lg/ml). IL-8 production was assayed by ELISA. Results: Primary cultures of human meniscal chondrocytes demonstrated the fibrochondrocytic nature with expression of collagen type I and II, but not CD34 by immunocytochemistry. COX-2 was significantly upregulated following IL-1beta stimulation from 6 to 24 h tested (p \ 0.05). COX-2 protein induction was inhibited by MG-132, but not SN-50. IL-1beta-induced IL-8 secretion by meniscal chondrocytes was also inhibited by MG-132 (control meniscus cells 585 ± 179 pg/ml, IL-1beta stimulation group 7,721 ± 2568 pg/ml, MG132 incubation only 432 ± 164 pg/ml, and IL-1beta/MG132 group 491 ± 176 pg/ml Taavon University, 3 Azad Medical University, 4 Ferdowsi University, 5 Purpose: Carbohydrate ingestion during prolonged strenuous exercise attenuates the leukocytosis and subsequent neutrophilia and lymphopenia by maintaining plasma glucose concentration and minimizing stress hormone and inflammatory cytokines responses. The purpose of this study was to determine the effect of pre-exercise high carbohydrate meals with high glycemic index (HGI) or low glycemic index (LGI) on blood leukocyte redistribution during subsequent endurance exercise. Methods: 24 male subjects (age 24.8 ± 0.35 years, body mass 76.1 ± 3.5 kg, height 1.77 ± 0.02 m, body fat percentage 11.1 ± 3.23, VO2max 51.18 ± 0.65 mL kg -1 min -1 ; mean ± SEM) performed two 90-min runs on a treadmill at 70% VO2max 2 h after ingesting a HGI or LGI meal. Each isocaloric test meal contained 1 g kg -1 body mass of carbohydrate and the glycemic index values were 94 and 40, respectively. Trials were separated by at least 7 days in counterbalanced order. Results were analyzed using a two-factor (trial 9 time) repeated measures ANOVA with post hoc (Bonferroni) comparison as appropriate. Results: Plasma glucose concentrations were significantly higher in LGI than HGI immediately after exercise. However, there were no significant differences in circulating leukocyte and neutrophil counts, plasma cortisol, interleukin-6 concentrations between trials. Conclusion: Ingestion of a LGI meal 2 h before sub maximal exercise maintains plasma glucose concentration during subsequent exercise when compared with ingestion of a HGI meal. However, it seems to have limited effects on circulating leukocyte and neutrophil counts, circulating stress hormone and cytokine responses to exercise. Keywords: High Glycemic Index (HGI), Low Glycemic Index (LGI), leukocyte redistribution, cortisol, interleukin-6(IL-6 Background: Inflammation is one pathology in the progression of heart failure. Here, we investigate the chemotactic properties of myofibroblasts as a stimulator for cardiac inflammation. Methods: Endomyocardial biopsies were obtained and analyzed in regard to inflammation and the number of myofibroblasts in patients with heart failure. Moreover, a human cardiac fibroblast cell culture system using cells from these heart failure patients was used to investigate the response of the fibroblasts to different stress stimuli in regard to the expression of chemokines, migratory activity and induction of inflammatory cell activation. Results: We show here that heart failure patients have increased numbers of myofibroblasts in their biopsies and importantly, S104 Inflamm. Res. increased numbers of inflammatory cells. Myofibroblasts did express chemokines (CCL2, CCL7 and CCL12) as well as proinflammatory cytokines (TNF-a und Il1b). Using supernatant from myofibroblasts in migration assays against inflammatory cells, we could document that the chemotactic properties of myofibroblasts were increased significantly by 300% compared to supernatant from unstimulated fibroblasts. Moreover, investigating the adhesion of inflammatory cells to myofibroblasts, we show an increased adhesion of inflammatory cells on myofibroblasts (+150%). These inflammatory cells were also activated by the supernatant of myofibroblasts and showed an increase in degradatory activity, allowing for easier transendothelial migration through the basal membrane. Conclusions: Myofibroblasts, known as the main contributor for extracellular matrix proteins were also highly chemotactic active and increased the number of inflammatory cells in patients with heart failure. This makes the myofibroblasts an inflammatory supporter cell and may be one explanation for cardiac inflammation in heart failure in general. Disclosure of interest: None declared. Introduction: High-risk type HPV is a marker for tumour progression from low-grade squamous intraepithelial lesion (LSIL) to an invasive squamous cell carcinoma (ICC) via a high-grade squamous intraepithelial lesion (HSIL). The host immune response is involved in viral persistence and progression of lesions. Most HPV infections likely involve a balance of Th1 and Th2 type immune responses. Objective: To measure serum and cervical levels of IL-2, IL-4, IL-6, IL-10, IL-12, IFN-c and TNF-a from women with LSIL, HSIL and ICC. Patients and methods: The study included 109 women with LSIL (n = 16), HSIL (n = 40), ICC (n = 13) and 40 women with suspected of HPV-induced disease, but no pathological changes on cervix biopsy (control group) attending in the Colposcopy Clinic of the Botucatu Medicine School (UNESP) and in the Preventive Gynecology, Hospital Amaral Carvalho, Jaú, SP. The cytokines concentrations were determined in cervical secretion and in serum by enzyme-linked immunosorbent assay. The search and genotyping for HPV in the fragments of the biopsies were performed by polymerase chain reaction using specific primers. The socio-demographic data was obtained by interview and study was approved by the local ethics committee. Results: A total of 78.9% of the patients were of Caucasian origin, 23.8% completed the second degree, 37.6% were smokers, 57.8% had at least three sexual partners lifetime and 8.3% reported a history of previous Sexually Transmitted Diseases. HPV DNA was detected in 90.0% in control group, 93.7% in LSIL, 100.0% in HSIL and 84.6% in the ICC. The HPV type 16 was the prevalent in all groups. The levels of IL-4, IL-6 and IL-10 in cervical secretion were significantly increased in patients with ICC. In the serum, the level of cytokines was not significantly different among the studied groups. Conclusion: These results suggest that the increased of cervical and serum Th2 cytokines of women with ICC is related to the progression of intraepithelial lesions. Chemotaxis, which is crucial for the trafficking of leukocytes during inflammatory and immune responses, is achieved by chemokine signaling through a chemokine receptor that belongs to the GPCR family. The precise mechanism underlying the connection between the chemokine-stimulated receptor and cytoskeletal reorganization is not fully understood. A cytoplasmic protein FROUNT binds to C-terminal region of chemokine receptor CCR2 and regulates monocyte and macrophage infiltration to sites of inflammation. Here we aim to reveal the molecular mechanism of FROUNT-chemokine receptor interaction in the regulation of chemotaxis signaling. We identified the minimum sequence in the C-terminal region of CCR2 for binding with FROUNT. We exploited the sequence to develop a novel screening method to identify inhibitors of FROUNT-CCR2 interaction. We identified selective inhibitors by screening small-molecule libraries. In a cell-based assay, some of such inhibitors tuned out to block CCR2-mediated chemotaxis. Our results indicate the potent possibility of this interaction between chemokine receptor and FROUNT as a therapeutic target for controlling the chemotaxis signaling. One appeal of our strategy is that it can avoid the undesired side effects caused by conventional approaches targeting receptor itself at the cell surface. Disclosure of interest: None declared. Methods: A study with 19 healthy elderly (12 women and 7 men) aged 84 ± 0.58 years and average co-morbidities of 5.31 ± 0.59 has been conducted at Federal University of São Paulo. It was evaluated the independence of instrumental activities of daily living (IAD), grip strength (GS), ability to sit and stand up in seconds (ASS) and IL-6 and TNF-a from monocytes stimulated with LPS. Data were expressed as mean ± SE and the Pearson' test between variables was used. Results: 63% of healthy elderly were independent IAD and 37% partial dependent, good GS (21.42 ± 1.69) and normal ASS (17.0 ± 1.21). TNF-a and IL-6 levels in culture supernatants of stimulated monocytes were 1,152.0 ± 113.70 and 1,045.0 ± 12.33 pg/ml, respectively. There was no correlation between cytokines levels and physical function or co-morbidities. Conclusion: Although low grade inflammation may be a marker of functional limitations in older people, we could not find any correlation between IL-6 and TNF from stimulated monocytes and physical function. It is possible that monocytes are not related to the functional activity of this elderly and that these healthy elders have a defense mechanism against the physical function disorders. Disclosure of interest: None declared. Introduction and objective: Studies have shown that supplementation with fish oil may modify cardiovascular risk and inflammatory markers. The studies with olive oil supplementation, however, are controversial yet. The aim of this study was to evaluate the effect of fish or olive oil supplementation on systolic blood pressure (SBP) and some mediators of hypertension and inflammation, using SHR. Procedures: Male SHR (12 weeks) received olive oil (Aceites Borges.) (OO, 1.5 g/kg), fish oil (Sigma) (FO, 1.5 g/kg) or vehicle (C) by gavage for 8 weeks, once a day. SBP was measured once a week by tail cuff plethysmography. ACE activity was determined in serum fluorimetrically and in cardiac and renal tissue using Hipuril-His-Leu (HHL) and Z-Phe-His-Leu (ZPhe-HL) as substrates. Cholesterol HDL and glucose were measured in serum and serum cytokines (TNF-a, IL-1b and IL-6) were determined by ELISA. Results: Both oils decreased SBP, ACE activity in serum and the ZPhe-HL/HHL ratio in the cardiac tissue. Only the FO decreased Cholesterol HDL but increased all studied cytokines. FO also increased Glucose levels in relation to C group. The Heart weight, however, was lower only in OO group. Conclusion: These data shows that FO and OO supplementation decrease the SBP associated with a reduction on ACE activity, in SHR. The decreased ZPhe/HHL ratio by both oils, however, may indicate decrease of N-domain ACE, able to cleave angiotensin 1-7, which has antiproliferative and vasodilatadores effects. The decrease of HDL and the increased glucose and cytokines levels in FO group can suggest insulin resistance, which could promote cardiovascular hypertrophy and hyperplasia. Disclosure of interest: None declared. We preciously reported the potent role of Amyloid b (Ab) on complement activation in pathogenesis of age-related macular degeneration (AMD) in WCI 2009. Using in vitro and in vivo studies, we further investigated the effect of Ab on complement alternative pathway (CAP) activation. Activities of CAP inhibitors; factor H and factor I were examined by cofactor assay. iC3b production was determined in Ab stimulated retinal pigment epithelium (RPE) and NEP mice which over accumulate Ab in subretinal space. Gene expression of CAP activator; factor B in RPE was analyzed by real-time PCR. Ab bond to factor H, factor I and inhibited activity of factor I to cleave C3b into iC3b. Ab stimulation decreased iC3b production in RPE. iC3b generation in NEP mice decreased compared to wild type mice. Ab did not alter factor B expression in RPE. In co-cultured RPE and microphage/microglia, Ab caused up-regulation of factor B in RPE, neutralizing antibodies against IL-1b and TNF-a abolished factor B up-regulation caused by Ab. finally, factor B was identified present in subretinal deposit of NEP mice. Combined mechanism of Ab-induced CAP activation: dysfunction of factor I and cytokine-induced up-regulation of factor B in subretinal space. Non-specifically activated CAP may result in the development of AMD. Disclosure of interest: None declared. We examined the pathophysiological roles of CC chemokines in antigeninduced arthritis (AIA) by using CCL3 -/-, CCR1 -/and CCR5 -/mice. Administration of methylated BSA (mBSA) into the intra-articular of the knee joint induced severe arthritis in wild-type (WT) mice, whereas the absence of CCL3 and CCR1, but not CCR5, attenuated the severity of AIA. Histopathologically, the infiltration of macrophages and T cells, in S106 Inflamm. Res. the mBSA-treated joint of CCL3 -/and CCR1 -/mice significantly reduced compared with WT mice. Furthermore, severe bone destruction was markedly suppressed in CCL3 -/and CCR1 -/mice with attenuated osteoclast differentiation, compared with WT mice. In vitro study, RANKL-induced osteoclastogenesis was significantly enhanced by CCL3 addition in WT-derived bone marrow cells, and anti-CCL3 Abs significantly suppressed these CCL3 effects on osteoclastogenesis. Supportingly, the enhancement failed to be observed in CCR1 -/-derived bone marrow cells. These observations indicated that CCL3-CCR1 axis would be essential for the osteoclastogenesis as a co-stimulator. Thus, the absence of CCL3-CCR1 interaction reduced macrophage recruitment from the bone marrow, and local osteoclastogenesis, and eventually ameliorated AIA. Collectively, these observations indicated that CCL3-CCR1 interaction might be a molecular target for the treatment of inflammatory arthritis. Disclosure of interest: None declared. Bone marrow (BM)-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a crucial role for CX3C chemokine receptor 1 (CX3CR1) in the recruitment of lung fibrocytes. An intratracheal injection of bleomycin (BLM) into wild-type (WT) mice caused a massive infiltration of leukocyte subsets, such as neutrophils, T lymphocytes, and macrophages, followed by the development of diffuse pulmonary fibrosis (PF) with accumulation of fibrocytes, characterized by CD45 + /collagen type I + cells. Intrapulmonary CX3CR1 and its ligand CX3CL1 expression were enhanced significantly and remained at elevated levels until PF developed. Compared with WT mice, collagen deposition was attenuated in CX3CR1 -/mice with reduced number of lung fibrocytes although BLM increased leukocyte infiltration to a similar extent in WT and CX3CR1 -/mice, implying that less intrapulmonary recruitment of fibrocytes directly correlated with decreased collagen deposition in CX3CR1 deficiency. Furthermore, BM transplantation from CX3CR1 -/to WT mice, but not that from WT to CX3CR1 -/mice, recapitulated the phenotypes in CX3CR1 -/mice. These results demonstrated that CX3CL1-CX3CR1 interaction was involved in BLM-induced recruitment of BM-derived fibrocytes and subsequent development of PF. Disclosure of interest: None declared. Infertility is an important health problem throughout the world and repeated in vitro fertilization (IVF) failure is distressing for the couples seeking reproductive assistance. Since among about onefourth of these couples no identifiable cause for infertility can be found, an immunologic basis has been attributed to unexplained losses for years. The aim of this study is to investigate the effects of antiinflammatory parameters (AIPs) as well as total antioxidative capacity (TAC) on the outcome of IVF therapy. Serum samples were obtained prior to and after IVF treatment from 26 women selected according to the appropriate criteria among 70 infertile women and 26 fertile women as the control group. Interleukin 10 (IL-10), IL-1 receptor antagonist, fetuin A, adiponectin as the AIPs were determined by ELISA. TAC was measured spectrophotometrically. SPSS was used for statistical analyses. IVF clinical pregnancy and live birth rates were determined as 30. 8 Background: Bacterial vaginosis (BV) is the most common type of abnormal vaginal flora. Although BV stimulates local cytokine production, it is still not well established its cytokine profile. It is believed that sialidase activity, a microbial hydrolytic enzyme, may modify the local innate immunity in BV and interfere in cytokine production. Objective: To determine whether differences in cytokine profile in BV cases are correlated with the sialidase activity. Methods: A total of 120 women attending outpatient clinics were included and were sampled for vaginal and cervical swabs, and vaginal wash with sterile buffer. All women had confirmed BV by microscopic classification of GRAM stained vaginal smears by Nugent criteria. Additionally, they were tested for other infections to assure that only cases of solely BV were included. Absence of Candidiasis was confirmed by microscopy, possible presence of Neisseria gonorrhoeae and Chlamydia trachomatis were excluded by culture and PCR, respectively. Sialidase activity was qualitatively determined by incubation of the samples of vaginal washes with the fluorogenic substrate 2 0 -(4-methylumbelliferyl)-aL-D-N-acetylneuraminic acid. The levels of IL-1b, IL-6, IL-8 and TNF-a were measured in vaginal washes by ELISA. All women provided written informed consent term and study was approved by the local ethics committee. Results: Among the 120 women with exclusive BV, the median values (range) of IL-1b, IL-6, IL-8 and TNF-a levels were 52. CCL25 is a homeostatic chemokine expressed in lymphoid organs and small intestine. CCR9 (CCL25 receptor) is mainly expressed on alpha 4 beta 7 -integrin + T lymphocytes and dictates their homing to intestinal mucosa. Here we show that CCL25 and alpha 4 beta 7 -integrin mediates gamma delta T cell migration into inflamed tissue during allergy. The intra-pleural (i.pl.) injection of ovalbumin (OVA) induced the migration of gamma delta T cells expressing CCR9 and alpha 4 beta 7 -integrin into the pleural cavities of immunized C57BL/6 mice. CCL25 levels increased in the pleural washes recovered from OVA-challenged mice (OPW). The in vivo neutralization of CCL25 did not inhibit gamma delta T cell migration to pleural cavities after OVA challenge; however, it selectively inhibited alpha 4 beta 7 -integrin + and IL-17 + gamma delta T lymphocyte migration into inflamed pleura. Accordingly, we observed that the majority of pleural alpha 4 beta 7 + gamma delta T cells from OVA-challenged mice express CCR9. Furthermore, CCL25 i.pl. injection induced the migration of alpha 4 beta 7 + gamma delta T cells to mouse pleura and triggered IL-17 production by those cells. CCL25 induced IL-17 + gamma delta T lymphocyte in vitro chemotaxis, even though it failed to alter IL-17 production by gamma delta T cells after in vitro stimulation. In addition, alpha 4 beta 7 -integrin blockade inhibited in vitro transendothelial migration of gamma delta T cells induced by CCL25 and OPW. Moreover, the in vivo blockade of alpha 4 beta 7 -integrin inhibited the migration of IL-17 + gamma deltaT lymphocytes into OVAchallenged mouse pleura. In contrast, anti-CCL25 mAb and antialpha 4 beta 7 -integrin mAb failed to inhibit the migration of alpha beta T lymphocytes, suggesting that the CCL25/alpha 4 beta 7 -integrin pathway is selective for gamma delta T cells. Our results reveal that, during the allergic reaction, CCL25 drives IL-17 + gamma deltaT cell mobilization to inflamed tissue via alpha 4 beta 7 -integrins and modulates IL-17 levels. Disclosure of interest: None declared. Caspase-4 has been proposed to be a member of the inflammatory caspases. We have previously found that caspase-4 mRNA is significantly upregulated in the brain of Alzheimer disease subjects. Other studies also implicate its role in the neuropathologies such as Alzheimer's disease. Despite numerous studies implicating the role of the gene in apoptosis and inflammation, the fact that the gene is only represented in the primate genome makes it difficult to characterize its physiological and pathological function in vivo. S108 Inflamm. Res. To study physiological function of caspase-4, we have generated transgenic mice expressing human caspase-4 under the control of its authentic promoter using bacterial artificial chromosome (BAC). Tissue expression analysis revealed the prominent level of caspase-4 expression in immune tissues such as spleen and thymus, while those in the brain and liver are relatively low, mirroring well the previous studies of mRNA distribution in human tissue. Unexpectedly we found significant portion of caspase-4 are in the processed form, although transgenic mice were viable and fertile. The role of caspase-4 in cytokine secretion becomes apparent upon lipopolysaccharide (LPS) challenge, as transgenic mice exhibited hyper-sensitivity to intraperitoneally injected LPS. At high dose, transgenic mice died within 24 h due to cytokine storm in the serum as well as in the brain. Quantitative gene expression analysis in the brain revealed the moderate upregulation of caspase-4 expression. At low dose, transgenic mice rapidly secreted higher levels of proinflammatory cytokines including interleukin-1b (IL-1b) and interleukin-18 (IL-18), both of which are known substrates of caspase-1. To investigate whether caspase-4 acts in a manner independent or dependent on caspase-1, caspase-4 transgenic mice are studied with caspase-1 null background. Our study indicates that caspase-4 acts in caspase-1 dependent manner for IL-1b and IL-18 secretion and endotoxemia. Disclosure of interest: None declared. Serum amyloid A (SAA) is a major acute-phase protein in mammals with proinflammatory and angiogenic activities. Although SAA is mainly synthesized by liver, extrahepatic synthesis occurs in several tissues including the placenta. In the early stages of pregnancy the hypoxia is essential to induce cell proliferation and angiogenesis in the placenta and organogenesis in the embryo. The hypoxia is also related to placental diseases. Interestingly, the serum levels of SAA are increased in preeclampsia. The possibility that hypoxia may control the expression of SAA was recently observed by our lab in adipose tissue cells. Herein we evaluated whether hypoxia affects the expression and intracellular concentration of SAA in placental villous explants. For this purpose, placental villous explants were cultured at \1% (hypoxia) and 21% (normoxia) of oxygen tension during 24 h. The placental villous explants from normal term placenta were lysate to measure SAA intracellular by commercial ELISA kit. The RNA total was extracted by Tryzol Ò and analyzed by RT-PCR using specific primers for SAA1 and SAA4. We also evaluated the mRNA and protein expression for hypoxia-inducible factor-1alpha (HIF-1a) by western blotting. In the experimental conditions used it was observed an increase mRNA and protein expression for HIF-1a. On the contrary, the hypoxia decreased the SAA1 and SAA4 mRNA levels. Also the intracellular concentrations of SAA in lysate of placental villous explants were diminished. According MTT test and consumption of glucose the viability of the cultured placental villous explants was maintained. Compared to adipocyte, in that it was observed that hypoxia increases SAA expression, the decrease of SAA induced by hypoxia in placental villous explants may contribute with a local physiological compensatory response in the first trimester of pregnancy or may be involved to abnormal remodeling of the placental vasculature found in preeclampsia. Sepsis remains an important clinical problem with a high mortality rate. Our long-term goals are to define new targets for therapy. We have previously shown that CD14-deficient mice are resistant to infection with clinical isolates of E. coli and that this resistance is accompanied by efficient clearance of the bacteria; in contrast, wild-type (WT) mice die from such infection and are unable to clear the bacteria. To define the mechanisms that regulate resistance, we have extended our studies to TLR4-deficient mice, the signaling molecule for the CD14/TLR4 receptor complex, and have designed experiments to define these mechanisms. Mice [wild-type (WT), CD14 -/or TLR4 -/-] were injected with live E. coli and parameters of their response to acute infection were measured, including survival, bacterial clearance, chemokine production and cell recruitment. Similar to CD14 -/mice, TLR4 -/mice were resistant to infection and cleared the bacteria more efficiently than WT mice. Bacterial clearance was due to an early recruitment of neutrophils to the site of infection that occurred in response to activation of a unique chemokine pathway that is activated independently of both CD14 and TLR4. Our results show a pathogenic role for the CD14/TLR4 pathway in severe Gram-negative infection resulting in high levels of proinflammatory cytokines; however this response may be secondary to a more important insidious role resulting in high levels of chemokines preferentially in blood, preventing PMN transmigration to infected tissue and facilitating the dissemination of bacteria from a localized site of infection. In contrast, activation of a second chemokine pathway that is independent of CD14 and TLR4 results in a chemokine gradient that promotes PMN recruitment to the site of infection, resulting in enhanced bacterial clearance. Supported by NIH/NIAID R01 AI023859 to SMG and by a grant to the RCMI program to The City College of New York from NIH/NCRR, G12RR03060. Disclosure of interest: None declared. Human odontoblasts are neural crest-derived, dentin-producing mesenchymal cells aligned at the periphery of the dental pulp. They become exposed to oral bacteria as these demineralise enamel then dentin to gain access to the pulp during the caries process. Given their situation at the dentin-pulp interface, odontoblasts are the first cells encountered by invading pathogens and/or their released components, and represent, in the tooth, the first line of defence for the host. We have focussed our study on the effects of Gram-positive bacteria as these largely dominate the carious microflora in initial and moderate dentin caries lesions. We have shown that odontoblast TLR2 engagement in vitro by lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, triggers TLR2 up-regulation, NF-kB nuclear translocation, production of chemokines and cytokines including CCL2, CXCL1, CXCL2, CXCL8, CXCL10, IL-6 and IL-10, while promoting immature dendritic cell recruitment. Responses to LTA were modulated by lipopolysaccharide-binding protein and CD14. Together these data suggest that odontoblasts may elicit innate immunity and related inflammation. Studies are ongoing to hinder Gram-positive bacteria sensing by odontoblast TLR2 and downstream signal transduction in order to prevent excessive inflammatory and immune responses that lead to the necrosis of the human dental pulp challenged with cariogenic bacteria. This work was supported by University Lyon 1, CNRS, IFRO and Région Rhone-Alpes. Disclosure of interest: None declared. Introduction: Interleukin-18 is a proinflammatory, proapoptotic, and proatherogenic cytokine belonging to the interleukin-1 family of cytokines. The cytokine exerts many unique immunologic and biological effects. It is produced as a biologically inactive and leaderless precursor protein, which must be cleaved into its mature form by caspase-1. The caspase-1 also exists in an inactive precursor in the cytosol and needs proteolytic auto-cleavage, which is catalyzed by the assembly of a multi-protein complex called inflammasome. During inflammation, interleukin (IL)-18 is produced by macrophages and other cell types such as keratinocytes and damaged endothelial cells. Objectives: To explore the role of IL-18 in inflammatory innate immune responses we investigated their impact on toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) expression by human peripheral blood monocytes, and its effect on TNF-alpha, IL-12, IL-15 and IL-10 production. Subjects and methods: Monocytes from healthy donors were stimulated or not with IL-18 during 18 h, and then TLR2, TLR4 and MR expression as well as intracellular TNF-alpha, IL-12 and IL-10 production were assessed by flow cytometry and the levels of TNFalpha, IL-12, IL-15 and IL-10 on culture supernatants were measured by ELISA. Results: IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-alpha and IL-10 was also increased by the treatment with the cytokine. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Introduction: Protection in tuberculosis has been associated with lymphocyte and macrophage activation, secretion of IL-12, IFN-gamma and TNF-alpha. On the contrary, IL-4 and IL-10 production, expansion and activation of regulatory T cells have been correlated with progression of infection. Our group showed that the frequency and activity of regulatory T cells during M. tuberculosis experimental infection are influenced by genetic background of infected host. Objectives: The aim of our study was to evaluate whether the expression of IFN-gamma receptor as well as IL-12 and IL-10 receptors would be differently expressed in infected C57BL/6 and BALB/c mice. Methodology: BALB/c and C57BL/6 mice were infected with 1 9 10 5 bacilli by intra-tracheal route. Thirty (initial phase) and 70 days (late phase) post infection, Colony-Forming Unit number and expression of cytokine receptors were evaluated. Results: C57BL/6 mice control the infection after 70 days while BALB/c mice did not. Day 70 infected C57BL/6 mice had a higher expression of IFN-gamma receptor (alpha chain) in lung CD11b + cells compared with day 70 infected BALB/c mice. The expression of IL-10 receptor (alpha chain) was up regulated in lung CD11b + and CD11c + cells obtained from day 30 infected BALB/c mice compared with C57BL/6 mice at the same period of infection. In addition, an increase in the expression of IL-12 receptor (beta1 chain) was found only in lung CD4 + cells of day 70 infected C57BL/6 mice. Conclusion: These data suggest that the differential expression of IFN-gamma, IL-12 and IL-10 receptors in hosts genetically different may represent one of immunological parameters which contribute to coordinate the induction of cellular immune response associated with the distinct capacity to control M. tuberculosis infection. Objective: To compare the viability of alveolar macrophages (AM) after in vitro infection with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Methodology: Male Wistar rats (n = 12) were anesthetized and tracheotomized in order to obtain AM from the bronchoalveolar fluid. Following the AM isolation procedures, four systems were set up: negative control containing AM (NC), positive control containing AM and 10 lL of lipopolysaccharide (PC), MSSA containing AM and 100 lL of a suspension of ATCC 29213 (OD = 0.15, corresponding to 10 6 CFU mL -1 ) and MRSA, containing AM and 100 lL of a suspension of ATCC 33591 suspension (OD = 0.15, corresponding to 10 6 CFU mL -1 ). Systems were then incubated at 37°C in a 5% CO 2 atmosphere for 24 h. AM viability was assessed based on the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl Leprosy is chronic infectious disease caused by Mycobacterium tuberculosis with wide clinical spectrum. Although the detection rates has fallen a lot in the last 20 years, Brazil is still considered an endemic country for leprosy by WHO and north region of the country has the largest coefficients. Despite its ancient history, leprosy still presents many aspects that need to be studied, in special regarding the chronic skin inflammation linked to the bacteria-induced lesions. In the last years, a novel lineage of CD4 T helper cells (Th), termed Th17, was characterized by the production of interleukin 17 (IL-17), involved in a plenty of autoimmune and chronic inflammatory reactions. However, its role in the leprosy immunopathology is still unknown. Then, this study aimed to identify whether the expression of IL-17 in leprosy patients. Tissue samples of leprosy patients were obtained from 20 patients at Alfredo da Matta Foundation, in Manaus, Amazonas State, Brazil. Leprosy diagnosis was clinically confirmed and associated with baciloscopy and histopathology. The tissue samples were collected before treatment under topical anesthetic inoculation. The samples were cryopreserved and used for immunohistochemistry analysis using rabbit anti-IL-17 antibody. IL-17 expression was confirmed after immunoperoxidase staining by microscopy analysis. IL-17 expression was consistently higher in leprosy patients than in normal cutaneous tissue from control individuals. IL-17 seemed to be detected specially in the mononuclear cells in the skin cellular infiltration, as visualized in the stained sections. The IL-17 expression was elevated in all clinical forms of leprosy evaluated. The results demonstrated that IL-17 + cells are present in all clinical forms of the leprosy skin lesions, suggesting that the IL-17 inflammatory response is restricted to the skin lesion site. A single dose of Crotalus durissus terrificus snake venom induces a long lasting anti-inflammatory effect on paw edema and cell migration induced by carrageenan in mice. Recently it was demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, is responsible for this effect. Cytokines are regulators of the acute phase of inflammation, increase the production of chemokines that attract inflammatory cells to the inflammatory site, and also stimulate the expression of adhesion molecules. Cell migration induced by carrageenan has been shown to be mediated by cytokines IL-6 and IL-1b. The aim of this study was to evaluate the action of crotoxin on the secretion of IL-1b and IL-6. A single dose of crotoxin (0.89 lg/50 lL s.c.) or saline (50 lL) was administered 7 days or 1 h before carrageenan injection in males Swiss mice. To evaluate the effect of cytokine secretion, after 4 h of intraperitoneal injection of Cg (300 lg/200 lL) or saline (200 lL), peritoneal exudate was collected and the assay of cytokines, such as IL-1b and IL-6 were determined by ELISA. The peritoneal exudate of animals pretreated with crotoxin showed a significant decrease in the secretion of all pro-inflammatory cytokines evaluated, such as IL-1b and IL-6 when compared to untreated animals. The decrease in cytokine secretion observed in exudates from animals pretreated either 7 days or 1 h with crotoxin was 49 and 71% for IL-1b, respectively, and 12% and 51% for IL-6, respectively. We could suggest that the long-lasting anti-inflammatory effect on cell migration involves the modulatory action of crotoxin on the secretion of important pro-inflammatory cytokines that participate in the inflammatory response induced by carrageenan. The functions of the pleiotropic cytokine IL-6 are mediated through a receptor system consisting of gp130 and soluble or membrane IL-6 receptor (IL-6R). Deregulation of this system is implicated in a variety of diseases, including rheumatoid arthritis (RA). Blocking IL-6R results in clinical benefit as shown with tocilizumab, an anti-IL-6R monoclonal antibody (mAb). Nanobodies (NB) are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, naturally occurring in Camelidae. They have a high degree of homology to human Ig VH domains but yet have distinct features that favor monovalent high affinity binding. ALX-0061 is a highly specific anti-IL-6R NB engineered to obtain half-life extension (HLE) in vivo, and high target affinity and potency. Monovalent interaction with IL-6R avoids unwanted cross-linking, while the HLE strategy excludes the need for an Fc-region, thus avoiding immune effector functions. These properties were considered important for a potentially improved safety profile compared to full IgG mAbs. ALX-0061 demonstrated primary pharmacology in targeting and neutralizing IL-6R with superior affinity and comparable in vitro potency to tocilizumab. ALX-0061 dose-dependently suppressed inflammation in an induced acute phase response model in cynomolgus monkey. Pharmacokinetic analysis demonstrated a favorable profile which was predicted to translate into convenient dosing in humans. Measurement of circulating target levels supported the calculation of the minimum pharmacological effect level and the first-in-human dose. No unexpected pharmacology has been demonstrated both in vitro and in vivo. In conclusion, ALX-0061 combines high potency with favorable PK properties in non-human primates and has the potential to be an effective treatment for multiple inflammatory conditions. A clinical trial application for the treatment of active RA patients has been filed and initiation of a Phase I study is planned for beginning 2011. Disclosure of interest: None declared. Background: Interferon alpha (IFNa) has been associated with the severity and disease activity of Systemic Lupus Erythematosus (SLE). Active immunization against IFNa with IFNa-Kinoid (IFN-K) has been shown to prevent severe renal lupus disease and to prolong survival in NZW/NZB mice models. Anti-huIFN antibodies neutralize all 13 subtypes of huIFNa and as well as against IFNa in sera of lupus patients. Objectives: We evaluate IFN-K in lupus patients in terms of safety, immunogenicity, clinical response and impact on IFNa-regulated serum chemokines levels and on IFNa-inducible gene signature. Material and methods: IFNa-Kinoid (IFN-K, Neovacs SA, Paris, France) is an immunotherapeutic composed of recombinant human IFNa conjugated to KLH as a carrier protein, inactivated and adjuvanted with ISA-51 emulsion. Patients with mild to moderate lupus (SLEDAI 4-10) and positive for anti-dsDNA antibodies are being enrolled in a double-blind, placebo-controlled, phase 1-2, dose escalation study to evaluate four different doses of IFN-K Immune responses are being evaluated through titration of anti-IFNa and anti-KLH antibodies with isotyping, evaluation of neutralizing capacity and of cellular responses. Clinical response is being assessed by regular evaluation of BILAG and SLEDAI scores and titration of serum auto-antibodies. Impact on IFNa regulated chemokines and neutralisation of IFNa-inducible gene signature as assessed by Affymetrix Ò are evaluated over the study period in comparison to the scores in healthy volunteers who had blood collected to determine baseline IFNa-regulated serum chemokines levels and IFNa-inducible gene signature. The CXCL12 chemokine plays an important role in inflammation. Our team identified a CXCL12 neutraligand (C05) belonging to the family of chalcones, that inhibits its interaction with CXCR4 and CXCR7 (JBC 2008) and the eosinophil infiltration in a model of asthma. Due to its low solubility (C05, 9 lM), we decided to facilitate its administration by adding three types of groups: phosphate (C05-P), L-serine (C05-Ser) and sulfate (C05-S), as prodrugs. The analytical characterization of the three compounds was performed on the Techmed ILL platform (http:// www.pcbis.fr): solubility and stability (t 1/2 ) were measured in serum, PBS and lung homogenate. The in vitro activity was evaluated by S112 Inflamm. Res. intracellular calcium assay and in vivo in a 8-day asthma model: Balb/c mice were sensitized (OVA+alum, i.p, D0,1,2) and challenged to OVA or saline (i.n, D5,6,7). Drugs or solvent were administered i.p. or i.n. 2 h before each challenge. Prodrugs are 5-10,000 times more soluble than the chalcone C05. None of these prodrugs modifies the calcium impulse induced by CXCL12. On the other hand, after incubation in mouse serum or lung homogenate, the prodrugs are transformed into C05 (HPLC). In vivo, at 350 lmol/kg i.p, C05-P (PBS) has the same activity as C05 (CMC 1%) with 80% inhibition of eosinophil recruitment in BAL. C05-P versus C05 administered i.n. at a dose of 22 lmol/kg (limit of C05 solubility in PBS) inhibits the eosinophil recruitment by 50 versus 0%, respectively. C05-P i.n. shows an activity 10,000 times higher (IC 50 = 10 ± 7 nmol/kg) than i.p. administration. A comparison between compounds C05-P, C05-Ser and C05-S (i.n.) at 30 nmol/kg shows similar activity (67, 69 and 64%, respectively). Use of soluble CXCL12 neutraligand prodrugs enable local administration and activity at low doses and offers an interesting strategy for the development of drug candidate for asthma treatment. Furthermore, our short model of asthma is highly efficient to estimate new therapeutic strategies. Disclosure of interest: None declared. We have also investigated their association with psoriasis severity and looked for the correlation between BAFF titres and the mRNA TNF-a intralesional ones. Patients and methods: Serum BAFF was measured in 62 patients with psoriasis and 50 healthy controls (HC). Skin biopsy specimens were taken from both lesional and non lesional skin of 24 among the 62 patients and from 4 HC skin. BAFF levels were detected by ELISA. TNF-a mRNA expression levels were analyzed by QRT/PCR. Disease activity was evaluated according to the Psoriasis Area and Severity Index (PASI) score. Results: The level of serum BAFF was significantly increased in patients with psoriasis compared to HC (p \ 10 -3 ). In addition, both elevation in serum BAFF and mRNA TNF-a intralesional expression levels were associated with disease activity (p \ 10 -3 in the two cases). In addition, BAFF serum and mRNA TNF-a intralesional expression levels were higher in PsA group when compared to all other psoriatic patients (respectively p = 0.002 and p \ 10 -3 ). More interestingly, the titres of serum BAFF were correlated with mRNA TNF-a intralesional expression levels (p = 0.008). Conclusion: Our results suggest that BAFF may be involved in the pathogenesis of psoriasis. Nevertheless, it is unclear which mechanisms contribute to the high BAFF levels. It is conceivable that an inflammatory process triggers, via the production of inflammatory cytokines notably TNF-a was incriminated. Disclosure of interest: None declared. Pharmacology, Gifu Pharmaceutical University, Gifu, Japan Bronchial asthma is characterized by allergic airway inflammation caused by various mediators. Many recent studies demonstrated the importance of lipid mediators in the onset and development of bronchial asthma. These studies promote our investigation to find out a new inhibitor of lipid mediator as a remedy for allergic bronchial asthma. Our purpose of this study is to evaluate the effects of dual antagonists for cystenyl leukotriene receptor and thromboxane A2 receptor (KP-496 and RS-601) on experimental allergic asthma in guinea pigs. Actively sensitized animals were repeatedly exposed to antigen, and drugs were administered [KP-496 (0.01 and 0.1%); inhalation for 5 min and RS-601 (3 and 10 mg/kg); per orally 2 h] before every antigen exposure. KP-496 (0.1%) and RS-601 (3 and 10 mg/kg) significantly inhibited an antigen-induced increase of airway resistance at 4 h after antigen challenge (late phase response) and airway hyperresponsiveness to acetylcholine. Furthermore KP-496 and RS-601 clearly suppressed the infiltration of inflammatory cells into airway. From above experiments, two dual antagonist, KP-496 and RS-601 showed the inhibitory action on both an asthmatic respiratory disorder and inflammatory changes of airway in guinea pigs. These findings suggest they will be beneficial agents in the treatment of bronchial asthma. (All experiments were undertaken following the guidelines for the care and use of experimental animals of the Japanese Association for Laboratory Animals Science.) Disclosure of interest: None declared. Background: Sphingosine 1-phosphate (S1P) is one of the cellderived lysophospholipid growth factors that signal diverse cellular functions such as proliferation, angiogenesis, and inflammation. We previously demonstrated that S1P signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. FTY720(FTY) is a high-affinity agonist of S1P receptors which induces internalization of S1P receptors, rendering the cells unresponsive to S1P. The present study we investigated the role of S1P signaling for osteoblast differentiation using C2C12, a murine myoid cell line, which differentiate into osteoblasts. Methods: Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence either S1P or FTY. Osteoblast differentiation was determined by osteoblastspecific transcription factor Runx2 mRNA expression after 2 days of culture, ALP activity in the cells after 7 days of culture, or osteocalcin production by the cells after 10 days of culture. Smad1,5,8 and ERK1/2 phosphorylation was examined by western blotting. Results: Runx2 expression, ALP activity and osteocalcin production by BMP-2-stimulated C2C12 was enhanced by addition of either S1P or FTY. This enhancing effect of FTY on osteoblast differentiation was stronger than that of S1P. Both S1P and FTY enhanced BMP-2 stimulated Smad1,5,8 phosphorylation of C2C12. These enhancing effects of S1P/FTY were inhibited by addition of MEK1/2/inhibitor (U0126), indicating that S1P signaling enhanced BMP-2-Smad axis via activation of MEK1/2, ERK1/2 axis. Conclusions: These results suggest that S1P signaling enhances BMP-2-Smad axis via activation of MEK1/2-ERK1/2 axis, thereby augmenting osteoblast differentiation. FTY may be useful for the treatment of RA by enhancing osteoblast differentiation as well as the inhibition of inflammatory cells infiltration in the synovium. Disclosure of interest: None declared. Asthma is a chronic inflammatory disease of the airways that can lead to airway fibrosis. One of the key events in this process is the local activation of fibroblasts to myofibroblasts, which expresses a-smooth muscle actin (a-SMA) and actively synthesizes and secretes type I collagen. PGE 2 is a lipid mediator that inhibits fibroblast activation by ligating the EP2 receptor. Idiopathic pulmonary fibrosis is associated with a defect in fibroblast PGE 2 synthesis and/or responsiveness. We investigated if the airway fibrosis in murine asthma is associated with a defect in the synthesis/response axis of PGE 2 by lung fibroblasts. Female BALB/c mice were immunized 29 with ovalbumin (OVA) in alum (ip) and subjected to 3, 7 or 12 airway challenges with OVA in PBS. Lung sections were stained for Collagen-1 and PAS. Fibroblasts were isolated from lung minces after the indicated number of challenges and grown in culture. Cells were incubated with PGE 2 , the selective EP2 analog butaprost free acid (BFA), and the direct activator of adenyl cyclase forskolin. Proliferation was measured by [ 3 H]thymidine incorporation and a-SMA and collagen-I synthesis by western blot. PGE 2 was measured by EIA and EP2 receptor expression by real time RT-PCR. Collagen deposition around airways was absent after 3 challenges, minimal after 7, and intense after 12. This was accompanied by a time-dependent impairment in the ability of the lung fibroblasts to upregulate PGE 2 under IL-1b stimulation, which was maximal after 12 challenges. At this time point we also observed a reduced expression of the PGE 2 synthetic enzymes COX-2 and mPGEs1. The responsiveness of fibroblasts to PGE 2 , measured as proliferation, a-SMA expression and collagen-I synthesis, as well as EP2 expression, was not different among the groups. Since PGE 2 inhibits fibroblast activation, a defect in the ability of fibroblasts from animals with chronic asthma to produce this molecule may be implicated in the pathogenesis of airway fibrosis. Disclosure of interest: None declared. Among pro-inflammatory lipids, platelet-activating factor (PAF) is a key mediator in the induction and progression of many pathophysiologic states such as atherosclerosis, where dendritic cells (DC) can act as regulators of disease progression. As some signalling pathways activated by PAF involve JAK kinases, we investigated the modulation of functional responses of DCs to PAF by one of the regulators these kinases, protein-tyrosine phosphatase non-receptor type 1 (PTPN1). Using DCs, generated from monocytes isolated from healthy volunteers, we down-regulated PTPN1 expression using siRNAs, which decreased PTPN1 protein expression by 25%. Here, we show that even if modest, this down-regulation of PTPN1 is enough to modify the profile of cytokine mRNA expression observed after PAF stimulation, without affecting maturation of DCs. In DCs transfected with PTPN1 siRNAs, as compared to those transfected with control siRNAs, PAF induced significantly higher mRNA levels of IL-6, IL-8 and CCL2 whereas IL-10 level was not significantly affected. On the other hand, PAF-induced mRNA levels of TNF alpha and TGF beta were drastically decreased in presence of PTPN1 siRNAs. Luciferase assay studies in PAF receptor-transfected HEK 293 cells showed that over-expressed PTPN1 down-regulated promoter activity of IL-6 and IL-8 in response to PAF. Our results suggest that the effects of PTPN1 on IL-6 promoter activity are dependent on Tyk2 and Src kinase activity, given that over-expression of mutants of either kinase partially rescues the activity of the IL-6 promoter in response to PAF. Together, our results suggest that PTPN1 is an important modulator of PAF-induced functional responses by influencing cytokine production. Me´decine, Universite´Laval, CRCHUQ, Que´bec, Canada LPA stimulates fibroblast-like synoviocyte migration in a woundclosing assay and the secretion of IL-8 and IL-6. As IL-8 is a potent chemoattractant involved in inflammatory cell influx into the human synovium and autotaxin-derived LPA has been detected in synovial fluids from rheumatoid arthritis (RA) patients, we assumed that LPA might recruit leukocytes to inflammatory sites either directly or indirectly by stimulating the release of cytokines/chemokines. In this study we used a murine air pouch model to study the influx of immune cells. To assess LPA receptor dependency and the mechanisms of LPA-induced leukocyte recruitment in vivo we used both a pharmacological and a genetic approach. We report that (1) LPA injected into air pouches recruited leukocytes in a concentrationand time-dependent manner; (2) in LPA3 -/mice, LPA-induced leukocyte influx into the air pouch was partially inhibited while LPA3 agonist OMPT-induced leukocyte influx was totally blocked. The LPA1/3 antagonist VPC32183 injected either locally into the air pouch or intravenously completely inhibited LPAinduced leukocyte recruitment in WT and LPA3 deficient mice; and (3) TNFa injected into the air pouch prior to LPA strongly enhanced LPA-mediated leukocyte influx as well as chemokine secretion, such as KC. Interestingly, the CXCR2 chemokine receptor antagonist SB22502 administered i.v. to mice reduced, in a concentration-dependent manner, LPA-mediated recruitment of leukocytes into the air pouch. The ability of LPA to recruit leukocytes and the enhanced production of cytokines/chemokines in TNFa-primed air pouches suggest that LPA, via LPA1 and LPA3 receptors, may contribute to the pathogenesis of RA. The results provide novel insights into the relevance of LPA receptors as potential therapeutic targets for the treatment of autoimmune diseases such as RA. Disclosure of interest: None declared. Background: Acute pancreatitis is an inflammatory process of the pancreatic gland that in the severe forms triggers the inflammation in remote organs. An additional characteristic of pancreatitis is the necrosis of peripancreatic adipose tissue due to the release of lipolytic enzymes. These areas of adipose tissue release in turn inflammatory mediators. Here we have evaluated the generation of halogenated free fatty acids by necrotic adipose tissue in a model of acute pancreatitis. Methods: Pancreatitis was induced in male rats by intraductal administration of 3.5% sodium taurocholate. We obtained samples of adipose tissue and ascitic fluid 3, 6 and 18 h after induction, and the levels of free fatty acids as well as fatty acid chlorohydrins were evaluated by GC-MS. In additional experiments we administered fatty acid chlorohydrins, generated by chlorination of adipose tissue lipid extracts, on the peritoneum of control animals. Three hours later we obtained peritoneal macrophages and the expression of TNFa on these cells was evaluated by RT-PCR. Results: During pancreatitis, necrotic areas of adipose tissue generate and release free fatty acids as well as its chlorohydrins. We identified oleic acid chlorohydrin and mono-and bis-chlorohydrin of linoleic acid in both adipose tissue and ascitic fluid. Administration of chlorinated lipids in the peritoneal cavity results in an increased expression of TNFa by peritoneal macrophages. The Low-density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/a-collagen (ShcA), which also binds to several tyrosine kinase receptors (TKRs) such as the insulinlike growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies TKRs signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Objective: Although an important role of sphingosine-1-phosphate (S1P) in arthritis has been suggested, its function in chondrocytes is still not fully known. On the other hand, an important involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of osteoarthritis (OA), probably through regulating angiogenesis has been speculated. We here investigated the in vitro effect of S1P on the expression of VEGF in human articular chondrocytes from OA patients. Methods: Human articular cartilage samples were obtained from patients with OA under informed consent. Chondrocytes were isolated using enzymatic procedure and then cultured in vitro by monolayer, and the cells were stimulated with S1P in the presence or absence of inhibitors of mitogen activated protein kinase (MAPK) signaling molecules and a Gi protein inhibitor pertussis toxin (PTX). VEGF expression and its secretion in culture supernatants were analyzed using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA It is has been suggested that COX2 is the primary source of the protective metabolite, PGI 2 (prostacyclin), because selective COX2 inhibitors lower urinary levels of its metabolites. In most healthy S116 Inflamm. Res. tissues, however, COX2 protein is rare in comparison to COX1 and we have previously shown that COX1-but not COX2-deficiency prevents PGI 2 release by isolated arteries. Using this bioassay approach, we sought to determine the relative role of COX1 and COX2 in PGI 2 synthesis in other tissues of healthy mice. Methods: PGI 2 release was determined in 10 mg pieces of heart, liver, kidney and spleen from wild type (WT), COX1 -/and COX2 -/mice (n = 4). Within 10 min of death, tissues were dissected and incubated in DMEM containing A23187 Ca 2+ ionophore (50 lM) with or without the COX1/2 inhibitor diclofenac (100 lM) for 30 min at 37°C. Medium was then removed and PGI 2 measured as its stable breakdown product, 6-keto-PGF 1a by ELISA. Results: In heart pieces, 6-keto-PGF 1a accumulation was not altered by COX2-deficiency (WT: 8.2 ± 2.8 ng/ml; COX2 -/-: 6.0 ± 3.3 ng/ml) but was abolished by COX1-deficiency (.3 ng/ml; p \ 0.05). In liver pieces too, 6-keto-PGF 1a accumulation was similar between tissue of WT and COX2 -/mice (WT: 4.0 ± 0.5 ng/ml; COX2 -/-: 3.5 ± 0.8 ng/ml) but absent in tissue from COX1 -/mice (.3 ng/ml; p \ 0.001). In kidney pieces, 6-keto-PGF 1a levels were not different between tissues of WT and COX2 -/mice (WT: 8.2 ± 1.3 ng/ml; COX2 -/-: 11.1 ± 4.6 ng/ml) but were undetectable in COX1 -/tissues (.3 ng/ml; p \ 0.001). Finally, in spleen pieces, 6-keto-PGF 1a accumulation from WT tissues ( Lipoxins (LXs), are endogenously produced eicosanoids, which possess potent antiinflammatory and proresolution bioactivities. The role of LXs in the endometrium is unknown. Our initial observations showed LXA 4 enhanced estrogen receptor (ER)-mediated transcriptional activation in Ishikawa endometrial epithelial cells. Furthermore, we demonstrated that LXA 4 possesses robust estrogenic activity through its capacity to alter cellular proliferation as well as the expression of estrogen-regulated genes implicated in cancer development. Interestingly, LXA 4 also demonstrated antiestrogenic potential in that it attenuated E2-mediated cellular proliferation, consistent with the effects of a partial ER agonist. Subsequent studies revealed that these actions of LXA 4 were directly mediated by ERa and appear to closely mimic those of the potent estrogen, 17b-Estradiol (E2). Using competitive radioligand binding assays, we confirmed that this lipid binds ER. We additionally demonstrated this estrogenic activity of LXA 4 in mouse uterus in vivo using a uterotrophic assay and the expression of E2dependent genes as readouts. Taken together our results establish a dual capacity of LXA 4 to modulate estrogenic activity in the endometrium. These findings highlight a previously unappreciated Prostaglandins play an important role in inflammatory processes. PGD2 and PGE2 are biological antagonistic, respectively, stimulating or inhibiting the removal of microorganisms in the host. However, these compounds possess poor hydro solubility and chemical instability, difficulty there in vivo administration. It is well known that microspheres (MS) are able to improve stability and sustain the release of substances and target their deliveries. The aim of this work was to prepare and characterize MS containing PGD2 or PGE2; in vitro evaluation of their ability to be phagocyted by alveolars macrophages (AMs); establish the involvement of PGD2 in Histoplasmosis in vivo. The PLGA [poly-(lactic acid-glycolic acid)] MS were prepared by the emulsification-solvent evaporation technique. Size and zeta potential were evaluated in aqueous media by Light Scattering, and morphology analyzed by Scan Electronic Microscopy. The size, zeta potential and shape of MS (MS-PGD2, MS-PGE2 and unload-MS) were proper to be administered through the intranasal route and able to attempt the lung. Phagocytosis were determined after 4 h with 2 9 10 5 AMs using 1 mg of each MS. Both Gamma delta T lymphocytes migrate into inflammatory sites and participate in inflammatory and infectious responses. We investigated the involvement of the 5-LO-derived lipid mediator LTB 4 in gamma delta T cell migration. When injected into the intrapleural (i.pl.) space of C57BL/6 mice, LTB 4 triggered gamma delta T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli (LPS) triggered increased levels of LTB 4 in pleural cavities. The in vivo inhibition of LTB 4 biosynthesis by the 5-LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS-induced gamma delta T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit gamma delta T cells into the inflammatory site after i.pl. LPS. Antagonists of the high-affinity LTB 4 receptor BLT1, CP105,696, and LY292476 also attenuated LPSinduced gamma delta T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS-simulated mice. LTB 4 /BLT1 also accounted for gamma delta T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS-recruited gamma delta T cells. Isolated gamma delta T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB 4 in vitro, confirming that gamma delta T lymphocytes can respond directly to LTB 4 . In addition to its direct effect on gamma delta T cells, LTB 4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that gamma delta T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB 4 Leukotrienes play an important role in innate immunity and are active participants in inflammatory diseases such as asthma, atherosclerosis and rheumatoid arthritis. 5-lipoxygenase (5-LO) is the crucial enzyme for leukotriene biosynthesis by catalyzing the transformation of arachidonic acid to leukotriene A 4 . We identified several splicing variants of 5-LO in lymphoid and myeloid cell lines and in human neutrophils. All cells that were tested expressed an mRNA encoding for 5-LO that contained the expected 14 exons of the full-length 5-LO. Additionally, we identified several variants of the 5-LO mRNA: one that retained intron 10 (a-10), another that lacked exon 13 (D-13), one that lacked exons 10 and 13 (D-10,13) and one that lacked the first 96 base pairs of exon 10 (D-p10). When the cDNA of these splicing variants were transfected in HEK293 cells, the resulting 5-LO isoforms were expressed but were devoid of catalytic activity. Immunoblot analyses with recombinant 5-LO isoforms demonstrated the expression of the D-13 isoform in Raji cells and in human neutrophils. Interestingly, when variant 5-LO isoforms were co-expressed with the active full-length 5-LO, biosynthesis of 5-LO products was inhibited. The focus of ongoing studies is to understand mechanisms regulating the expression 5-LO isoforms and to determine the molecular interactions involved in their inhibition of the biosynthesis of 5-LO products. We are currently investigating the subcellular distribution of the different 5-LO isoforms and their putative interactions with 5-LO-activating protein (FLAP) and coactosin-likeprotein (CLP). Overall, these variant 5-LO isoforms likely represent a novel endogenous mechanism for the regulation of the 5-LO pathway and lipid mediator biosynthesis. (Supported by the Heart and Stoke Foundation of Canada and the Canada Research Chairs (MES); the Natural Sciences and Engineering Research Council of Canada (NF); a scholarship from the Canadian Arthritis Network (LHB)). Disclosure of interest: None declared. Aim: To investigate the anti-tumor effect of lipoxin A4 (LXA4), a endogenous pro-resolution lipid mediator, on hepatocellular carcinoma in vitro, in vivo and its mechanism. Methods: HepG2, U937, THP-1, RAW264.7 and H22 cells were cultured. To assess the effect of tumor-assassinated macrophages (TAM) on HCC cells, in some of the experiments, supernatant from activated macrophages was isolated and applied to stimulate HepG2. Cytotoxicity was first screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, immunofluorescence, flow cytometry analysis, western blot were employed to elucidate the anti-tumor mechanism of lipoxin. Anti-tumor activities against subcutaneoulsy implanted solid tumor induced by H22 cells in mice were evaluated. Results: Lipoxin could significantly suppress hepatocellular carcinoma, inhibit the proliferation, angiogenesis and invasive ability of HepG2 cells, promote its apoptosis via blocking the activation of macrophages, and in vivo experiment, BML-111, LXA4 receptor (ALX) agonist could inhibit the growth, invasion and angiogenesis in H22 bearing mice. Conclusion: LXA4 effectively suppressed hepatocarcinoma. It could be a possible candidate for liver cancer therapy, and blocking macrophages' activation will be an effective drug target for liver cancer therapy. Disclosure of interest: None declared. The human monocyte-like Mono Mac-1 (MM1) cell line is a recognized model to study monocyte/macrophage differentiation. In this study, we investigated whether their differentiation with LPS would impact polyunsaturated fatty acid (PUFA) metabolism. Upon treatment of MM1 cells with LPS, the expression of the monocyte marker CD14 was enhanced and this correlated with a significant increase in 5-lipoxygenase expression (assessed by qPCR and immunoblot). Importantly, major changes in the cellular fatty acid profile were also observed. The 16-carbon monounsaturated to saturated fatty acids ratio was greatly decreased in differentiated cells, which would indicate a decrease in stearoyl-CoA desaturase-1 expression during differentiation. Interestingly, we found that differentiated MM1 cells' phospholipids were enriched in 20:4n-6 (arachidonic acid, AA) and 20:5n-3 (eicosapentaenoic acid, EPA) which are the precursors of pro-and anti-inflammatory lipids mediators. Additional experiments were performed to elucidate the putative mechanisms involved in AA and EPA enrichment. When incubated with their 18-carbon precursors, differentiated MM1 cells accumulated twice as much AA and EPA compared to undifferentiated cells. Similarly, the retroconversion to AA and EPA from their 22-carbon counterparts was also more efficient in differentiated cells. Finally and in agreement with the enhanced 5-LO expression and the AA and EPA buildup, we observed that differentiated MM1 cells also acquired the capacity to release AA and to synthesize leukotriene B 4 following ionophore stimulation. In conclusion, MM1 cells represent a good model to investigate changes in fatty acid metabolism and incorporation into phospholipids and in eicosanoid metabolism that accompany human monocyte-macrophage differentiation. ( In this study, we demonstrate that the naturally occurring caffeic acid phenethyl ester (CAPE), a component of propolis from honeybee hives, is a potent inhibitor of LTs in human neutrophils. The calculated IC 50 for the inhibition of LTs biosynthesis by CAPE and the known 5-LO inhibitor zileuton were 0.51 and 3.49 lM, respectively. Synthesis of some structural analogues of CAPE was also performed to investigate their structure-activity relationship as free radical scavengers, antioxidants and 5-LO inhibitors. Both ester and amide analogs of CAPE were designed with the rational that esters may be more susceptible to chemical and enzymatic degradation compared to the corresponding amide. Our results show that while these compounds are effective free radical scavengers and antioxidants, certain structural features are required for effective inhibition of LTs biosynthesis in human neutrophils. Hepatitis caused by hepatitis C virus is a serious global public health, the main route of transmission is related to contact with contaminated blood or intravenous drug use. Various evidences have suggested that the formation of LBs during some infections. This is a phenomenon that may have implications in the pathogenesis of these infections and is related to the formation of LTB4 and PGE2. The purpose of this study was to characterize the presence of LBs and LTB4 and PGE2 synthesis in peripheral blood mononuclear cells of individuals infected with hepatitis C. The study included 70 candidates for blood donation, which in the period of August/2009 at July/2010 showed reactivity in serological screening tests (ELISA and immunoblot) in the Foundation of Hematology of the Amazon. Peripheral blood mononuclear cells were cultured from under different stimulis for subsequent staining and counting of LBs. The LTB4 and PGE2 in the cell culture supernatant was determinated. We observe that the core and NS5 proteins of HCV stimulates the formation of lipid bodies in cells of HCV positive patients stimulated in vitro, but not induces PGE2 production in this group. However LTB4 is increased in patients infected with HCV compared with HCV negative individuals. HCV positive individuals have increased concentrations of IL-12 and IL-10 in peripheral blood. Our data suggest that in individuals infected with hepatitis C there is a weak signal for the Th1 response and a high number of LBs in PBMC and high concentration of LTB4, IL-12 and IL-10 when compared to HCV patients negative. These results suggest one possible mechanisms of viral escape and may be associated with chronic infection. Alteration in skin blood flow has been implicated in a variety of clinical conditions associated with inflammation such as urticaria and diabetes. Hydrogen sulfide (H 2 S) has recently been identified as an endogenous gaseous transmitter with ability to induce both vasodilatation and vasoconstriction in a variety of vascular beds. There is little known about the contribution of hydrogen sulfide in modulation of skin blood flow. The present study was designed to investigate the effect of local and systemic administration of hydrogen sulfide on skin blood flow in anesthetized rats. Male Wistar rats were used in this study. Cutaneous blood flow was recorded from soles skin using a laser Doppler flowmeter. Local inhibition of cystathionine-c-lyase (the enzyme responsible for synthesis of H 2 S in vasculature) was associated with a dose-dependent increase in skin blood flow. Cutaneous application of sodium sulfide (a H 2 S donor) induced a biphasic response in presence of cystathionine-c-lyase inhibitor. While low concentration of sodium sulphide made a significant decrease in skin blood flow, higher concertinos induced vasodilatation. Intravenous administration of sodium sulfide induced a significantly less cutaneous vasodilatation in presence of cystathionine-c-lyase inhibitorThis study is the first study to show a biphasic effect for local H 2 S in modulation of skin blood flow in an experimental model. Disclosure of interest: None declared. Objective: Exogenous H 2 S delivered to rat knee joint significantly inhibited carrageenan (CGN)-induced synovitis; supporting the role of H 2 S donors as therapeutic alternatives for arthritis. We now aimed to identify the existence of H 2 S producing enzymes in rat knee and their role in synovitis model. Methods: Under approval of USP Animal Ethics Committee, anaesthetized rats pretreated with cystathionine-c-lyase (CSE) inhibitor (DL-propargylglycine; PGly), an H 2 S donor (Lawesson's reagent; LR) or saline were intra-articular injected with carrageenan (CGN, 3%) or saline. After 4 h,leukocyte counts were performed in bone marrow (BM), knee samples were collected and, cytokines, protein expression and activity of CSE, cystathionine-b synthase (CBS), caspase-1, NF-kB and AP-1 were measured. The kinetic cycle of H 2 S synthesis in synovia was determined based on methylene blue formation method. Data are mean ± SEM for n animals. Statistical analysis was by ANOVA + Bonferroni's test. Results: Injection of CGN evoked synovitis and increased leukocyte concentration in BM, IL-1b and IL-10 in joint fluid and activity of caspase-1 and NF-kB. Treatment with LR increased IL-10 and attenuated cell infiltration and caspase-1 activity without affecting NF-kB activition. PGLy treatment only augmented AP-1 activity. Quantitative assay shows that CBS and CSE, the key enzymes for H 2 S synthesis, were identified in synovial membrane, and H 2 S generation augmented in a time-dependent manner. CSE and CBS inhibitors reduce H 2 S generation by 50 and 75%, respectively. Conclusion: The protective effects of H 2 S on CGN-induced synovitis might be mediated by increase of IL-10 and down-regulation of caspase-1 activity. This occurs independently of H 2 S-producing enzymes found to be constitutively expressed in the rat synovial membrane; reinforcing the importance of exogenous H 2 S in arthritis. Acknowledgments: FAPESP, CNPq, University Agostino Neto. MA Barreto and IM Gouvea for technical assistance. Disclosure of interest: None declared. S120 Inflamm. Res. Hydrogen sulfide (H 2 S) has been shown to be a potent inhibitor of leukocyte infiltration, and appears to contribute to resolution of inflammation in the GI tract. This study investigated both the synthesis and functional characteristics of H 2 S in four phenotypically different primary macrophage populations. Bone marrow-derived macrophages are differentiated ex vivo with macrophage-colony stimulating factor, representing a 'classical', inflammatory macrophage. Polyacrylamide-gel beads (BioRad) injected i.p elicited an 'alternative' macrophage phenotype. Finally resident/tissue macrophages were collected from the peritoneal cavity or dissociated from colon lamina propria (LP). Resident peritoneal cells are the first line of host defence, acting to prime the inflammatory response via pattern recognition receptors. LP macrophages, on the other hand, lack many of these receptors due to their proximity to local gut flora. The capacity for macrophages to synthesize H 2 S, assessed by methylene blue formation, was shown to be variable across a wide concentration range with LP and resident peritoneal macrophages being the most abundant source. Induction of inflammation, both in vitro and in vivo, reduced the ability of macrophages to synthesize H 2 S, paralleled by reduced cystathionine gene and protein expression. The addition of H 2 S to macrophage populations modulated cytokine release (TNF-a, IL-10) following inflammatory stimuli, increased phagocytic capacity, and also up-regulated chemotactic responses to fMLF in a concentration dependent manner. Mechanisms of H 2 S signaling were assessed by intracellular cAMP/cGMP levels and NFjB activation. This study provides the first phenotypic characterisation of H 2 S synthesis in different primary macrophage populations. Together, these data suggest an important role for macrophage-derived H 2 S in maintenance of tissue homeostasis and resolution of inflammation. Disclosure of interest: None declared. Hydrogen sulphide (H 2 S) is increasingly being recognized as an important pro-inflammatory mediator in various inflammatory conditions including sepsis. The production of H 2 S from L-cysteine in the cardiovascular system is catalysed primarily by cystathionine gammalyase. H 2 S has been shown to induce transient receptor potential vanilloid 1 (TRPV1)-mediated neurogenic inflammation in polymicrobial sepsis. However, endogenous neural factors that modulate this event remain unclear. Here, we examined whether substance P is one important neural element that implicates in H 2 S-induced neurogenic inflammation in sepsis-associated lung injury in a TRPV1-dependent manner. Male Swiss mice were subjected to cecal ligation and puncture (CLP)-induced sepsis and treated with TRPV1 antagonist capsazepine 30 min before CLP. DL-propargylglycine, an inhibitor of H 2 S formation, was administrated 1 h before or 1 h after CLP, whereas sodium hydrosulfide, an H 2 S donor, was given at the same time as CLP. Capsazepine significantly attenuated H 2 S-induced substance P production, inflammatory cytokines (TNF-a, IL-1b, IL-6) and chemokines (MIP-1a, MIP-2) expression, edema formation, and neutrophil sequestration in septic lung. Administration of sodium hydrosulfide further enhanced substance P production and exacerbated lung inflammatory damage but capsazepine reversed these deleterious effects. In the absence of H 2 S, capsazepine had no effect on DL-propargylglycine-mediated abrogation of substance P levels, inflammatory cytokine and chemokine production, edema, and myeloperoxidase activity in the lungs of septic mice. Taken together, the present findings reveal that H 2 S regulates TRPV1-mediated neurogenic inflammation in sepsis-associated lung injury through enhancement of substance P production. The anti-inflammatory effects of capsazepine may have therapeutic potential for the treatment of sepsis. Disclosure of interest: None declared. Introduction:The septic responses including thrombocytopenia and increased production of reactive oxygen species (ROS) can be reproduced by LPS in experimental animals.The Src kinase and PI3 K has been involved the ROS production, and may also mediate some effects of LPS. In the present study, we aimed to identify the sources of ROS formation, and to investigate the role of Src and PI3K in modulating ROS formation and aggregation in platelets obtained from LPS-treated rats. Methods: Wistar rats were injected i.p. with saline or LPS (1 mg/kg), and at 6 or 48 h thereafter arterial blood was collected (Ethical Committee No. 2097-1) . ROS production in platelets was measured by flow cytometry DCFH-DA (5 lM). ADP (20 mM)-induced platelet aggregation was evaluated using a two-channel aggregometer. Results: The ROS production in platelets from LPS-treated rats was 2.5-fold higher (P \ 0.05) than in saline-injected rats. Incubation of platelets with NADPH oxidase inhibitor DPI (5 lM) significantly attenuated the increased ROS production in LPS group (27 and 60% at 6 and 48 h, respectively). Similarly, the PI3K inhibitor wortmannin (100 nM) and Src kinase inhibitor PP2 (5 nM) significantly attenuated the increased ROS production in ADP (20 lM)-activated platelets in LPS group, whereas acetylsalicylic acid (100 lM) and allopurinol (100 lM) had minimal effects. The ADP-induced platelet S122 Inflamm. Res. aggregation was significantly reduced in LPS group compared with saline (P \ 0.05), but incubation with wortmannin or PP2 failed to modify this inhibitory aggregatory response. The incubation of platelets from saline-treated group reduced 60% with wortmannin and 30% with PP2. Conclusion: Our data indicate that NADPH-oxidase is the main source of ROS formation in platelets isolated from LPS-treated rats, and that activation of Src kinase and PI3 K takes place in the response. On the other hand, Src kinase and PI3 K are not involved in the reduced platelet aggregation seen in LPS-treated rats. Disclosure of interest: None declared. Introduction: Some clinical studies speculate that the pathophysiology of malaria associated-acute kidney injury (AKI) may occurs by nephrotoxicity of products of oxidative stress, element present in Plasmodium infection, as well as pro-inflammatory response induced by the parasite. This resume briefly summarized our progress in the understanding of mechanisms involved in malaria associated-AKI development during experimental model of severe malaria. Methods: The parasite load and renal function were monitored in Balb/c mice infected with 10 5 parasitized erythrocytes with P. berghei ANKA. To assess renal function, blood urea nitrogen and serum creatinine was evaluated. The presence of parasitized erythrocytes and profile of pro and anti-inflammatory molecules was quantified by real time PCR (kidney tissue) and Bioplex (serum). Products of oxidative stress were estimated by ELISA in plasma of animals described above. Results: So far, we observed an increase of serum levels of creatinine and urea, as well as a decrease of ratio of creatininuria and proteinuria during acute phase of infection. Furthermore, it was found the adhesion of parasitized erythrocytes in the kidneys of infected mice. These data are consistent with the increased expression of mRNA of iNOS, HIF-1a, IFN-gand ICAM-1, associated with a decrease in the expression of mRNA HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizabilityalso showed a significant increase in plasma and urine of infected animals. Conclusions: Together, our findings bring some elucidation about mechanisms of acute kidney injury associated with oxidative stress and structural changes during acute phase of plasmodium infection. Homocysteine (Hcy) is a metabolic risk factor for cardiovascular disease. High levels of homocysteine promote oxidant stress in vascular cells and tissue because of the formation of reactive oxygen species (ROS), which have been strongly implicated in development and progression of diabetes mellitus by the activation of matrix metalloproteinase-9 (MMP-9). Thus, we investigated the sources of ROS generation and mechanismsn of MMP-9 production in murine macrophages stimulated with homocysteines. Homocysteine enhanced protein expression and gelatinolytic activity of MMP-9 in murine macrophages. The enhanced MMP-9 expression and activity was significantly attenuated not only by PD98059, a ERK inhibitor but also by SH-5, an Akt inhibitor, respectively. In line with these results, homocysteine exclusively increased the phosphorylation of ERK and AKT, suggesting a role for the ERK and AKT pathway in homocysteineinduced MMP-9 expression and activity. Furthermore, homocysteine stimulated macrophage cells to showed an increased production of ROS, which was significantly attenuated by cyclooxygenase inhibitors such as indomethacin, but not affected by other oxidase inhibitors involving xanthine oxidase, lipoxygenase and mitochondrial, suggesting a potential role of cyclooxygenase in homocysteine-induced ROS generation. Taken together, it is suggested that homocysteine enhances MMP-9 production in macrophages via cyclooxygenase generation of ROS-mediated activation of ERK/Akt signaling pathways, consequently leading to vascular remodeling in diabetes. Disclosure of interest: None declared. Objective: Kinin B 1 receptors are highly induced in the course of inflammation. The present study analyzed the Nitric Oxide (NO) production in primary cultured endothelium obtained from lung explants of B 1 receptor knockout (B 1 -/-) and wild type (WT) mice. Methods: Cells (n = 3-4 for each group) were pre-incubated with DAF-2 DA (10 lmol/L), fluorescence was detected in a confocal microscope and quantified by optic densitometry. Images were obtained before and after 5 min of acethylcoline [ACh (1 mmol/L)] stimulation, with 15 s intervals between each image capture. Cells were pre-treated with the NOSynthase (NOS) co-factor tetrahydrobiopterin [BH4 (0.1 mmol/L)] or the substrate L-arginine (1 mmol/L). Control experiments were performed in WT cells in the presence L-NAME (1 mmol/ L) or B 1 antagonist [des-arg 9 -leu 8 -BK (0.1 mmol/L)]. Results: ACh caused a gradual increase in fluorescence that was lower in B 1 -/cells during the whole 5 min of image capture. In all experiments, maximal responses (expressed as arbitrary units) were reached 270 s after ACh stimulation, thus, this point of the time curves was chosen to be compared in all treatments. At this point NO release was markedly reduced in B 1 -/-(35.8 ± 3.1*) in comparison to WT cells (66.9 ± 3.2). B 1 -/responses were reversed by BH 4 (54.3 ± 1.7), but not by L-arginine (26.2 ± 8.5*). In WT cells, BH 4 , L-arginine and des-arg 9 -leu 8 -BK had no effect (55.0 ± 9.2, 51.1 ± 11.9 and 55.3 ± 4.8, respectively), whereas NO production was blunted by L-NAME (35.8 ± 4.7*) (* P \ 0.05 vs. WT/ACh). Conclusions: Targeted B 1 receptor deletion impairs endothelial NO production by mechanisms involved in the reduction of the co-factor BH 4 availability. Since deficient levels of BH 4 induce uncoupling of NOS and production of reactive oxygen species, a relevant role of endothelial B 1 receptors in modulating vascular oxidative stress during inflammation can be suggested. Background and objectives: Inflammatory responses management such as Reactive Oxygen Species (ROS) formation, lipid peroxidation and reduction in conjugating capacitance of hepatocytes are so important in preventing cell necrosis and death due to some injuries like acetaminophenoverdose which causes severe hepatic necrosis or exposure to some oxidant metal ions.Studies on this field remain a very active area since some of current data are still uncertain. In this study, freshly isolated rat hepatocytes were used to determine the effects of garlic extract on the acetaminophen and metal ion -induced cell cytotoxicity and to compare it with the effect of N-Acetyl Cysteine (NAC) as a standard treatment. Materials and methods: Male Sprague-Dawley rats (200-250 g) wereanaesthetized and after hepatocyte isolation, the cell suspensions were studied and the amount of ROS formation, lipid peroxidation and glutathione depletion were determined using spectrofluorometry, spectrophotometry and analytical HPLC methods respectively, in different groups of cell suspensions. Results and conclusion: The results of this study indicate that, garlic extract was significantly effective (P \ 0.05) effective in decreasing ROS formation, lipid peroxidation and glutathione depletion induced by acetaminophen overdose, as well as the first two factors in Ferric ion exposure; but was not significantly effective (P [ 0.05) in glutathione depletion caused by Ferric ion. NAC was completely effective (P \ 0.01) in reducing all 3 necrotic factors. In conclusion, we can suggest garlic extract as an anti inflammatory agent in preventing lipid peroxidation and ROS formation. It seems that more studies are necessary on different cell types to confirm this hypothesis. Introduction: Acute kidney injury (AKI) is a clinical condition associated with high levels of morbidity and mortality. The ischemia and reperfusion injury (IRI) is one of the most relevant conditions that lead to AKI. The presence of inflammatory cytokines, chemokines, adhesion molecules and the recruitment of leukocytes into the postischemic kidneys characterizes the inflammatory process. Apocynin is a pro drug that is converted to a dimer form through the oxidation of mieloperoxidase. It prevents the translocation of the cytosolic oxidase components to the membrane fraction of NADPH-oxidase. The aim of this study is to evaluate the ability of apocynin in preventing the inflammatory process associated to renal IRI. Methods: C57/BL6 mice were subjected to renal IRI, having their renal pedicles clamped bilaterally for 45 min, and after that, renal blood flow were restored. Sacrifice was performed 24 h after reperfusion. Animals were divided in three groups: sham, IRI and apocynin (4 mg/kg i.p. just before reperfusion). During sacrifice, blood and renal tissue were collected. Renal function was evaluated by serum creatinine and urea S124 Inflamm. Res. levels. Histological alteration was observed by HE staining. RT-PCR was performed for TNF and IL-6 and IKK expression were assessed by western blotting. Results: We observed that IRI group presented higher creatinine and urea levels when compared to sham group; whereas in apocynin group it was restored. Histological evaluation showed less inflammatory infiltrates on treated group. Also, IRI showed a significant increase on gene expression of TNF and IL-6, when compared to the other groups. Finally, IKK protein expression was higher on IRI group and apocynin treatment was able to decrease it. Conclusion: Treatment with apocynin reduced renal dysfunction and preserved histological architecture. Moreover, it also modulated the inflammatory process, leading to renoprotection. Methodology: male Wistar rats (n = 12) were breastfed by rats whose diet contained 17% and 8% of protein during lactation (N and NM group, respectively). After weaning, both groups were administered a normoproteic (23% of protein) diet. AM were obtained from the brochoalveolar fluid after tracheotomy. Following the isolation procedures, three systems were set up: negative control containing AM (NC), positive control containing AM and lipopolisaccharide (PC) and SA containing AM and a suspension of ATCC 29213 (OD = 0.15, corresponding to 10 -6 CFU.mL -1 ). Systems were then incubated at 37°C in a 5% CO 2 atmosphere for 24 h. During incubation, samples of 100 lL from the supernatant were collected at intervals of 2 h and subsequently nitric oxide was measured with the Greiss reaction. Statistical analysis were performed using Student t test and Mann-Whitney assuming a significance level of p \ 0.05. Results: malnutrition led to the decrease of macrophageal production of NO in all systems (p \ 0.005). Major production of NO was reached at the intervals between 4 and 8 h in both experimental groups, N and MN (p \ 0.001) in the SA system. In addition, a decrease in nitrite concentration was observed after 10 h with similar values as determined in NC system (p [ 0.005). Conclusion: the model of neonatal malnutrition promoted disorders to the function of phagocytic cells, as well as interfered in the macrophageal nitrosative stress. Moreover, S. aureus possibly has developed a mechanism for reducing the NO concentration in the extracellular fluid) in order to preserve its cellular integrity. Disclosure of interest: None declared. Introduction: Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in airways. FA is emitted by building materials, furniture carpets, wallpaper, plywood, floor coverings and sterilizing agents. We recently demonstrated that FA effects on airways are partially due to an increased oxidative stress. However, the source of reactive oxygen species (ROS) remained to be determined. It is investigated herein the role of antioxidant and oxidant enzymes on the release of ROS after FA exposure. Material and methods: Male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for three consecutive days. After 24 h of the last FA inhalation, the activities and gene expression of glutathione peroxidase (GPX), glutathione redutase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD) 1 and 2, catalase (CAT), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 were determined in lung tissue. Results: FA inhalation did not modify the activity of GPX, GR, GST and CAT, but reduced the activity of SOD when compared to the naïve group. A significant increase in the SOD 1 and 2, CAT, iNOS and COX-1 expression were observed in FA group when compared to the naïve group. Leukotriene B4 (LTB 4 ), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent released in many inflammatory situations and it is able to activate biological responses in human neutrophils (PMN) as well as reactive oxygen species (ROS) generation by the NADPH oxidase complex. LTB 4 delays neutrophils spontaneous apoptosis through the activation of classical pro-survival signaling, which in turn may corroborate to the onset of a chronic inflammatory condition. Recently, ROS have emerged as secondmessengers, coordinating intracellular signaling cascades, and thus modulating several biological phenomena, including apoptosis. In this study, we aim to elucidate the putative role of NADPH oxidasederived ROS in LTB 4 antiapoptotic effect. PMN were isolated from whole blood of healthy volunteers by Ficoll-Paque TM density gradient. ROS production was evaluated by cytochrome c reduction as well as lucigenin and luminol enhanced-chemiluminescence. Apoptosis was determined by cell morphology and annexin V-phosphatidylserine binding. Mitochondrial membrane potential was assessed by flow cytometry of JC-1-stained cells. Protein expression was evaluated by western blot analysis of total or nuclear extracts. Our data show that NADPH oxidase-derived ROS are critical to LTB 4 prosurvival effect on neutrophils. This event depends on redox modulation of NF-jB translocation and IjB-a phosphorylation/ degradation. We have also observed that LTB 4 -induced Bad degradation and mitochondrial stability requires NADPH oxidase activity. Our results strongly indicate that LTB 4 -induced antiapoptotic effect in neutrophils occurs via ROS-dependent signaling routes and we do believe that a better knowledge of molecular mechanisms underlying neutrophils spontaneous apoptosis may contribute to design better strategies to control chronic inflammation. Objectives: Relaxin (RLX), a member of the insulin/relaxin family of structurally related hormones, has an influence on many physiologic processes, such as collagen turnover, angiogenesis, and antifibrosis. The periodontal ligament (PDL) contains fibroblasts, blood vessels, nerves, and a large amount of collagen, primarily of type I. The homeostasis of the PDL implicates towards intensive and subtle transcriptional and translational regulation of the collagens and the matrix metalloproteinases (MMPs) genes. In response to mechanical stress during orthodontic tooth movement, the PDL produces inflammatory cytokines and MMPs in vivo. Therefore, it is considered that thishormone may have an influence on orthodontic tooth movement through alterations of PDL. In the present study,we investigated the effects of RLX on the release and gene expression of MMP-1 and 8 by hPDL cells in vitro. Methods: hPDL cells were cultured with 100 ng/ml RLX for 0-72 h. The mRNA levels of the MMP-1 and 8 were determined by using real-time PCR and the protein levels were determined by using ELISA. Effects of specific inhibitors of p38 mitogen-activated protein kinase (MAPK) activity, extracellular signal-related kinase (ERK) activity and c-Jun N-terminal kinase (JNK) activity on RLX-induced MMP-1 and 8 expression in hPDL cells were also investigated by real-time PCR. Results: Real-time PCR analysis revealed that RLX increased MMP-1 and 8 mRNA expression in a time-dependent manner. The release of MMP-1 and 8 also increased in a time-dependent manner. ERK inhibitor and JNK inhibitor considerably decreased the RLX-induced MMP-1 expression, whereas p38MAPK inhibitor markedly inhibited MMP-8 expression. Conclusion: These results indicate that RLX stimulated the expression of MMP-1 and 8 in hPDL cells via MAPK signaling pathways. Therefore, RLX may contribute to periodontal connective tissue and bone matrix remodeling during orthodontic tooth movement via MMP-1 and 8. We clarified the mechanism of elastinolysis by granulocytes. Proteases in granulocytes that have been reported to have elastinolytic activity, such as neutrophil elastase, proteinase-3 (p29b, AGP7, proteinase-4), showed negligible elastinolytic activity when orceinelastin was used as a substrate. Although medullasin alone showed negligible elastinolytic activity, it expressed significant elastinolytic activity in the presence of the granulocyte protein, azurocidin, in the incubation mixture. The mechanism by which the product of partial digestion of azurocidin by medullasin induced the elastinolytic activity of medullasin was studied. Neither the low nor highmolecular weight fractions produced by the digestion of azurocidin by medulllasin showed elastinolytic activity by themselves. However, medullasin expressed significant elastinolytic activity in the presence of high-molecular-weight fraction produced by the digestion of azurocidin by medullasin. The product of partial digestion of azurocidin by medullasin forced the protease to adsorb to elastin fibers, thereby, inducing elastinolytic activity. These results indicate that elastinolytic activity of granulocytes is not derived from single proteases such as medullasin, neutrophil elastase, proteinase-3, but from the cooperation between medullasin and azurocidin in granulocytes. Disclosure of interest: None declared. Matrix metalloproteinases (MMPs), a family of Zn-dependent neutral proteolytic enzymes, are known to play a critical role in erosion of cartilage during arthritis. Moreover, levels of these enzymes are increased in inflamed articular joints. MMPs and snake venom metalloproteinases (SVMPs) share common catalytic domain organization and exhibit identical Zn-binding motif. Therefore, studies on SVMPs may provide insights into the functions of MMPs. In this study we investigated the effects of BaP1 on isolated synoviocytes, the main articular cells involved in production of inflammatory mediators, evaluating: (a) release of prostaglandin E 2 (PGE 2 ); (b) gene and protein expression of ciclooxigenase-2(COX-2); (c) participation of NF-kB in COX-2 protein expression, and (d) expression of EP4 receptor. B type synoviocytes isolated from rat knee joint synovial membranes were used. Levels of PGE 2 were measured by EIA, and gene and protein expression of both COX-2 and EP4 receptor determined by real time RT-PCR and W.blot, respectively. Results showed that BaP1 induced release of PGE 2 from B type synoviocytes after 1, 3 and 6 h, but not after 30 min incubation, in comparison with control cells. BaP1 induced COX-2 gene and protein expression by synoviocytes (30 min-3 h) without modification on constitutive COX-1. Inhibition of NF-kB by TPCK or SN50 compounds significantly decreased BaP1-induced COX-2 protein expression. Increased levels of EP4 receptor expression (52 and 65 kDa) were also detected in synoviocytes stimulated by BaP1 (3 h). In conclusion, BaP1 can directly stimulate synoviocytes to synthesize PGE 2 and express COX-2. This effect is mediated by NF-kB. In addition, BaP1 is able to increase expression of EP4, a PGE 2 receptor.This may contribute for amplification of PGE 2 -induced effects. These findings suggest novel regulatory mechanisms for metalloproteinases in B type synoviocytes, which may be relevant for inflammation and pain in the joints. Support: FAPESP, CNPq. Disclosure of interest: None declared. Proteinase-activated receptor (PAR) 2, a G-protein-coupled receptor activated through N-terminal cleavage by serine proteinases, has been implicated in both intestinal inflammation and epithelial proliferation. Cyclooxygenase (COX)-2 is over-expressed in the gut during inflammation as well as in colon cancer. We hypothesized that PAR 2 drives COX-2 expression in intestinal epithelial cells. Treatment of Caco-2 colon cancer cells with the PAR 2 -activating peptide 2-furoyl-LIGRLO-NH 2 (2fLI) for 3 h led to an increase in intracellular COX-2 protein expression that was not observed following treatment with a reverse-sequence PAR 2 -inactive peptide. COX-2 expression was also increased in Caco-2 cells following a 3 h treatment with either trypsin, cathepsin G or proteinase 3, all serine proteinases known to cleave the N-terminus of PAR 2 . Treatment of Caco-2 cells with 2fLI for 30 min significantly increased metalloproteinase activity in the cell culture supernatant. Increased epidermal growth factor receptor (EGFR) phosphorylation was observed in cell lysates following 40 min of treatment with 2fLI. The broad-spectrum metalloproteinase inhibitor marimastat inhibited both COX-2 expression and EGFR phosphorylation. The EGFR tyrosine kinase inhibitor PD153035 also abolished 2fLI-induced COX-2 expression. Although PAR 2 activation increased ERK MAPK phosphorylation, ERK pathway inhibitors failed to affect 2fLI-induced COX-2 expression. However, inhibition of Rho kinase signalling by Y27632 and inhibition of PI3 kinase signalling by LY294002 each prevented 2fLI-induced COX-2 expression. In conclusion, PAR 2 activation drives COX-2 expression in Caco-2 cells via metalloproteinase-dependent EGFR transactivation and activation of Rho kinase and PI3 kinase signalling. Our findings provide a mechanism whereby PAR 2 participates in the progression from chronic inflammation to cancer in the intestine. Disclosure of interest: None declared. PARs and TLRs receptors are involved in innate immune response. Aim of this study was to evaluate the possible cross-talk between PAR-2 and TLR4 in vascular district in physiological condition by using in vivo and ex vivo models. In this study we used thoracic aortas harvested from both naïve and endotoxemic rats for in vitro studies. Arterial blood pressure was monitored in anesthetized rats in vivo. In aortic homogenates immunoprecipitation of TLR4 receptor followed by PAR-2 immunoblot and western blot analysis for TLR4 and PAR-2 were performed. PAR-2, but not TLR4, expression was enhanced in aorta of endotoxemic rats. PAR2-AP-induced vasorelaxation was increased in LPS-treated rats aortic rings. TLR4 inhibitors, curcumine and resveratrol, reduced PAR-2-AP-induced vasorelaxation and PAR-2-AP-induced hypotension in both naïve and endotoxemic rats. Immunoblotting with anti-PAR-2 of anti-TLR4 immunoprecipitates revealed PAR-2/TLR4 association in both naïve and endotoxemic rats. Western blot performed on surnatants of anti-TLR4 immunoprecipitates showed the presence of PAR-2 exclusively in aorta of endotoxemic rats. Finally, in TLR4 -/mouse aortic rings, PAR2-AP-induced vasodilatation was impaired compared to wild type. PAR-2 and TLR4 are constitutively associated and contribute to vascular homeostasis. Disclosure of interest: None declared. Patients with clinically active Crohn's Disease exhibit increased intestinal permeability. However, apical addition of serine proteases like trypsin to intestinal epithelial cell monolayers reduces permeability as shown by increased transepithelial resistance (R TE ). This effect of trypsin is independent of activation of protease-activated receptors, but the underlying mechanism is unknown. Given the central role of epidermal growth factor receptor (EGFR) in epithelial cell biology and homeostasis, we hypothesized that trypsin increases R TE by activating EGFR. Methods: R TE of confluent SCBN monolayers and mouse colonic tissue was measured in Ussing chambers. To determine a role for EGFRassociated signaling, cells were pretreated with inhibitors of EGFR (PD153035), ErBb2 (AG879), ERK-1/2 (PD98059), PI3 K (LY294002) and MMPs (marimastat, MMT) andthen stimulated apically by trypsin or EGF. Flux of FITC-dextran across the monolayer was measured following treatment with PD153035 or AG879 and trypsin. Results: Apical trypsin increased R TE by 300% (p \ 0.05) within 60 min. PD153035 or AG879 caused a significant dose-dependent decrease in trypsin-induced DR TE , with IC 50 s of 0.38 and 2.73 lM, respectively. PD98059, LY294002 and MMT also reduced trypsininduced DR TE by 66, 63 and 25% respectively. Pretreatment of cells with PD153035 or AG879 prevented trypsin-induced reduction of dextran flux. Apical EGF increased R TE by 80%. PD153035 and AG879 reduced this increase by 99 and 84% respectively. Also, soybean trypsin inhibitor decreased R TE in mouse colon in vitro, suggesting that luminal serine proteases maintain barrier function. Conclusions: Our data suggest that trypsin increases R TE via activation of EGFR and ErBb2 and partially via activation of ERK-1/2, PI3K and MMPs. Serine proteases may strengthen epithelial barrier to enhance the efficiency of electrolyte and nutrient transport, and may represent a method to counter barrier defects caused by intestinal inflammation. Disclosure of interest: None declared. Tea has various beneficial effect on health. Green tea contains catechins, theanine, and vitamins such as folic acid and ascorbic acid which exert healthy effect. In contrast to green tea, black tea contains polymers of catechins such as theaflavin or thearubigin other than catechins. In this study we examined the effect of black tea extract, theaflavin, and thearubigin on the activity of neutral serine proteases in granulocytes. Neutral serine proteases in granulocytes such as medullasin and cathepsin G were shown to play important roles in biophylaxis. Both proteases stimulate lymphocyte function and enhance NK cell activity. Medullasin causes inflammation leading to the development of atherosclerosis. Mice fed with diet containing black tea components such as theaflavin or thearubigin revealed decreased medullasin activity dose-dependently, but cathpsin G activity decreased only slightly. In contrast to black tea components, addition of green tea components such as epicatechin, epigallocatechin, and epigallocatechin gallate to the diet of mice caused an increment of both medullasin and cathepsin G activity. From these results described above drinking of black tea is considered to be useful for the prevention of atherosclerosis by decreasing medullasin activity in granulocytes. Disclosure of interest: None declared. Pulmonary emphysema has become epidemic worldwide. The mechanisms by which extracellular matrix degradation occurs are not yet fully understood. However, literature described both inflammatory and oxidative processes as critical to parenchyma destruction. Thus, this study focused to investigate the partipation of oxidative stress during the emphysema establishment process induced by elastase in mice. C57BL/6 mice were submitted at a protocol with pancreatic porcine elastase (PPE) instillation (0.05 U, 0.5 U (i.t.) or 3 U (i.n.) of PPE per animal), to induce pulmonary emphysema in vivo (n = 6). Lung tissue was collected on days 1, 7, 14 and 21 after PPE instilation. Vehicle treated mice were used as controls. Emphysema typical morphological alterations were seen just at 21 days after PPE 0.5 U. TNF-a was elevated at 7 and 14 days after PPE 0.5 U, and was accompanied by a reduction in IL-10 levels at the same time-points. MPO was elevated at the three time-points with PPE 0.5 U. The levels of Nitrite, TBARS and the SOD activity were also increased at 7 days after PPE 0.5 U, while CAT activity was decreased. When the dose was enhanced to 3 U of PPE and a group with just 1 day after stimulus was performed, CAT activity presented reduced at 1 day, while SOD activity was increased at the same time-point. These results are corroborated with mRNA expression, with an increase in Nrf2, Sirt-1, CAT and GPx. However, there was no difference in mRNA SOD expression between any groups (control, 1, 7, 14 and 21 days). ELISA for TGF-b1 showed a reduction just at 21 days after 3U of PPE. These results indicate that the proteolytic pathway has a link with the oxidative pathway. These data suggest that redox imbalance generated by proteolytic stimulus may be crucial in the early stage of the injury, and has no effect in later stages, when in fact, emphysema is installed. But the stimulus for the antioxidant response remains enhanced, as seen by the RT-PCR. Disclosure of interest: None declared. Biochemistry, Latrobe University, Bundoora, 2 Biochemistry, Cooperative research centre for biomarker translation, Melbourne, Australia Heparanase is a b-D-endoglucuronidase which degrades heparan sulfate, a key component of the extracellular matrix and basement membrane. The degradation of the ECM is essential for both S128 Inflamm. Res. physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Heparanase is also known to have non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM and basement membrane, its role in generating an inflammatory response by mediating the release of chemokines, cytokines and growth factors have not been determined. In this study, the role of heparanase in regulating the expression of these factors in innate immune cells has been studied. Peripheral blood monocyte cells were stimulated in vitro with endotoxin-free heparanase and cytokine release was examined. Heparanase treatment of cells resulted in the release of a range of pro-inflammatory cytokines including IL-8, IL-10, TNF, IL-6 and IL-1b. A similar pattern of cytokine release was observed after addition of soluble heparan sulfate, implying that the role of heparanase in induction of inflammation may be due to the enzymatic role of cleaving of its substrate. This result was validated via an abolishment of cytokine release following the treatment of MyD88 -/mouse spleen cells with heparanase, suggesting that cytokine release occurs via the toll-like receptor pathway, which heparan sulfate fragments are known to signal through. These data suggests that heparanase can promote inflammation via the release of pro-inflammatory cytokines. Disclosure of interest: None declared. Animal models suggest that proteolytic balance at the surface of inflamed mucosa is broken towards an increased proteolytic activity. This increase could be explained by an important release of proteases, a decreased expression of endogenous inhibitors, or both. We have investigated here the proteolytic activity released by fresh colonic tissue biopsies of IBD patients (Crohn's disease: CD, and Ulcerative Colitis: UC) and non-IBD patients. The expression of elafin, a serine protease inhibitor naturally expressed at mucosal surfaces was investigated. Methods: Non-IBD, CD and UC patient colonic biopsies were harvested and incubated (1 h, 37°) in culture media (HBSS). Arginin cleavage specific activity was measured in biopsy supernatants using a chromogenic substrate (tosyl-GPR-pNa). Other biopsies were paraffin-embedded and used for the detection of elafin mRNA by a fluorescently labeled riboprobe. Results: A significant increase in proteolytic activity was detected in supernatants from CD, and UC biopsies compared to control patients. In non-inflamed biopsies, we detected a strong expression of elafin mRNA in the epithelium, while only few cells in the sub-mucosa expressed this mRNA. In both UC and CD biopsies, elafin mRNA was detected in the sub-mucosa in a zone that correlated with infiltrated immune cells. However in the intestinal epithelium itself, elafin mRNA expression was clearly reduced. Conclusions: Our results showed that in IBD patients, proteolytic balance of the mucosa is disrupted towards an increased proteolytic activity. Elafin mRNA was constitutively expressed by epithelial cells in non-IBD patient tissues, but in CD and UC patient tissues, a down-regulation of elafin was observed in the epithelium. This study showed that the intestinal protease-antiprotease balance is altered in IBD and proposes that deficient expression of elafin by the epithelium could participate to explain this unbalance. Disclosure of interest: None declared. We have previously reported that human intestinal epithelial cells (IECs) released from their basolateral side significant proteolytic activity upon stimulation by LPS. Further, we have observed that LPS-induced increased permeability of intestinal epithelium could be prevented in vitro by treatment with a large spectrum serine protease inhibitor. Here, we investigated the effects of pathogenic (EPEC) or commensal (HB101) forms of Escherichia. coli (E. coli) on the expression of the trypsinIV by IECs. We also investigated the role of proteolytic activity in E. coli-induced increased permeability. Methods: Caco-2 cells (human IECs) were polarized for 15 days and apically stimulated for 5 h with HB101 and EPEC at 2 9 10 8 CFU/ well, in the presence or not of the serine protease inhibitor FUT-175 (50 mg/ml). Epithelial permeability was evaluated by dextran-FITC apical to basal passage method. We have evaluated the expression of the trypsin IV mRNA by qRT-PCR and trypsinogen IV by westernblot. Results: We have observed that EPEC, but not HB101 was able to break the barrier function of IECs, increasing the passage of dextran-FITC. The serine protease inhibitor FUT-175 was able to completely prevent this increased permeability in response to EPEC. Trypsin IV mRNA was expressed in Caco-2 cells infected with EPEC, while it was significantly down-regulated when cells were exposed to commensal HB101. The trypsinogen IV protein was also detected in supernatants of IECs exposed to EPEC, but not in supernatants of IECs exposed to HB101. Conclusions: The intestinal epithelium infected by a pathogenic E. coli (EPEC), but not by a commensal form (HB101) becomes leaky and the release of proteases seems to play a major role in this event. Epithelial trypsin IV, which is differentially regulated by pathogenic or commensal forms of E. coli, could play an important role in this permeability changes and overall in alerting the immune system to pathogen exposure. Disclosure of interest: None declared. Introduction: In CF, the protease/antiprotease imbalance results in the degradation of lung tissue but therapies aiming at reestablishing this balance by the administration of inhibitors gave limited results. The different distribution of neutrophil serine proteases (NSPs) in CF secretions may explain that they are differently regulated by their natural inhibitors. The aim of this work was to investigate the contribution of neutrophil extracellular traps (NETs) to the regulation of NSPs activity. Methods: Proteases activities and DNA were quantified in CF sputum and in suspensions of neutrophils isolated from sputum or control blood. We analysed the inhibition of proteases by natural inhibitors in total sputum before and after DNase treatment. NETs secretion was triggered by incubation of isolated neutrophils with S. aureus and P. aeruginosa and NETs were observed by scanning electron microscopy and confocal microscopy. Results: Elastase (HNE), protease 3 (Pr3) and cathepsin G (CG) are active in CF sputum and resist to inhibition because of their binding to extracellular DNA and other macromolecular compounds. Part of sputum DNA belongs to NETs as deduced from the presence of NETotic neutrophils in sputum. HNE activity in whole sputum increases dramatically after DNase treatment and can be fully inhibited whereas Pr3 and CG activity increase only slightly and still resist inhibition. CF pathogens induce a higher secretion of NETs by sputum neutrophils than by control blood neutrophils but the activity of the 3 NETs-bound proteases can be measured only after their solubilization from NETs by DNase. Conclusion: We conclude that NETs contribute to DNA load in CF sputum and constitute a reservoir of active proteases that can be mobilized by DNase. The improvement of anti-inflammatory therapies by protease inhibitors requires a solubilization of the three major NSPs in lung secretions in parallel to the administration of inhibitors. Disclosure of interest: None declared. The pathophysiological function of proteinase 3 (PR3) is not well understood mainly because of its close structural and functional resemblance with neutrophil elastase and the absence of a specific inhibitor able to target its active site in vivo. Based on the structural analysis of the active sites of human neutrophil elastase (HNE) and PR3, we have raised recombinant serpins derived from the polyvalent inhibitor serpinB1 (monocyte, neutrophil elastase inhibitor), that specifically inhibit PR3 and differ from wild-type serpinB1 by only few residues in their reactive center loop. The rate constant of association between the best serpinB1 mutant and purified PR3 was 1.4 9 10 7 M -1 s -1 which is about 100-fold higher than that observed with wild-type serpinB1, and compares with that of alpha1-PI towards HNE. Proteolytic cleavage of the mutant serpinB1 by HNE does not impair the formation of the SDS stable, irreversible complex with PR3 when a molar excess of HNE is present in the reactional mixture. Mutant serpinB1inhibits soluble PR3 as well as membrane-bound PR3 at the surface of activated neutrophils and clears induced PR3, but not constitutive PR3, from the cell surface. Such a specific inhibitor should help investigating the biological function of this protease and possibly serve as a therapeutic agent in PR3-related inflammatory diseases such as Wegener granulomatosis. Introduction: Liver fibrosis is the result of a complex interaction between different cell types and is characterized by an inflammatory process in response to chronic injury and by the activation of hepatic stellate cells (HSCs), leading to the accumulation of extracellular matrix. We investigated the involvement of inflammasome pathway, the role of P2XR7 and of matrix metalloproteinases (MMP and TIMP-1) in the CCL4-induced inflammatory process and liver fibrosis in mice. Methods: CCL4-induced liver injury was obtained by intraperitoneal injection of 0.35 ml/kg CCL4 dissolved in a 1:12 ratio with mineral oil in C57BL/6 males. Animals were injected once with CCL4 and liver were harvested. In another set of experiments, mice were treated twice a week for 3 weeks and were sacrificed 2 days after the last injection. Livers were harvested either into 10% buffered formalin for histological evaluation or snap-frozen into liquid nitrogen for mRNA or cellular lysate preparations. Collagen deposition was analysed by red Sirius staining. Components of Inflammasome, MMPs and TIMP-1 were analysed by ELISA and/or immunostaining and/or QPCR Results: CCL4 induced a significant collagen deposition in liver after 3 weeks but not at 24 h, as determined by red sirus staining and increased alpha1-collagen expression. Pro-MMP-9 and TIMP-1 were increased both at 24 h and 3 weeks, but MMP-2 only at 3 weeks. An increase in Nlrp3 inflammasome transcription was observed at 24 h and 3 weeks but purinergic receptor P2rx7 and Pannexin 1 were only significantly elevated at 3 weeks. In control mice, immunostaining showed a low expression of P2RX7 and PANX1 in the portal area, NLRP3 was not detectable, whereas CCL4 induced a significant increase expression of NLRP3, PANX1 and P2RX7 at 24 h but more markedly at 3 weeks. Conclusions: These results showed that CCL4-induced an early activation of inflammasome pathway inducing an imbalance between MMP and TIMP-1 and leading to collagen deposition and fibrosis. Disclosure of interest: None declared. Nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs) were initially described as cytosolic receptors for bacterial components while toll-like receptors mediated bacterial recognition at the cell surface. Mutations in NOD1 and NOD2 genes are associated with hyperinflammatory responses and stimulation of Nod2 in osteoblasts synergistically enhances osteoclast formation induced by LPS, IL-1a, and TNF-a; suggesting that Nod proteins play an important role modulating signaling pathways associated with the immune response. Since NF-kB and p38 MAPK are signaling pathways involved in the expression of inflammatory mediators, we examined the role of Nod1, Nod2 and MyD88 proteins on the activation of these signaling pathways by western-blot. The involvement of Nod1, Nod2 and MyD88 in the regulation of bone turnover was assessed by determining their role on the modulation of receptor activator of NF-jB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression by RT-PCR. Bone marrow stromal cells (BMSCs) were obtained by flushing the marrows of long bones of MyD88, Nod1 and Nod2 KO mice. BMSCs obtained from wild-type C57/Bl6 mice were used as controls. Cells were stimulated with Escherichia coli (TLR4 agonist) and Porphyromonas gingivalis (TLR2 and -4 agonist) lipopolysaccharide and to interleukin-1beta. MyD88 -/-cells showed decreased activation of NF-jB, which was completely abrogated in cells lacking either Nod1 or Nod2. Activation of p38 MAPK in MyD88 -/-BMSC was preserved, but was impaired in Nod1 -/-and Nod2-/cells. MyD88, Nod1 and Nod2 were all required for LPS-induced RANKL expression. Inhibition of OPG mRNA induced by LPS and IL-1 required MyD88 and Nod1, but not Nod2. Our results suggest that MyD88, Nod1 and Nod2 modulate activation of NF-jB and p38 MAPK and may play important roles in regulation of bone turnover in inflammatory and infectious conditions. Disclosure of interest: None declared. IL-12 p70/IL-23 balance is central to the development of the Th1 and Th17 responses. IL-12 p70 and IL-23 share a common chain and differ by another chain, IL-12 p35 (il12a) in IL-12 p70 and IL-12 p19 (il23a) in IL-23. The molecular mechanism explaining the IL-12 p70/IL-23 balance relies on the regulation of il12a and il23a. Human monocyte-derived dendritic cells were obtained by differentiation with GM-CSF and IL-4. Phosphorylation of c-Rel and histone H3 by mitogen-and stress-activated kinase (MSK) and protein kinase A were studied in cells and on recombinant proteins. Chromatin immunoprecipitation was used to study the transcriptional regulation of il12a and il23a. Coimmunoprecipitation assays were used to address the association of the Notch family proteins with acetylated-K14-histone H3. The transcriptional regulation of il23a depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and S10histone H3 phosphorylation. Zymosan blocked the transcription of il12a induced by other stimuli and triggered the nuclear translocation of the transcriptional repressors Hes1, Hes5, Hey1, and TLE. In addition, zymosan induced the interaction of Hes1 and TLE with histone H3 phosphorylated on S10 and deacetylated on K14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70. The selective induction of IL-23 by b-glucans is explained by the activation of c-Rel associated with S10-histone H3 phosphorylation in the il23a promoter and inhibition of il12a transcription by a mechanism involving corepressors with ability to bind TLE and to promote histone deacetylation. Disclosure of interest: None declared. During phagocytosis, the engulfment of the particle begins with the nascent phagosome formation, which occurs at 5 min. At nascent phagosomes an increase on tyrosine phosphorylation and actin polymerization are observed, this polymerization leads to phagosome maturation. Previous studies showed that Crotalus durissus terrificus snake venom inhibits the phagocytic activity of macrophages and neutrophils and that crotoxin, the main component of the venom, is responsible for this effect. In macrophages, crotoxin cause reorganization of the actin cytoskeleton and inhibition of phosphotyrosine. The aim of this study was to investigate the effect of crotoxin on tyrosine phosphorylation and actin polymerization on nascent phagosome of neutrophils. Neutrophils were obtained from peritoneal cavity of rats 4 h after the intraperitoneal administration of carrageenan (4.5 mg/kg) and then incubated (1 h) with crotoxin (0.08 lg/mL) and submitted to phagocytosis of opsonized zymosan for 5 min. For in vivo assays, neutrophils obtained from rats treated with crotoxin (0.1 mg/kg), 2 h before carrageenan, were also submitted to phagocytosis. Tyrosine phosphorylation and actin polymerization were analyzed by immunocytochemical after incubation with anti-phosphotyrosine and staining with phalloidin. The results showed that crotoxin induced a marked reduction in staining of phosphotyrosine and F-actin on neutrophils during phagocytosis, when compared to the controls. Unlike what occurs in macrophages, our results demonstrate that crotoxin inhibits tyrosine phosphorylation and consequently actin polymerization which may contribute to explain the inhibitory effect of crotoxin on phagocytosis by neutrophils. Furthermore, taking into account the importance of these phagocytes on inflammatory response, these results contribute to the elucidation of the mechanisms involved in the anti-inflammatory effect of crotoxin. Respiratory syncytial virus (RSV) is the most important respiratory pathogen in infants and young children. 2-3% of primary RSV infections result in severe cases of bronchiolitis and pneumonia requiring hospitalization and supportive care. Treatment options are very limited. Excessive inflammation triggered by the host is believed to play a major role in the development of the clinical manifestations. The underlying molecular mechanisms are not well defined but mainly involve the activation of the transcription factor NF-jB. Hence, elucidating the mechanisms of NF-jB activation after RSV infection will likely help to develop new anti-inflammatory therapies. Here, we used transfected HEK 293-derived cell lines that overexpress selected human Toll-like receptors (TLRs) and harbour a NF-jB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, and measured SEAP reporter activity upon infection with live RSV or stimulation with purified TLR ligands (controls). In addition, parental cells that harbour the SEAP reporter gene but generally lack TLR expression were used to control for endogenous responses. We demonstrate that HEK 293 cells are permissive for RSV; however the live virus fails to activate human TLRs 3, 4 and 7. Moreover, live RSV is unable to interfere with TLR3, -4 and -7 activation in HEK 293-derived cells mediated by their specific, purified agonists poly(I:C), E. coli LPS, and R848, respectively. This suggests that RSV can evade recognition by these innate immune receptors and may explain why in some children the immune system fails to control primary infection with this virus. Studies are ongoing to determine which pattern recognition receptors are essential for the human innate immune system to sense RSV infection. Disclosure of interest: None declared. TLR4 plays fundamental roles in provoking a robust anti infectious inflammatory reaction. Recognition of LPS by TLR4 could trigger generally two signaling pathways, the MyD88-dependent and -independent pathways. Additionally, this recognition could also induce the internalization of LPS/TLR4 complex, which is considered as an interacted process with signaling events. To deeply investigate the role of LPS/TLR4 complex internalization in macrophage activation, we did the following experiments. The localization of LPS and TLR4 as well as the changes of major proinflammatory cytokines and signaling molecules were observed or detected in macrophages treated with or without internalization inhibitors such as clathrin inhibitor (MDC), dynamin inhibitor (dynasore) and endosome acidifying maturation inhibitor (chloroquine). These results showed LPS down-regulated cell surface TLR4 level and both LPS and TLR4 were endocytosed into cytoplasm and colocalized at same local site. On the other hand, inhibitors of clathrin/dynamin, MDC and dynasore, could maintain higher TLR4 on the cell surface and less LPS/TLR4 within the cells because of blocking LPS/TLR4 internalization. However, CQ, an inhibitor of endosome acidification, markedly decreased TLR4 amount on the cell surface and increased LPS and TLR4 amounts within the cells. Furthermore, the data from ELISA, cytokine antibody array and western blot assays S132 Inflamm. Res. demonstrated that only MyD88-independent pathway was involved in early phase of LPS internalization; but in later phase, TLR4 back to cell surface could mediate the MyD88-dependent pathways again, and start to the next round of internalization. Taken together, this study clarifies that internalization of the LPS/TLR4 complex not only maintained the TLR4 recycle, but also took part in the macrophage activation by the MyD88-independent pathway. This finding might provide a new approach to treat the over-inflammatory diseases. Disclosure of interest: None declared. Glucocorticoids (GC) are of extraordinary therapeutic value in a wide range of inflammatory, autoimmune and inflammatory diseases. Their therapeutic activity is due to regulatory effects on activation, cell growth and differentiation in a number of cells and tissues, including cells of the immune/inflammatory system. With the aim to deeply analyze the molecular mechanisms of GC action, we have identified a number of GC-induced genes including glucocorticoid-induced leucine zipper (GILZ), a protein rapidly induced by GC treatment. Using different experimental models of inflammatory diseases, we show GILZ is an important mediator of the anti-inflammatory and immunosuppressive effects of GC. Moreover, we identified a new GILZ isoform, L-GILZ, involved in mediating the effects of GC on inflammation and on cell differentiation. Furthermore, our study demonstrates that both GILZ and L-GILZ are crucial mediators of GC-induced effects in that mediate the anti-inflammatory/immunosuppressive activity. In particular, GILZ regulates T cell activation and differentiation, cytokines, including pro-inflammatory cytokines, production and inflammatory process development. Results could provide new means to predict sensitivity of normal and tumor cells to treatment with GC and to outline new therapeutic approaches. Disclosure of interest: None declared. GILZ is an ubiquitous protein whose expression is induced by glucocorticoids in haematopoietic cells. GILZ regulates signal transduction pathways central to inflammation, such as AP-1, NF-jB and FOXO. Indeed, Forkhead transcription factors have been increasingly recognized to play a role in immune homeostasis, especially in chronic inflammation. Nuclear import of FOXO factors follows stress signals such as oxidative stress, leading to up-regulation of target genes, whereas nuclear export is favoured by phosphorylations, mainly by Protein Kinase B, and subsequent 14-3-3 recruitment. We recently showed that GILZ inhibits FOXO factors transcriptional activities in the acute promyelocytic leukaemia HL60 cells independently of PKB phosphorylation but through a Crm-1 dependent nuclear exclusion of FOXO3 leading to its relocalization to the cytoplasm. The aim of this work was to elucidate the mechanism of FOXO factors relocalization by GILZ. First, we showed that the cytoplasmic localization of FOXO3 in the presence of GILZ was not due to an association of FOXO3 with shuttling GILZ. Our hypothesis is that GILZ may promote phosphorylation of FOXO factors on regulatory sites affecting their nucleo-cytoplasmic localization, possibly through new 14-3-3 binding sites creation. ERK, JNK or p38 Mitogen Activated Protein kinases activities were inhibited by GILZ, as assessed in GILZ-expressing HL-60 clones. We invalidated MAPK phosphorylation sites (S284, S294, S425) on FOXO3-WT and FOXO3-TM (triple mutant with mutated PKB sites) and observed that these mutants were still significantly inhibited by GILZ. Interestingly, mutation of Ser 413, a target of AMP-activated protein kinase (AMPK) located in a consensus 14-3-3 recognition motif, along with substitution of Ile 394 to Ala in the NES of FOXO3, strongly impaired the inhibitory effect of GILZ, suggesting that GILZ could promote the association of an export complex on FOXO factors independently of PKB sites. Disclosure of interest: None declared. Introduction: Acute kidney injury (AKI) remains an independent risk factor for mortality and morbidity and is well established that inflammation plays an important role in the pathophysiology of the disease. An organelle that suffers cellular stress is the endoplasmic reticulum (ER), leading to altered protein folding. The accumulation of unfolded proteins generates a cell response, which tends to return to homeostasis, but may cause the production of inflammatory factors and even cell death. The Heme oxygenase-1 (HO-1) is an enzyme with cytoprotective and anti-inflammatory properties. Thus, its induction may lead to a modulation of endoplasmic reticulum stress (ERS). The aim of this study is to evaluate if ERS generated by AKI can be modulate by HO-1 induction. Also, we tested if ERS can be induced in ox-LDL stimulated macrophages. Methods: C57/BL6 mice were subjected to bilateral ischemia and reperfusion injury (I/R) for 45 min and sacrificed after 0, 15 and 30 min, and 6, 12 and 24 h of reperfusion. A group of animals was also treated with Hemin, an HO-1 inducer. We collected blood samples for renal function assessment and renal tissue to determine the gene expression of HO-1, TNF-a, HIF-1a, BiP and CHOP. Moreover, it was also done immunoblotting for ERS markers. Further, macrophages were stimulated with ox-LDL (50 lg/mL) for 2, 6 and 24 h, and RT-PCR was performed for inflammatory molecules Results: Animals treated with Hemin had lower serum urea and creatinine levels when compared with I/R group. Moreover, treated group had higher gene expression of HO-1 and lower expression of TNF-aand HIF-1a. Also, ERS molecules had lower gene and protein expression in treated animals. Finally, ox-LDL stimulated macrophages had a significantly increase on ERS, with concomitant higher levels of IL-6. Conclusion Delphinidin is the major anthocyanidin present in many pigmented fruits and vegetables, which has antioxidant, anti-inflammatory, and antiangiogenic properties. Their properties have been studies in number cell types, but is unknown their effect on T cell. In this study, delphinidin was found to increase the interleukin-2 (IL-2) production and expression in Jurkat cells via Calcium Release Activated Calcium (CRAC) and activation of the Nuclear Factor of Activated T cells (NFAT). By spectrofluorimetric experiments, we tested the efficacy of putative CRAC inhibitors, BTP-2 and gadolinium (Gd 3+ ) on Ca 2+ entry induced by delphinidin. BTP2 and Gd 3+ reduced the calcium entry stimulated by delphinidin in a dose-dependent manner. In experiments of patch clamp, we observed that delphinidin was able to stimulate a calcium release-activated Ca 2+ current (I CRAC ), which was inhibited in part by BTP-2 and Gd 3+ . Moreover, in assays of bioluminiscence imaging of NFAT activity in single living Jurkat F6 cells stably expressing the luciferase reporter gene under control of three NFAT response elements, we showed that delphinidin induced the NFAT activity at 2 h post stimulation. Meanwhile, we observed that delphinidin induced the IL-2 production and expression in Jurkat cells after 24 h. To asses if CRAC and NFAT contributes to the IL-2 production and expression induced by delphinidin, we treated the Jurkat cells with BTP-2 or an inhibitor of calcineurin, Cyclosporine A (CsA). Both, significantly decreased the IL-2 production and expression induced by delphinidin. Finally, we observed that delphinidin induced ERK1/2 phosphorylation at 120 min and that inhibitors of MAPK/ERK kinase, PD98059 and U0126, reduced the IL-2 production induced by delphinidin, not so the inhibitor of JAK2, AG490. With this results, we concluded that delphinidin is able to induces IL-2 through of CRAC, NFAT and MAPK/ERK activation in Endothelin-1 (ET-1) is a vasoconstrictive peptide, as a key mediator of vasoconstriction and tissue repair. It is found that patients with asthma have significantly higher ET-1 levels as compared with normal subjects. Connective tissue growth factor (CTGF) is an immediate early gene, and overproduction of CTGF underlies the development of lung fibrosis. Previous studies have shown that ET-1 induces expression of matrix-associated genes in lung fibroblast through MEK/ERK pathway. However, little information is known about the signaling pathway of CTGF expression in lung fibroblasts caused by ET-1. In this study, we found that ET-1-induced CTGF expression was inhibited by BQ123 (ET A R antagonist), but not BQ788 (ET B R antagonist). Pretreatment of SP600125 (a JNK inhibitor) and transfection with JNK1/2 DN significantly reduced ET-1induced CTGF expression. ET-1-induced CTGF luciferase activity was predominately controlled by the sequence -747 to -408 bp upstream of the transcription start site on the human CTGF promoter, suggesting ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. Pretreatment of curcumin (AP-1 inhibitor) inhibited ET-1-induced CTGF expression. Furthermore, ET-1 caused increases in c-Jun phosphorylation, the formation of AP-1-specific DNA-protein complex, and the recruitment of c-Jun to the CTGF promoter. These results suggest that ET-1 acting through ET A R, activates the JNK/AP-1 signaling pathway, which in turn initiates AP-1 activation and recruitment of AP-1 to the CTGF promoter, and ultimately induces CTGF expression in human lung fibroblasts. Disclosure of interest: None declared. Blood platelets are central to haemostasis and exert important effects on the regulation of certain elements of the immune system, as they interplay in innate (including inflammation) and adaptive immunity. The purpose of this study was to further investigate TLR2 stimulation and NF-jB/MyD88-dependent signalling machinery. Platelet-rich plasma (PRP), from healthy donors, was stimulated with the TLR2 ligand Pam3CSK4 (w or w/o human TLR2 blocking MoAbs and w or S134 Inflamm. Res. Initially identified as cyclosporin A-binding proteins, cyclophilins A and B (CyPB) are inflammatory mediators that induce migration of T lymphocytes and monocytes/macrophages. Cyclophilin A was also reported to induce expression of pro-inflammatory mediators as TNF-a, IL-1b or IL-8 in monocytes/macrophages. However, these last findings are controversial and no data are available concerning the responses of macrophages to a long-term exposure to cyclophilins. We recently demonstrated that a pre-treatment of macrophages with CyPB reduced the secretion of TNF-a induced by LPS, suggesting that CyPB can regulate inflammatory responses in macrophages. Based on this hypothesis, we examined the molecular mechanisms responsible for desensitization of LPS-induced TNF-a expression. So we used macrophages derived from human peripheral blood monocytes and THP-1 cells, which can be efficiently differentiated into macrophage-like cells. Quantitative real time PCR analysis allowed us to demonstrate that CyPB strongly inhibits the expression of TNF-a mRNA. We also found that CyPB induced in macrophages expression of Bcl-3, a protein known to enhance binding of p50/p50 homodimer, a nuclear factor devoid of transcriptional activity, to the promoter of TNF-a gene. We therefore analyzed the molecular events that down-regulate transcriptional activation of TNF-a gene expression in CyPB-treated cells. By using chromatin immunoprecipitation, we confirmed that pre-treatment of cells with CyPB induced the accumulation of p50/p50 NF-jB, which bound to TNF-a gene promoter in place of p65/p50, thus reducing transcriptional activation. Finally, silencing the expression of Bcl-3 by RNA interference efficiently restored LPS-induced TNF-a expression in CyPB-treated cells, thus confirming the involvement of Bcl-3 in the inhibitory activity of CyPB. Altogether, our results support the hypothesis that CyPB is a regulatory factor, which may take part in the modulation of the inflammatory response. Disclosure of interest: None declared. Results: Initially, we observed higher gene expression of MyD88, Nod-1 and Nod-2 in renal tissue after different periods of IRI. After 24 h of reperfusion we observed that WT and Nod-1KO animals showed a significant renal dysfunction assessed by serum creatinine and indices of acute tubular necrosis while knockout animals for MyD88, Nod-2, RIP-2 and Nod1/2 showed a significant protection of renal function when compared to WT. Also, WT animals showed extensive areas of hypoxia compared to different KO, and mRNA expression of HIF-1a was expressed 2.59 more than Nod-2, RIP-2, Nod1/2 and MyD88 KO animals. Interestingly, the gene expression of Bcl-2 was reduced in WT mice while in KO animals it was up regulated. We also verified that KO mice presented significantly less serum levels of pro-inflammatory molecules, compared to WT animals. Conclusion: Besides the TLR, the intracellular sensors Nod-1 and Nod-2 play a role in the pathogenesis of AKI by different mechanisms that result in an exacerbation of the inflammatory process, leading to a renal dysfunction. Fapesp/CNPq/INCT Disclosure of interest: None declared. High amounts of glutamate are found in the brains of people with multiple sclerosis. Glutamate might affect neuroinflammation via effects on cell types within and peripheral to the central nervous system. Metabotropic glutamate receptors (GRM) comprise a unique family of G-protein coupled receptors that are promising targets for therapeutic interference. The binding of glutamate to GRMs induces signal transduction cascades that lead to gene-specific transcription. However, the molecular mechanisms that mediate and regulate GRM-induced gene expression are still largely unclear. We are studying intracellular signal transduction in response to type I GRMs (GRM1 and GRM5). Group I agonists are known to activate phospholipase C (PLC), leading to release of calcium and activation of protein kinase C (PKC). We could show that overexpression of GRM1 and GRM5 in HEK293T cells induces the activation of an NF-jB dependent reporter gene. Coexpression of the ubiquitin-editing protein A20 or the GRM1 and ubiquitin-binding protein optineurin (OPTN) significantly prevented GRM1-and GRM5-induced NF-jB activation in a synergistic manner. Interestingly, mGluR1 triggered the generation of a shorter OPTN fragment, whose formation could be inhibited by A20. In conclusion, our results indicate an important role for NF-jB activation and its regulation by A20 and OPTN in type I GRM signaling. Disclosure of interest: None declared. Cell death mechanisms Dendritic cells (DC) are professional antigen presenting cells playing a major role in the induction of primary immune response. After the exposure of DC to skin contact sensitizers, such as nickel (NiSO 4 ) or dinitrochlorobenzene (DNCB) an up-regulation of phenotypic markers, cytokine secretion and activation of signaling pathways such as Mitogen Activated Protein Kinases (MAPK) are observed. These compounds generate a chemical stress that can be perceived as a danger signal by DC, leading to their maturation. Among signaling pathways known to be redox-sensitive, the Nrf2/Keap1 pathway is central for the detection of electrophilic molecules. In the absence of a chemical stress, Keap1 associates with Nrf2 and targets it to degradation. In the presence of an electrophilic compound, Keap1's conformation is modified leading to Nrf2 translocation to the nucleus and transcription of its target genes. To study the role of the transcriptional factor Nrf2 in response to chemical sensitizers, DC derived from monocytes (Mo-DC) were treated with NiSO 4 , DNCB or cinnamaldehyde (CinA) for different times and Nrf2 expression was measured by Western blot. Accumulation of Nrf2 protein was observed after 4 h of stimulation. To evaluate the role of Nrf2 in cell survival, Mo-DC have been transfected with small interfering RNA (siRNA) to invalidate in vitro nrf2 transcripts and were then treated with NiSO 4 or DNCB or CinA for 18 h. AnV and 7-AAD staining were used to measure apoptotic and necrotic cells respectively. Results showed an increase of secondary necrotic cells (AnV + /7-AAD + ) and total apoptotic cells (AnV + /7-AAD -) when Nrf2 is invalided. Otherwise, living cells decreased in absence of Nrf2. These results suggest that the transcriptional factor Nrf2 would play a role in cell survival and would protect DC from contact sensitizers. Now, we are focusing our work on caspase signaling pathway in order to know if Nrf2 controls caspase activation, known to be involved in DC maturation. Disclosure of interest: None declared. It was originally believed to be important for NF-jB activation and its protein kinase activity is dispensable. Previous studies also reported that RIP1 and subsequent c-Jun N-terminal kinase (JNK) activation may mediate DNA damager N-methyl-N 0 -nitro-N 0 -nitrosoguanidine (MNNG)-induced necrotic cell death. However, the detail mechanism was not well-investigated. MNNG can cause excess DNA strand breaks that lead to poly (ADP-ribose) polymerase-1 (PARP-1) overactivation and cell death (parthanatos). In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the regulatory roles of RIP1 in MNNG-induced parthanatos. We found that MNNG-induced cell death can be abrogated in RIP1 deficient cells or by using silence RNA technique to knockdown RIP1 expression. MNNG can also induce rapid PARP-1 and JNK activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1 min and peaking at 5-15 min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30 min and time-dependently increasing up to 6 h after MNNG treatment was generated by mitochondria. Results further indicated the involvement of RIP1 in sustained late phase ROS production and calcium increase. The interactive roles of RIP1 with ROS, JNK and calcium elevation in MNNG-induced parthanatos were characterized. Moreover, we found that RIP1 is also involved in oxidative stress-induced NF-jB and AMPK activation. Taken together, RIP1 is a key mediator for oxidative stress-induced cellular signaling and death. Disclosure of interest: None declared. Terminal differentiation of epidermal keratinocytes to corneocytes is a unique model of cell death, which is indispensable for the homeostasis of skin. Despite earlier disputation, cornification has been distinguished from other types of programmed cell death, especially apoptosis. The previous report revealed that differentiating keratinocytes induced by cell confluence can be resistant to tumor necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis and indicated differentiation is distinct from death receptor-mediated apoptosis. In our study, we report during confluence-induced differentiation, TRAIL mRNA is up-regulated in HaCaT and normal human epidermal keratinocytes (NHEKs). After treating dividing NHEKs with TRAIL, markers of keratinocyte differentiation such as involucrin and type 1 transglutaminase can be induced in addition to the induction of apoptosis. However, under the pretreatment of calcium in NHEKs, expression of differentiation markers cannot be further enhanced by TRAIL but cell death-induced by TRAIL is diminished. When blocking the activity of caspase 3 and 8, both differentiation and death of NHEKs triggered by TRAIL are abolished but differentiation induced by calcium and phorbol 12-myristate 13-acetate are barely affected. Moreover, TRAIL-evoked signaling of ERK and p38 and degradation of p63 are also attenuated by inhibiting the activation of caspase 3 and 8. Comparing floating cells with adherent NHEKs after the treatment of TRAIL, we find much more abundant expression of differentiation markers including involucrin, keratin 10 and loricrin is detected in floating cells than adherent NHEKs. These findings provide a new insight into the complicated relationship between apoptosis and cornification, and also unravel the novel role of apoptotic caspases in keratinocyte differentiation. Disclosure of interest: None declared. Introduction: Asthma is a chronic lung inflammatory disease that modifies bronchial structure, causing remodeling and increased responsiveness. Remodeling is recognized by fibrosis of sub-epithelial basement membrane, hypertrophy of bronchialglands, goblet cell hyperplasia, thickening of airway epithelium. In this study, we investigated the role of female sex hormones in airway remodeling and responsiveness in a model of chronic lung inflammation. Material and methods: Female C57B/6 mice were subjected to ovaries removal (OVx) and were OVA-sensitized (OVA 1 mg/ml SC 7 and 14 days later). Control group consisted of sham OVx-OVA. After day 21, OVA-challenge (aerosol, 15 min, OVA 1%) was performed three times a week during three weeks (OVx-OVA). After 5 days of the last OVA-challenge, part of the animals (OVx-OVA and sham OVx-OVA) was submitted to in vitro tracheal maximal contractile response (E max ) to methacholine (MCh) evaluation and histological analysis (picrusirius red and PAS staining) to identify collagen deposition and mucus production on bronchial compartment. The other part of the animals (OVx-OVA and sham OVx-OVA) was anesthetized, mechanically ventilated and respiratory mechanics assessment (total lung resistance, R, and elastance, E) was performed. Results: Measurements of bronchial mucus and collagen were significantly lower (6 and 2 times lower respectively) after chronic antigen exposure of OVx-OVA compared with their sham OVx-OVA counterparts. OVx-OVA showed reduced tracheal responsiveness in vitro to MCh (E max : 3.65 g) in comparison to sham OVx-OVA (E max 1.6 g). Total lung resistance (R) and elastance (E) were higher and lower respectively in sham OVx-OVA. Conclusions: Removal of ovaries affected the magnitude of lung inflammatory response after chronic exposure to antigen suggesting that female sex hormones could mediate the airway responsiveness and remodeling in chronic allergic lung inflammation. The mast cell is an important inflammatory effector in both innate and adaptive immunity. Adenosine, an endogenous nucleoside, modulates a large number of cellular systems in inflammation. While activation of mast cells through IgE receptors (initiation of allergic reaction in adaptive immunity) is known to be modulated by adenosine, the effects of adenosine on toll like receptors (TLRs) induced mast cells activation (initiation of inflammation in innate immunity) has not been thoroughly studied. We pharmacologically characterized the actions of adenosine on TLR induced IL-8 release from human cultured mast cells (HCMC). Treatment of bacterial peptidoglycan (PGN) and lipopolysaccharides (LPS), specific ligands of TLR2 and TLR4 respectively, significantly increased IL-8 production from HCMC. Adenosine and NECA at subnanomolar concentrations (10 -8 -10 -7 M) potentiated PGN-mediated IL-8 production which was not observed in LPS treated cells. A 1 receptor agonists, CCPA and 2 0 MeCCPA, both markedly potentiated the IL-8 production by PGN dose-dependently. Moreover, a similar effect was also found with A 3 receptor selective agonists 2Cl-IB-MECA and HEMADO. A 2 receptor was not involved in the enhancing effect as A 2 receptor specific CGS21680 and CV1808 were found not to affect the PGN effect. The potency of adenosine agonists was found in rank order of 2 Nonspecific airway hyperresponsiveness (AHR) is one of cardinal features of bronchial asthma. We have described TNF-a-induced late AHR in a murine model of asthma, in which protocol, the mice received twice intraperitoneal immunization, followed by bronchoprovocation twice with aerosolized ovalbumin with a 1-week interval. The AHR occurs at 10-14 after the second airway allergen challenge. In this study, we examined the kinetics of late AHR up to 60 h in a 6 h intervals We found that the specific airway resistance increased 4-fivefold at 12 h after airway challenge of allergen, followed by the second phase response being larger and broader in resistance at 24-48 h, peaking at 36 h with 3.5 to 4.5-fold increase, indicating that biphasic late AHR occurred in this murine model of asthma. The first phase of late AHR was abrogated by pretreatment of the sensitized animals with anti-TNF-a. Intratracheal administration of IgE as well as IgG immune complexes (ICs) were able to induce first phase of late AHR via producing TNF-a in the airways in normal animals. The second phase of late AHR was abrogated by pretreatment of the sensitized animals with CpG-ODN, which inhibited Th2 asthmatic phenotypes, such as airway levels of IL-4 and IL-5 and eosinophils, but it affected neither airway levels of TNF-a, nor the magnitude of the first phase of AHR. These data indicate that biphasic late AHR occurs in a murine model of asthma, in which TNF-a and Th2 response are responsible for the development of the first and second phases of late AHR, respectively. Disclosure of interest: None declared. In order to find the anti-inflammatory flavonoid derivatives having optimum chemical structures, various 8-heterocyclic-substituted chrysin derivatives were previously synthesized and their effects on prostaglandin E 2 (PGE 2 ) production from lipopolysaccharide (LPS)-treated mouse macrophage cell line, RAW 264.7 cells were evaluated. Through this screening procedure, 5,7-dihydroxy-8-(pyridine-4yl)flavone (C-721) among the derivatives was selected for further pharmacological study. Contrary to the parent molecule, chrysin, C-721 was found to potently inhibit PGE 2 and NO production from LPS-treated RAW cells. The IC 50 values of C-721 were 6.2 and 22.6 lM, respectively for COX-2 mediated PGE 2 and iNOS-mediated NO production. Western blotting and reverse transcriptase-polymerase chain reaction analysis proved that this compound inhibited PGE 2 production at least in part by COX-2 down-regulation and COX-2 inhibition, while C-721 inhibited NO production mainly by down-regulation of iNOS expression. In addition, C-721 inhibited TNF-a and IL-6 production at 10-50 lM. In vivo study has revealed that oral and intraperitoneal administration of C-721 showed 25.2-44.3% inhibition against k-carrageenan-induced paw edema in mice. Furthermore, this compound inhibited collagen-induced arthritis in mice by intraperitoneal injection. All these results suggest that C-721 may have a potential for a synthetic lead compound as new antiinflammatory agent. Disclosure of interest: None declared. The mechanisms underlying airway remodeling in asthma remain unknown and ultrastructural changes of asthmatic airway might reveal novel mechanisms. To investigate the histopathological and ultrastructural features of the airway in a chronic ovalbumin mouse model of asthma, two groups of BALB/c mice [Sham and ovalbumin (OVA) n = 6 each] were sensitized and challenged with 1.5% OVA in PBS [OVA group (30 min per day, alternative days)] or PBS (Sham group) for 4 weeks. After determining airway hyperresponsiveness (AHR) to methacholine, mice were sacrificed and lungs were taken for both histopathological and electron microscopic studies. AHR in response to methacholine was enhanced in OVA mice. Histopathological studies revealed the presence of denuded bronchial epithelial cells, goblet cell metaplasia and mucus hypersecretion and cellular infiltration in perivascular and peribronchial area. The infiltrated cells were composed mainly of eosinophils, lymphocytes and macrophages. Transmission electron microscopy (TEM) revealed that there were inactive granules in chronic compared to partial active granules in acute asthma. There were signs of epithelial apoptosis such as shrinkage of bronchial epithelia, widening of intercellular space between adjacent bronchial epithelial cells, and subepithelial fibrosis. Basement membrane were thickened and composed mainly of fibroblast and collagen. Interestingly, we found mucus granules in both ciliated and Clara cells. In addition, there is increase in the number of mitochondria in smooth muscle as well as evidence of mitochondrial injury in asthmatic mice. In conclusion, TEM revealed the interesting and crucial ultrastructural changes of asthmatic airway in relation to airway remodeling and thus it appears to be a useful tool in understanding its pathogenesis. Disclosure of interest: None declared. Allergic contact dermatitis (ACD) resulting from skin sensitization is a common occupational and environmental health problem, in which dendritic cells (DCs) play an essential role. Indeed, after hapten binding, internalization and processing, DCs acquire a mature phenotype and migrate from the skin to the draining lymph nodes where they present hapten-peptide complexes to naive T cells. Haptens need to link with proteins to form a stable antigenic complex, able to induce an immune response. Our hypothesis is that chemical reactivity is necessary for a chemical to be a sensitizer allowing both covalent binding to proteins and stimulation of DC maturation by mimicking ''danger signals'' signalling. To date there is no evidence that a positive correlation exist between the capacity of a chemical to bind to a protein and the amount of stress it induces in DC allowing its maturation. As it is now well accepted that chemical stress will induce the up-regulation of detoxification systems, allowing DC to handle the perturbation of cellular redox homeostasis, the aim of this study is to evaluate sensitizer potency based on their chemical reactivity using the activation of the Nrf-2 pathway. For this purpose, dendritic (Mo-DCs) and dendritic-like cells (THP-1) were exposed to six ''true'' haptens with different reactivity (two haptens with high cysteine affinity, two haptens with high lysine affinity and two haptens with mixed cysteine/lysine affinity) and two pro-haptens and pre-haptens. Activation of the Nrf-2 pathway was assessed by measuring the mRNA expression of 4 Nrf-2-dependent genes: nqo1, hmox, il-8 and gpx by qPCR. Our results show that chemical sensitizers were able to induce the transcription of Nfr-2 target genes in both models, in correlation with CD86 and/or CD54 surexpression. These studies will allow developing a new approach for detecting chemical sensitizers in integrating data coming from peptide reactivity tests and data on activation of dendritic cells. Disclosure of interest: None declared. The aim of this study was to investigate the effect of CTLA4-Ig on the induction of contact hypersensitivity (CHS). CHS is a T cell mediated immune reaction in response to epicutaneous application of a hapten and consists of two phases; sensitization-and elicitation phase. Mice were sensitized with DNFB or oxazolone and 5 or 6 days later, a challenge dose was applied. The inflammatory response is quantified by measuring the increase in ear-thickness at various time-points after challenge and the treatment groups are compared to isotype-treated control groups. Firstly, CTLA4-Ig was given in the doses 25, 5 and 1 mg/kg a day prior to sensitization. This study revealed a dosedependent suppression of the ear-swelling response in both DNFBand oxazolone-induced CHS, and the effect was most marked in the DNFB model. To obtain full inhibition in the oxazolone model, the doses were increased up to 125 mg/kg which reduced the response very close to background level. Mice treated with CTLA4-Ig were resensitized and re-challenged 3 weeks after the first sensitization without any further treatment with CTLA4-Ig. In both models, CTLA4-Ig still had a suppressive effect 3 weeks after administration although no CTLA4-Ig could be detected in serum. The effect was again most significant in the DNFB model. Based on these data, we can conclude that CTLA4-Ig induce a long-term immunosuppression of both DNFB-and oxazolone-induced CHS. The effect was most marked in the DNFB model, which could indicate quantitative difference between the two models. Although the effects of CTLA4-Ig on CHS have been studied previously, this study is the first to compare the effect of this compound in both DNFB-and oxazoloneinduced CHS and to show a long-term effect of CTLA4-Ig in the oxazolone model. Introduction: The renal ischemia-reperfusion injury (IRI) is a systemic inflammatory reaction with predominance of a Th1 response, which affects other organs, modulating a local inflammatory response, including the lung. The lung inflammation is a complex disease that involves the participation of inflammatory mediators and Th2 lymphocytes. Objectives: To evaluate the phagocytic and microbicide capacity of macrophages in rats with lung inflammation that underwent IRI. Materials and methods: The alveolar macrophages of rats sensitized to OVA were evaluated for testing phagocytosis and killing, 24 h after antigen challenge the animals were submitted or not to 60 min of renal ischemia. The reperfusion injury were analyzed 24 h after the re-establishment of renal blood flow. Serum creatinine, urea, renal histology and lung tissue were evaluated. Results: Bronchoalveolar lavage has a high cellular infiltrate in immunized animals (420%) compared with control animals, IRI significantly reduced this infiltration (52%). Macrophages from animals immunized and challenged with OVA presented a 109 increased phagocytic capacity of red cells compared to control group, whereas immunized animals subjected to IRI showed a reduction in phagocytic index of 68%. Killing of K. pneumoniae by macrophages from immunized animals is (56%) higher compared with control group, being reduced in animals submitted to IRI (45%). The immunized and challenged group showed an increase in gene expression of IL-10 (450%), HO-1 (259%), IFN-c (460%) and MCP-1 (370%), when compared to the immunized group subjected to IRI. The levels of urea and creatinine were only altered in animals that underwent IRI showing increased urea (239%) and creatinine (324%) levels in serum compared to control. Conclusions: Renal ischemia and reperfusion injury alters the phagocytic and microbicide capacity of macrophages, reducing lung inflammation to OVA. The aim of this study was to identify proteins which can be responsible for cross-reactivity in patient's sera being positive for wheat and reacting also with rice and pollen allergens and explore the effect of thermal processing on the IgE reactivity of rice proteins. We stained also levels of total IgG, IgA, IgE, ECP and presence of autoantibodies. Total IgG, IgA were measured by nephelometry (Dade-Behring BNII TM ). The total IgE, specific IgE and ECP were measured by IMMULITE 2000 Ò . Antinuclear (Anab) antibodies were detected by indirect immunofluorescence (The Binding Site Ltd.) Sera were then processed by immunoblotting using proteins from wheat flour, raw and cooked rice. We have analyzed sera of 138 patients which were divided into three groups according positivity to specific IgE. Thirty-three patients were positive only for wheat and were marked as a group I. Patients positive for both wheat and pollen allergens were marked as a group II (68 patients) and group III (37 patients) were patients positive only for pollen allergens. We have found a strong cross-reactivity with broad spectrum of rice components (116-7 kDa) in patients (group II) having both, wheat and pollen specific IgE Abs. Patients with specific IgE against wheat or pollen allergens only, (group I and III) reacted with limited number of rice components. We have found out the same level of total IgE and ECP in group I and II but the levels of IgA and IgG were higher in group II. The most common clinical diagnose in group I was atopic eczema (17 patients). In group III we found the lowest level of total IgE. On the other hand the level of total IgA, IgG and ECP were the highest as a presence of autoantibodies. Conclusion: Interindividual variation in the IgE antibody response may involve the clinical expression and reactivity of the each patient. There is also evidence that patients from group I and II differ at the level of total IgG, IgA and ECP form patients allergic only to pollen allergens. Introduction: The Th1/Th2 balance represents an important factor in the pathogenesis of the renal ischemia-reperfusion injury (IRI). Besides, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. Objectives: Investigate the ability of renal IRI in modulating the pulmonary function in a specific model of allergic lung inflammation. Materials and methods: C57Bl/6 mice were immunized with ovalbumin/alumen on days 0 and 7 and challenged with ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 min of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analyses. Results: Serum creatinine levels increased in similar extension in allergic and non-immunized animals subjected to IRI. However, BAL presented reduction in total cells (46%) and neutrophils (58%) when compared with control allergic animals not submitted to IRI. In addition, OA challenge induced phosphorylation of ERK and Akt, and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, phosphorylation of ERK, and expression of COX-2 and iNOS were markedly reduced, however there was no difference for phosphorylation of Akt between sham and ischemic OA challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down regulated in immunized/ challenged mice subjected to IR. Conclusions: These results suggested that renal IRI modulate lung allergic inflammation probably by altering the Th1/Th2 balance, at DMTI-II (23-kDa trypsin inhibitor purified from Dimorphandra mollis seeds) promotes acute inflammation accompanied by an early infiltration of eosinophils, a critical cell type involved in allergic diseases. We have evaluated here the capacity of DMTI-II to enhance the allergic pulmonary inflammation, looking over time to the leukocyte trafficking from bone marrow to peripheral blood, and their recruitment into the allergic airways. Male Wistar rats were sensitized and challenged with ovalbumin (OVA). At 2-16 h prior to OVA challenge, animals were exposed to DMTI-II (10 lg). Bronchoalveolar lavage fluid (BAL), circulating blood and bone marrow were examined at 24 h post-OVA challenge. Challenge with OVA significantly increased the influx of total inflammatory cells, neutrophils S140 Inflamm. Res. and eosinophils in BAL and lung tissue. Pre-exposure to DMTI-II potentiated the total inflammatory cells and neutrophil recruitment (p \ 0.05). Neutropoiesis and neutrophilia accompanied the pulmonary cell influx. Pre-exposure to DMTI-II also significantly increased the eosinophil recruitment to BAL, an effect starting at 4 h, remaining markedly elevated at 16 h (p \ 0.05). Eosinopoiesis and eosinophilia (seen within 2-4 h) were also observed. Exposure to DMTI-II alone increased the IL-4 levels, and further increased the IL-4 levels in OVA-challenged rats. The levels of IgE, LTB 4 and eotaxin in OVA-challenged rats were greater compared with non-sensitized rats, but DMTI-II exposure failed to further enhance such levels. In summary, our study shows that DMTI-II itself presents granulocytopoietic activity, and enhances allergen-induced neutrophil and eosinophil mobilization from bone marrow to lung tissues that is accompanied by enhanced IL-4 production. Disclosure of interest: None declared. Clinical evidences have shown a strong association between Staphylococcus aureus and pathogenesis and/or exacerbation of bronchial asthma. However, few studies have attempted to evaluate whether SEA and SEB exposure enhances the allergic cell pulmonary infiltration. In the present study, we evaluated the effects of pulmonary SEA and SEB pre-exposure on the eosinophil (EO) recruitment of allergic mice. At 4-48 h prior to OVA challenge, mice were intranasally instilled with SEA or SEB (1 lg). Pre-exposure to SEA significantly increased the eosinophil recruitment to BAL (at 48 h) and pulmonary tissues (12 and 48 h). SEB also potentiated the EO influx to BAL (at 4 h pre-exposure) and pulmonary tissues (at 12 and 24 h pre-exposure). The eotaxin levels in lung homogenates of OVAchallenged mice pre-exposed to SEA (4 h: 1,593.59 ± 261.57 pg/g) or SEB (4 h: 1,598.86 ± 156.45 pg/g) were greater compared with OVA-challenged mice instilled with saline (1,067 ± 68.26 pg/g; P \ 0.05). No significant differences were detected for IL-4 and IL-6 in lung tissue. The lung EO infiltration in OVA-challenged mice preexposed to SEA was accompanied by marked increases in the EO forms in bone marrow (24 h: 0.33 ± 0.07 9 10 6 cells/ml) and circulating blood (24 h: 0.45 ± 0.07 9 10 6 cells/ml) compared with saline-instilled mice (0.15 ± 0.04 and 0.14 ± 0.03 9 10 6 cells/m, respectively). The levels of IL-5 and eotaxin in bone marrow were 180 and 125% higher (P \ 0.05) in OVA-challenged mice preexposed to SEA compared with saline-instilled mice. Mice preexposed to SEB also exhibited eosinopoiesis and blood eosinophilia, along with increased levels of IL-5 and eotaxin in bone marrow. In conclusion, airways pre-exposure to SEA and SEB exacerbates the pulmonary eosinophilic inflammation and promotes eosinopoiesis in OVA-challenged mice by mechanisms involving eotaxin and IL-5 production. Disclosure of interest: None declared. Introduction: In previous studies we have shown that antigen sensitization 7 days after ovariectomy (OVx) reduces pulmonary inflammation in an acute model of experimental asthma, suggesting that the course of lung inflammation (IAP) depends on female sex hormones (FSH) circulating levels. By measuring the levels of IL-1b, TNF-a, IL-4, IL-13 and IL-10 in pulmonary explants from a murine model of asthma, in this study we investigated how FSH could possibly influence the mechanisms triggering the allergic lung inflammation. Methods: Female Balb/c mice were ovariectomized (OVx) or falsely operated (Sham-OVx) on day 0, sensitized with ovalbumin (grade V OVA/alum) on day 7 and challenged with OVA (grade V) once a day, for 15 min. on the days 14, 15 and 16. Twenty-four hours after the last challenge, mice were killed in excess of ketamine and xylazine. Upon lungs removal, the cytokines IL-1b, IL-4, IL-10 and IL-13 were measured in samples of pulmonary explants using Duo Set (R&D System Ò ) kits for ELISA. Results: There was a lung tissue increase of IL-1b, IL-4, TNF-a and IL-13 in Sham-OVx and OVx group (with or without lipopolysaccharide or OVA stimuli) with regard to their corresponding basal controls. Overall, the rise in these cytokines levels were significantly less in OVx than in Sham-OVx mice. Explants from Sham OVx animals had significantly higher levels of IL-10 regarding to basals, and these were even higher in the OVx group. Conclusion: It is presumable that FSH mediate inflammatory cytokines release by the lungs and can thereby modulate the development of pulmonary allergic inflammation. Introduction: Formaldehyde (FA) is a common indoor and outdoor pollutant that is found in many products including particle board, plywood, floor coverings and office furniture. FA causes lung inflammation and may impair lung cell recruitment after an allergic stimulus. Considering the positive relationship between female sex hormones (FSH) and asthma, we evaluated the role of FSH on allergic lung inflammation after FA inhalation. Material and methods: Female Wistar rats were ovariectomized (OVx) or not (Sham-OVx) and after 7 days subjected or not to FA exposure (1%, 3 days, 90 min/day). Subsequently the rats were sensitized and challenged with ovalbumin (OVA). After 24 h the bronchoalveolar lavage (BAL) and the myeloperoxidase enzyme activity (MPO) were determined. In a parallel set of experiments, groups of rats 7 days after OVx were treated with estrogen (280 mg/ kg, sc) or progesterone (200 mg/kg, sc) 4 h before each FA inhalation; after this the rats were sensitized and challenge with OVA. After 24 h the BAL and MPO were evaluated. Results: OVx in FA/OVA group did not alter the total cells and the MPO activity in BAL when compared to Sham-OVx group, but in theOVA/OVA group there were reductions of both total cells and MPO activity. The treatment with estrogen or progesterone increased the neutrophils and eosinophils in BAL whereas reduced the MPO activity of FA/OVA OVx group when compared to the untreated group. Treatment with estrogen, but not with progesterone increased the neutrophils and eosinophils in BAL of OVA/OVA OVx group when compared to the untreated group. A more targeted therapy to control allergy symptoms requires a better understanding of its molecular mechanisms. The RBL-2H3 basophilic cell line (RBL) is the reference model for the study of FceRI, the high-affinity receptor for IgE. We have recently shown that (1) RBL cells express the substance P (SP) and its receptor, NK1R, (2) binding of IgE to FceRI increases the expression of NK1R and (3) aggregation of IgE-FceRI complex induces autocrine activation of the NK1R, which in turn amplifies the response of FceRI (e.g. degranulation). However, molecular mechanisms regulating the interaction between the tachykinergic system SP/NK1R and FceRI remains unknown. Hypothesis: Syk, a proximal element of the FceRI signaling pathway, is involved in IgE binding-increased NK1R expression, and the autocrine activation of NK1R resulting in the amplification loop responsible of FceRI-induced basophil activation. Objective: To demonstrate the role of Syk in the molecular processes described. Methodology: A stable cell line RBL expressing the NK1R conjugated to the fluorescent protein eGFP (NK1R-eGFP) was established. The expression of the NK1R-eGFP after incubation with IgE, in the presence or absence of a specific Syk inhibitor (BAY61-3606, 250 nM) was measured by flow cytometry. The level of internalization of NK1R-eGFP after IgE-FceRI complex aggregation was determined by confocal microscopy. Results: Cytometric analyses indicated that the increased expression of NK1R resulting from the binding of IgE to FceRI is reduced when Syk activity is inhibited. Moreover, confocal microscopic analyses showed that the internalization of NK1R-eGFP in response to IgE-FceRI complex clustering is Syk dependent. Conclusion: Our study demonstrates a novel role for Syk as a regulator of the interaction between the tachykinergic system SP/NK1R and FceRI to control both the expression of NK1R and an autocrine pathway responsible of the amplification of basophil-mediated allergic responses. Disclosure of interest: None declared. Inflamm. Res. Introduction: Allergic lung inflammation is characterized by onset airway constriction, increased vascular permeability, mucus secretion, and recruitment of inflammatory cells, contributing to pulmonary dysfunction Leptin, an adipokine of the obesity (ob) gene, is synthesised and secreted by adipocytes in proportion to their mass; besides suppressing satiety and increasing metabolism, leptin is considered a proinflammatory cytokine. Objective: To investigate the role of cytokines/chemokines in the development of the allergic lung inflammation in leptin-deficient mice (ob/ob). Materials and methods: C57Bl/6 and ob/ob mice were immunized with ovalbumin/alumen on days 0 and 7 and challenged with ovalbumin (OVA) aerosol on days 14, 16, 18, 20 and 22. After 24 h of the last antigen challenge, the bronchoalveolar lavage (BAL) was performed, blood was collected and lungs were harvested for analyses. Results: Significantly fewer cells were recovered from OVA-challenged ob/ob versus OVA-challenged C57BL/6; mucus and collagen production were also reduced in OVA-challenged ob/ob versus OVAchallenged C57BL/6 (83 and 28%, respectively). In OVA-challenged ob/ob, the decreased BAL cytokines/chemokine levels were observed, when compared to OVA-challenged C57BL/6 (73% IL-4; 39% IL-12; 36% RANTES). Compared with OVA-challenged C57BL/6, OVAchallenged ob/ob also presented a reduced expression of IL-13 (63%) and TGF-b (66%) in lung tissue. Conclusions: These results demonstrate that attenuation of the allergic lung inflammation in leptin-deficient mice is strongly influenced by down-regulation of proinflammatory cytokines/chemokines expression. Eosinophils (EOS) in bone marrow are derived from myeloid precursors in response to cytokine activation. Following antigen challenge, EOS are released into the circulation and recruited to tissues. Recently, we showed that a 10-week diet-induced obesity in mice stimulates eosinophilopoesis and enhances EOS trafficking from BM to lung tissues (Calixto et al. in Br J Pharmacol 159:617-625, 2010) . The present study aimed to investigate the expression of adhesion molecules and chemokine receptor, as well as the influence of cytokines on bone marrow eosinophilopoesis of obese mice. In obese mice, ovalbumin (OVA) challenge largely increased the bone marrow EOS counts at 48 h post-challenge (2.2 ± 0.4 9 10 6 /ml; P \ 0.05) compared with lean group (0.6 ± 0.2 9 10 6 /ml). Expression of VLA-4 in bone marrow EOS from obese mice was significantly lower (P \ 0.05) compared with lean animals, as assessed by flow cytometry assays. In addition, the adhesion of bone marrow EOS to ICAM-1 and VCAM-1-coated plates in vitro was significantly lower in obese compared with lean mice. The levels of eotaxin in bone marrow supernatant did not differ between obese and lean groups. No differences in the CCR-3 expression in bone marrow EOS surface were observed. Treatment of obese mice with antibodies anti-IL-5 (2 mg/kg) and anti-TNF-a (2 mg/kg) normalized the number of immature and mature form of bone marrow EOS, as compared with lean mice treated with these antibodies. In conclusion, the increased bone marrow EOS in OVA-challenged obese mice is likely to reflect a down-regulation of VLA-4 in EOS surface, and hence a decreased interaction with the ligands ICAM-1 and VCAM-1. The bone marrow EOS differentiation in obese mice may involve TNF-a and IL-5 production, with no involvement for eotaxin or its receptor CCR-3 (experimental protocols were approved by the Ethics Committee of UNICAMP; No. 1496-1). Introduction and objective: Considering the importance of cytokines on allergy symptoms exacerbation and oral tolerance development, this study aimed to verify whether dietary treatment may induce a balance in the production of cytokines pro (IL-4, IL-13) and antiinflammatory (IL-10, TGF-b). Methods: Fourteen infants fed hydrolyzed soy protein (HSF) and 5 fed amino acid free (AA) formula (Alergomed, ComidaMed from Germany) during 120 days were evaluated. The CMA diagnosis was made with IgE levels, clinical parameters, elimination diet, challenge test and skin prick test. Afterward, the children were treated with cow's milk and derivatives of elimination diet and their replacement by HSF or AA. Serum cytokines were determined before the dietary treatment, in the presence of symptoms (T0) and after 120 days without allergy symptoms (T1). Data were presented as mean ± SE. Statistical analysis was performed by paired t test with p \ 0.05 adopted as significant level. Results: 54% of infants with CMA were IgE-mediated and 46% were non IgE-mediated. The pro-inflammatory cytokine, IL-13 was significantly reduced by both treatments 2.02 ± 1.45-0.33 ± 0.06) (p \ 0.05). Regarding IL-4, a significant decrease was also observed from T0 to T1 (4.093 ± 1.73-0.34 ± 0.13) (p = 0.01). By the other hand, the anti-inflammatory cytokines, IL-10 and TGF-b, were significantly increased by both treatments (IL-10 from 13.56 ± 3.77-24.08 ± 5.08 (p = 0.05), (TGF-b from 44.98 ± 4.89-57.03 ± 3.17) (p = 0.05). Conclusion: These data show that HSF and AA increase the antiinflammatory cytokines, IL-10 and TGF-b and decrease the proinflammatory cytokines, IL-13 and IL-10. As the imbalance between these cytokines play an important role in the symptoms exacerbation hindering the development of oral tolerance, this study shows that proper dietary treatment is able to alter inflammatory mediators leading to reduction of clinical symptoms. Disclosure of interest: None declared. S144 Inflamm. Res. Background: Sensitization to cockroach allergen ranks among the most significant world-wide risk factors for asthma in inner city communities. Cockroach allergen extract (CE) is known to contain proteolytic activity, and we find that sensitization to cockroach allergens in mice is dependent on the activation of PAR 2 in the airway by CE serine proteinases. Our aim was to identify, isolate and characterize proteolytic allergens in the cockroach allergen extract. Methods: Trypsin-like activity in the CE (Greer, Lenoir NC) was quantified with a fluorogenic trypsin substrate (Gln-Ala-Arg [QAR]aminomethyl coumarin [AMC] ) and visualized with an activity-based probe (ABP). Three ABP-labeled CE enzymes were detected and purified using ion-exchange chromatography. Each isolated enzyme was distinguished by (a) its Km for different fluorogenic peptides, and (b) its relative K i for inhibition by different trypsin inhibitors. Additionally, each enzyme was tested for (1) Identification of new target molecules through which eosinophils activate and secrete their stored proteins may be highly significant for our understanding about the pathobiology of host immune responses to parasites and allergic inflammation, as well as reveal new therapeutic targets for the control of the eosinophilic disorders. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils (Neves et al. in JACI, 2010) , however its contributions to eosinophilic inflammation remains to be elucidated. The P2Y12R was recently described as crucial in mediating the proinflammatory actions of the leukotriene E4 (LTE4) and appointed as a new therapeutic target for pulmonary allergic diseases (Paruchuri et al. in JEM, 2010) . A better understanding of the components of the immune response that mediate response to helminthes and allergens is still needed. In this work we evaluated the expression and functional roles of the purinergic P2Y12R in isolated human eosinophils. We isolated eosinophils from blood of normal and allergic donors by negative selection. By flow cytometry we confirmed the expression of the P2Y12R on isolated human eosinophils. After ADP stimulation, eosinophils secrete eosinophil peroxidase (EPO) as measured in the supernatants by a colorimetric assay. Eosinophil EPO secretion in response to ADP was differentially inhibited by the blockage of P2Y12 and P2Y1 receptors, both known to be responsive to ADP and expressed in eosinophils. Eosinophils treatment with ADP or LTE4 did not induce or prevent eosinophil apoptosis as assed by flow cytometry after labeling with annexin V. It is well established the involvement of T cells subsets in the pathogenesis of allergic airway inflammation. Schinus Terebinthifolius Raddi is a native plant from America widely used in folk medicine. We previously described the anti-allergic effect of acetate fraction obtained from their leaves in mouse pleurisy model inhibiting eosinophil influx. In this study we aimed to determinate the effect of Anacardiaceae fraction (ST fraction) in lymphocyte influx to the pleural cavity, its action in subcutaneous lymph nodes cells and in cytokines modulation. Intrathoracic OVA challenge (12.5 mg/cavity) triggered a CD3 + CD4 + and CD3 + CD8 + T cells accumulation at 24 h, concomitant with an increase of CD3 + CD25 + and CD3 + CD4 + CD25 + cells as well as of CD3 + CD4 + CD44 + cells into pleural cavity of sensitized mice. Oral pre-treatment (ST fraction, 100 mg/kg) significantly inhibited the increase of CD3 + CD4 + and CD3 + CD8 + T lymphocyte and the expression of CD44. The treatment failed to inhibit the expression of CD25 but was able to attenuate these cell numbers. The IL-2 and IL-4 production by T cells from subcutaneous lymph nodes was inhibited after in vitro treatment of ST fraction (100 lg/mL) or phenolic acid (1-100 lg/mL). We can suggest that the diminished eosinophils observed previously, seems to be also directly related to the lower number and activation of lymphocytes reaching the inflammation site suggesting an anti allergic and an immunoregulatory effect to Anacardiaceae fraction, observed after the mouse antigen challenge. Disclosure of interest: None declared. The prevalence of asthma worldwide is increasing for reasons that are unclear. Eosinophilic inflammation is a characteristic feature of asthma. Members of leukocyte integrin family (CD18) are present in eosinophils and are involved in allergic response. The CD11d integrin is expressed on most human leukocytes, including eosinophils. The surface expression of CD11d in eosinophils can be regulated by various stimuli and can function as ligand for VCAM-1. However the role of CD11d in asthma and in other allergic diseases is largely undefined. In this study, we analyzed the role of CD11d in in vivo models of eosinophilic inflammation. In this study, CD11d knockout mice (KO) and wild type (WT) mice were sensitized intraperitoneal injection of ovalbumin (OVA) and Al(OH) 3 in saline on days 1 and 10. From days 19-24 after sensitization, mice were challenged daily with either OVA (5%) or PBS by aerosol. Six hour after the last aerolization, we observed that KO mice shown lower infiltration of eosinophils in bronchoalveolar lavage (BAL) and less intense pulmonary inflammatory process than WT mice, after allergen challenge. Moreover, the airway responsiveness (AHR) analyses in a plethysmography suggest a decrease in AHR in KO mice. Several bowel diseases, such as colitis and Crohn's disease, affect the equilibrium of intestine tissue by promoting a high inflammatory context. The control of the inflammation by immune cells in the gut is crucial in the prevention of these disorders. In particular, dendritic cells (DC) play a key role both in innate and in adaptative immunity. Depending on the state of maturation and on cytokine secretion, DC can induce either specific immune response or immune tolerance. During the maturation process, for example, in the presence of zymosan (TLR2 agonist), LPS (TLR4 agonist) or fermented product, DC change their morphology and the expression pattern of their maturation markers. Here, we investigated the effect of a bacteria-free supernatant fermented by Bifidobacterium breve C 50 (BbC50sn) on the maturation markers of human DC (hDC). BbC50sn was able to mature hDC after 48 h of stimulation by inducing the expression of the same profile of maturation markers such as CD25 and CD83 compared to other maturation agents. In addition, hDC stimulated by BbC50sn secreted more IL-10 pro-tolerogenic cytokine than IL12, a pro-inflammatory cytokine. The S146 Inflamm. Res. maturation pathway activated by BbC50sn was shown to be through at least Toll like receptor 2 (TLR2). Nevertheless hDC stimulated by BbC50sn demonstrated a specific kinetics of maturation markers expression compared to hDC stimulated by other TLR2 agonist such as zymosan. The precise mechanism of the anti-inflammatory pathway induced by TLR2 engagement by BbC50sn remains currently unknown. One possibility could be that TLR2 pathway could modulate innate immunity receptors. These data show that BbC50sn was able to induce the maturation of DC and to skew towards a pro-tolerogenic profile by inducing IL10 secretion. These properties of BbC50sn are thought to act via TLR2. The BbC50sn could have anti-inflammatory and anti-allergic effects in the gut environment and might therefore protect against inflammatory bowel disease. Disclosure of interest: None declared. Under healthy condition cardiac rhythm exhibits a complex dynamic which is due to a non-linear interplay between autonomic nervous system and cardiac pacemaker cells. Systemic inflammation is associated with decreased heart rate variability which is a strong predictor of survival in patients with sepsis. However, underlying mechanism of this phenomenon is not understood. We hypothesized that cardiac pacemaker cells may act as a target for inflammatory cytokines leading to alteration in heart rate dynamics during systemic inflammation. Systemic inflammation was induced by intraperitoneal injection of endotoxin (1 mg/kg) in rats. Cardiovascular signals were recorded in conscious animals using telemetric system. Spontaneously beating atria were isolated and beat-to-beat variation in isolated atria was studied using an organ bath system. Sample entropy was calculated to assess the degree of cardiac cycle regularity. Short-term fractal scaling component of the cardiac cycle were calculated by using detrended fluctuation analysis (DFA). Endotoxemia was associated with a biphasic increase in heart rate in conscious rats. A significant increase in cardiac cycle regularity was also observed in endotoxemic animals starting 3 h after endotoxin administration. Beat-to-beat variation of isolated spontaneously beating atria exhibited a scale invariant (fractal-like) dynamics in both healthy and endotoxemic rats. However, short-term fractal scaling component in control atria was compatible with white noise (a 1 = 0.53 ± 0.07) which was significantly altered to a different (anti-correlate) type of fractal dynamics (a 1 = 0.19 ± 0.02, p \ 0.05) in endotoxemic atria. These data propose that altered cardiac cycle dynamics during systemic inflammation does not necessarily reflect the altered input from the autonomic nervous system, but rather a remodeling of the cardiac pacemaker dynamics by inflammatory mediators. Disclosure of interest: None declared. Cardiac cycle is regulated by a complex interplay between autonomic nervous system and cardiac pacemaker cells. Decreased heart rate variability (HRV) and increased cardiac rhythm regularity is associated with poor prognosis in patients with sepsis. However, the underlying mechanism of decreased HRV in sepsis is unknown. In complex systems, uncoupling of the regulatory centers is known to be related to greater regularity and less variability. The present study was aimed to test the hypothesis that impaired responsiveness of cardiac pacemaker to autonomic nervous system may lead to uncoupling of the cardiovascular regulatory mechanisms during systemic inflammation. Systemic inflammation was induced by intraperitoneal injection of endotoxin (1 mg/kg) in rats. Cardiovascular signals were recorded in conscious animals using telemetric system. Heart rate dynamics was analysis using Poincaré plot. Spontaneously beating atria were isolated and chronotropic responsiveness to adrenergic and cholinergic stimulation was assessed using standard organ bath. Vagal modulation of cardiac cycle (as assessed by SD1 in Poincaré analysis of HRV) exhibited a significant reduction in endotoxemic rats. Endotoxemia was also associated with a significant hypo-responsiveness of isolated atria to cholinergic stimulation (IC50 of charbacholine changed from 5.8 ± 0.3 9 10 -7 M in controls to 9.3 ± 0.9 9 10 -7 M in endotoxemic rats, P \ 0.05). The chronotropic responsiveness to adrenergic stimulation was identical in controls in comparison with endotoxemic rats. These data propose that altered cardiac responsiveness to cholinergic stimulation may lead to uncoupling of cardiac pacemaker cells from autonomic neural control. et al. in 41:420-432, 2007) . However, some Aristolochia species have been reported to cause nephrotoxicity due to aristolochic acid (AA) and its derivatives (Tran et al. in 2008; 6) . Here, we have investigated whether AA administered intravenously or intragastrically causes nephrotoxicity in vivo. Male Wistar rats (200-210 g) were randomly divided into three groups in which the rats were intragastrically administered water or 100 mg/kg AA, and another group was intravenously administered 100 mg/kg AA. Serum urea and creatinine were measured before and 7, 14 and 21 days after a single treatment. There was no significant difference in serum urea and creatinine between groups either at baseline, 14 or 21 days after the treatment. A great rise in serum urea and creatinine was observed at day 7 after the treatment. In addition, the intravenous AA significantly increased lipopolysaccharide induced-liver damage measured with serum alanine transaminase (ALT) on day 22 after the treatment. These results highlight that (i) the intravenous aristolochic acid significantly caused nephrotoxicity and precipitated liver damage induced by lipopolysaccharide, and that (ii) these effects were not observed in rats received AA through intragastric gavage. Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance changes inflammatory response and many studies have shown that tolerant mice have higher survival rates after a LPS challenge. To study potentially new mechanisms of immune regulation in endotoxemia, we examined macrophage function in experimental tolerance. LPS tolerance was induced in Balb/C mice exposed to 1 mg/kg of LPS/day during 5 days. The injections were subcutaneous to avoid direct effect on peritoneal cells. Naïve mice received distilled water. Peritoneal macrophages were isolated and we observed that LPS treated mice presented a larger number of peritoneal macrophages (1.6 ± 0.4 9 10 6 vs. 3.7 ± 0.6 9 10 6 ). We then used opsonized sheep erythrocytes, opsonized zymosan and non-opsonized particles of Candida albicans respectively, for Fc-, C3b-or manose-receptor mediated phagocytosis evaluation. We found a reduction in the phagocytosis by Fc (41% in control vs 23% in tolerant group) and manose receptors (23% in control vs 16% in tolerant group). On the other hand, ROS production was increased in macrophages from tolerant mice, both in basal (0.1 ± 0.08 vs. 1.3 ± 0.6 lM H 2 O 2 ) and after PMA stimulation (0.25 ± 0.07 vs. 5.9 ± 1.5 lM H 2 O 2 ). These differences can be due to a macrophage (reprogramming) activation as demonstrated by the expression of CD80 and CD86. We observed an increase of CD80 and decrease of CD86 expression in tolerant mice of 9 and 7%, respectively. These data suggest that although phagocytic activity of macrophages is suppressed in tolerant mice, bacterial clearance may be more effective due to increased ROS production and antigen presentation to other leukocytes. Financial support: FAPESP. Disclosure of interest: None declared. Background: The tracheal cilia epithelium prevents via its highly effective clearance functions the contamination of the lower airways by pathogens. These specialist epithelium cells are not only in contact with the gas phase, in addition they can also be influenced by inflammatory mediators carried in the blood stream. These mediators alter the protective function of the cilia epithelium (Jain et al. 1995) . Since TNF-a plays a key role within the inflammatory cascade, we investigated its effect onto the cilia-driven particle transport (CPT). Material and methods: Adult male mice (15 weeks old) were sacrificed by an overdose isoflurane, subsequently the trachea was rapidly removed under sterile conditions and transferred into an incubation chamber of an Olympus upright brightfield microscope. CPT and cilia-beat-frequency (CF) were continuously recorded over a period of 130 min as described previously ( Candida albicans is an opportunistic dimorphic fungus that exists as a commensally and colonizes mucosal surfaces of the oral and vaginal cavities and the digestive tract. It is the most common pathogen of human fungal infection and can cause both superficial and serious systemic disease. The prophylactic and curative treatments with antifungal drugs can cause the appearance of Candida resistantstrains. The aim of our study was to evaluate the antifungal activity of two anti-inflammatory drugs, aspirin and diclofenac sodium, and investigate the effect of combination of these drugs with ketoconazole. Two strains were isolated from pharyngeal secretions. Strains were identified by biochemical and molecular methods (RFLP of ARN ribosomal 5.8S gene) as Candida albicans using Candida albicans ATTC 10231 as reference strain. Viability cells tests were done in the presence of 2 mg/ml aspirin and diclofenac sodium and 0.32 lg/ml ketoconazole. Results showed that aspirin alone and aspirin-ketoconazole combination have the same effect on C. albicans strains and cell viability decrease with 70-74%. Unlike aspirin, diclofenac sodium-ketoconazole combination induced higher antimicrobial activity (76.5-80% inhibition) than diclofenac sodium alone (40-73% inhibition). The effect of aspirin and diclofenac was similar with activity of ketoconazole at MIC breakpoint. Antifungal activity of non-steroidal anti-inflammatory drugs was strain depending. According to our results aspirin and diclofenac sodium alone or in combination with ketoconazole have a remarkable antifungal activity against Candida albicans strains. Association of anti-inflammatory drugs with antifungal compounds could be more efficient for treatment of candidiasis Many documents suggested that the synergic effect of MD-2 to LPS stimulating on TLR4 might be critical step during LPS-TLR4 response signaling pathway. MD-2 has been considered as one of the most important and potential targets to interfere the activation of excessive inflammation caused by LPS. Varied efforts are being made to disable MD-2. In this research, we combined bioinformatics and the technique of phage peptide display to try to obtain peptides that would antagonize against MD-2. Based on our bioinformatics analysis of MD-2 protein, NH2-FSKGKYKCV-COOH (K128-132) might be the key sequence for MD-2 binding LPS, thus random phage display peptide library Ph.D.-C7C was used for selecting antagonistic peptide against the sequence. After three round selections, three positive clones binding to the sequence were picked out. Further results showed that all three peptides can bind directly to the MD-2 protein identified by Sulfo-SBED biotin Label transfer. In addition, all three peptides can inhibit the productions of TNF-alpha and IL-6 in U937, THP-1 macrophage cell lines and health human peripheral blood monocytes stimulated by LPS in dose dependent manner. Furthermore, BALB/C mouse challenged by LPS (O111:B4) were used to estimated the protection of these peptides from sepsis, but only two of the peptides can reduce mortality of the infected mice from 100 to 55.6% (5/9), and 54.5% (6/11), meanwhile the levels of TNF-alpha and IL-6 in serum and homogenized lung were profound reduced in peptides treated group, moreover the pathological examination showed that the pulmonary inflammatory responses were alleviated in peptides treated group. These studies demonstrated that interference focused at binding site between MD-2 and LPS might be one potential therapeutic strategy for LPS induced sepsis, and the two peptides might be considerable agents against sepsis. NO produced by iNOS is responsible for endotoxin-induced vascular hyporeactivity and hypotension resulting in multiple organ failure. Endotoxic shock is also characterized by decreased expression of COX-1, CYP4A, and eNOS. Our previous studies demonstrated that dual inhibition of iNOS and COX with a nonselective inhibitor indomethacin restores blood pressure presumably due to increased production of 20-hydroxyeicosatetraenoic acid (20-HETE) derived from arachidonic acid by CYP4A in endotoxemic rats. The aim of this study was to investigate the effects of a selective COX-1 inhibitor, piroxicam, on the endotoxin-induced changes in blood pressure, expression of COX-1, COX-2, CYP4A1, eNOS, iNOS, and hsp90, and production of PGI 2 , PGE 2 , 20-HETE, and NO. Injection of endotoxin (10 mg/kg, i.p.) to male Wistar rats caused a fall in blood pressure and an increase in heart rate associated with elevated renal 6-keto-PGF 1a and PGE 2 levels as well as an increase in COX-2 protein expression. Endotoxin also caused an elevation in systemic and renal nitrite levels associated with increased renal iNOS protein expression. In contrast, systemic and renal 20-HETE levels and renal expression of eNOS, COX-1, and CYP4A1 were decreased in endotoxemic rats. The effects of endotoxin, except for renal COX-1 and eNOS protein expression, were prevented by piroxicam (10 mg/kg, i.p.), given 1 h after injection of endotoxin. Endotoxin did not change renal hsp90 protein expression. These data suggest that a decrease in The aim of the present study was to determine which toll-like receptor(s) (TLRs) were involved in triggering the inflammatory response responsible for the death due to B. cenocepacia pneumonia. Indeed, we previously showed that MyD88, a key downstream adapter for most the TLRs, was involved the pathogenesis. We specifically focus on the TLRs 4 and 5, as these two receptors are the main ones involved in the recognition of P. aeruginosa, a flagellated Gram-bacterium like B. cenocepacia. Mice deficient for TLR4, TLR5 or both were infected intratracheally with a suspension of B. cenocepacia. Animals were then observed daily for signs of morbidity. Alternatively, bronchoalveolar lavages (BAL) were collected at different time points to further determine cytokine concentrations and the number of CFU of B. cenocepacia. We observed that the innate immune response of the host to B. cenocepacia lung infection was due to TLR4 and not to TLR5. As for the MyD88 -/strain, TLR4 -/mice were protected from death and cytokine and chemokine synthesis to infection were reduced. By contrast, we observed a reduced pathogen burden in the case of TLR4 -/mice compared to the enhanced ( Conclusions: The colonizing bacterial cells and their DNA were detected in fracture callus but not other deep tissues. Contamination was precluded by lack of isolates in disinfected cutis, subcutis, muscles and materials used for sampling cultured after surgery. We suggest that certain strains of bacteria dwell in normal tissues of lower limbs and may cause inflammation upon stimulation by trauma. Their source may be tissue fluid, superficial and deep lymphatics, and lymph serving the physiological transport to the regional lymph nodes of microorganisms penetrating foot skin during microinjuries. Disclosure of interest: None declared. Adhesion proteins, degradative enzymes and other proteins ensure bacterial parasitism as well as virulence. The aim of our study was to compare characteristics of S. aureus strains isolated from patients with ischemic (IFU) and neuropathic (NFU) diabetic foot ulcers. Methods: Study was done on 36 S. aureus isolates obtained from ulcer base curettage (UC) and skin biopsy (SB) of patients divided into IFU and NFU group. For detection of fnbA and cna genes (encoding binding proteins for fibronectin and collagen) bacterial DNA was extracted and PCR with specific primers was done. For detection of gene expression bacterial RNA was extracted and mRNA production was estimated in RT-PCR. Ability of biofilm formation was determined using Congo red agar (CRA) method and for production of polysaccharide intercellular adhesin (PIA) the staining with antiserum was used. For testing of in vitro enzyme production Difco spirit blue agar with lipase reagent and Difco DNase test agar were used. Gelatinase activity was detected using 3% gelatin medium. Results: The prevalence rate of fnbA gene and its expression were more often associated with strains originating from UC and SB of NFU than IFU. Expression of cna gene was higher in strains originating from NFU than IFU. The frequency of strains producing biofilm was higher among isolates originating from NFU than IFU when tested by CRA method. Activities of gelatinase, DNase and lipase were found in higher rate of strains yielded from UC of IFU than NFU. Conclusions: It can be suggested that the high rate of PIA producing strains with hydrolytic enzyme activity present in IFU can be responsible for the worse outcome of this type of ulcers in contrast to NFU. Disclosure of interest: None declared. Background: Post-operatively acquired immune dysfunction is associated with a higher mortality rate in case of septic complications. As details of this severe clinical problem are still unknown and intervention strategies are lacking, animal models are essential to characterise the mechanisms involved. Materials and methods: Isolated splenocytes were stimulated ex vivo with LPS and the release of cytokines was determined. The degree of surgical trauma was analysed by detection of high-mobility group box 1 (HMGB1) and IL-6 in serum. Results: We have adapted the previously described animal model of intestinal manipulation (IMM) so as to provide a model of surgically induced immune dysfunction (SID). Following intestinal manipulation mice showed elevated serum levels of HMGB1 and IL-6. Ex vivo cytokine release by splenocytes was suppressed at early and late time points in the post-operative period and the degree of suppression correlated with the extent of surgical trauma. Conclusions: In this study we describe a surgically-induced immune dysfunction (SID) animal model in which a significant surgical trauma is followed by an immune dysfunction syndrome. This model may be qualified for the characterisation of the post-operative immune dysfunction syndrome and to analyze its inflammatory components. Disclosure of interest: None declared. Endogenous glucocorticoids (GC) have a protective role in septic shock. GCs exert their effects via the glucocorticoid receptor (GR) that alters gene expression by binding to GC responsible elements (GRE) as a dimer in the promoter region of target genes or by interacting with and thus interfering with other transcription factors as a monomer. We show that the GR in Macrophages (Mphs) is important for the repression systemic inflammation. Mice lacking the GR in myeloid cells (GR LysMCre ) are highly susceptible to LPS induced septic shock. Also binding as a dimer to DNA is important for protection from septic shock. Mice with impaired GR dimerization (GR dim ) display a strong lethality in septic shock models. Mphs from GR dim mice are resistant to suppression of some inflammatory mediators (IL1b, NO). We identified GR dimer controlled genes, possibly essential in inhibition of inflammation in a micro array analysis from Mph mRNA isolated from wt and GR dim mice. One of the genes encoded Sphingosine kinase 1 (Sphk1). Sphk1 mRNA and activity of the enzyme was found to be up regulated synergistically in wt Mphs co-treated by lipopolysaccharide (LPS) and the GC analogue dexamethasone (Dex). In GR dim Mphs the up regulation was significantly attenuated, indicating that GR dimerization was required for this process. The mechanism for the Sphk 1 mRNA regulation by LPS and Dex is protein synthesis and p38 dependent. Sphk 1 and Sphk 2 are key enzymes for production of sphingosine-1phosphate (S1P). In primary LPS activated Mphs S1P is important for the repression inflammatory cytokines and seems to function as anti-inflammatory mediator of the dimerized GR. The impact of Sphk1 in antiinflammatory actions of the GR is currently exploited in vivo. We demonstrate that GR dimerization in Mphs is required for immunosuppression in systemic inflammation. Mphs of GR dimer deficient mice serve as an S152 Inflamm. Res. Serum amyloid A (SAA) is an acute phase protein involved in homeostasis of inflammation. SAA is necessary in host defense and has beneficial properties in protection against viral and bacterial infections. Urothelial infections have been especially difficult to treat since latent intracellular pools of bacteria remain in urothelial cells, also after treatment with antibiotics, and cause episodes of recurrent cystitis without evident re-infections. To date it has been unclear whether SAA is present in infected urothelium and whether it could be protective against bacterial infections. The major aim of this study was to better understand the role of SAA in murine infections of the urothelium with E. coli and determine the differential expression of SAA isoforms in the urothelium and in the serum (and whether SAA could act in a protective manner). Urothelial SAA protein was detected by immunofluorescence in mouse tissue. SAA1 and SAA3 mRNA expression in the liver, urothelium and urinary bladder were quantitated using real-time PCR from infected and noninfected mice. Serum SAA1/2 and SAA3 were detected by ELISA. The biofilm inhibition assay was performed using crystal violet to stain adherent/biofilm-associated bacteria adhered to plastic. Our experiments revealed that SAA is highly expressed in urothelial cells of E. coli infected mice versus non-infected mice. Especially, there is a high increase in cytoplasmic SAA during infection as compared to the nuclear SAA in urothelial cells. We also have evidence that following E. coli inoculation into bladders, SAA1 mRNA expression is higher than that of SAA3 in the urothelium. SAA1/2 can act protectively, by inhibiting E. coli biofilm formation. Pathophysiology of osteoarthritis was associated with the accumulation of inflammatory mediators in the joint space. Accumulation of immunoglobulins secondary to chronic inflammation or exposure of antigen in the joint space has been well described. Parvovirus B19 (PVB19) is a ubiquitous pathogen normally causes a mild short-lived illness. However, this virus is potentially capable of causing significant pathology and of long-term persistence. Certain inflammatory disorders have been linked to PVB19 infection. Enhanced IL-6 production in hematopoietic cell lines transfected with NS1 protein of PVB19, providing evidence that NS1 protein may be involved with transactivation of the IL-6 gene promoter. IL-6 is a ubiquitous and pleomorphic cytokine, multicellular in origin that acts on numerous cells and tissues. Hence, NS1 protein expression could play a role in the ongoing inflammation and cell damage with persistent PVB19 infections in nonpermissive cells such as synovial membrane cells. Aims: To provide more insight into the possible role of this virus in the etiology of osteoarthritis. Methods: Synovial fluid samples from 42 patients with osteoarthritis and 10 healthy control (totally 52) were participated to this study. Anti-PVB19 IgG, anti-PVB19 IgM, IL-6 levels and PVB19 DNA were assayed by ELISA and PCR. Results: PVB19 DNA was detected in 28 of 42 (66.66%) and in 3 of 10 (30%) in patients and controls. Anti-PVB19 IgG and IgM response were detected in 21 of 42 (50.00%) and in 2 of 42 (4.76%) patients. Anti-PVB19 IgG and IgM were also detected in 1 of 10 (10.00%) and in 0 of 10 (0.00%) controls. IL-6 was positive in 15 of 42 (36%) and in 3 of 10 (30%) patients and controls. All IgG ( The strategies for treatment of systemic inflammation are based on neutralization of LPS or cytokines induced by it. Heat shock proteins (HSPs) inhibit production of pro-inflammatory cytokines. Carbenoxolone induces the production of HSPs. The aim of this study is the evaluation of the effect of Carbenoxolone in reduction of the severity of systemic inflammation induced by LPS in a murine model. 80% lethal dose of Salmonella typhimurium LPS was determined in balb/C mice, also appropriate dose of drug for efficient reduction in mortality was determined. LPS with drug were injected to different groups, in different times. LPSCarb group (injected with LPS + D-GalN and Carbenoxolone simultaneously) and LPS1Carbi.p. (Received Carbenoxolone by i.p. injection 1 h after LPS + D-GalN), after 24 h the mortality rate was calculated and the severity of necrosis measured in the livers of survived mice. Then mice were injected with LPS + D-GalN and drug in different times, after 3 h serum samples were collected and the concentrations of TNF-a and IL-10 were measured. Administration of carbenoxolone with LPS + D-GalN reduced the lethality rate from 80 to 20%. The severity of necrosis in the liver of LPSCarb and LPS1Carbi.p. groups were significantly decreased in compare with LPS group (injected by LPS + D-Gal) (P = 0.01 and P = 0.04 respectively). The concentration of TNF-a in mice received LPS were significantly increased in compare with control group (P = 0.032). The changes in the IL-10 concentrations were accompanied with changes in TNF-a concentration. Carbenoxolone significantly reduced TNF-a concentration, inflammation and damage due to LPS, however changes observed in IL-10 concentration was in response to TNF-a concentration changes. Disclosure of interest: None declared. CD163 is a transmembrane scavenger receptor for haptoglobinhemoglobin complexes expressed by monocytes and macrophages with a role in the resolution of inflammation. Inflammatory stimuli induce ectodomain shedding of soluble CD163 (sCD163). High serum levels of sCD163 predicted mortality in some inflammatory diseases. We investigated sCD163 release and predictive power towards outcome in critical illness. This is a secondary analysis of 2 randomized clinical studies. Critically ill patients (n = 1657) received conventional (insulin administered only for blood glucoses[215 mg/dL) or intensive insulin therapy (glucose maintained at 80-110 mg/dL). Matched healthy volunteers (n = 24) were included as controls. We measured serum levels of sCD163 as well as gene expression levels in liver biopsies. On day 1, critically ill patients had twofold higher sCD163 levels than controls (p \ 0.001). Day 1 sCD163 levels were higher in patients with an ICU stay[5 days (long-stay) versus short-stay patients (p \ 0.001). sCD163 further increased by day 5, 10 and 15 in long-stayers and remained elevated until the last day, the latter irrespective of short or long stay. Non-survivors and patients who developed new kidney injury, liver dysfunction, polyneuropathy or bacteremia had higher sCD163 levels from day 1 onwards. Moreover, even after correction for baseline risk factors, elevated day 1 sCD163 levels remained independently predictive for organ damage, ICU stay, and mortality. Avoiding hyperglycemia with insulin reduced sCD163 levels. Gene expression of CD163 in liver was strongly elevated in critically ill patients as compared with controls. Circulating sCD163 levels were elevated during critical illness, with the liver as a potential source. This study hallmarks elevated sCD163 as marker of disease severity and predictor of outcome in critical illness. Reduced sCD163 with intensive insulin therapy supports reduced inflammation and may contribute to improved outcome with this therapy. Disclosure of interest: None declared. Systemic inflammatory response induced by over expressing inflammatory mediators is the main pathogenic mechanism of septic shock. Glutamine (Gln), a non-essential amino acid, has been demonstrated to inhibit pro-inflammatory cytokine release through enhanced heat shock protein (HSP) expression. The aim of this study was the evaluation of the effect of co-administration of Gln and ciprofloxacin in reduction of septic shock severity caused by Pseudomonas aeruginosa in mice. Balb/c mice were divided to five groups. Mice of the control negative group injected only with PBS and those of the positive control group received 75% lethal dose (LD75) of Pseudomonas aeruginosa. Mice of the remaining three groups were administered by ciprofloxacin, glutamine or both 30 min after injection of LD75 of the bacteria. Five hours after injection of the bacteria, serum samples were collected and concentrations of TNF-a, IL-10 and HSP-70 were measured. Eleven hours after injection of the bacteria, livers of the mice were removed and the severity of necrosis was determined. The survival rate was considered for 10 days. The results showed that injection of glutamine in combination with ciprofloxacin significantly increased the survival rate and the serum HSP-70 and IL-10 concentration and significantly decreased the serum TNF-a concentration and the liver necrosis in comparison with the positive control group. The findings indicated that Gln has a synergistic effect with ciprofloxacin in reduction of Pseudomonas aeruginosa-induced septic shock. Disclosure of interest: None declared. Background: Streptococcus anginosus is a part of the normal flora found in human dental plaque. Although it has emerged that S. anginosus infection could be associated with oral, esophagus and gastric cancers, its pathogenic factors are still unclear. We previously identified fibronectin binding protein gene, fbp62, which could associate the adherence of S. anginosus to mucosal epithelial cells. In this study, by using the fbp62 knockout mutant we investigated the pathogenic involvement of Fbp62 in an S. anginosus-infection model. Materials and methods: An fbp62 knockout mutant (Dfbp62) was prepared by homologous recombination from S. anginosus NCTC 10713. The adhesive ability of Dfbp62 to the immobilized fibronectin and HEp-2 cells was analyzed by using [ 3 H]-labeled bacteria. The survival rate and the abscess formation were investigated in an S. anginosus-infection mouse model with ip injection of Dfbp62. The mRNA expression of inflammatory cytokines (TNF-a, IL-1b, IL-6) of mouse macrophages was analyzed by real-time PCR. Results: The adhesive ability of Dfbp62 to HEp-2 cells as well as the immobilized fibronectin was considerably reduced. In the mice infected with Dfbp62, the mortality and the abscess formation were substantially lower than those in the mice infected with the wild type of bacteria. However, no difference was observed in the mRNA expression of inflammatory cytokines between the macrophages from Dfbp62and wild type of S. anginosus-infected mice. Conclusions: The present findings indicated that the fibronectin binding proteins, Fbp62, of S. anginosus could play an important role in the adherence to mucosal epithelial cells, and suggested that the adhesive ability could be involved in the pathogenicity of S. anginosus. Disclosure of interest: None declared. Natural killer (NK) cells play a pivotal role in the effector arm of host innate immune defense system. The major functions of NK cells in killing tumor transformed or virus-infected cells were well established. However, whether NK cells can directly kill bacteria remains poorly understood. In this study, using the pathogenic mycobacterium Mycobacterium kansasii as the study model, our results indicated that human NK cells directly kill mycobacteria in a contact dependent manner, using the cytolytic proteins, perforin and granulysin as the weapons. These results indicate that the antibacterial activity is constitutively expressed in NK cells, in which it requires prior activation, granulysin and perforin, plays the dominant role in NK cell antibacterial activity. Moreover, the expression of NKG2D, NKp30, NKp44 and NKp46 upstream signaling molecule of the MAP kinases pathway, are also enhanced. M. kansasii-enhanced granulysin and perforin expression and antibacterial activity are effectively inhibited by pretreatment with the inhibitors of JNK (SP600125), ERK-1/2 (PD98059), p38 (SB203580), or by siRNAs against JNK-1, ERK-2 and p38. A similar killing phenomenon by perforin and granulysin was also observed in Mycobacterium tuberculosis, suggesting a conserved killing mechanism. In conclusion, results from this study showed NK cells directly kill mycobacteria. Disclosure of interest: None declared. Y Toll-like receptor (TLR) is a major family of pattern recognition receptors (PRRs) and plays a crucial role in innate immunity. Microbial pathogens can activate TLRs signaling and trigger dramatic immune responses. The Spleen tyrosine kinase (Syk) presents in innate immune cells and plays a key initiation of ITAM-containing immunoreceptors signaling and TLRs signaling. However, the precise role of Syk in TLRs signaling is still controversial. Here we show the dual role of Syk in TLR4-mediated signaling cascade and inflammatory gene expression in primary murinemacrophages. We found that Syk-deficient macrophages after LPS stimulation can induce higher expression levels of inflammatory genes and proteins. Loss of Syk also increases TAK1 downstream signaling cascades activation after LPS stimulation. In contrast, less LPS-induced TBK1 activation and type I IFN (IFNb) gene expression in Syk-deficient cells than wild type cells were observed. In HEK293T overexpression system, we also found Syk interacts with The neuropeptide substance P (SP) has been shown to be an important mediator in lung injury during polymicrobial sepsis. SP exhibits its proinflammatory effects via increased capillary permeability and neutrophil extravasation. In a mouse model of polymicrobial sepsis, animals were protected against lung injury after administration of a specific SP receptor antagonist. For the neuropeptide galanin no data concerning septic conditions are available so far. However, in murine skin, galanin has been shown to inhibit plasma extravasation induced by SP and calcitonin-gene related peptide (CGRP). This suggests galanin as an opponent of SP and CGRP in neurogenic inflammation. To study a possible role of galanin during systemic inflammation we investigated the expression of the galanin system, SP and TNF-a mRNA in organs of septic mice. Sepsis was induced in C57/BL6 mice by colon ascendens stent peritonitis (CASP), because this animal model mimics closely human septic conditions. Lungs were removed 12 h after surgery. Quantitative real time PCR was performed to determine the relative expression of galanin, TNF-a and SP mRNA. We were able to detect a significant up regulation of SP (60-fold) and TNF-a (110-fold) expression and a 50% down regulation of galanin in lungs of septic mice compared to sham operated animals. The expression of the galanin receptors R1, R2 and R3 was not changed. These results implicate that galanin is down regulated in the initial immune response to favour pro-inflammatory mediators. In further course galanin might be increased as an opponent of SP to avoid an overshooting immune response, including vasodilatation, neutrophil accumulation and organ dysfunction. Septic shock is characterized by severe hypotension and reduced response to vasopressor agents, called vascular hyporeactivity. The hypotension and vascular hyporeactivity are associated with the development of multiple organs dysfunction which causes death eventually. Thus, it is important to investigate the mechanism of vascular hyporeactivity in septic shock. Among the cell signaling pathways that are crucial to control vascular tone, Ca 2+ -sensitization contraction has become more and more important. The small GTP-binding protein, RhoA, plays a crucial role in mediating smooth muscle contraction. Activation of RhoA leads to inactivation of myosin light chain phosphatase via activation of Rho-kinase. It has been shown that Src family tyrosine kinase (SFK) can activate RhoA via phosphorylation of guanine nucleotide exchange factor. In addition, it is reported that activation of protein kinase C (PKC) can lead to Ca 2+ -independent vasocontraction through activation of RhoA. The purpose of this study was to investigate the role of PKC and SFK in RhoA activity in thoracic aortae from endotoxemic rats. Rats received an intravenous injection of lipopolysaccharide (LPS, 10 mg/kg) for 4 h. After then, rats were sacrificed and the thoracic aortae were excised and immediately incubated in GF-1092039 (a PKC inhibitor) and PP2 (a SFK inhibitor). We found that RhoA activity was decreased significantly in aortae from endotoxic rats. GF-1092039 and PP2 had inhibitory effect in aortae from endotoxic rats only. In addition, the phosphorylation of SFK was decreased by GF-1092039 in endotoxemic rats. In conclusion, PKC and SFK might play a more crucial role in RhoA activation and PKC might play a more important role in activation of SFK endotoxemic rats. These results suggest that RhoA is related to vascular hyporeactivity caused by LPS-induced septic shock. In addition, PKC and SFK might be targets for improving vascular hyporeactivity. Disclosure of interest: None declared. Methods: Pneumonia-induced sepsis was induced by intra-traqueal inoculation of Klebsiella pneumoniae (4 9 10 8 CFU/animal) in Swiss mice. Sham-operated mice were used as control. Mice received lipoxin receptor (FPR2/ALX) antagonist BOC-1 (1 lg/kg; i.p.) soon after surgery and every 12 h in survival experiment. Six hours after sepsis induction, animals were sacrificed and bronchoalveolar lavage (BAL) and tissues were obtained for analysis. Results and discussion: Neutrophils (PMN) migration to lungs from septic animals increased when compared to sham group. Animals exhibited a high degree of bacterial load in BAL, spleen and heart. Compared to septic mice, animals treated with BOC-1 exhibited increases of PMN number to the infectious focus and consequently a reduced bacterial load and dissemination. Interestingly, LXA 4 levels in BAL from septic mice were higher compared to sham animals. Treatment with BOC-1 also increased survival rate. Therefore, LXA 4 levels increased in sepsis and FPR2/ALX antagonist BOC-1 reduced bacterial load and improved survival. These data suggest for the first time that a reduction in anti-inflammatory mechanisms in the beginning of the sepsis process could be an interesting therapeutic alternative. The innate immune system provides the first barrier against pathogens. It has evolved to recognise a broad range of pathogens and discriminate between self and non-self. Toll-like receptors (TLRs) are the crucial sensors of pathogen associated molecular pattern or PAMPS. Intracellular Toll-like receptors (TLR3, 7 and 9) localise in endosomes and recognize single stranded RNA and nucleotides from viruses and bacteria. This interaction induces their conformational changes resulting in the production of proinflammatory cytokines and up regulation of cell surface molecules in dendritic cells (DCs). In the absence of stimulation, TLRs are retained in the endoplasmic reticulum (ER) together with another ER resident protein, UNC93B. Upon stimulation, they relocate to the endo-lysosomal compartment, allowing the recruitment of the adaptor molecule, MyD88, and thereafter, signal. It has been shown that mouse TLR9 requires proteolytic cleavage in the endosomes to be functional. This is an important step, as it is the activation of the MyD88 signaling pathway that leads to the production of pro-inflammatory cytokines and maturation in DCs. Indeed, upon stimulation, full length TLR9 is cleaved into a C-terminal-fragment and this processing is highly dependent on a cysteine protease named asparagine endopeptidase (AEP). We have now evidence that TLR7 is also cleaved by AEP into a C-terminal fragment competent for signaling. Like TLR9, a boost in AEP activity, induced shortly after TLR7 stimulation, promotes TLR7 cleavage and correlates with an increased acidification in endosomes. Moreover, mutating a putative AEP cleavage site in TLR7 strongly decreases its signaling in DCs. In contrast, TLR3 processing and signaling seems to require a different set of proteases. As endocytic TLRs recognize DNA/RNA from various parasites, bacteria, viruses and are likely to play an important role in many inflammatory diseases, we are now developing infection models in AEP deficient mice. Introduction: The functioning of the immune system is finely balanced by the activities of pro-inflammatory and anti-inflammatory mediators or cytokines. Unregulated activities of these mediators can lead to development serious inflammatory diseases. Therefore, anticytokine agents that target biosynthetic pathways of pro-inflammatory cytokines would offer an attractive alternative to the treatment of inflammatory diseases. Recent data showed the capacity of VGX-1027 [(S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid] to inhibit the increase of circulating levels of tumor necrosis factor a and interleukin-1b in lipopolysaccharide (LPS)-challenged mice. In this study, we evaluated the role of DIC, a 5-(4-pyrildil)-4,5-dihydroisoxasole derivate, as a potential anti-cytokine compound. Results: DIC significantly decreased the levels of inducible TNF-a, IL-1b and interleukin-6 (IL-6) in RAW 264.7 macrophages stimulated with LPS. DIC also inhibits the activity of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages with consequently decrease of prostaglandin-2 (PGE2) levels. Electrophoretic mobility shift assay revealed that DIC attenuated the LPS-induced DNA binding of NF-kB. In addition, it was found that treatment with DIC significantly inhibited the activation of p38 and ERK MAP kinase. Taken together, these results suggest that anti-inflammatory effect of DIC in LPS-treated RAW 264.7 macrophages is associated with the inhibition of NF-kB transcriptional activity, possibly via the MAP kinase pathway. Furthermore, in vivo, DIC conferred increase in the survival of mice that have been subjects to cecal ligation and puncture, an experimental sepsis model. Conclusions: We believe that the anti-cytokine activity of DIC could be due to the NF-kB/MAPK pathway inhibition. In addition, our data suggest that DIC may potentially play a role in the therapy of inflammatory disease as sepsis. Keywords: Pro-inflammatory cytokines, Sepsis, 4,5-dihydroisoxasole, NF-kB. Disclosure of interest: None declared. Inflammation is a host response to infection and is characterized by elevated inflammatory mediators. Two of the major mediators during inflammation, i.e. nitric oxide (NO) and prostaglandin E2 (PGE2), are released by macrophages and neutrophils. Overt inflammation can lead to detrimental consequence, such as septic shock, which is known due to excessive NO and proinflammatory cytokines production. Chlorella has been shown to have various remarkable biological effects, including anti-inflammatory effect through inhibition of iNOS pathway and proinflammatory cytokines (e.g. IL-6 and TNF-a production), however its protection against LPS-induced sepsis has not been studied. In this study, we investigate protective effects of Chlorella extract against LPS-induced sepsis in mice. Endotoxin-induced septic shock was initiated by injecting 20 mg/kg of lipopolysaccharide (LPS) in mice. Mice were given different dosages of Chlorella extract (10, 20, 30 mg/kg, orally) 30 min after the treatment of LPS. Serum NO production was prepared and measured as nitrite (using Griess reagent), IL-6 and TNF-a were concurrently monitored by using commercial ELISA kits. The mortality rate was observed 12, 24, and 36 h after the LPS treatment. Chlorella extract administration decreased LPS-induced mortality as evidenced by increased survival rate in a dose dependent manor. The survival rate remained as high as 95% in those mice treated with 30 mg/kg of Chlorella extract compared to 40% of survival rate in the control (vesicle treatment) 36 h after LPS injection. Chlorella extract treatment also significantly suppressed LPS-induced serum NO, IL-6 and TNF-a levels. In conclusion, Chlorella extract effectively prevents LPS-induced septic mortality by reducing inflammatory mediator and proinflammatory cytokines production. Disclosure of interest: None declared. Introduction: The airways mucus gel performs a critical function in the defense of the respiratory tract against pathogenic and environmental challenges. In respiratory infections, mucins such as Muc5AC could be protective and able to sequester pathogens [1] . Aims: (1) To determine whether IAV (seasonal and pandemic Influenza A strains) is able to modulate Muc5AC production in airway epithelial cells. (2) To dissect the molecular pathways involved in IAV modulation of Muc5AC synthesis. Results: We demonstrate here that seasonal and pandemic strains infection of NCI-H292 cells (MOI = 1) induced significantly MUC5AC gene expression. Influenza A/Scotland/20/74 (H3N2), the most virulent strain, lead to a significant Muc5AC increase compared to mock-treated cells, and this was dependent on viral replication. Moreover, using the same strain, we showed that in vivo infection of C57Bl/6 mice with 150 or 300 pfu lead to a threefold increase of Muc5ac RNA expression at day 3 and 4 post-infection compared to PBS-treated mice. We demonstrated that the Sp1 transcription factor is involved in IAV-induced MUC5AC gene expression and that this is mediated by the MEK/ERK signaling pathway. Next, we showed that this pathway was initiated upstream by a matrix metalloproteinase (TACE)-mediated epidermal growth factor receptor (EGFR) activation and that TGF-a is one of the ligands implicated in EGFR activation [2, 3] . Conclusion: We show for the first time that IAV up-regulates Muc5AC through a TACE/EGFR/MAPK pathway and that this induction is IAV-replication dependent. We are studying now if upregulation protects against IAV infection or whether Muc5AC overexpression is deleterious in IAV-induced deleterious exacerbations in chronic lung diseases, such as asthma or cystic fibrosis. It remains controversial whether prophylactic antibiotics are necessary after lower third molar removal (LTMR) and whether a surgeon's clinical experience impacts this possible need. This study evaluated and compared postoperative complications of patients who were operated on by surgeons ranging in clinical experiences. These surgeons included an oral surgeon specialist, a PhD student in Oral Biology, and a senior dental student. In the preoperatory period and on the second and seventh postoperative days, the following parameters were analyzed: pain, infection, swelling, trismus, body temperature, C-reactive protein levels and salivary neutrophil counts. During surgery the following parameters were analyzed: systolic, diastolic and mean arterial pressure, oximetry, heart rate, quality of anesthesia, amount of local anesthetic, bleeding, surgery difficulty and duration of surgery. There were some differences in the surgery duration, local anesthetic amount, anesthesia quality, bleeding, pain experienced, trismus, C-reactive protein levels and salivary neutrophil counts. However there were no changes in hemodynamic parameters, rescue analgesic medication, wound healing, swelling, body temperature, confirmed case of dry socket or any other type of local infection in the patients. Taken Introduction: This study is designed to explore the differential inflammatory responses in macrophages by the different resistant strains of vancomycin-susceptible Staphylococcus aureus (VSSA) and vancomycin-intermediate Staphylococcus aureus (VISA). Materials and methods: Macrophages (RAW264.7 cell line) were infected using two strains of VSSA and VISA isolated from a patient with septic arthritis. The time course was 0, 6, and 12 h and the doses were 1 9 10 3 , 1 9 10 4 , and 1 9 10 5 CFU/ml, respectively. Gene regulation including cyclooxygenase 2 (COX-2), 5-lipoxygenase, IL-1beta, IL-6, and TNF-alpha was investigated by reverse transcriptasepolymerase chain reaction (RT-PCR). COX-2 protein expression and signal transduction were evaluated by western blotting and electrophoretic mobility shift assay (EMSA), respectively. Results: COX-2 and 5-lipoxygenase gene expression did not influenced by starvation within 12 h. COX-2 gene induction by VSSA and VISA infection in macrophages was in a dose-dependent and time course-dependent manner. VSSA yielded an intense, significant response compared to VISA in early infection (6 h) and also produced similar effects by dosage experiments (p \ 0.05). The 5-lipoxygenase pathway did not demonstrated similar responses. VSSA also produced a higher level for IL-1beta and IL-6 gene expression than VISA did. TNF-alpha showed an increase tendency but no significant difference. COX-2 protein expression showed significant difference at a concentration of 1 9 10 5 CFU/ml VSSA and VISA. Signal transduction involved the phosphorylation of extracellular regulated kinase 1/2 (p-ERK1/2) and NF-kB activation. Conclusion: Our data may suggest that changes of Staphylococcus aureus strains to the antibiotic sensitivity may induce differential inflammatory responses in macrophages and play an important role in the host inflammatory adaptation. Disclosure of interest: None declared. Conclusions: After traumatic injury, in the absence of shock and other activators of inflammation, circulating MtDNA levels reflected injury severity. These molecules may be trauma alarmins. Nucleosomes were not released by tissue injury alone. HMGB1 levels increased in proportion to the base deficit and not injury severity. Our study findings suggest that HMGB1 may not be passively released by pure tissue damage in human trauma. Disclosure of interest: None declared. Medicine, Federal University of Sa˜o Paulo, 2 Immunology, University of Sa˜o Paulo, Sa˜o Paulo, 3 Pathology, Federal University of Triangulo Mineiro, Uberaba, Minas Gerais, Brazil Introduction: Sepsis is considered one of the main causes of acute kidney injury (AKI) in ICU patients, mainly due to bacteria infection. TLR2 e TLR4 are important innate immunity receptors responsible for pathogen-associated molecular pattern structure recognition that when activated, they trigger, with the adaptor molecule MyD88, a signaling cascade that results in the expression of pro-inflammatory cytokines/chemokines. Objective: To study TLR2, TLR4 and adaptor molecule MyD88 role in sepsis-induced AKI. Methodology: C57BL/6, TLR2 KO, TLR4 KO and MyD88 KO male mice were subjected to sepsis by cecal ligation and puncture (CLP) with two perforations using 23G needle. Blood and kidney tissue samples were collected after 24 h for analysis. Results: After 24 h, TLR2 KO, TLR4 KO e MyD88 KO animals subjected to CLP presented preserved kidney morphology, with lower acute tubule necrosis scores. We observed less areas of hypoxia when compared with WT animals (WT 3.07 ± 1.2; TLR2 KO 1.89 ± 0.9; TLR4 KO 1.76 ± 1.02; MyD88 KO 0.01 ± 0.02) and less areas of apoptosis (WT 0.01 ± 0.003; TLR2 KO 0.006 ± 0.004; TLR4 KO 0.008 ± 0.003; MyD88 KO 0.001 ± 0.0006). TLR2 KO and TLR4 KO animals presented a tendency towards less injury, while MyD88 KO animals were completely protected compared to WT animals (Urea WT 237.57 ± 28.9; MyD88 KO 91.71 ± 4.9) (Serum Cr WT 1.86 ± 0.54; MyD88 KO 0.44 ± 0.02). We also observed a lower expression of proinflammatory cytokine mRNA (IL1-b, TNF-a, IL-6, IL17 e KC), in TLR2 KO, TLR4 KO and MyD88 KO kidneys when compared to WT. We observed less neutrophil infiltration in MyD88 KO kidneys 24 h after sepsis. MyD88 KO animals also presented higher survival scores when compared to WT animals. Conclusion: Here, we concluded that the innate immunity participates in sepsis-induced polymicrobial AKI, mainly through MyD88 pathway. Background: It has been demonstrated that ovarian hormones modulate immune response during inflammatory conditions, acting in production of cytokines, as well as nitric oxide (NO) synthesis. The aim of this study was to evaluate the participation of estradiol in the resistance to infection and also, in nitrate and tumor necrosis factor alpha (TNF-a) production in rats submitted to Enterococcus faecalis infection. Materials and methods: Experiments were performed in female Wistar rats ovariectomized and treated for 3 consecutive days with two doses of estradiol (50 or 100 lg/kg) or vehicle. On the third day, 2 h after the last injection, each rat received an intraperitoneal injection of bacterial suspension of Enterococcus faecalis. Blood was collected by cardiac puncture at 6 and 24 h after bacterial inoculum administration, for blood culture, plasma nitrate and TNF-a measurement. Nitrate was detected by chemiluminescence, while TNF-a were determined by ELISA sandwich kit. Furthermore, the microbiological S160 Inflamm. Res. tests were determined in blood and liver at the same experimental periods. Results: Estradiol at the higher dose was effective in decreasing bacterial dissemination in the liver (p \ 0.05), but not in blood at 6 and 24 h after bacterial challenge. Moreover, estradiol also increased significantly plasma nitrate concentration only at 24 h (p \ 0.001), while TNF was increased at both time evaluated (p \ 0.05). The febrile response was observed 3 h after Enterococcus faecalis administration, but it did not demonstrate statistical difference among the experimental groups. Conclusion: These data suggest that estradiol does not have effect in body temperature of ovariectomized rats submitted to Enterococcus faecalis infection. However, it seems to be effective to oppose the bacterial spread in liver, and also modulate the production of inflammatory mediators, such as NO and TNF-a. Disclosure of interest: None declared. Administration of lipopolysaccharide (LPS) in low levels induces a resistance state called tolerance, with a silencing of inflammatory genes. Endotoxin-tolerant cells and leukocytes from septic patients exhibit alterations of the cellular signaling that can protect against a lethal challenge of LPS. This condition is regulated by several mechanisms, included changes in chromatin structure, an epigenetic process of gene reprogramming. Nitric oxide (NO) participates of the tolerance conditions. It is known that the interaction of NO with cysteines residues, the S-nitrosylation, could alter the structure of enzymes and consequently its functions. We investigated whether the tolerance could modulate the S-nitrosylation of enzymes involved in the epigenetic regulation such as the Histone Deacetylases (HDAC), responsible by a repression in the gene expression, and consequently modulate the production of the proinflammatory mediators. THP-1 human promonocytes were cultivated in RPMI (C group), submitted to tolerance to LPS (T group 500 ng/mL during 24 h) or challenge (D group 1 lg/mL during 24 h). We used tricostatin (TSA 30 nM) to inhibit the HDAC activity, LNAME (100 lM) to inhibit the NO release and SGNO (0.5 mM) as NO donor. The HDAC activity and cytokine levels (IL-6, IL-10 and TNF) were measured with ELISA kits. S-nitrosylation was assessed by biotin switch and the NO levels by Griess Reaction. Tolerance increased the total amount of S-nitrosylated proteins. We found reduced HDAC activity in the T group that was associated to increasing of S-nitrosylated HDAC-2. Endotoxin tolerance cells demonstrate a reduced levels of IL-10 (p \ 0.05) and IL-6 (p \ 0.05). However, the administration of HDAC or NO inhibitors repressed the effects of the tolerance in HDAC activity as well as cytokines production.Our results demonstrate an important role of endotoxin tolerance in the epigenetic regulation and suggest that the S-nitrosylation may have implication for inflammatory repression. Disclosure of interest: None declared. R. S. Saia 1, *, G. Bertozi 2 , F. Q. Cunha 2 , E. C. Cárnio 3 1 Physiology, P harmacology, 3 General and Specialized Nursing, University Of Sa˜o Paulo, Ribeira˜o Preto, Brazil Background: Nitric oxide (NO), tumor necrosis factor-a (TNF-a) and interleukin (IL)-10 play a key role in innate immune system controlling microbial infection. However, during septic shock their exacerbated formation is associated with several deleterious complications, as hypotension. Cholecystokinin (CCK) was firstly described as a gastrointestinal hormone, but immune cells express their receptors, suggesting a possible involvement of this hormone in modulation of inflammatory response. Our aim was to evaluate the role of CCK on NO, TNF-a and IL-10 production during endotoxemia in rats as well as lipopolysaccharide (LPS)-induced hypotension. Materials and methods: Male Wistar rats received an intravenous injection of CCK (0.4 or 40 lg/kg) 10 min before LPS (1.5 mg/kg) administration. The mean arterial pressure and cardiac output were monitored continuously during 6 h after endotoxin injection. Blood was collected for plasma nitrate, TNF-a and IL-10 determinations at 2, 4 and 6 h after LPS. Nitrate was detected by chemiluminescence, while cytokines were determined by ELISA sandwich kits. Results: The LPS induced-hypotension was reverted by the pretreatment with CCK only at the lower dose tested (p \ 0.01). Moreover, CCK reduced plasma nitrate levels at 2 and 6 h after LPS administration (p \ 0.05), but did not alter TNF-a production. The plasma IL-10 concentration was increased (p \ 0.05) by CCK administration at 2 and 4 h after endotoxemia. Conclusion: These data suggest that CCK improves hypotension and reduces NO formation during endotoxemia in rats. Furthermore, CCK regulates positively anti-inflammatory cytokine IL-10 synthesis, however do not modify TNF-a. Disclosure of interest: None declared. Medicine, Infection and Immunity, Background: Early life immune challenge has been considered an adaptive defense strategy against pathogens when the innate immune system is not completely developed. Our aims were to evaluate whether neonatal lipopolysaccharide (LPS) challenge alters body temperature response in adult female rats during endotoxemic shock and whether ovarian hormones may participate in this response. Methods: Female Wistar rats were intraperitoneally injected with LPS (100 lg/kg) (nLPS) or saline (nSal) at post-natal day 14. Then, experiments were performed in adults submitted to endotoxemic shock (10 mg/kg), surgically implanted with biotelemetry probes to body temperature monitoring. Blood were collected to measure tumor necrosis factor (TNF)-a and interleukin (IL)-10 levels at 1 and/or 2 h after LPS administration. Results: The LPS injection in adult nSal rats caused an initial hypothermia, followed by a febrile response. However, nLPS showed an increased hypothermia and an attenuation of fever (p \ 0.05). The bilateral ovariectomy abolished the difference in body temperature between the nLPS and nSal rats. Ovariectomized rats treated with estradiol cypionate (ECP) restored hypothermia and the suppressed febrile response (p \ 0.01). Moreover, the same results were not obtained when the animals were supplemented with ECP and medroxyprogesterone acetate (MPA). The nLPS rats displayed a significant reduction in TNF-a levels and an increase in IL-10 levels when compared to nSal animals (p \ 0.01). The ECP injection significantly enhanced IL-10 and suppressed TNF-a in nLPS (p \ 0.001), but it did not change the inflammatory response in the nSal rats. The ECP + MPA regiment in the nLPS rats reduced TNF-a, but eliminated IL-10 stimulation in comparison to the nSal group. Conclusion: These data suggest that neonatal LPS challenge alters the thermoregulatory response during endotoxemic shock in adulthood and estradiol could mediate this difference. Disclosure of interest: None declared. PKCH is required for the development of experimental cerebral malaria but dispensable for associated lung pathology Cerebral malaria is the most severe neurological complication in children and young adults infected with Plasmodium falciparum. In mice T-cell activation is required for the development of Plasmodium berghei ANKA (PbA)induced experimental cerebral malaria (ECM). In order to characterize the T cell activation pathway involved, we addressed the role of protein kinase C-Theta (PKC-h) in ECM development. Here we report that PKC-h deficient mice are resistant to ECM development. In the absence of PKC-h no neurological sign of CM developed after blood stage PbA infection. Resistance of PKC-h deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA), while wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8+ T cells, and ICAM-1 expression were reduced in the brain of resistant mice while the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-h. PKC-h deficient mice however developed high parasitemia and succumb at 3 weeks with a severe anemia. Therefore, PKC-h signaling is crucial for the development of brain microvascular pathology resulting in fatal ECM but not for pulmonary inflammation and edema. Disclosure of interest: None declared. Some bacterial species present in fermented foods are regarded as probiotics because of their ability to modulate the prevention of some intestinal infections and the modulation of the inflammatory immune response. Spore-forming bacteria such as the genus Bacillus make convenient probiotics as they can survive transit through the stomach intact and in the gut. The present study was carried out for the isolation of bacteriocin-like substance (BLS) producing Bacillus subtilis from a traditional Korean fermented soybean paste which is consumed commonly as a low cost source of high protein food and safe food. All B. subtilis and B. licheniformis isolates demonstrated inhibitory activity against S. aureus using agar spot inhibition assay. Interestingly, one strain B. subtilis (BS1800) displays a wide range of antibacterial activity against S. aureus, E. coli, E. coli O157:H7 (EHEC) and S. typhi, but not K. pneumoniae, P. aeruginosa and S. marcescens. Furthermore, BS1800 completely killed E. coli, EHEC and S. typhi after 3 days coculture in LB broth using viable cell count on MacConkey agar. The bactericidal effect on EHEC and S. typhi was strain specific. BLS of BS1800 was purified using ammonium sulfate precipitation and was shown to have a molecular mass of 8 kDa as determined by SDS-PAGE analysis. We therefore propose strain BS1800 as a suitable candidate probiotics for use in the prevention and treatment of infections caused by EHEC and S. typhi. Disclosure of interest: None declared. Platelets are anucleated cells that play a major role in primary haemostasis but their role in inflammation has now been recognized. Platelets contain and secrete various immunomodulatory factors, which behave as ligands for immune receptors. Platelets express several transcription factors that exert non-genomic functions. We tested for the relevance and the finality of platelets TLR4 pathways. Expression of TLR4 on the platelet surface was determined by flow cytometry analysis. Western blot was performed to detect platelet intracellular protein: MyD88, TBK-1, IRAK-1, JNKs, MAPk, TRAF3, TRAF6, IRF-3, IKK-i, IkB-a, NF-jB p65 subunit or phospho-tyrosine antibody 4G10 and a-tubulin. TLR4 is expressed on human platelets and the percentage of CD41 + TLR4 + platelets increase after TRAP stimulation compared to unstimulated control. Following the addition of E. coli or S. minnesota LPS there was no significant alteration of CD62P expression. There were striking differences in the levels of the sCD40L, sCD62P, PF4, RANTES and PDGF-AB induced by two different LPS stimuli. Western blot analyses using antibodies to MyD88 and TRIF subunits showed that both proteins were consistently present in purified human platelet lysates. We indeed observed that E. coli or S. minnesota LPS platelet stimulation modulated phosphorylation protein profiles by using a phosphotyrosine specific antibody 4G10. Finally, supernatants from short term cultured platelets stimulated with two different LPS stimulate differentially blood mononuclear cells with different patterns of cytokine secretion. These data demonstrate a different panel of these chosen secreted cytokines that may reflect consistent variation in the composition of platelet supernatants secreted after TLR4 engagement. This data question the sentinel role of platelets in their environment, their capacity of sensing differentially even through one given PRR, and their subsequent immunoregulatory function. Disclosure of interest: None declared. Host immune responses to xenografts show similarities to those of fighting infection. In this study we investigated the effect of xenogeneic stimulation and xenoantibody induction on bacterial infection. Lewis rats were immunized with different protocols of hamster blood and assessed how these procedures affected the development of sepsis induced by cecal ligation and puncture (CLP). Rats that received three injections of hamster blood at 2-week intervals (IgG Group) elicited very high titers of anti-hamster IgG and relatively low specific IgM antibodies at day 40 after initiating the immunizations. Day 40 was thus selected to conduct CLP in this rat cohort. Another set of rats received three injections within a week inducing high titers of antihamster IgM and relatively low IgG levels at day 5 after the first injection (IgM Group). They were assigned for CLP at day 5. Multiple hematological, biochemical and general-health parameters were monitored during the study. IgM Group showed a significant increase in blood neutrophils (two, sixfold; p \ 0.05) and monocytes (one, fivefold; p \ 0.05) just prior to CLP, whilst IgG Group did not show changes. A CLP procedure that produced a mild septic state was not associated with any mortality in IgG Group, whereas in IgM Group resulted in higher mortality [8/17 (47%) , p \ 0.05]. In a more severe CLP model IgG Group showed an improvement of rat general-health parameters that did not reduce mortality (68.7 vs. 76.5% controls). We also determined xenoantibody reactivity to bacteria isolated from blood of septic rats. Interestingly, we observed a threefold increase in IgM and IgG reactivity to Enterococcus faecalis using sera from Group IgM rats collected just prior to CLP, but not to Escherichia coli. In conclusion, the generalized immune response induced by a short xenogeneic stimulation had a detrimental effect on the septic process, despite the induction of antibodies that cross-react with some of the microorganisms isolated in the blood. Disclosure of interest: None declared. Hypertonic saline solution (HS solution, NaCl 7.5%) has shown to modulates immune function favorably and decrease organ injury triggered by endotoxic shock. Therefore, our objective was to investigate the effects of HS solution on the mechanism involved in acute respiratory injury, in an experimental model of endotoxemic shock. Wistar rats (220-260 g) received lipopolysaccharide-LPS (10 mg/kg, intraperitoneal, ip) and volume after 15 min. The animals were assigned in four groups (n = 6 per group): control group (not subjected to LPS); LPS group (injected with LPS 10 mg/kg i.p); HS group (injected with LPS and treated with hypertonic saline, 4 mL/ kg) and NS group (injected with LPS and treated with normal saline, 34 mL/kg). At 24 h after treatment, pulmonary mechanics, pulmonary edema, and lung histology to evaluate collagen deposition were measured. Hypertonic solution improved pulmonary resistance, compared to LPS and NS groups. HS presented a reduction of 52 and 65% in the resistance. Collagen deposition showed reduced on HS group compared to LPS and NS groups, 61.5 and 82.2% respectively. There were no differences in pulmonary elastance and pulmonary edema with HS. Therefore, we concluded that treatment of endotoxemic shock with HS solution can decrease pulmonary damage and improve the respiratory function. Disclosure of interest: None declared. Introduction: Alzheimer's disease (AD) physiopathology is recognized as multifactorial and remains unclear. This disease which presents two characteristic lesions, senile plaques and neurofibrillary degeneration is specific to humans. Until now, there is no relevant animal model at our disposal. Recent evidence suggesting that inflammation may be a third important component which may contribute actively to disease progression, we hypothesized that repeated infections during a lifetime facilitate Alzheimer's disease via neuroinflammation and oxidative stress. Aim: The aim of this study was to mimic an infectious state in rats using acute or repeated LPS administration (1 mg/kg; i.p.). Methods: After LPS administration, reactive oxygen species (ROS), nitrites/nitrates (NOx), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were used as inflammatory and oxidative stress markers in different brain regions and/or blood. Moreover cognitive, cholinergic system and memory function have been assessed on animals after peripheral inflammation (LPS). Results: We have shown memory impairment and cholinergic system disorders after LPS treatment. LPS also induced a disruption of the blood-brain-barrier (BBB). We also observed that LPS exposure significantly increased ROS and NO cortex production (26 and 63%, respectively) associated with a significant decrease in blood vitamin E (40% Hypertonic saline solution (HS, NaCl 7.5%) promote immediate blood volume expansion, restore cardiac output, improve microcirculation and modulate immune responses. Therefore, our objective was to investigate the effects of HS solution on the S164 Inflamm. Res. systemic inflammation and heart damage in a rodent model of endotoxemic shock. Wistar rats (220-260 g) received lipopolysaccharide-LPS (10 mg/kg, intraperitoneal, ip) and volume after 15 min. The animals were assigned in four groups (n = 6 per group): control group (not subjected to LPS); LPS group (injected with LPS 10 mg/kg i.p); HS group (injected with LPS and treated with hypertonic saline, 4 mL/kg) and NS group (injected with LPS and treated with normal saline, 34 mL/kg Survival during sepsis requires both swift control of infectious organisms and tight regulation of the associated inflammatory response. The aim of this study was investigate the role of CCL2 and NO in clearance of bacteria after CLP model. For this, mice were subjected to CLP model. After 6 h, the numbers of CFU counts and measured of mediators were determined. To investigate the role of CCL2 in clearance of bacteria, the animals were treated with anti-CCL2 and 6 h after CLP the peritoneal fluid were collected. The numbers of CFU were significant increased in anti-CCL2 group. Peritoneal macrophages treated with CCL2 or anti-CCL2 and stimulated with E. coli presented the significant increased in numbers of CFU after anti-CCL2 treatment and decreased in numbers of bacteria after CCL2 treatment. These results suggest an important role in the elimination of CCL2 in models of bacterial sepsis. To investigate the involvement of NO in more bacterial elimination, we submitted the deficient mice of iNOS (iNOS-/-) to CLP. We observed the significant increased in numbers of CFU in iNOS-/-group. In order to examine the relationship between CCL2 and NO in clearance of bacteria, peritoneal macrophages of deficient mice to CCL2 (CCL2-/-) and WT mice were stimulated with LPS, IFN-g or LPS + IFN-g, and the NO production was determined. We observed the significant decreased in NO levels into CCL2-/-group after all treatments when compared to the control group. In order to investigate the signaling pathway involved in NO production by macrophages treated with CCL2, we analyze the ERK1/2 pathway by immunolocalization technique. After CCL2 treatment we observed the increase in fluorescence of pERK and iNOS. We conclude that the CCL2 and NO are important mediators involved in better elimination of bacteria in models of sepsis. We observed that CCL2 promotes the release of NO by a mechanism associated with ERK1/2 pathway signaling. Introduction: Acute respiratory distress syndrome is a severe complication of acute lung injury (ALI) characterized by hypoxemia, lung neutrophil infiltration and edema. Lipopolysaccharide (LPS) exposure is a major cause of ALI induction. Female sex hormones (FSH) may exert protective or deleterious effects on Th1 and Th2-mediated lung inflammation. We investigated herein the modulatory role of FSH on Th1 lung inflammation induced by LPS notably on cell recruitment. Material and methods: Seven days after ovariectomy (OVx), female mice (C57BL/6, n = 5-8/group) were subjected to intranasal instillation of LPS (Escherichia coli, 100 lg/ml, 1 ll/g) or saline (0.9% NaCl, 1 ll/g). The animals were euthanized 24 h thereafter, and the cells present in bronchoalveolar lavages (BAL), peripheral blood and bone marrow lavages were quantified. Furthermore, lung myeloperoxidase (MPO) activity was also analyzed. Otherwise intact animals were subjected to the same procedures and served as controls (Sham-OVx group). Integrins are adhesion molecules that play a pivotal role during inflammatory process. The most relevant integrins for leukocyte are members of the CD18 subfamily. The leucointegrins subfamily includes CD11a, CD11b, CD11c and CD11d molecules. CD11d integrin is expressed on most human leukocytes, including neutrophils and macrophages. CD11d integrin has been associated with pathological events in some inflammatory diseases, as rheumatoid arthritis. Despite the existing data, the pathophysiological role of this molecule remains unknown. In this study, we aimed to investigate the central role of CD11d integrin in the inflammatory processes in model of Salmonella infection. In this study, we used CD11d Knockout mice (CD11dKO) and wild type (WT) mice infected by an intraperitoneal injection of Salmonella Typhimurium and S. aureus. Our results showed that CD11dKO mice were more susceptible to infection. In CD11dKO mice we observed higher bacteremia, decreased migration of leukocytes to the peritoneum and increased levels of TNF-a, IL-6 and MIP-1a, 24 and 72 h after infection, when compared to the WT. Macrophages seem to be important cells in the onset of the observed responses, which are mediated by specific factors of Gram negative and independent of the CD11b and TLR4 expression. Moreover, our results showed problems in the migratory ability of granulocytes from Cd11dKO mice toward chemotactic stimuli. We did not observe any correlation between the expression of CD11d integrin and nitric oxide production and presence of apoptotic cell death, after infection. However, we have shown correlation between CD11d integrin and IL-1b and IL-18 production. Our results indicate an important role of CD11d molecule in the inflammatory response after Salmonella infection and suggest a different role of CD11d integrin in Gram negative and Gram positive bacterial infections. Moreover, our results suggest for the first time the involvement of CD11d integrin in inflamossoma activation. Disclosure of interest: None declared. Introduction and objectives: IL-18 is a proinflammatory cytokine and its role in paracoccidioidomycosis, a deep mycosis, is not fully understood. The effect of IL-18 on monocyte activity against P. brasiliensis (Pb) was studied by evaluating the fungicidal activity, H 2 O 2 production, the modulation of TLR2, TLR4 and mannose receptor (MR) expression, and IL-18, IL-10, TNF-a, IL-12 and IL-15 production after challenge in vitro with fungus. Methods: Monocytes were stimulated with IL-18 and treated with anti-TLR2, anti-TLR4, anti-CD14 and/or anti-MR before challenge with Pb18 (virulent) and Pb265 (avirulent). Fungal recovery (FR) and H 2 O 2 production were assessed. TLR2, TLR4, MR and CD14 expression were analyzed by flow cytometry and IL-18, IL-10, TNFa, IL-12 and IL-15 production by ELISA. Results: The results showed that IL-18 increased FR from infected monocytes and the blockage of receptors with specific neutralizing antibodies showed that this effect of IL-18 was mediated by MR. The challenge with Pb18 also increased MR, TLR2 and TLR4 expression. No alterations on H 2 O 2 levels were observed. The challenge with Pb18 and Pb265 induced monocytes to produce IL-18, TNF-a, IL-10, IL-12 and IL-15. IL-18 production was associated with MR increase, while TNF-a and IL-10 production was associated with TLR4 and TLR2 respectively. The high IL-12 production was not related with none of receptors studied. Conclusions: Although IL-18 was able to induce high levels of proinflammatory cytokines, the effect of IL-18 on the increase of FR from monocytes would be due to modulation of MR that in addition to facilitate the entry of the fungus in the cell, will activate a new molecular pathway that links engagement of MR with peroxisome proliferator-activated receptor (PPARc) activation, which regulates the macrophage inflammatory response, thereby playing a role in paracoccidioidomycosis pathogenesis. Severe malaria, or cerebral malaria (CM), is responsible for 2 millions deaths each year, and is characterized by fever, cerebral commitment and respiratory distress. Even though pulmonary edema and acute respiratory distress syndrome are described during CM, the inflammatory response in the lungs has not been characterized yet. The aim of this study was to characterize lung inflammation during experimental severe malaria. One day after the infection, P. berghei (5 9 10 6 infected RBC)-infected C57BL/6 mice presented increased lung elastance, transpulmonary pressure and viscoelastic pressure when compared to non-infected mice. Morphometric and histological analysis revealed edema formation and leukocyte accumulation in the interstitial tissue (but not in the alveolus) 5 days after the infection. A significant increase in the numbers of total leukocytes 3 days (mainly due to neutrophils) and 5 days (mainly due to gd and ab T cells) after infection was observed. Lung tissues of P. bergheiinfected mice presented increased levels of the cytokines TNF, IFN-c and IL-6, at day 3 and 5 post-infection. It has been demonstrated that Salmonella are able to infect and survives within B-lymphocytes but there is no information regarding B-lymphocyte ability to produce pro-inflammatory cytokines in response to Salmonella infection via TLR or NLR pathways. S168 Inflamm. Res. Objectives: To analyze the ability of Salmonella infected B lymphocyte to produce pro-inflammatory cytokines by TLR or NLR pathway. Results: TNFa and IL-6 was secreted in response to magnitude of infection: TNFa (pg/mL), non-infected {NI} 0 ± 0, MOI 1/10, 28 ± 2, MOI = 50 84 ± 5, LPS 54 ± 2, CpG, 0 ± 0; IL-6 (pg/mL), NI, 50 ± 6, MOI = 10, 53 ± 8, MOI = 50, 100 ± 11, LPS, 78 ± 9 and CpG, 48 ± 8. RANTES secretion (pg/mL) was not different among different magnitude of infection: NI, 165 ± 0, MOI = 10, 300 ± 10, MOI = 50, 295 ± 4, LPS, 297 ± 14 and CpG, 97 ± 20. In regards of NLR pathway, B lymphocyte were able to secrete IL-1b (pg/mL) in response to Salmonella infection (NI 0 ± 0 and MOI = 10, 88 ± 20). Conclusions: These data show that B-cells are not impaired to produce pro-inflammatory mediators in response to Salmonella infection. These mediators are produced by TLR pathway (TNFa and IL-6) as well as by NLR pathway (IL-1b), however in contrast to macrophages B-cells are able to resist the cytotoxic effect of these cytokines. In severe sepsis neutrophils present a state of unresponsiveness to chemotactic stimuli. This correlates with a poor prognosis and may contribute to worsen the clinical outcome in sepsis by precluding an efficient bacterial clearance. Experimental data have implicated nitric oxide (NO) in this phenomena. However NO synthesis inhibition in sepsis leads to increased mortality despite the restored neutrophil migratory function. This could be at least in part due to an inhibition of the microbicidal effects of NO, suggesting that a better understanding of the roles of NO in sepsis is required. Here we investigated the molecular mechanisms triggered by NO that lead to decreased neutrophil chemotactic responses in sepsis. Our results show that stimulation of human neutrophils with lipopolysaccharide, a Toll-like receptor (TLR) 4 ligand, as well as lipotheichoic acid, a TLR2 ligand, decreased in vitro neutrophil responsiveness to classic chemoattractants as interleukin-8, leukotriene B 4 and fMLP. These effects involved the activation of the NO/guanylate cyclase (GC)/protein kinase G (PKG) signaling pathway. Further we found that NO-GC-PKG signaling induces both CXCR2 and BLT1 internalization which was paralleled by increased expression of the GPCR kinase, GRK-2. Based on these findings we reasoned that GC inhibition could restore neutrophil migration without impairing NO microbicidal activity. In fact, we found that pharmacological inhibition of GC activity in an experimental model of sepsis resulted in increased survival, restored neutrophil migration and increased bacterial clearance, while inhibition of NO synthesis rendered mice susceptible to infection and increased bacterial counts. Finally we show that GC inhibition increased survival even after the onset of sepsis, suggesting its potential clinical application. Together our results suggest a mechanism by which NO interferes with neutrophil chemotaxis and point to GC as a potential therapeutical target in sepsis. Disclosure of interest: None declared. Pseudomonas aeruginosa (P.a.) induces lung injury in pulmonary diseases including cystic fibrosis (C.F.). P.a. is known to activate the host cytosolic phospholipase A2a (cPLA2a), which catalyzes the release of arachidonic acid (AA) and its metabolite prostaglandin E2 (PGE2). However, the mechanisms and role of cPLA2a in P.a.induced lung injury have not been investigated. Here, we investigated lung inflammation and mortality in mouse models of pulmonary infection by a laboratory (PAK) and a clinical strain (CHA) of P.a. CHA revealed to be more potent than PAK in inducing lung inflammation and animal mortality. cPLA2a-null mutation attenuated animal mortality caused by both PAK and CHA but failed to reduce lung inflammation or bacterial proliferation. CHA infection led to increased mortality in CF compared to non-CF mice. Pharmacological inhibition of cPLA2a attenuated CHA-induced CF mice mortality without interfering with lung inflammation. CHA triggered AA and PGE2 release and cPLA2a phosphorylation in lung epithelial cell line A549 at higher levels than PAK. This was accompanied by increased p38 MAPK phosphorylation. Inhibition of cPLA2a and p38 attenuated both PAK-and CHA-induced AA and PGE2 release by A549 cells. However, inhibition of cPLA2a had partial effect on IL-8 secretion. CHA caused LDH release by A549 cells at higher extent than PAK both of which were reduced by cPLA2a inhibitors. Together these finding suggest that cPLA2a plays a role in P.a.-induced lethality in pulmonary infection models including CF. This effect seems to occur via a process related to cPLA2a-induced toxicity independently from lung inflammation and bacterial proliferation. Disclosure of interest: None declared. Endothelins (ETs) are pro-inflammatory peptides involved in pain, fever, edema and cell migration. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by joint inflammation, as well as local and systemic increased ET-1 levels. The present study demonstrates the effect of ET antagonism in articular inflammation on zymosan-and collagen-induced arthritis and also show the modulation of ET-1 levels by lipoxin (LX)A 4 , an endogenous anti-inflammatory mediator. Two hours after induction of zymosan (500 mg/cav, i.a)-induced arthritis, ET-1 mRNA expression levels were increased in synovial extracts, which was followed, at 6 and 24 h, by massive edema formation and neutrophil influx into inflamed joint. Pre-treatment with bosentan (10 mg/kg; i.v.), a dual pharmacological ET receptor antagonist, significantly impaired 6 h zymosan-induced leukocyte accumulation and edema formation. ET receptor blockade also decreased zymosan-induced production of TNF-a, CXCL1 and leukotriene (LT) B 4 within 6-24 h. Bosentan (10 mg/kg; i.p.) post-treatment effectively attenuated clinical score of established collagen-induced arthritis in mice and caused a significant decrease on paw edema. LXA 4 in vivo pre-treatment (20 ng/cav, i.a.) significantly impaired zymosan-induced preproET-1 mRNA expression (80% of inhibition) with concomitant suppression of zymosaninduced edema formation and neutrophil influx. In addition, in vitro pre-treatment of neutrophils with LXA 4 (1-100 nM) inhibited ET-1(100 nM)-induced activation and chemotaxis, evaluated by shape change and boyden chamber assay, respectively. Our results suggest that pharmacological blockade of ET receptors, or the modulation of ET-1 expression, may serve as a possible novel therapeutic tool for the treatment of inflammatory and autoimmune articular diseases. Disclosure of interest: None declared. S170 Inflamm. Res. Background: Lymphocytes immunephenotyping is a reflection of the functional level of immune system. It was found that serum soluble b2microglobulin (b2 M) may modulate the function of T lymphocytes. Our aim in this work is to determine the activation state of T cells after major burn by detection of CD25 as an early activating marker of T-lymphocytes and HLA-Dr expression as a late one and their relation to b2-microglobulin. Methods: T lymphocytes of 26 major burn patients complicated with infection were analyzed in 24 h, 1 and 2 weeks after burn injury, using monoclonal antibodies of CD3, CD4, CD8, CD25 and HLA-Dr by flowcytometry and comparing them with those of 26 apparently healthy donors. Serum level of b2 M was also tested using ELISA technique. Results: There was a statistically significant reduction in CD3 + T lymphocytes (p \ 0.000), CD4/CD8 ratio (p = 0.01) in the first 24 h compared to controls. The CD3 + cells increase after 2 weeks (p = 0.03). HLA-Dr was markedly increased after 2 weeks (p = 0.001), while absolute number of CD25 + T lymphocytes was lower than controls. The b2 M of patients samples of the study were significantly higher than that of the controls (P values: .000, .001 and .000 respectively). The Nlrc4 inflammasome is triggered in response to contamination of the host cell cytoplasm with bacterial flagellin, which induces pyroptosis, a form of cell death that accounts for restriction of bacterial infections. Although induction of pyroptosis has been extensively investigated in response to Salmonella typhimurium and Legionella pneumophila, little is known regarding the role of the inflammasome for restriction of nonpneumophila Legionella species. Here, we used five species of the Legionella genus to investigate the importance of the inflammasome for restriction of bacterial infection in vivo. By infecting mice deficient for inflammasome components, we demonstrated that caspase-1 and Nlrc4 contribute to restriction of pulmonary infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. Legionella longbeachae, a non-flagellated bacterium that fails to trigger pyroptosis, was not restricted by the inflammasome and induced death in the infected mice. In contrast to L. longbeachae, flagellin mutants of L. pneumophila did not induce mice death; therefore, besides bypassing the Nlrc4 inflammasome, L. longbeachae may employ additional virulence strategies to replicate in mammalian hosts. Collectively, our data indicate that the Nlrc4 inflammasome plays an important role in host protection against opportunistic pathogenic bacteria that express flagellin. Disclosure of interest: None declared. Classical (M1) and alternative (M2) polarization of mononuclear cells such as monocyte and macrophages, is known to occur in response to challenges within a micro-environment, like the encounter of a pathogen. Lipopolysaccharide, also known as endotoxin (LPS), is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in mononuclear cells known as endotoxin tolerance, defined as the reduced capacity of a cell to respond to LPS activation following an initial exposure to this stimulus. Using systems biology approaches in peripheral blood mononuclear cells and monocyte derived macrophages involving microarrays and advanced bioinformatic analysis, we determined that gene responses during endotoxin tolerance were similar to those found during alternative polarization, featuring gene and protein expression critical for the development of key M2 mononuclear cell functions, including reduced production of pro-inflammatory mediators, expression of genes involved in phagocytosis as well as tissue remodeling. Moreover, expression of different metallothionein gene isoforms, known for their role in the control of oxidative stress and in immunomodulation were also found to be consistently up-regulated during endotoxin tolerance. These results demonstrate that after an initial inflammatory stimulus, mononuclear cells undergo an alternative polarization probably to control hyper-inflammation and heal the affected tissue. Disclosure of interest: None declared. A major immunomodulatory molecule is soluble CD40L (sCD154) and activated platelets are estimated to produce 95% of the plasma sCD154 pool. It has been recently shown in mice that HIV-1 Tat induced activation of platelets resulting in sCD154 release. In this work we investigated platelet activation state and their ability to release sCD154 upon stimulation in HIV-1 infected patients under HAART. We noted that membrane expression of the platelet activation marker CD62P did not differ significantly between HIV-1 infected-patients and healthy donors (respectively 14.1 ± 9.2 and 10.9 ± 2.7%) suggesting that platelets were not activated in HIV patients. However, we evidenced a significant difference in plasma soluble CD62P (sCD62P) levels between HIV-1 patients (82.7 ± 27 ng/ml) and healthy donors (43.9 ± 20.4 ng/ml) that could result from CD62P shedding from the membrane of activated platelets. Then we examined sCD154 plasma levels and we noted that they were significantly higher in HIV-1 patients compared to healthy donors (p \ 0.01), i.e. 2.4 ± 1 and 1 ± 0.9 ng/ml, respectively. Next, we tested whether thrombin was able to induce the release of sCD154 by platelets from HIV patients. We observed that platelet isolated from HIV patients respond as well as platelets from healthy donors to thrombin stimulation by releasing comparable amounts of sCD154 (respectively 2.6 ± 1.5 and 2.3 ± 1.3 pg/ml for 3 9 10 8 platelet) whereas only healthy donor platelets could release a significant amount of sCD62P upon stimulation (55.4 ± 24 vs. 25.6 ± 9.9 ng/ml for 3 9 10 8 platelet for HIV patients). This suggests that while activated platelets of HIV patients are not able anymore to release sCD62P, they remain able to release sCD154 and could therefore boost an inflammatory response in case of treatment failure. Disclosure of interest: None declared. The mitogen-activated protein (MAP) kinase phosphatase (MKP) family of proteins plays an important role in regulating inflammation by inactivating MAP kinases. MKP-1/DUSP-1 is a nuclear, dual specificity phosphatase known to dephosphorylate MAP kinases. We are interested in TNF-induced systemic inflammation, because we believe that a tight control of this inflammation might increase the therapeutic value of TNF in cancer therapy. Since glucocorticoids protect very well against TNF-induced inflammation, and since MKP-1 is strongly induced by glucocorticoids, we hypothesize that MKP-1 can also regulate TNF-induced inflammation. In general, our results demonstrate that MKP-1% mice are more sensitive to TNF-induced inflammation in the liver, and show high levels of apoptosis in intestinal cells. These results can be mechanistically explained by a prolonged JNK activation and consequently stronger induction of AP-1 dependent genes and induction of apoptosis. To go deeper into the mechanism behind this sensitivity, the sensitivity of JNK1% and JNK2% mice to the in vivo effects of TNF was studied. In contrast to JNK1%, JNK2% mice are protected against TNF-induced lethal shock. Furthermore, the sensitivity of MKP-1% mice could be reversed by knocking-down JNK2, since double knock-out MKP-1/ JNK2% mice showed enhanced resistance to TNF. Disclosure of interest: None declared. Results: Supplementing culture media with murine BDL plasma reduced LPS-induced IL-1/6/10 & TNF-a production, which was completely reversed by Indomethacin, with no effect of naive plasma (p \ 0.01). Lipidomics revealed rises in PGF2a/E2. Human macrophage LPS-induced TNF-a secretion was significantly increased by supplementation of media with healthy volunteers' plasma (n = 8) compared to culture media alone (p \ 0.001). In contrast TNF-a secretion from macrophages co-incubated with cirrhosis plasma showed a bimodal distribution with profound immune suppression observed with 4/10 of the cirrhosis samples. However, the mechanism of action of DC on the exacerbation of lung inflammation induced by particulate matter is unclear. Objectives: We evaluated in vivo the role of residual oil fly ash (ROFA) in the exacerbation of inflammation in a murine model of asthma and in in vitro assay the effect of ROFA in the maturation of BMDC in response to allergen (OVA); and using a cell transfer assay the capacity of ROFA-pulsed BMDC to modulate the allergic inflammation. Methods and Results: In vivo: We evaluated in lung, lymph nodes and spleen of the OVA-sensitized BALB/c mice challenged with OVA and ROFA the subsets of DCs, the number of Treg, IFN-g + and IL-5 + T lymphocytes and the expression of Ly6C and CD40 in CD11b + F4/ 80 + macrophages. ROFA exposition exacerbated lung inflammation induced by OVA, increasing eosinophilia and goblet cells as well a decreasing the number of Treg, IFN-g + and IL-5 + T lymphocytes in lung tissue. In spleen, ROFA maintains elevated the number of mDC, IFN-g + and IL-5 + T lymphocytes and activated macrophages. Using an adoptive transfer model, we also showed that ROFA-pulsed Objective: Neonatal mice exposure to ambient pollutant 1,2-naphthoquinone (1,2-NQ) enhances animal susceptibility to inflammation at adulthood. Since Toll-like receptors (TLRs) in mammals function as crucial sentinels of infectious and non-infectious antigens throughout the respiratory tract, this study was undertaken to examine TLRs as a potential signaling pathway of this response. Methods: Under approval of USP Animal Ethics Committee, TLR4 -/and MyD88 -/knockout (KO) neonate mice and their wild type (WT) were exposed three times via nebulization to 1,2-NQ or its vehicle. Eight weeks later, animals were sensitized and challenged with ovalbumin (OVA). Bronchial hyperresponsiveness (BHR) was assessed and samples collected 24 h after the last challenge. Eosinophils in bronchoalveolar lavage (BAL) and bone marrow (BM), cytokines in BAL, IgE in serum and mRNA expression of TLRs and adaptors molecules in lung were measured. Results are mean ± SEM for n = 6 animals. Stats were by ANOVA plus Bonferroni's test. Results: Increased number of eosinophils was seen in BAL and BM of WT mice exposed to 1,2-NQ and OVA compared to allergic WT mice exposed to vehicle. The lung mRNA expression of TLR2, TLR4 and TLR7 or TRIF molecules was higher in WT mice exposed to 1,2-NQ, with or without OVA, compared to WT mice exposed to vehicle. TLR4 -/and MyD88 -/-KO mice exposed to 1,2-NQ and OVA exhibited a significant decrease in BHR, cytokines concentration (but not IgE) and eosinophils counts in BAL (2.5 ± 0.5 and 1 ± 0.1 9 10 4 cells/BAL) compared to TLR4 -/and MyD88 -/-KO mice exposed to vehicle and OVA (39 ± 8 and 9 ± 1 9 10 4 cells/BAL, respectively). Conclusion: TLRs may serve as a signal transduction pathway increasing susceptibility to allergic response in mice exposed to ambient pollutant during neonatal period. This might be one mechanism that contributes to common pulmonary illnesses in children living in heavy traffic areas. Acknowledgments: FAPESP, CAPES and CNPq for financial support. Disclosure of interest: None declared. S174 Inflamm. Res. Due to their unique physico-chemical characteristics, nanoparticles (NPs) are now one of the leading technologies. As a non-metal oxide, silica (SiO 2 ) NPs have found extensive applications in industry and biomedicine, hence leading to possible occupational, medical and/or environmental exposures. Aiming at evaluating their toxicological impact, we asked the question of SiO 2 NPs acting as immune adjuvants. Experiments were carried out on dendritic cells (DCs) which immunological role is to initiate an adaptative immune response when antigens and ''danger signals'' are concomitantly present within their surrounding environment. Under these conditions, DCs undergo a maturation process leading to phenotypical modifications. We therefore investigated whether SiO 2 NPs may alter this process. Primary cultures of both human monocyte derived DCs (MoDCs) and murine bone-marrow DCs (BMDCs) were exposed to 100 nm SiO 2 particles. Particle size and zeta potential were characterized using photon correlation spectroscopy (PCS) and zetametry, respectively. After 24 h, NPs internalization, cell viability and markers of cell maturation were studied. Using fluorescent NPs, microscopic observations revealed that NPs were found within the DCs after 24 h of exposure. Cytotoxicity evaluation indicated that both DCs types showed about 20% cell death after 24 h of exposure to 100 lg/mL SiO 2 NPs. Phenotypic analyses, carried out at this subtoxic concentration, showed that both MoDCs and BMDCs undergo a maturation process after SiO 2 NPs exposure as evidenced by significant upregulation of maturation markers at their surface (CD40, CD86, and CCR7 or CD83) as well as by a significant release of cytokines (IL-6, IL-8, IL-10, TNF, CCL5, CXCL10). Our results suggest that SiO 2 NPs may have an impact on the immune system function through the maturation of MoDCs and BMDCs. Further experiments will be dedicated to the understanding of signaling pathways involved in such a maturation process. Disclosure of interest: None declared. Objective: Whereas prior to puberty cases of asthma is higher in boys than girls, post puberty there are similar numbers in both genders. Moreover, it is known that children living close to high density traffic areas have enhanced susceptibility to respiratory diseases. The purpose of this study was to evaluate whether early-lifetime exposure to 1,2-naphthoquinone (1,2-NQ), a diesel exhaust contaminant, induces differential lung inflammation in juvenile male and female mice. Methods: Under approval of USP Animal Ethics Committee, neonate mice were exposed three times via nebulization to 1,2-NQ or its vehicle. Sixteen days later, animals were sensitized and challenged with ovalbumin (OVA). Bronchial hyperresponsiveness (BHR) was assessed and samples collected 24 h after the last challenge. Leukocytes in bronchoalveolar lavage (BAL) and bone marrow (BM) or cytokines in BAL were measured. Results are mean ± SEM for n animals. Stats were by ANOVA plus Bonferroni's test. Results: The OVA response in prior exposed 1,2-NQ male mice induced a significant increase of leukocytes, characterized by eosinophils and neutrophils, in BAL (71 ± 18 9 10 4 cells/BAL) and BM (38 ± 3 9 10 6 cells/BM) compared to BAL (19 ± 4 9 10 4 cells/ BAL) and BM (14 ± 2 9 10 6 cells/BM) of female groups. In the BAL male group, the Th1/Th2 cytokine concentration was significantly higher compared to the female group, but the BHR was similar in both groups prior to exposure to 1,2-NQ. Conclusion: Our results show, for the first time, that upon neonatal exposure of male and female mice to 1,2-NQ pollutant, the allergic response to OVA during puberty was still greater in male mice compared to female, thus suggesting that males are at greater risk than females. This may be related to increased differences of cytokine concentration in males compared to females. Acknowledgments: Fapesp, CNPq and CAPES for financial support. We thank IMG for the technical assistance. Disclosure of interest: None declared. Carbon nanotubes (CNT) are promising engineered nanomaterials. However, nanosized particles are likely to exhibit pro-inflammatory activity and increase immune response to allergens. Thus, in the present study, we evaluated the effects of CNT on the secretion of cytokines by peripheral blood mononuclear cells (PBMC) from healthy subjects or mite-allergic patients. Freshly prepared PBMC, stimulated or not with innate [Toll-like receptor (TLR) 1-9 agonists], polyclonal T-cell (phytohemagglutinine A, PHA) or adaptative (mite allergen extract) stimulus were cultured for 48, 72 h or 5 days, respectively, in the presence or absence of multi-walled CNT (0-100 lg/mL). The cytokine secretion was quantified in the culture supernatants by ELISA. Basal secretion of all the cytokines assessed (TNF-a, IL-2, IL-5, IL-6, IL-12/23p40 or IFN-c) was not altered by CNT in PBMC from both healthy subjects or allergic patients. In PBMC from healthy subjects, secretion of TNF-a, IL-6 and IL-12/ 23p40 evoked by the TLR-4 agonist, lipopolysaccharide (LPS), was however increased in a dose-dependent manner by the nanomaterials. Significant increases in the release of these cytokines were also observed in PBMC from healthy subjects stimulated with a TLR-2 or TLR-3 agonist, suggesting a pro-inflammatory activity of CNT. In PBMC from mite-allergic patients, CNT inhibited allergen-induced IL-5 production, although they increased TNF-a secretion evoked by LPS. However, IL-2 and IFN-c secretion induced by PHA in PBMC from both healthy and allergic subjects was increased by the presence of CNT. These data demonstrate that multi-walled CNT interfere differently with innate and adaptative immune responses in human PBMC and suggest differences in sensitivity to multi-walled CNT among cells of the immune system. Disclosure of interest: None declared. Introduction: Hydroquinone (HQ) is an environmental contaminant, present in the cigarette smoke and an important benzene metabolite, which cause detrimental effects on immune cells. The toxicity of in vivo HQ exposure on neutrophil mobilization into the E. coli lipopolysaccharide (LPS)-inflamed lung was investigated. Methods: Male Swiss mice were exposed to aerosolized vehicle (control) or 12.5, 25 or 50 ppm HQ (1 h/day/5 days). One hour after last exposures, animals inhaled LPS (0.1 mg/ml/10 min). Three hours later, number of circulating leukocytes and in the bronchoalveolar lavage fluid (BALF) were quantified using Neubauer chamber and stained smears; adhesion molecules on circulating neutrophil and lung microvessels endothelial cells expressions were quantified by flow cytometry and immunohistochemistry, respectively; myeloperoxidase activity was measured in the pulmonary tissue by colorimetry; cytokines in the BALF were determined by enzyme immunoassay; neutrophil oxidative burst was quantified by flow cytometer; plasma malonaldehyde (MDA) levels were measured by high performance liquid chromatography. Results: In vivo HQ exposure increased plasma MDA levels and oxidative burst in circulating neutrophils, and reduced LPS-induced neutrophil migration into the BALF, as these cells were maintained in the pulmonary tissue. The reduced number of cells in the BALF was not dependent on alterations on circulating leukocytes number, cytokines secretions in the BALF and lung endothelial adhesion molecules expressions, but elevated b2 and b3 integrins and plateletendothelial cell adhesion molecule-1 (PECAM-1) expressions in circulating neutrophils, which were not further enhanced by fMLP in vitro stimulation. Conclusions: Low levels of HQ exposures affect circulating neutrophils, which may contribute to the impaired host defense during intoxications. Aims: To assess the effect of smoking and the HLA-DR shared epitope (SE) copy number on aryl hydrocarbon receptor (AHR) activation and the expression of inflammation-related genes in rheumatoid synovium. To consider the effects of the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP), on MMP9 gene expression in human U937 cells. Methods: Synovial membrane tissues were obtained at joint replacement surgery from patients fulfilling ACR criteria for RA. Patient smoking status was established retrospectively. Patients were typed for HLA-DR SE allotypes using sequence specific-primer-PCR. AHR, CYP1A1, AHRR, IL17A, CXCL12 and MMP9 gene expression in synovial tissue was assessed using qRT-PCR. U937 cells were treated with phorbol 12-myristate 13-acetate (PMA) and/or BaP. Western blotting was used to assess AHR knockdown by AHR-targeted siRNA. Statistics utilised paired or the multirange Dunnett's t tests and variance of ANOVA regression analysis. Results: Smoking was responsible for AHR activation in synovia ''reported'' by significantly increased expression of both CYP1A1 (P \ 0.001) and AHRR (P \ 0.01) genes. Although not significant, there was a trend towards more IL17A expression in synovia from non-smoking RA patients (P = 0.06) with double copies of the SE (P = 0.08). Synovial MMP9 expression was affected by both smoking (P = 0.02) and SE copy number (P = 0.007). In vitro, PMA-activated U937 cells responded to BaP treatment with increased expression of AHR and MMP9 genes. AHR knock-down through RNA interference reduced MMP9 expression consistent with a role for AHR in transcriptional regulation of this gene. Conclusions: Smoking impacts directly on joint synovial tissue in RA. MMP9 is the first identified gene that is influenced by the combined effect of smoking and the SE copy number in RA. PAHs, such as BaP have a role in regulating MMP9 gene expression in U937 cells. The data suggest reciprocal and synergistic roles for smoking and the SE in rheumatoid inflammation. Disclosure of interest: None declared. With the increased use of carbon nanotubes (CNTs), public concern about their potential risk to human health has also risen. Although recent studies showed the in vitro and in vivo pro-inflammatory potential of CNTs, the inflammatory signaling pathways triggered by these particles in human monocytes remain unknown. Here, we showed that CNTsfree LPS did not induce synthesis and secretion of TNFa, IL-1b and IL-6 pro-inflammatory cytokines by human monocytes from healthy donors. However, CNTs increased IL-1b secretion in a dose dependent manner when monocytes were primed with LPS Toll-like receptors ligands. Moreover, we demonstrated that CNTs-increased IL-1b secretion was specifically caspase-1 dependent. Furthermore, by a siRNA approach targeting Nrlp3 receptor in human monocytes, we highlighted a decrease of IL-1b secretion in response to CNTs. Altogether these data demonstrate for the first time the involvement of the Nlrp3 inflammasome pathway in the pro-inflammatory effect of CNTs. Then, we explored the signaling pathway leading to the Nlrp3 inflammasome activation in response to CNTs. We showed that CNTsinduced IL-1b secretion was dependent on potassium efflux. Moreover, using pharmacological approaches, we demonstrated that the increase of IL-1b secretion after stimulation with CNTs was linked to their phagocytosis associated to cathpesin-B protease activation and phagosomal vacuolar acidification. These findings provide real evidence that CNTs trigger inflammasome signaling pathway and hence their use could present a potent health risk. Disclosure of interest: None declared. Species of the red algae genus Laurencia elaborate an astonishing variety of structurally unusual secondary metabolites frequently presenting antiinflammatory properties. From Laurencia grandulifera, we have isolated and characterised, by means of spectral data analysesneorogiotriol, a new tricyclic brominated diterpene. The aim of this study was to evaluate the anti-inflammatory properties of this compound. Methods: The anti-inflammatory effect of Neorogiotriol was evaluated both in vivo using carrageenan-injected rats in the paw and in vitro on lipopolysaccharide (LPS)-treated RAW 264.7 macrophages stably transfected or not with the NF-kappaB-dependent luciferase reporter. Results: The in vivo study demonstrated that intra-peritoneal injection of neorogiotriol (1 mg/kg) resulted in the significant reduction of carregeenan-induced rat oedema in the paw. In vitro, our results showed that neorogiotriol treatment decreased the luciferase activity in LPSstimulated RAW264.7 cells independently of MAPK pathways. Neorogiotrioltreatment significantly decreased LPS-induced tumor necrosis factor-alpha (TNF-a) secretion in RAW 264.7 cells. Further study indicated that this compound also significantly inhibited the release of nitric oxide (NO) and the expression of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 cells. Conclusions: The results demonstrated that neorogiotriol could play a significant role in the inhibition of inflammatory processes. Disclosure of interest: None declared. Short-chain fatty acids (SCFAs) are produced by bacterial fermentation of dietary fiber and bind to GPR43 modulating inflammatory responses. Gout is an inflammatory disease characterized by release of uric acid crystals into the joint cavity and neutrophil infiltration that leads to tissue damage. Our aims are understand the mechanism of SCFAs as well as, the role of GPR43 in gout. Treatment with SCFA protected mice from injury by intra-articular injection of Monossodium Uric Acid crystal showing reduction of pro-inflammatory cytokines and increasing anti-inflammatory mediators, such as, IL-10 and TGFbeta inducing neutrophils's apoptosis. Interestingly, we observed that GPR43ko mice presented reduction in inflammatory parameters after gout induction due deficiency on recruitment of cells, when compared to MSU-injected WT mice. Germ-free mice, which no express SCFAs, showed similar ''hiporresponsive'' and inhibited recruitment of cells. However if we treated GF with acetate before challenged with MSU, this ''phenotype'' was reversed. Thus, microbiotainduced SCFA and GPR43 activation are necessary for proper neutrophil recruitment. However, SCFA treatment during an established inflammatory response promoted resolution of the inflammation. We suggest that endogenous microbiota shapes the host's ability to respond to inflammatory stimuli. The presence of SCFAs provides a link between diet, gastrointestinal bacterial metabolism and inflammation. Disclosure of interest: None declared. In the interaction culture and nature is remarkable use of wildlife for medicinal purposes in different human societies. The oil extracted from subcutaneous fat of capybaras (Hydrochaeris hydrochaeris) has certain properties that help in curing respiratory diseases, arthritis, hypercholesterolemia, and also acts as a powerful healing for external wound (although not proven in clinical and experimental research). The aim of this work is assess cutaneous wound healing in Swiss mice with the topical application of capybara oil. Forty-two Swiss mice, male, adults were allocated into two groups (N = 42): capybara oil group (N = 21, divided into three subgroups with N = 7) oil treated; and control group (N = 21, divided into three subgroups with N = 7) who received no treatment. The daily application of oil was about standardized circular wound of 0.6 mm diameter on the dorsum of each animal. At days proposed (7, 14 e 21 days after injury), mice were deeply anesthetized with Thiopental; were then fixed to the surgical table to collect morphological data and surgical specimens. Wound excised with 1 cm margin of healthy skin around the lesion, deep to the fascia muscle, were fixed and prepared accordingly the histological routine to be analyzed on light microscopy. Statistical analyses were realized (ANOVA test, p \ 0.0001). In Swiss mice, 7 days after wounding, the capybara oil group presented a thicker neoepidermis than control group. In all groups mast cells were present, but the capybara oil group showed a higher amount of mast cells than the control group in 7 days after wounding. Wound contraction was evaluated showed a progressive reduction in wound area during the experiment, but 7 days after injury, the wound area was less in the capybara oil group. The capybara oil seems to exert a beneficial effect on the cutaneous wound healing. Disclosure of interest: None declared. Evaluation of the respiratory burst induced by receptors FccR (for the Fc portion of IgG) and CR (for complement) was carried out in rheumatoid arthritis (RA) patients presenting the active disease or not. Simultaneously, the cooperation between these receptors and their expression, the chemotaxis of peripheral blood neutrophils, and the systemic activity of the complement system were also investigated. Neutrophils were stimulated with IC-IgG (immune complex of IgG) opsonized with normal human serum (NHS) or not, or with IC-IgG opsonized with RA human serum (RAHS). ROS (reactive oxygen species) production by RA neutrophils of patients with active or inactive disease responded to IC-IgG opsonized with NHS. However, these RA neutrophils presented poor FccR/CR cooperation, as indicated by decreased ROS production upon stimulation with IC-IgG opsonized with RASH. Neutrophils of active RA patients presented significantly augmented CR1 and CR3 expression, as well as slight elevation in FccRII and FccRIII expression. Only patients with active disease exhibited positive correlations between FccR and CR. The complement alternative pathway was activated and the chemotaxic activity of RA serum was increased only in the case of patients with active disease. Taken together, these results indicate that several abnormalities of the complement system exist at the systemic level, as reflected by the neutrophil function, particularly in RA patients with active disease. Namely, these abnormalities are impaired membrane receptor cooperation, activation of the alternative pathway, and presence of pre-existing chemoattractant factors in the serum, which together may contribute to RA pathogenesis. Disclosure of interest: None declared. Collagen induced arthritis (CIA) rodent models have been extensively used in rheumatoid arthritis (RA) research. An RA model in non-human primate (NHP) is particularly demanded because of the close phylogenesis that provides the cross-reactivity to human for different development compounds using most modern drug technologies. However, NHP RA model has been reported extremely difficult because of the low and inconsistent disease incidence. We studied type II collagen induced arthritis in Cynomolgus monkeys. Following immunization with collagen, the disease progression was monitored for 8 weeks. Overall the arthritic incidence reached 87% and the average arthritic incidence of proximal interphalangeal (PIP) joint reached near 90%, significantly higher than what was previously reported. The average swelling of PIP joint increased approximately by 45%. Radiography, histopathology and histomorphometry analysis of the joint bones well supported the arthritic disease with the similar characteristics of human RA joints. The average arthritic score was significantly reduced with the single agent treatment of methotrexate or dexamethasone. Our results demonstrated the successful establishment of an reliable CIA in Cynomolgus monkeys, providing a valuable tool for studies of RA disease in pathogenesis, biomarker, translational research, and most importantly, anti-arthritic therapeutics as well as other relevant diseases, such as anemia of chronic disease and arthritic pain. Disclosure of interest: None declared. Inflamm. Res. angiogenesis; however, its role in tumor immunity of host remains unclear. In this study, we investigated effect of MIF on tumor progression and generation of tumor regulatory T cells (Treg). The results showed that tumor growth rate was significantly lower in MIF knockout (KO) mice than in wild type (WT) mice when syngenic colon cancer cells (CT26 cells) were injected. The infiltration of CD4 (+) and CD8 (+) T cells in tumor mass was significantly higher in MIF KO mice than in WT mice. Flow cytometric analysis of both spleen and tumor cells revealed that MIF KO mice had significant lower levels of tumorassociated Treg than WT mice. In parallel, the expression of GITR and CTLA-4 was also reduced in MIF KO mice. Interestingly, there were three cases of tumor rejection among twenty MIF KO mice, but not in WT mice. The number of CD4 (+) and CD8 (+) T cells in these mice with rejection was further increased as comparable to tumor-naïve mice. Moreover, the frequency of Treg was much lower in spleen of tumor-rejected mice than in those of tumor-bearing MIF KO mice. In contrast, when induced by anti-CD3 + IL-2 + TGF-b, the number of the Treg in the spleen was significantly higher in MIF KO mice than in WT mice, suggesting that these stimuli may be associated with the decreased Treg in MIF KO mice. As expected, MIF KO mice showed significant lower levels of IL-2 production by spleen cells stimulated with anti-CD3 than WT mice. In the mean time, both IFNg production by spleen cells and the frequency of CD8 (+) and IFNg (+) cells were not different between the two groups. Taken together, our data suggest that MIF promotes tumor growth in mice by increasing tumor Treg through the regulation of IL-2 production. Thus, anti-MIF treatment might be useful to enhance adaptive immunity of host to colon cancers. Disclosure of interest: None declared. Introduction: The rising awareness of the importance of long-lived antibody-secreting cells (ASC) for the maintenance of protective and autoimmune humoral memory generates research efforts to gain more insight into the biology, migratory behavior and mainly survival mechanisms. We previously demonstrated that Thalassophryne nattereri fish venom induces a humoral memory immune response in mice with the differentiation of ASC B220 neg dependent on IL-5 and IL-17A secreted by T cells. However, how and when CD4+ T cells participate of the generation of ASC still not understood. Objective: Evaluate the importance of effector/memory CD4+ T cells in the maintenance of ASC. Methods and Results: To investigate the phenotypic features of effector/ memory T cells, BALB/c mice were immunized three times with 10 mg of venom in alum and killed at days 15, 49 and 121. Our results show an early formation of effector CD4 + T cells in the peritoneum and bonemarrow (BM) and memory CD4 + CD44 + T cells expressing both CD40L and Ly6C in spleen and peritoneum and expressing Ly6C only in BM. In the memory phase, the effector CD4 + T cells migrate to spleen and the CD4 + CD44 + CD40L + Ly6C + memory T cells exit from the spleen remaining in the peritoneum. Bone-marrow is the appropriated niche for CD4 + CD44 + Ly6C + central memory T cells. To confirm the importance of memory CD4 T cells in memory Ab production, adoptive transfer of memory cells from spleen and BM was conducted. Bone-marrow cells from longlasting venom immunized mice, but not control or splenic cells, induced high and persistent levels (80 days after transfer) of specific antibody production (IgG1 and IgG2a) in naïve recipients that was impaired in CD4 or CD28 KO recipients. Conclusions: Thus, in the memory phase of immunity, central memory helper T cells are maintained in BM as resting but rapidly expressed CD40L and efficiently induced the production of highaffinity antibodies by B lymphocytes. Many autoimmune diseases (ADs) are characterized by the production of autoantibodies (autoAbs) specific for self-antigens. However, the precise mechanism for the pathogenesis of autoimmunity is unknown. On the other hand, Intravenous Immunoglobulin (IVIg) exhibits therapeutic effects in the treatment of variety of ADs in clinical practice, while the precise mechanisms have also been left unclear. Innate B cells or B-1 cells, which produce mainly natural antibodies including weakly autoreactive antibodies, have a distinct lineage different from conventional B cells or (B-2 cells). Recently, B-1 cells are shown to play a role in development of ADs when they are activated, and the class-switch is induced upon stimulation of their toll-like receptor (TLR)9 with unmethylated CpG oligonucleotide. Here we show that IVIg treatment in mice injected with CpG inhibits the proliferation and activation of B-1 cells. Upon stimulation of B-1 cells with CpG in vitro, IVIg attenuated the autoantibody production as well as the IL-10 production that is necessary to induce CSR, while it did not reduced the IL-6 production. The inhibitory effect of IVIg was dependent on the F(ab 0 ) 2 but independent on the Fc. In addition, IVIg upregulated the expression of CD22, a B cell inhibitory receptor, on B-1 cells. However, IVIg treatment exhibited a comparable inhibitory effect on B-1 cells from wild-type and CD22-knockout mouse. Inspections of IVIg-induced modulation of intracellular signaling revealed that the IVIg attenuated several TLR9-initiated signaling pathways, such as phosphorylation of TAK1, NF-kB and ERK, but not IRAK-1 and p38 MAPK. We propose a novel inhibitory mechanism of IVIg in ADs, in which IVIg inhibits TLR9 signaling that leads to production of autoreactive antibody in a F(ab 0 ) 2 -dependent manner. The roles of inflammatory versus neurodegenerative processes in the pathology of multiple sclerosis (MS) and in its animal model experimental autoimmune encephalomyelitis (EAE) are controversial. Novel treatment strategies aim to act within the CNS to reduce inflammation and induce neuroprotection and repair processes. In this study we analyzed and compared the in situ pathological manifestations of relapsing-remitting PLP-and chronic MOG-induced EAE models, using transmission electron microscopy (TEM) and immunohistochemistry. The effect of immunomodulatory treatment by glatiramer acetate (GA, Copaxone) on myelin damage/repair and on motor neuron loss/preservation was studied in both models. TEM analysis revealed multiple spinal white matter damages, demyelination, axonal degeneration and axonal loss, with different occurrence in the two EAE models. Hence, in the relapsing-remitting model most of the axons manifested myelin aberration, whereas in the chronic model axonal degeneration was more widespread. Remyelinating fibers with thin compact myelin sheathes were detected mainly in the PLP model and their number increased with time. Loss of motor neurons was manifested only in the chronic model. In GA-treated mice, smaller lesions and higher amounts of normal appearing axons were found, with preservation of motor neuron number and formation. Furthermore, for each time point, significantly more remyelinating fibers were counted, three to sevenfold elevations from untreated mice. These effects were obtained even when GA-treatment was applied in a therapeutic schedule after the appearance of clinical symptoms. In conclusion, demyelination and remyelination are characteristic to relapsing-remitting PLP-induced EAE whereas in the chronic MOG-induced model axonal degeneration and somatic neuronal pathology are mainly manifested. The remyelination and neuronal preservation induced by GA are in support of neuroprotective consequences of immunomodulatory treatment in these models Inflammatory cytokines are implicated in the pathogenesis of different autoimmune diseases like Type 1 diabetes, rheumatoid arthritis and inflammatory bowel disease in humans as well as in animal models. IL-21, and IL-15 are pro-inflammatory cytokines and they exert their biological effects at various stages of the immune responses. We have shown previously that IL-21 can synergize with IL-15 or IL-7 to reduce the threshold of TCR signaling, while simultaneously increasing the production of effector cytokines such as TNFa and IFNc. IL-21 maps to one of the shared autoimmune disease susceptibility loci in humans and mice. In non obese diabetic (NOD) mouse that develops spontaneous autoimmune type 1 diabetes (T1D), IL-21 is a candidate gene in the Idd3 locus. C57BL/6 derived Idd3 allele protects NOD mouse from T1D. As IL-2 also maps to the same locus, the relative contribution of IL-2 and IL-21 to the disease process is not well understood. It has been shown previously that NOD mouse lacking the receptor for IL-21 does not develop autoimmune diabetes. Here we report that the absence of IL-21 protects the 8. Objective: To identify a relationship between existing TPOAb among pregnant woman and its relation with their outcome of pregnancy, morbidity of autoimmune thyroiditis, to justify screening, when would be the best time for doing it. Materials and methods: The study included 120 pregnant, romanian women, ages 18-40. We used one-time screening TPOAb, one-time screening TSH and FT4. We performed also a cost-effectiveness analysis of the screening. When probabilities were not available, we were estimated using expert clinical judgment. Results: At initial presentation all the women were euthyroid. 10.83% had positive TPOAb level. The complications were found to be more significantly raised among positive TPOAb cases. It seems that autoimmune disease can increase the risk of reproductive failure. The administration of L-T4 prevent the risk for developing hypothyroidism during gestation and reduces pregnancy loss and preterm delivery. Conclusions: The prevalence rate for thyroid autoimmunity in young women seems high enough to justify the screening. Screening only high-risk women, however, would fail to diagnose woman with thyroid disease. Screening pregnant women with TFTs in the first trimester of pregnancy is cost-saving compared with no screening, while screening using TPO-Ab is an economically favorable screening strategy. Disclosure of interest: None declared. Immune system stands on 'trade-off' between infection-induced and defense-induced tissue injuries. To avoid 'too much battle against invasive organisms', immune system tries to suppress but not to eradicate invading antigens. Thus, our immune system is constantly exposed to small but continuous stimulation with antigen. We test here the integrity of immune system, i.e., how immune system responds to the overload of antigen, by stimulating maximally the immune system repeatedly with the same antigen. Methods: BALB/c, DO11.10 TCR Tg, b 2 m-deficient and rag1/gfp knock-in mice were i.p. immunized with SEB, OVA or KLH. Cells were transferred to naïve BALB/c or b 2 m-deficient mice via i.p. or i.v. cDNA was amplified by PCR and subjected to run-off reactions with labeled Jb primers, Jb1.1, Jb1.3 and Jb2.4. To test antigen crosspresentation, CD11c + DCs were incubated with OVA followed by cocultured with DO11.10 CD8 + T, and expression of CD69 on DO11.10 CD8 + T was examined, using chloroquine as inhibitor. Results: Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4 + T cells led to the development of autoantibody-inducing CD4 + T (aiCD4 + T) cell which had undergone TCRa but not TCRb revision and was capable of inducing autoantibodies. aiCD4 + T cell was induced by de novo TCR revision but not by mere cross-reaction to antigen, and subsequently overstimulated CD8 + T cells, driving them to become antigen-specific CTL. These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury identical to SLE. The \CD4 + T cell was specifically detected in SLE. Conclusions: Autoimmunity is the inevitable consequence of overstimulating host's immune 'system' by repeated immunization with antigen, to the levels that surpass system's self-organized criticality. SLE is induced by normal, but not autoimmune, response to antigen. Disclosure of interest: None declared. Cochin Institute, Paris, France Proteinase 3 (PR3) is a granular serine-proteinase, which is also present at the plasma membrane in a stable neutrophil subset. ANCA binding to PR3 at the surface of primed neutrophils results in their activation. In neutrophils, PR3 has been shown to be associated with a number of protein partners but the function of these associations remain to be defined. CD177 is a GPI-anchored glycoprotein, which is expressed on the same neutrophil subset than PR3. It has been proposed that CD177 is essential for PR3 membrane expression in the basal state. The rat basophilic cell line (RBL) has been used to study the molecular mechanisms leading to PR3 membrane expression but unfortunately RBL-PR3 do not display PR3 membrane expression. We hypothesized that expression of CD177 would ''reconstitute'' the molecular ''scaffold'' of PR3 partners. We generated stable RBL transfectant expressing either hCD177 or hPR3 or both. Membrane expressions of CD177 or PR3 were studied by flow cytometry under basal conditions or following degranulation or apoptosis. Stable hCD177-transfected cells show a membrane expression under the basal state in WT-RBL as well as RBL-PR3. However, in RBL-PR3 CD177 failed to trigger PR3 to membrane under the basal state. After degranulation PR3 membrane expression was similar in RBL PR3 and RBL-PR3 CD177. We found the same result in apoptotic cells; membrane expression of PR3 was similar in RBL-PR3 and RBL-PR3 CD177 in flow cytometry analysis. These cells have similar intracellular PR3 storage. In keeping with data from other groups, we also found that CD177 is a receptor for extracellular PR3, as demonstrated by the binding of exogenous PR3 to RBL-CD177 in the basal state contrary to RBL WT. CD177 appears to be a partner of PR3, which could play a role as a receptor for extracellular PR3, but it seems to be insufficient for PR3 membrane targeting in the basal state in a cell model of neutrophils Background: Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract, which clinically present as one of two disorders, Crohn's disease (CD) and ulcerative colitis (UC). In a previous study, we have shown correlation between NO production and intestinal tissue damages in patients reached by Crohn disease and ulcerative colitis. In this study, we investigate in vitro and in situ the modulation of nitric oxide production by TNF-a and/or IL-17A in the presence of IL-10 or retinoic acid in cultures of PBMC, macrophages and colonic mucosa from patients (CD and UC). We also evaluated IL-17 expression in inflamed colonic mucosa treated by retinoic acid. Patients and methods: Twenty Patients with UC, 30 patients with CD and 10 healthy controls were enrolled in this study. Freshly isolated peripheral blood mononuclear cells (PBMCs) and macrophages were resuspended in RPMI 1640 and DMEM culture medium, respectively. Multiple colonic biopsies were taken from patients who underwent colonoscopy. Colonic biopsies were immediately placed in DMEM culture medium. The cultures were stimulated by human recombinant TNF-a, IL-17A in presence of IL-10 or retinoic acid. Cultures supernatants were harvested after 24 h of incubation; NO measurement was performed by modified Griess. IL-17 expression was evaluated by standard immunohistochemical procedure. Results: Our results show that TNF-a and IL-17A up regulated NO production by PBMC, macrophages and colonic mucosa. In contrast, exogenous IL-10 and retinoic acid down regulated NO production in vitro and in situ for the two groups of patients. Retinoic acid downregulates IL-17 expression in colonic mucosa. Conclusion: These results suggest that proinflammatory cytokines (TNF-a, IL-17A) play a pivotal role in IBD pathogenesis disease through nitric oxide pathway. Collectively our study suggests that IL-10 and retinoic acid seem to be a useful tool for development of therapeutic strategies in IBD. Disclosure of interest: None declared. Background: In giant cell arteritis (GCA), immunological studies suggest the existence of a triggering antigen of unknown nature activating T-cell in the arterial wall (1) . In experiments performed in animal models, a proliferation of T-cells from the xenotransplant suggests a possible recognition of a locally expressed antigen (2) . S182 Inflamm. Res. Objectives: We thus decided to use a proteomic approach in order to identify the target antigens of auto-antibodies directed against endothelial cells (EC) and vascular smooth muscle cells (VSMC). Patients and methods: Sera from 15 GCA patients were tested in pools of 3 sera and compared to a sera pool from 12 healthy controls (HC). Serum IgG reactivity was analyzed by use of a 2-D electrophoresis and immunoblotting technique with antigens from normal human umbilical vein endothelial cells (HUVEC) and mammary-artery-VSMC. Targets antigens were identified by mass spectrometry (MALDI-TOF-TOF). Results: Serum IgG recognized 162 ± 3 and 100 ± 17 protein spots from HUVEC and VSMC, respectively, whereas serum IgG antibodies from HC recognized 79 and 94 protein spots, respectively. Thirty spots from HUVEC were recognized by at least 2/3 pools and not by HC. Nineteen spots from VSMC were recognized by at least 3/5 pools and not by HC. Among identified proteins we found lamin A/C, voltage dependent anion selective channel protein 2, annexin V and other protein involved in cell energy metabolism and key cellular pathways. Conclusion: IgG anti-EC antibodies and anti-VSMC antibodies are present in the serum of patients with GCA. These antibodies recognize cellular targets playing key roles in cell biology and maintenance of homeostasis. The function of these antibodies and their possible role in pathology have to be further explored. Disclosure of interest: None declared. Purpose: In an experimental model, immunization of splenocytes or CD4 T cell with vascular smooth muscle cell (VSMC) is responsible for a pulmonary vasculitis after adoptive transfer (1, 2) . Objectives: We thus decided to identify the target antigens of anti-VSMC in patients with ANCA-associated vasculitis. Patients and methods: Sera from 33 ANCA-associated vasculitis patients, 15 with Wegener's granulomatosis (WG), 9 with microscopic polyangiitis (MPA), 9 with Churg Strauss syndrome (CSS), tested in pooled sera for groups of 3, were compared to a sera pool from 12 healthy controls (HCs). Serum IgG reactivity was analyzed by use of a 2-D electrophoresis and immunoblotting technique with immortalized mammary artery-VSMC antigens. Target antigens were identified by mass spectrometry. Results: Serum IgG antibodies in the pools of patients with WG, MPA, CSS recognized 121 ± 70, 57 ± 29 and 66 ± 14 protein spots, respectively, whereas healthy controls recognized 94 protein spots. Thirty one, 18 and 22 protein spots were specifically recognized by at least 3/5, 2/3 and 2/3 pools of WG, MPA and CSS, respectively. Among identified proteins are lamin A/C, vimentin, annexin and alpha-enolase. Target antigens are involved in cytoskeleton, cell energy metabolism, and other key cellular pathways. Material and methods: we have used a two-dimensional electrophoresis and immunoblotting technique with protein extracts of human umbilical vein endothelial cells (HUVEC). We have tested sera from 18 SSc patients with (n = 9) or without PAH (n = 9), 9 sera from iPAH patients (3 sporadic, 3 familial and 3 dexfenfluramin-associated PAH) and 12 sera from healthy blood donors as negative controls. These sera were tested at a dilution of 1/100 by pools of 3 for patients and in a pool of 12 for healthy controls. Targeted spots were identified by mass spectrometry. Results: Serum IgG from SSc patients with or without PAH recognized 42 ± 4, 37 ± 7 protein spots, respectively. IgG from patients with sporadic, familial or dexfenfluramin-associated PAH recognized 46, 30 and 30 protein spots, respectively, whereas 53 protein spots were recognized by IgG from healthy donors. 64 protein spots were recognized specifically by IgG from SSc patients including 17 spots which were recognised specifically by PAH patients. Only six protein spots were recognized by IgG from more than 75% of patients' pools of sera and not by the IgG from healthy donors. Antigens targeted by IgG AECA from SSc and/or PAH patients have been identified by mass spectrometry (MALDI-toff) including triosephosphate isomerase, peroxiredoxin, cathepsin D, prohibitin and phosphoglycerate mutase 1. Introduction: Approximately 10% of SSc patients have no routinely detectable antinuclear antibodies (ANA), usually detected by indirect immunofluorescence on HEp-2 cells. For 20 to 40% of those with detectable ANA, there is no identified specificity. In this work, we aimed to identify new target autoantigens in SSc patients. Methods: Using a proteomic approach combining 2-D electrophoresis and immunoblotting with HEp-2 cell total and nuclear protein extracts, we systematically analysed autoantibodies in the sera of 45 SSc patients tested in 15 pools from groups of 3 patients with the same phenotype. A sera pool from 12 healthy individuals was used as a control. Proteins of interest were identified by mass spectrometry and analysed using Pathway Studio software. Results: We identified 974 and 832 protein spots in HEp-2 cell total and nuclear protein extracts, respectively. Interestingly, alpha-enolase was recognised by IgG from all pools of patients in both extracts. Fourteen and 4 proteins were recognised by IgG from at least 75% of the 15 pools in total and nuclear protein extracts, respectively, whereas 15 protein spots were specifically recognised by IgG from at least 4 of the 10 pools from patients with non-identified ANA. The IgG intensity for a number of antigens was higher in sera from patients than in those from healthy controls; these antigens included triosephosphate isomerase, superoxide dismutase mitochondrial precursor, heterogeneous nuclear ribonucleoprotein L and lamin A/C. In addition, peroxiredoxin-2, cofilin-1 and calreticulin were specifically recognised by sera from phenotypic subsets of patients with nonidentified ANA. Interestingly, several identified target antigens are involved in the transforming growth factor-beta pathway. Conclusions: We identified several new target antigens shared among SSc patients or specific to a given phenotype. These autoantibodies could represent new diagnostic and/or prognostic markers for SSc. Disclosure of interest: None declared. Pulmonary arterial hypertension (PAH) is defined as an increased mean pulmonary arterial pressure with normal pulmonary artery wedge pressure. It is widely accepted that immune and inflammatory mechanisms could play a significant role in PAH genesis or progression, but the pathophysiology is still unclear. Recent evidence has demonstrated a detrimental involvement of leptin in promoting the pathogenesis of various autoimmune diseases by controlling the regulatory T cells (Treg) onset. Despite this knowledge, the role of leptin in PAH is unknown. Here, we considered whether leptin promotes the immunopathogenesis of PAH by regulating Treg. To test this hypothesis, we withdrew blood samples from patients with PAH with normal body mass index (BMI) (idiopathic (I), heritable (H) and connective tissue disease-related (CTD)) and healthy control subjects. To measure serum leptin levels, we performed an ELISA test on serum samples. To detect circulating Treg and those expressing leptin receptor (ObR), we isolated the peripheral blood mononuclear cells, stained the latter with surface fluorescently tagged antibodies and selected Treg expressing ObR as CD4 + CD25 + CD127 low ObR + cells by flow cytometry (FACS). Leptin was higher in PAH patients compared to controls. Furthermore, leptin was higher in IPAH and CTD-PAH patients compared to HPAH patients, whereas it did not differ between IPAH and CTD-PAH patients. FACS analysis revealed that Treg number was comparable in PAH patients and controls. However, the percentage of Treg ObR + was higher in PAH patients compared to controls and interestingly, it was even higher in IPAH and CTD-PAH compared to HPAH patients. IPAH and CTD-PAH patients were comparable. No correlation was found with BMI. Our findings show for the first time that leptin and its receptor are increased in PAH patients. Interestingly, IPAH and CTD-PAH are comparable. Therefore, leptin and its receptor could play an important role in the immunopathogenesis of PAH. Disclosure of interest: None declared. Immunology department, 2 Rheumatology department, 3 Pathology department, La Rabta Hospital Al Manar University, 4 Epidemiology department, Faculty of Medecine, Al Manar University, Tunis, Tunisia Objective: Anti-citrullinated protein antibodies (ACPA) seem to be produced locally at the site of joints inflammation in the first stage of rheumatoid arthritis (RA). A strong correlation between serum ACPA (S-ACPA) and ACPA in the synovial fluid (SF-ACPA) is now suggested. A case-control study was conducted to evaluate the usefulness of ACPA determination in SF of patients with RA. Methods: A total of 52 patients with a knee-joint effusion (25 RA, 18 peripheral spondyloarthropathies (SPA) and 9 osteoarthritis (OA)) were included in our study. SF samples were obtained by performing therapeutic arthrosynthesis. IgG S-ACPA and SF-ACPA levels were determined by the enzyme-linked immunosorbent assay (ELISA). We have also determinate IgG levels in serum and SF by nephelometry. Results: Higher levels of IgG ACPA in SF (p = 0.045) and serum (p = 0.045) were found in patients with RA respect to SPA and OA patients. There was a significant and positive correlation between ACPA in serum and SF (rho = 0.516; p = 0.007) not only in the RA group but also in patients with SPA. Serum ACPA discriminated RA from non-RA at cut off value of 2.7 U/ml (sensitivity 69%; specificity 78%; area under the curve (AUC) 0.72) whereas SF-ACPA discriminated RA from non-RA at a higher cut off value of 4.95 U/ml (sensitivity 73%; specificity 61%; AUC 0.71). Conclusion: Our study suggests that the determination of IgG SF-ACPA didn't give complement information to serum ACPA in patients with RA. Disclosure of interest: None declared. S184 Inflamm. Res. T cell activation is an important event in autoimmune diseases such as rheumatoid arthritis. Transformed malignant cells are known to greatly alter their metabolism of fatty acids as a mechanism that allows rapid cell proliferation. In this study, fatty acid metabolism was evaluated in rapidly proliferating T cell receptor-stimulated primary human T-cells. The distribution of fatty acids in these cells changed significantly following cell stimulation and resembled that measured in the human Jurkat T cell line. The cellular content of saturated and monounsaturated fatty acids was significantly increased in proliferating cells, suggestive of an increased expression of fatty acid synthase and steroyl-CoA desaturase as is seen in cancer cells. Additionally, cellular arachidonate (20:4n-6) was redistributed in a pattern that was unlike any other fatty acid. This was characterized by an accumulation in 1-acyl-PI, 1-alkyl-PC and 1-alk-1-enyl-PE species and a loss from 1-acyl-PC species in stimulated cells. This arachidonate mass redistribution is accompanied by enhanced CoAindependent transacylase-mediated arachidonate-phospholipid remodelling. When the stimulus was removed from proliferating T cells, they stopped proliferating and the fatty acid distribution largely reverted back to that of resting cells. To begin to identify enzymes involved in this remodeling, we measured significant changes in the expression of phospholipases A 2 (IVA, IVB, IVC) and lysophospholipid acyltransferases (LPEAT2, MBOAT7) by qPCR. Overall, these results suggest that metabolic pathways regulating the cellular content and distribution of fatty acids may be potential therapeutic targets for disorders associated with T cell proliferation. Pristane-induced arthritis (PIA) is an MHC dependent polyarthritis with infiltrating activated CD4+ T cells, erosion of peripheral joints and greater susceptibility in females. Unlike collagen induced arthritis it has a chronic relapsing course. A dose-ranging study was carried out to determine the pristane dose that would induce relapsing arthritis without excessive adverse effects as defined by preset humane endpoints and aimed to track disease progression on all individual peripheral joints. Pristane (75, 100 or 150 ll) was administered under light isoflourane anaesthesia, i.d. to the tail base to DA/OlaHsd rats. Controls received no injection. Rats were scored with a 16 point system recording swollen or red digits, knuckles, midfoot and ankle/ wrist between day 5-25 every 2 days and between day 25-100 twice every week. Paw swelling was measured with 0.02 mm graduation callipers. Adverse effects were monitored to Institutional ethical guidelines and approved by the UK Home Office. Paws were frozen or fixed with paraformaldehyde. For all pristane doses, inflammation followed an acute phase followed by a chronic relapsing phase. Front and hind paws responded with signs of disease by day 20 followed by a decline finishing by day 40. Ankle swelling persisted to day 70. Relapse occurred in front and hind digits with some wrist involvement, as opposed to ankles and midpaws which did not. Hind paw swelling only occurred in the acute phase, fully declining by day 40. In the chronic phase, front paws were often characterised by swollen knuckles and there was a high incidence of severe distortion of front digits. Pristane was best tolerated at 75 and 100 ll. Conclusion: In PIA in DA/OlaHsd rats, chronic relapsing disease follows a course that differentiates between digits, wrists, ankles, and foot thickness. Doses of 75ul and 100ul elicited a less severe and more manageable disease. Numerous studies highlight that monocytes are key-mediators of tissue damage in nephritis, including lupus nephritis. Previous published work in our lab has shown that CX 3 CR1 hi Gr1monocytes in mice and CD14 dim CD16 + monocytes in humans patrol blood vessels. These patrolling monocytes may scavenge dead cells and/or immune complexes. Furthermore, it has been shown that murine Gr1monocytes selectively expand and accumulate inside renal capillaries during nephritis, and that they are activated by immune complexes in an FccR-dependent way. Our lab recently published that CD14 dim CD16 + monocytes detect nucleic acids and mediate kidney inflammation in lupus patients. Nevertheless, little is known about the events that govern monocyte recruitment and their activation in the kidney. By using intravital single-and two-photon microscopy and fluorescentlabelled high and low molecular weight dextran we have been able to identify and image in real time the glomerulus and associated structures in CX 3 CR1 gfp/+ Rag 2 -/c c -/mice under basal non-inflammatory conditions. Our hypothesis is that Gr1monocytes patrol glomerular capillaries, detect immune complexes and are selectively recruited into the kidney, where they induce tissue damage. Using the novel imaging techniques developed in our lab we will be able to test our hypothesis and assess if the targeting of monocytes or a specific type of monocytes would be beneficial in kidney inflammation. Disclosure of interest: None declared. Introduction: Rheumatoid arthritis (RA) is an autoimmune disorder that affects a considerable number of populations during reproductive ages. RA is characterized by symmetric and erosive synovitis causes progressive joint destruction and disability. Anti-cyclic citrullinated peptide (CCP) antibody is a more sensitive and specific test than Rheumatoid factor for diagnosis of patients with RA. Moreover, Anti-CCP antibody titer predicts the severity of erosive arthritis and disease outcome. The objective of this study was to provide data regarding the prevalence of Anti-CCP antibodies in Iranian RA patients. Method: A total of 112 RA patients and 206 age and sex matched healthy controls from Kermanshah province (west of Iran) were enrolled in this study. All Patients fulfilled the American College of Rheumatology diagnostic criteria for RA and healthy controls did not suffer from any known rheumatic diseases. 5 ml of blood samples were collected and sera separated by centrifugation. Anti-CCP IgG antibody was measured using specific ELISA kits (Euroimmune). The data were analyzed using SPSS version 11.5 statistical software package. Results and discussion: Six of the controls (2.91%) and Sixty-eight out of 112 patients (60.71%) were positive for Anti-CCP antibodies. Sensitivity and specificity were 60.71 and 97.09%, respectively in this study. The results of this study showed that Anti-CCP antibody is prevalent in RA patients in west of Iran at 60.71%. Moreover, its prevalence seems to be higher in healthy controls in comparison to other countries populations. Further investigations with higher sample numbers may be helpful in determination of precise prevalence, sensitivity and specificity of Anti-CCP in both healthy control and RA patients in west of Iran. Disclosure of interest: None declared. Th1 systemic deficiency exerts pro-inflammatory effects in rheumatoid arthritis (RA) pathogenesis. A role of IL-17 in Th1 defects in RA patients has been recently suggested. We investigated pro-inflammatory and anti-inflammatory T helper phenotypes including Th17 and Th1 in order to clinical response to methotrexate (MTX) therapy. Methods: Eighteen patients with active RA and 15 healthy controls were enrolled in the study. The frequencies of Th17 and Th1 cells in peripheral blood (PB) from individuals studied were determined by flow cytometry. IFN-gamma as well as IL-17 intracellular contents were measured by mean fluorescence intensity (MFI) estimation. Disease activity score (DAS28) and C-reactive protein (CRP) levels were also determined before and after at least 4 months of therapy with MTX in a stable dose 15 mg/week. Results: Before therapy, we showed that clinical and biochemical signs of disease activity were present in all patients. Th17 population and intracellular contents of IL-17 were markedly heightened in RA patients compared to controls. In addition, imbalance in Th17/Th1 ratio with predominance of pro-inflammatory Th17 cells was found in RA. After MTX treatment, we observed a significant decrease in DAS28 and CRP levels in 17 out of 18 patients. Clinical improvement was accompanied by normalization in Th17 population as measured by both Th17 cell frequencies and IL-17 intracellular levels. In contrast, a significant increase in spontaneous IFN-gamma production in CD4+ T cells was found in RA patients treated with MTX. Conclusions: We show that expansion of PB Th17 cells parallels the inflammation in active RA. Normalization of Th17 population after successful treatment with MTX contributes to Th1 systemic improvement, what is in agreement with IFN-gamma anti-inflammatory effects in RA. The balance between these Th cell subsets would attenuate the inflammation levels and could thus be related to the remission period of RA. Disclosure of interest: None declared. Early identification of patients with undifferentiated arthritis (UA) destined to develop RA among all UA cases, enabling appropriate treatment at early stages of the disease, still remains a challenge. We attempted to find out if the analysis of lymphocyte proliferation dynamics could be a good diagnostic approach distinguishing prospective RA from non-RA patients prior to final diagnosis. Forty early UA patients were included based on routine clinical criteria and measurement of standard diagnostic markers: anti-CCP (anti-cyclic citrullinated peptide), RF (rheumatoid factor), ANA-Hep2 (antinuclear antibodies), ESR. The inclusion criteria were considered to define the population of patients with UA, for whom the early classification criteria is the main target especially in order to define patients with early rheumatoid arthritis. Peripheral blood mononuclear cells were isolated from blood samples taken at UA diagnosis, stained with CFSE, mitogen-stimulated and analyzed by FACS. CD4 + CD28 + cell cycle and G0-G1 time, % of dividing cells and number of divisions per cell were calculated. Final assignment of UA patients into RA and non-RA rheumatic diseases' groups based on EULAR criteria, was done during 2-year follow-up and confronted with laboratory and clinical findings at initial UA diagnosis in search of parameters with predictive capacities. We found strong, significant differences in all analyzed proliferation features between RA and non-RA groups. By thorough regression analysis we found that proliferation parameters were more relevant than disease activity and serological markers for distinction of RA from non-RA, with both high specificity and sensitivity. Our results highlights a new innovative diagnostics tool for distinguish early RA among early UA, at the beginning of diagnosis procedure, early in the disease course, what allow to gain EULAR/ACR recommendations. Disclosure of interest: None declared. Purpose: Multiple sclerosis (MS) is a T-cell mediated, autoimmune disease targeting central nervous system myelin. Auto-reactive T cells reacting to myelin antigens presented by antigen presenting cells drive the immune response. Expansion of CD4 + FoxP3 + T cells (Tregs) is being considered as a means to restore tolerance. Methods: Using myelin oligodendrocyte glycoprotein (MOG)induced experimental autoimmune encephalomyelitis (EAE), the animal model of MS, we investigated the effects of the recently discovered regulatory T cell epitopes (Tregitope). Tregitopes are promiscuous MHC Class II T-cell epitopes naturally located in the Fc and framework regions of Fab from IgG. Treated mice received 6 consecutive injections of human Tregitope 167 + 289 (200 lg/ mouse) i.p. in PBS between days 0-10 post-immunization. Control mice received 6 9 200 lg doses of either ovalbumin (Ova, negative control) or 6 9 16 mg intravenous immunoglobulin (positive control). Results: Using MOG-TCR transgenic XFoxP3-GFP mice, we found that, in agreement with the tolerogenic effects of IVIG, S188 Inflamm. Res. Tregitope 167 + 289 administration expanded Tregsin vitro and in vivo and ameliorated EAE compared to Ova-treated controls. Tregitope decreased the frequency of IFNg-producing T cells and induced IL-9+ T cells in the periphery. We also found upregulation of Jagged2 on CD11b+CD11c-macrophages. Jagged 2 is a Notch ligand that has been shown to induce murine Treg cell expansion. To understand the significance of the enhanced Jagged2 expression, we treated EAE mice with anti-Jagged2 monoclonal signaling antibody. We found that anti-Jagged2 treatment expanded Tregs and suppressed IFNg-positive T cells suggesting that Tregitopes effects are at least partially mediated by Jagged2. Mast cells play a key role in allergy and inflammation. When stimulated they secrete a host of mediators including histamine, arachidonic acid metabolites, cytokines, proteases, and growth factors such as the angiogenic factor, VEGF. Studies show mast cells accumulate in and around many human tumors including colorectal, breast, lung, and gastric. Angiogenesis is required for tumor growth and there is a positive relationship between the number of mast cells around tumors, microvasculature density, tumor growth and decreased patient survival. These results suggest that mast cells, via the release of angiogenic factors like VEGF, support tumor-required angiogenesis. The peptide NT is secreted by a number of human cancer cells and is a potent stimulus of mast cell secretion in vivo and in vitro. Rat and human mast cells express NT-type 1 G-proteincoupled receptors (NTS-1) and NT's stimulation of mast cells, in vitro and in vivo, is blocked by pretreatment with the NTS-1 antagonist, SR48692. The accumulation of mast cells around tumors, the secretion of VEGF by mast cells and the secretion of NT by human tumors, prompted us to ask if NT would stimulate VEGF secretion from human mast cells. HMC-1 mast cells withdrawn from serum were incubated at 37 C for up to 24 h with various concentrations of NT or with media alone. The media was then assayed for VEGF via ELISA. NT stimulated a significant, concentration-dependent increase in VEGF secretion that was blocked by pretreatment of the cells with SR48692 before stimulation by NT. VEGF secretion was also significantly reduced by pretreatment of the cells with inhibitors of protein kinase C (bisindolmelamide), P-I-3 kinase (wortmannin), MAPK (U0125), and p38 MAPK (SB202190 Chemokines and chemokine receptors not only act as a potent chemoattractant for inflammatory cells, but also have the powerful ability in cancer metastasis and tumorigenesis. CrkL and c-ABL interaction is reported to transform into many malignant cells. We examined and evaluated the effect of CCR7 and CrkL, c-ABL on pulmonary adenocarcinoma specimens and the relationship of their mRNA expression in pulmonary adenocarcinoma. 120 patients with pulmonary adenocarcinoma were included in this retrospective analysis. The expression of chemokine receptors CCR7, ligands CCL21, CCL19, CrkL and c-ABL mRNA expression in pulmonary adenocarcinoma were examined and evaluated the relation to prognosis. High mRNA expression of CCR7 in lung cancer patients indicated significantly good prognosis than the groups of low expressions. The expression of CrkL and c-ABL mRNA had relationships with CCR7 mRNA expression in pulmonary adenocarcinoma. The expression of CCR7, CrkL and c-ABL were confirmed with immunohistochemical analysis of pulmonary adenocarcinoma specimens. In conclusion, we propose CCR7 as clinical good prognostic factors and the strong relation between chemokine receptor CCR7 and CrkL, C-ABL kinase mRNA expression in pulmonary adenocarcinoma. Disclosure of interest: None declared. Solanum nigrum Linn (SNL), also known as black nightshade, is commonly used in traditional Chinese medicine for curing inflammationrelated diseases and various kinds of cancers. However, the mechanism of anti-inflammatory action of SNL is still unclear, thus in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of SNL, which would provide the basis of its medical usage. We measured the pressure-temperature relationship of CO2 dissolved in saline for different concentrations, plotting the first against the second. We found that in each diagram there are several inflex points and that the first derivatives of all of them have one maximum always near the 37°C, independently of CO 2 content. Thus, we discovered a physical constant, explaining the human body temperature. On that base we created the following theory: (1) The capillary blood flow is promoted using the Stirling-Malone engine cycle; (2) The working medium is the containing dissolved CO 2 blood plasma, expanding by pressure fall, temperature elevation or by factors reducing the CO 2 solubility in it or producing additional CO 2 , like acids; (c) The capillary glycocalyx chains (banded forward by the RBC passage) and the parachute formed RBC act as ratchet and pawl, determining their one-way motion-a capillary pump; (3) The plasma expansion is on the account of the nanobubbles at the liquid/solid site of the glycocalyx chains, being maximal at 37°C. All inflammatory agents have great solubility in blood (histamine) or react with its buffers or both, as formic acid, displacing the contained CO2 or producing new amounts of it. This raises the pressure in venules by capillary pumping and direct venules' CO2-induced pressure. We use this theory to treat inflammation and cancer, using, as well the discovered by us: (1) In this study, we examined the effects of jacalin on the initial stages of mouse colon carcinogenesis induced by Methyl-N 0 -Nitro-N-Nitroso-Guanidine (MNNG). Balb/c mice were given 4 intrarectal deposits of 0.2 ml solution of MNNG (5 mg/ml) twice a week for 2 weeks. Starting 2 weeks before carcinogen administration, animals were treated orally (0.1 ml/mouse) with jacalin (0.005 and 0.05 mg/ml) twice a week for 10 weeks. At sacrifice, the colons were removed, fixed and processed for immunohistochemical analysis. When compared to animals submitted to carcinogenic action, mice additionally treated with jacalin showed results similar to those of control animals. Upon lectin treatment, we observed a decrease in cell proliferation in the colonic crypts, in the rate of stromal cell apoptosis, in the stromal expression of COX-2 and in the number of macrophages, CD4 and CD8 T lymphocytes associated with preneoplastic lesions. Intriguingly, the number of preneoplastic lesions increased when the lectin was administered to mice submitted to the carcinogenic action. These observations indicate that jacalin exerts protumorigenic effects on the colon and might reflect the manner in which it affects elements of the inflammatory tumor microenvironment. Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34 + 38 -123 + progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast niche components, CD34 + 38 -123 + progenitors survive through an integrin-dependent activation of GSK3b by serine 9-dephosphorylation. Strikingly, GSK3b-mediated survival was restricted to female leukemic progenitors. The scaffolding protein RACK1, activated downstream of the a 5 b 1 integrin and the proinflammatory TNF receptor, was specifically upregulated in female but not male leukemic progenitors, and controlled GSK3b activation through the phosphatase PP2A. In a mirrored fashion, male but not female normal adherent progenitors (CD34 + 38 -) 0 survival was partially dependent upon this pathway. Thus, the GSK3b-dependent survival pathway might be gender-specific in normal immature population and flip-flopped upon leukemogenesis. In search for compounds able to reduce cell adhesion-mediated drug resistance of leukemic progenitors, we previously found that the anti-inflammatory flavonoid rutin, but not the structurally-related compound quercetin, induces apoptosis specifically in adherent leukemic cells. Importantly, we show that rutin inhibited survival of adherent female leukemic progenitors (CD34 + 38 -123 + ) but spared normal female progenitors (CD34 + 38 -). The pro-apoptotic effects of rutin were correlated with a decrease of active GSK3b and inhibitors of GSK3b reproduced rutin-induced cytotoxicity. GSK3b inhibition by rutin restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent female leukemic progenitors. Together, our results strengthen GSK3b as a promising target for leukemic stem cell therapy and reveal gender of patients as a new parameter in anti-leukemia therapy. Disclosure of interest: None declared. Melanoma is the most aggressive form of skin cancer and its incidence has increased in the last decade. One important target identified in melanoma tumor progression is the Nuclear Factor-jB (NF-jB) pathway. In vitro studies have shown that IKK is constitutively active in human melanoma cells as compared to normal melanocytes, leading to NF-jB activation which in turn regulates the expression of anti-apoptotic proteins. These events are implicated as major molecular mechanisms for melanocytes transformation [1] . It has been found that a short cell-permeable peptide spanning the IKKb NEMO binding domain (NBD), disrupted the association of NEMO with IKKs in vitro and blocked TNFa-induced NF-jB activation in vivo [2] . In the present study we investigated the effect of the NBD peptide on survival of several human melanoma cell lines (A375, WM115, SK-Mel-5) as well as on the IKK/NF-jB signalling pathway. We report that NBD peptide inhibits the proliferation of all human melanoma cell lines used. Inhibition of cell growth was associated with direct inhibition of (1) constitutive IKK activity; (2) NF-jB DNA-binding activation; (3) induction of apoptosis in all cell lines tested. Using the A375 melanoma cell line, we show that inhibition of IKK/NF-jB signalling pathway by NBD peptide leads to down-regulation of the expression of several NF-jB-dependent antiapoptotic gene products and to the activation of caspase-3. Our studies suggest that a selective inhibition of IKK/NF-jB activation can be an effective strategy to be developed in the fight against cancer. Arachidonic acid (AA) derived metabolites produced by lipoxygenase (LOX) pathways are synthesized by intestinal epithelial cell (Caco-2), and are involved in cell growth [1] . 15-LOX also converts the abundant linoleic acid to 13-hydroxyeicosatetraenoic acid (13-HODE), a metabolite produced by Caco-2 cells cultured in 10% fetal bovine serum (8.22 ± 1.73 nM). The biological effects of 13-HODE on cell growth/cancer development are controversial. Thus, Hsi et al. [2] described a mitogenic effect in combination with EGF in human colorectal cells whereas Nixon et al. [3] reported an anti-tumorigenic effect by 13-HODE. Considering these findings we aimed to study the role of 13-HODE on Caco-2 cell growth. Our results show that exogenous addition of 13-HODE (10-1,000 nM) induced a concentration dependent cell proliferation and DNA synthesis in Caco-2 cultures. 13-HODE has two enantiomeric forms. Interestingly, 13 (R)-HODE presents higher mitogenic effects than 13 (S)-HODE, and these effects were blocked by ketoprofen, a cyclooxygenase inhibitor, suggesting that prostaglandins are involved in the effects of 13-HODE on Caco-2 cell growth. allowing or not cell-to-cell contact between the different cell types. Cytokines secretion was measured using membrane array assays. Cytokines and CD47 receptor expression was assessed by RT-PCR. Mast cells increase the growth of U87 and CCF-STTG1 cell lines by about 50 and 40%; this effect required cell-to-cell contact between the 2 cell types. Several pro-inflammatory cytokines such as IL6, IL7, IL15, IFNc and MCP-1 were highly secreted from CCF-STTG1 and U87 cells lines in the presence of mast cells. Histamine increases the proliferation of both U87 and CCF-STTG1 human astrocytomas cell lines. Moreover, IL6 expression was dose-dependently increased by histamine in these cells. The CD47 receptor is expressed by mast cells, U87 and CCF-STTG1 cell lines. His activation by the agonist 4N1 induces the secretion of histamine by mast cell and increases the level of pro-inflammatory cytokines RNAm expression by human astrocytomas. Our data indicate that mast cells and CD47 receptor activation induce the growth of human astrocytomas cells lines and increase the level of pro-inflammatory cytokines by these cells, thereby likely contributing to tumour aggressiveness by enhancing the proliferation. Disclosure of interest: None declared. Objective: Thromboxane A 2 (TXA 2 )is known to be stimulator concerning vasoconstriction and angiogenesis via thromboxane prostanoid receptor (TP) activation. In this study we evaluated the role of TXA 2 in different physiological states using wound healing and cancer models. Materials and methods: Mouse TXA 2 synthase (mTXAS) gene was retrovirally introduced into C57 BL/6 J (C57) cells, then designated as C57-mTXAS. Only retrovirus vector introduced C57 cells were designated as C57-EV and used for control. Mouse bladder carcinoma cells (MBT) were subcutaneously injected to C3H mice for bladder carcinoma model. One x 10 6 cells of C57-mTXAS or C57-EV were directly inoculated to the tumor as the size of 0.5 9 0.5 cm. Tumors were extracted after 14 days of cell inoculation for thrombus rate measurement and vascular endothelial growth factor (VEGF) in sera was also measured. The urethane sponges were implanted to the back of C57 mice for wound healing model. C57-mTXAS or C57-EV inoculated to the sponges after 4 days of the implantation and sponges were extracted then used for VEGF measurement and microvascular density (MVD). The TXAS synthase inhibitor (OKY-046) or TP antagonist (S-145) were also used. Results and discussion: C57-mTXAS cell therapy successfully suppressed for the bladder tumor growth rate followed by induction of thrombonecrosis in vessel inside of tumors but not outside. Production of VEGF in sera was suppressed for 1-2 weeks after therapeutic cells inoculation. Secretion of VEGF in C57-mTXAS was significantly increased after arachidonic acid administration. Inoculation of C57-mTXAS into implanted sponges, significantly induced VEGF and MVD after 6 and 12 days, respectively. The induction of VEGF and MVD were abolished with administration with OKY-046 and S-145. These result suggested that TXA 2 may play different physiological role and its signal pathway such as stromal derived factor-1 (SDF-1) would be clarified in the future. 3 Monash University, Melbourne, Australia The immune system is critically involved in multiple stages of tumour growth and spread and the outcomes of this involvement can be both beneficial and harmful. Although neutrophils are abundant in many human and animal tumours, their role in cancer pathogenesis remains controversial. This is likely due to the fact that neutrophils can play a dual role in primary tumours: they can mediate tumour rejection but also promote angiogenesis and tissue remodelling which favour tumour growth. Furthermore, emerging evidence suggests that neutrophils may also have the ability to promote metastasis. Hence there is great potential for novel neutrophil-based therapies in the treatment of cancer if the contrasting roles of neutrophils in tumour growth are properly understood. We have designed a system which utilizes 2-photon imaging together with a photoswitchable transgenic mouse Kaede to track neutrophils in real time inside tumours deposits and also to follow their fate as they egress from the tumours. Using this method we show that neutrophils form dynamic swarms inside primary tumour deposits. This swarming behavior may lead to tissue remodeling, as was previously observed in infections, and promote tumour growth and metastasis. These results provide the first information about the dynamics of neutrophil behaviour inside solid tumours and their fate as they egress primary tumour deposits. Disclosure of interest: None declared. Inflammatory breast cancer is characterized by clinical symptoms including inflammation is the hallmark. This type of cancer remains poorly understood from a biological point of view: both the process of carcinogenesis than inflammation. In Tunisia, the incidence of inflammatory breast cancer is 5.7% and non-inflammatory breast cancer is 25-27%. Interleukin-10 has immunosuppressive properties, anti-inflammatory and antiangiogenic. To test whether IL-10 promoter region polymorphisms are associated with susceptibility to inflammatory breast cancer, we examined the contribution of interleukin-10 (IL-10) gene polymorphisms in Tunisian patients with localized or metastatic breast cancer and patients with Inflammatory breast cancer IBC. In this study we looked SNP at position -627 (C [ A) at the promoter regulatory region of IL-10 that has the ability to modulate the expression of IL10. The methodological approach to answer our question is the case-control study. We analyzed by RFLP, SNP-627 (C [ A) in peripheral blood samples collected from two groups of patients 22 with Inflammatory cancer (IBC) and 37 with non-inflammatory breast cancer (non-IBC) belonging to different histological stages. Genotypic and allelic frequencies were compared between groups of patients and 55 healthy controls; and analyzed according to the presentation of the disease. In Tunisian population, comparison of allele frequencies showed a contribution of the A allele in the IL-10 promoter as a risk allele for the inflammatory form of breast cancer, the C allele in the IL-10 promoter was found protective against this type of cancer (p = 0.015). The expression level of IL-10 differs between individuals and this polymorphism in relation to the promoter region of the gene where several variations in the level of expression of the gene have been described. The low expression alleles may be risk factors for inflammatory breast cancer. Disclosure of interest: None declared. The acute phase protein serum amyloid A (SAA) has been proposed as a marker of tumor progression and there are evidence sustaining its participation in processes that favor carcinogenesis and metastasis, in spite of this, the possibility of direct activities of SAA on tumor cells remains to be addressed. Here, we have investigated the effect of SAA on two human glioma cell lines, A172 and T98G. SAA stimulated the proliferation of both lines, but had different effects on migration and invasiveness behavior depending on cell line. SAA increases migration and invasion of T98G, whereas it decreased these in A172. Both lines expressed all the three isoforms of SAA and the production the protein was detected intracellularly. SAA affected the production of compounds present in the tumor microenvironment that can orchestrate tumor progression, such as cytokines, nitric oxide (NO) and matrix metalloproteinases (MMPs). The effect of SAA on IL-8 production was remarkable for A172; whereas these cells did not produce basal amounts of this cytokine, they were extremely responsive to SAA. On the other hand, T98G produced large amounts of IL-8 that was unaffected by SAA addition. SAA induced the production of (NO), increased the expression of (MMPs)-2 and -9, and down regulated the expression of the MMPs regulator gene RECK. These data based on in vitro assays are suggestive of the contribution of SAA in cancer progression depending on the cell type and concentration of SAA. In the case of gliomas, the most common brain cancer in adults, our data support previous findings indicating a role for the inflammatory process in tumor susceptibility and therapy. Inflammatory cells and mediators are components of the tumor microenvironment and inflammation has been proposed to represent a mechanism to cancer development. Actinic cheilitis (AC) is an oral pre-malignant lesion characterized by discrete chronic infiltrate and cellular abnormalities that might develop into oral squamous cell carcinoma (OSCC). The characterization of inflammatory mediators in the blood serum of pre-cancerous and tumor lesions has not been fully investigated. Besides, little is known about the role of elastase (ELA) and myeloperoxidase (MPO) activities, nitric oxide (NO) and IL-6, IL-10, IL-13, IL-17, TGF-b and GM-CSF in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of these molecules in the blood of patients with actinic cheilitis and oral squamous cell carcinoma. MPO, NO, ELA, IL-6, IL-10, IL-13, IL-17, TGF-b and GM-CSF levels were increased in OSCC, while in the AC only ELA was elevated. Therefore, oral tumors strongly induced the production of inflammatory mediators related to neutrophils and macrophages functions. Further studies are necessary to establish the significance of these cells in human oral cancer. The understanding of the differences between a pre-malignant lesion and multiple mechanisms of OSCC-induced immune suppression may provide a range of novel targets for cancer prevention and therapies. Disclosure of interest: None declared. The prothrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported, yet the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle generation. Consistently, DOX-treated platelets and generated microparticles induced thrombin generation, a typical feature of procoagulant activity. DOX-induced PS exposure was determined to be from intracellular Ca 2+ increase and ATP depletion which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (DW), cytochrome c release, Bax translocation and caspase-3 activation. Ca 2+ chelator (EGTA), caspase inhibitor (QVD) and antioxidants (Vitamin C and Trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that ROS generation, Ca 2+ and apoptosis were associated in DOX-enhanced procoagulant activity. Rat in vivo thrombosis models confirmed the prothrombotic effects of DOX through the mediation of platelet procoagulant activity, where DOX administration resulted in increased venous thrombus formation along with increased PS exposure and DW dissipation, reflecting that DOXenhanced procoagulant activity of platelets may lead to increased thrombus formation, indeed. Disclosure of interest: None declared. Within the context of tumors, macrophages have been increasingly recognized as central regulators. These cells are able to dramatically affect the course of the disease and depending on their functional orientation, can present both pro-and anti-tumor activities. The aim of this study was to analyze the modulation of macrophage tumoricidal activity by the lectin jacalin. We show that in vitro, jacalin (2.5-40 lg/ml) induced the production of both pro-and anti-inflammatory mediators by human macrophages. Lower concentrations of the lectin (up to 5 lg/ml), when compared to the higher (from 10 to 40 lg/ml), induced the secretion of higher levels of the anti-inflammatory cytokines IL-10 and TGF-b. Similar amounts of the proinflammatory cytokines TNF-a and IL-6 were secreted by cells stimulated with jacalin at all of the concentrations tested. For IL-12, high concentrations of the lectin determined the maximal responses. As assessed by MTT assays, when supernatants from macrophages stimulated with higher, but not with lower concentrations of the lectin were added to cultures of human colon adenocarcinoma cells (HT-29), up to 25% reduction of cell viability was observed. These results indicate that jacalin, through its ability to exert a pro-inflammatory activity, can direct macrophages to an anti-tumor phenotype. Prosopis cineraria (L) Druce, commonly called as Khejri, widely distributed and used in Rajasthan, India, as folk medicine and food. Cancer chemopreventive potential of Prosopis cineraria leaf extract was investigated using two stages skin carcinogenesis model system. Animals were divided into five groups (control, throughout, pre, peri and post). A significant reduction in tumour incidence, tumour yield, and tumour burden, weight and size of tumour were observed in all treatment groups as compared to control. Average latent period was also increased significantly in all treatment groups. Significant increased in GSH, SOD, CAT and decrease in LPO was also observed in all treatment groups as compared to control. Total phenolic, flavonoid and flavonol contents were found significantly higher in leaf followed by flower, pod and stem bark. The leaf extract showed significant free radical scavenging activity followed by flower, pod and stem bark as evidenced by low IC 50 for DPPH and high percentage inhibition of DPPH and ABTS. The results from the present study suggest significant chemopreventive potential, higher polyphenolic contents and antioxidant activity of Prosopis cineraria. Breast cancer is the major reason of female death, it could take different forms, the most fatal type is inflammatory that can grow and spread rapidly. In Tunisia, the incidence of inflammatory breast cancer (IBC) is 5.7% and non-inflammatory breast cancer (non-IBC) is 25-27%. Chemokines regulate many biological processes such as proliferation, apoptosis, angiogenesis, leukocyte migration; they are also heavily involved in inflammation and in the development of metastases. Our aim in this study was to evaluated and compared CCL2 and CCL5 and their receptors major CCR2 and CCR5 genes expressions in Tunisian patients, with localized or metastatic breast cancer and patients with IBC. We analyzed by RT-PCR the expression of two chemokines CCL2 and CCL5 genes and their receptors in biopsy samples collected from two groups of patients 19 with IBC and 34 with non-IBC. Results obtained allowed us to assess the correlation of expression of these chemokines to the histological classification and their prognostic implications especially in IBC. Our results show that an almost universal expression of CCL5 and CCR5 mRNA with 100% of IBC tumors express at least one of these markers, and 95% of these tumors express both markers at once, whereas this expression is less frequent in non-IBC. A statistically significant difference between the two groups of breast cancer for the expression of chemokine CCL5 (p value = 0.037) and its receptor CCR5 (p value = 0.049). These results suggest that expression of CCL5 and its receptor CCR5 might be a factor associated with IBC. Disclosure of interest: None declared. associated-diarrhea in up to 25% of patients. However, there is not a pattern clinical management of this side effect. Calotropis procera (CP), a plant found in Africa, Asia and South America, and abundant in the Northeast of Brazil shows anti-inflammatory activities in animal models. Thus, we aimed to evaluate the anti-inflammatory effects of a protein fraction from CP in CPT-11-induced IM. Swiss mice (n = 10, 23 ± 2 g) were treated for 4 days with saline (Sal, 5 mL/kg, i.p.) or i.p.) . In other experimental groups, CP (1, 5 and 50 mg/kg/day, i.v.) was administered for 6 days, 30 min before the CPT-11. On the 7th day, we evaluated the total leukocyte count (910 3 /mL) and diarrhea (by scores). After sacrifice, the duodenum was collected for measurement of myeloperoxidase activity (MPO), morphometric analysis (villi/crypt), IL-1b level (pg/mL), and in vitro contractility (% contraction in relation to KCl 60 mM Cancers promote suppressive mechanisms hampering protective antitumor immunity. Reversing tumor-associated immune dysfunction is an attractive cancer immunotherapy strategy. Although most patients with cancer are elderly, most preclinical studies are performed in young animals. We studied two immunotherapies for B16 melanoma in young (\6 months) versus aged ([20 months) C57BL/6 and FoxP3-IRES-RFP mice: depleting regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs), two immune regulators known to impede anti-tumor immunity. Treg numbers were comparable at baseline and following tumor challenge in young and aged mice. Denileukin diftitox (DT) reduced Treg numbers equivalently in young versus aged animals. However, Treg depletion reduced tumor growth and improved tumor-specific immunity only in young mice. Thus, either aged anti-tumor immunity is ineffective even with reduced Tregs, or other suppressive mechanisms exist. Aged mice had higher MDSC numbers both at baseline and upon tumor challenge. Depleting MDSCs with anti-Gr-1 antibody improved tumor-specific immunity and reduced tumor growth significantly better in aged versus young mice. Strikingly, only in aged mice, Treg depletion led to an unexpected and undesired increase in MDSC, suggesting that Treg depletion could fail in older patients from increased MDSCs. Tregs depletion also resulted in myeloid cells increase in naïve young and aged mice. Combining Treg depletion with MDSC depletion increased clinical response in aged tumor-bearing mice, consistent with the hypothesis that Tregs negatively regulate MDSC in aged but not young tumor-bearing mice. These data demonstrate that antitumor immunity can be effective in aged individuals provided that underlying immune dysfunction is correctly identified and reversed. Treg depletion efficacy could be enhanced in aged individuals by simultaneous MDSC depletion. Cancer immunotherapies must be tailored to account for age-dependent immune differences. Disclosure of interest: None declared. The secondary lymphoid organs (SLO) are classically described as the place of initiation of adaptive immune responses. Aside from the classical SLO, there is a rapidly growing body of evidence that under inflammatory condition, the infiltrating immune cells are able to organize into structures called ''tertiary lymphoid structures'' presenting a high degree of similarity with the SLO. Accumulating evidence indicates that adaptive immunity can be initiated independently of SLOs. Here, in human lung cancer, we have characterized the immune cell infiltrate as well as their in situ organization. We have described that immune cells can be organized into tertiary lymphoid structures in some tumors. We called these structures ''Tumor-induced Bronchus-Associated Lymphoid Tissues'' (Ti-BALT) as they were not observed in non-tumoral lung. More importantly, we demonstrated that a high density of these structures was associated with long-term survival in lung cancer patients. Thus, we wanted to explore the structural and cellular organization of these structures compared to canonical secondary lymphoid organ, as well as to determine which chemokines and receptors are involved in Tand B-cell recruitment into Ti-BALT. By flow cytometry and confocal microscopy, we showed that Ti-BALT are predominantly composed of CD4+ TIL with naive and central-memory phenotypes. The ratio CD4/CD8 T cells is unchanged in Ti-BALT whereas the ratio CD45ro/CD45ra T cells is much more in favor of memory T cells in Ti-BALT compared to lymph nodes. We also demonstrated that TIL in or out Ti-BALT have a distinct chemokine receptor profile. This data is in accordance with the expression of chemokines on micro-dissected Ti-BALT versus non-Ti-BALT areas. A better knowledge about the immunologic function of tertiary lymphoid structures in a tumor context is an important prerequisite for the development of immunological intervention strategies involving creation or suppression of these structures Disclosure of interest: None declared. S198 Inflamm. Res. Today, inhibition of angiogenesis is an important approach for cancer treatment and prevention. Epidemiological and animal studies have indicated that consumption of Allium plants like shallot is associated with a reduced risk of cancer development. Here, we investigated the effects of aqueous extract of shallot on critical steps and mediators of in vitro angiogenesis. Human umbilical vein endothelial cells were treated with aqueous extract of shallot at 100-1,500 lg/ml for antiproliferation, anti-migration and anti-tubulogenesis. In addition the effect of the extract on VEGF secretion, matrix metalloproteinases (MMP-2, 9) expression was assayed using Elisa, gelatin zymography and RT-PCR, respectively. Treatment with aqueous extract of shallot at 500 lg/ml and higher concentrations resulted in significant decrease of endothelial cells proliferation, migration and tubulogenesis. Moreover this extract showed a dose dependent inhibitory effect on VEGF secretion and MMP-2, 9 expression. Altogether, these results indicated that shallot is a potent anti-angiogenic herb of genus Allium. Aqueous extract of shallot exerts its inhibitory effect through reducing production of essentials mediators of angiogenesis including VEGF and MMPs. Disclosure of interest: None declared. Oral mucositis (OM) is a common side effect of anticancer agents. Amifostine (AMF) is an antioxidant that has being studied for the prevention of cancer chemotherapy toxicity. This study evaluated the effect of AMF on inflammation and xerostomia observed in 5-Fluorouracil (5-FU)-induced OM and its modulating effect on cytokine and inducible nitric oxide synthase (iNOS). Golden siriam hamsters were divided into groups: I: Saline (10 mL/kg, i.p.); II: 5-FU (60 and 40 mg/kg, i.p., days 1 and 2, respectively) + Mechanical trauma (MT) of cheek pouch on day 4; III: MT; IV: AMF (12.5, 25 or 50 mg/ kg, s.c) daily/10 days + 5FU. Animals were weighed daily. The salivary rate was recorded (days 4 and 10). They were killed on the 10th day to observe the macroscopic damage to the mucosa and samples were collected for histopathology, immunohistochemistry, myeloperoxidase activity (MPO), TNF-a and IL-1b tissue concentration. Data were analyzed with ANOVA/Student Newman Keul or Kruskal-Wallis/Dunn's test. P \ 0.05 was accepted. Ethics Committee (01/11). 5-FU was able to induce a significant (p \ 0.05) increase in tissue TNFa (1471 ± 452.7) and IL-1b (6870 ± 2417) compared to saline (0.0 ± 0.0, 787.5 ± 356.1, respectively). AMF (50 mg/kg) significantly reduced (p \ 0.05) the levels of TNFa (428.6 ± 246.3) and IL-1b (395.9 ± 86.06) when compared to 5-FU group. In addition, AMF (50 mg/kg) significantly reduced the MPO activity (1.79 ± 0.32), macroscopic (2.5 [1] [2] [3] [4] [5] ), and microscopic (2[0-3] ) injury, immunoexpression for TNF-a(0[0-1]), ) and iNOS (1[0-3] ), in comparison to ; 3 [2] [3] ; 2 [1] [2] [3] ; 2 [1] [2] [3] ; 2 [2] [3] , respectively). AMF, at all doses used, reversed the salivary rate reduction due to the 5-FU injection, but did not alter weight loss (p [ 0.05). In conclusion, AMF attenuates the inflammatory events observed in the OM and xerostomia induced by 5-FU. This inhibitory effect seems to occur via modulation of TNF-a, IL-1band iNOS. Support: CNPq/CAPES/FUNCAP. Disclosure of interest: None declared. Integrins expressed by tumor cells are strongly implicated in various cell functions including those promoting metastasis and have also been linked to chemoresistance. Produced by activated T cells, natural killer cells, and dendritic cells, IFN-g has a pleiotropic role in the immune response. In addition, IFN-g is a key player in Inflammation as it activates macrophage and neutrophil that then secret Reactive oxygen species and other cytokines. Inflammation is often associated with hypoxia which is link to metastatic, aggressive and resistant cancers. Our studies demonstrated that hypoxia increases the expression of integrins at the cell surface of lung cancer cells. In this study, we describe a new anti-cancer role for interferon gamma (IFNg); we show that IFN-g markedly inhibits the expression of aVb5, aVb3, and b1 integrins on lung cancer cells both in normoxia and in hypoxia, inhibits metastases and invasion, and chemosensitise lung cancer cells in vitro and in vivo. Our studies revealed the anti-invasion and anti-metastatic activity of IFN-g on lung cancer cells as treated cancer cells lose their ability to adhere, proliferate, migrate, invade, form colonies, and are significantly less efficient at infiltrating the lungs. We also demonstrate that IFN-g significantly increases the efficacy of several drugs on resistant cancer cells both in vitro (normoxia and in hypoxia) and in vivo. We propose to combine IFN-g with currently used low efficient chemotherapeutic agents to potentiate their antitumor efficiency for the treatment of chemoresistant tumors. This would allow the use of lower doses of chemotherapy, and consequently prevents their associated side effects. Such IFN-g combined therapies would efficiently eradicate primary and metastatic resistant lung cancers. Disclosure of interest: None declared. Manual therapy applied on knee for 30 min daily for a total of 10 sessions in 2 weeks. Eighteen patients (mean age 50 ± 0.9 years; range 40 to 60 years) received a total of 6 sessions of taping for a total of 2 weeks. Pain with numerous rating system and functional performance using the WOMAC questionnaires was assessed before and immediately after each treatment. The patients were evaluated before and after treatment in terms of pain by using of numerical pain scale (NRS) and functionality using the WOMAC (Western Ontario and McMaster Universities) osteoarthritis index. Results: The scores of all instruments showed significant improvements following treatment in both groups (p \ 0.001). Post treatment changes in relation to baseline scores differ significantly between the two groups. (p \ 0.05).manual therapy was better than taping in treatment of osteoarthritis of knee. Conclusion: Both manual therapy and taping had significant effect on osteoarthritis of knee but manual therapy is better than taping on improvement of pain and disability of this patients. Manual therapy is a noninvasive, complication-free, and cost-effective alternative modality for the conservative treatment of knee osteoarthritis. Keywords: Knee pain, knee osteoarthritis, manual therapy, taping. Disclosure of interest: None declared. Introduction: The inflammatory response induced by Dieffenbachia picta, san ornamental plant, is a result of tissue mechanical lesion caused by calcium oxalate needles. The toxicity arrises from its ability to cause painful edema of oral mucous membranes after chewing on the stem. Angioneurotic tongue edema caused by D. picta sap in live mice was standartized (DIP et al. 2004 (DIP et al. , 2011 . Methods: The tongue edema measures were done with a digital tachymeter, 2 h after topical application (0.1 mL) of D. picta sap. The mice were treated 15 min after edema induction, by injection or topical application of lidocaine 2-10%, bupivacaine 0.5%, ropivacaine 1% (50 ll/tongue). Neonatal mice received daily subcutaneously administration of capsaicin (1% 50 mg/kg) for 5 weeks; bilateral sections (5 mm) of lingual nerve were done in rats, 21 days before tongue edema induction. D. picta-induced mechanical hyperalgesia was assessed in mice by measuring hind paw withdrawal thresholds to an increasing pressure stimulus using an analgesymeter with a wedgeshaped probe. Paw withdrawal thresholds were measured before and up to 30 min after an intraplantar injection of 0.01-0.1 mL of the plant sap associated with anesthetic solutions. Results: All local anesthetic solutions showed anti-edematogenic effects. Partial reduction of tongue edema was observed after pre treatment with capsaicine and after lingual denervation. Sap-induction hipernociception was dose response and completely abolished by ropivacaine. Conclusions The key to reduce D. picta inflammatory reaction is inhibit antidromic vasodilation and axon reflex flare, reducing mastocyte degranulation and release of tachykinins from nerve endings. Ropivacaine maybe showed better results because it seems to function not only as a local anesthetic, blocking neurotransmission in the damaged tissue, but also leading to a local vasoconstriction, as well as an unspecific antagonist response on nociceptors involved in peripheral pain. Disclosure of interest: None declared. We have developed a model to screen for anti-inflammatory effects in the oral cavity. A range of doses (25-1,000 mcg in 0.1 ml) of carrageenan (CG) were injected into the upper lips of anesthetized animals (mixture of ketamine/xylazine, 15/90 mg/kg, subcutaneous), or intraplantarly (ipl) in Holtzman rats (150-180 g, male) at time zero. Control rats (C) received the same volume locally (0.1 ml), either in the lip or the hind paw of physiological saline at time zero. Increased thickness of the lip or hind paw was measured with a digital caliper (Mytutoyo, Japan) for up to 6 h following injections. CG induced a significant and dose-dependent increase in lip thickness in comparison with controls (Delta CG at 500 mcg/site = 4.12 ± 0.05 mm; Delta C = 0.023 ± 0.005 mm) with a maximal effect occurring at 1 h after injection, in contrast to 3 h (Delta CG at 500 mcg/site = 1.68 ± 0.04 mm; Delta C = 0.08 ± 0.004 mm) in rat paws. At these times, non-selective (2 mg/kg indomethacin; 20 mg/kg ibuprofen), selective (12 or 30 mg/kg celecoxib) non-steroidal anti-inflammatory drugs (NSAIDs) or dexamethasone (1 mg/ kg), given s.c. 30 min before CG, reduced oral and paw oedema to a similar extent. Although hind paw oedema is highly predictive of the Osteoarticular diseases (OD) such as arthritis and osteoarthritis are characterized by inflammation leading to articular cartilage destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs) targeting cyclooxygenases (COXs), improve rheumatic symptoms by reducing the production of prostaglandin E 2 (PGE 2 ), the main eicosanoid contributing to pain and inflammation in OD. However these drugs are associated with various side effects (gastrointestinal and cardiovascular toxicities) caused mainly by an impaired balance between COX-1 and COX-2. Thus, the strategy of novel anti-inflammatory drugs development could be to inhibit production of COX substrates without modifying the COX-1 to COX-2 ratio. Group IIA secreted phospholipase A2 (GIIAPLA2) is involved in the synthesis of eicosanoids upstream COXs. This enzyme is known to be involved in the pathogenesis of many inflammatory diseases including OD. We have developed novel GIIAPLA2 specific inhibitors such as the C8 oxadiazolone derivative. C8 inhibits IL-1b-stimulated PGE2 secretion in rabbit articular chondrocytes without modifying the COX-1 to COX-2 ratio. Interestingly, oxadiazolone derivatives have structural homology with PPAR ligands like rosiglitazone. Peroxisome proliferatoractivated receptors (PPARs) are ligand-dependent transcription factors. They have an anti-inflammatory activity in several inflammatory diseases, including OD. We hypothesize that oxadiazolone derivatives could activate PPAR. In order to test our hypothesis, we transfected rabbit articular chondrocytes with a PPAR-responsive construct (PPRE-luc) with/without overexpression of PPARs or PPAR dominant negative mutants. We show that C8 activates the PPREdependent transcription more powerfully than rosiglitazone. C8 effect on PPRE involves at least in part PPARc activation, but not PPARa nor PPARb/d. In conclusion C8 appears as candidate for new antiinflammatory drugs with double potentiality (GIIAPLA2 inhibition and PPAR activation). Disclosure of interest: None declared. Trigeminal ganglion C-fiber neurons bearing TRPV1 channels are selectively destroyed by resiniferatoxin (RTX), a potent capsaicin analogue (Karai et al., 2004) . The current study, approved by UFPR's Animal Use Ethics Committee, assesses the effects of RTX injection into the trigeminal ganglion in inflammatory and neuropathic models of thermal hyperalgesia. Methods: RTX (200 ng) or vehicle (2 ll) were microinjected into the right trigeminal ganglion of anesthetized male Wistar rats (200 g). After injection, rats were submitted to surgery for placement of two loose silk 4.0 ligatures around the right infraorbital nerve. Ipsilateral thermal stimulation of snout was conducted daily by application of either heat (*50°C, from source placed 1 cm from vibrissal pad) or cold (1-s tetrafluoroethane spray) stimuli. Decreased latency for head withdrawal or vigorous snout flicking, or increased facial grooming behavior duration indicated heat or cold hyperalgesia. One week after RTX, other rats received a 50 ll injection of carrageenan (50 lg), formalin (2.5%) or vehicle into the upper lip and carrageenan-induced heat hyperalgesia or formalin-evoked nociception were recorded. Results and conclusion: The eye wipes test confirmed the success of trigeminal ganglion C-fiber deletion in RTX-treated rats (4.1 ± 1.2 wipes) versus vehicle-treated rats (22.8 ± 2.6 wipes). Nerve injury surgery induced heat and cold hyperalgesia in saline-treated rats, starting on Day 2 and lasting until Day 9, but these responses on Days 3, 6 and 9 were abolished in RTX-treated rats. Carrageenan induced heat hyperalgesia in vehicle-treated, but not RTX-treated, rats. Nociceptive responses to orofacial formalin were unchanged by RTX treatment. Thus, trigeminal ganglion neurons expressing TRPV1 are crucial for the development of orofacial inflammatory and neuropathic thermal hyperalgesia. Thiazoles are important class of heterocyclic compounds exhibiting a wide range of biological properties, such as anti-inflammatory, analgesic, antimicrobial, antitumor, anticancer and found in many potent biologically active molecules such as fentiazac, meloxicam (antiinflammatory agent), nizatidine (antiulcer agent), sulfathiazole (antimicrobial agent), ritonavir (antiretroviral drug) and bleomycine and tiazofurin (antineoplastic agents). Literature survey reveals that 1,2,4triazoles represent an important pharmacophore, and play a vital role as medicinal agents showing diverse biological activities such as antiinflammatory, analgesic, antibacterial, antifungal, antitubercular and anticancer. Furthermore, condensed heterocyclic compounds having a thiazolo [3,2-b] [1, 2, 4] triazolering system have also been reported to possess anti-inflammatory and antibacterial activities. Arylalkanoic acids are an important class of NSAIDs, but their use has been associated with gastro-intestinal toxicity. It has been reported in literature that modification of carboxyl function of representative NSAIDs results in retained anti-inflammatory activity and reduced ulcerogenic potential. To reduce their toxic effects a number of derivatives have been prepared and in some of these carboxylic group has been replaced by 5-membered heterocyclic moieties such as thiazole and 1,2,4-triazole. In the present study we have synthesized some newer analogues of 2-(4isobutyl-phenyl)-propionic acid (ibuprofen) having thiazolo-triazole moiety. The tested compounds showed potent anti-inflammatory activity as compared to standard drug ibuprofen at the same oral dose. The compounds showing significant anti-inflammatory activity also showed maximum reduction in severity index along with minimum lipid peroxidation, with no hepatocyte necrosis or degeneration. Thus these compounds represent a fruitful matrix for development of new class of non acidic anti-inflammatory agents. Disclosure of interest: None declared. Introduction/aim: Riparin I (ripI) is an alcamid compound that was isolated from unripe fruit of Aniba riparia. We decided to evaluate the anti-inflammatory effect of ripI in models of inflammation based on a prior anti-inflammatory effect shown in the second phase of the formalin test (Araujo et al., Naunyn-Schmied Arch Pharmacol, 2009) . Methods: Male mice (20-25 g) were pre-treated with vehicle (tween 80 3%, i.p.), ripI-25 mg/kg, i.p., ripI-50 mg/kg and indometecin 10 mg/ kg, i.p. or cyproheptadine 10 mg/kg, i.p. One hour after treatment, the paw edema was induced by intraplantar injection of 50 lL of carrageenan 1%, dextran 1.5%, histamine 4 lg/lL or serotonin 4 lg/lL. The edema volume was measured in microliter s(lL). Data were analyzed by two-way ANOVA followed by Bonferroni as the post hoc test. This work was approved by the local ethics committee (protocol number 15/09). Results: RipI decreased carrageenan-induced paw edema at 4 h (ripI-25: 35.71 ± 9.72; ripI-50: 44.29 ± 12.12) as compared to control (95.71 ± 10.66). RipI inhibited dextran-induced paw edema at 3 h (ripI-25: 60.0 ± 7.99; ripI-50: 60.5 ± 5.84) and 4 h (ripI-25: 43.5 ± 7.56; ripI-50: 45.0 ± 6.7) comparing to controls (3 h: 90.5 ± 2.73; 4 h: 72.0 ± 4.95) . Also, ripI decreased the edema induced by histamine in all measure times, 15 min (ripI-25: 35.5 ± 4.56; ripI-50: 40.0 ± 9.63), 30 min (ripI-25: 26.0 ± 3.23; ripI-50: 21.00 ± 3.39), 60 min (ripI-25: 17.50 ± 5.97; ripI-50: 26.00 ± 5.36) and 90 min (only ripI-25: 11.50 ± 3.16) as compared to controls (15 min: 64.0 ± 4.46; 30 min: 56.0 ± 4.87; 60 min: 47.50 ± 6.29; 90 min: 43.00 ± 6.37) but did not alter serotonin-induced paw edema. Conclusion: RipI is more effective on dextran-induced edema whose main mediators are serotonin and histamine. RipI decreased the histamine-induced paw edema but not serotonin-induced paw edema showing that its anti-inflammatory effect is mediated, in part, by inhibiting the actions of histamine. There is accumulating evidence that substance P (SP) released from trigeminal ganglia neurons participates of orofacial inflammatory pain. Here, we have investigated the ability of SP to induce orofacial nociception and thermal hyperalgesia and the role of NK1 receptors on two models of orofacial inflammatory pain. Male Wistar rats (200-250 g) were used in all protocols, which were previously approved by the UFPR's Committee on the Ethical Use of Animals (authorization # 424). Rats, pre-treated or not with captopril (5 mg/kg, i.p.), received SP (1-100 lg/50 ll, into the upper lip) or vehicle and were placed in observation cages for evaluation of the facial grooming behavior up to 15 min. Thirty minutes later, rats were submitted to ipsilateral application of either heat (*50°C heat source placed 1 cm from vibrissal pad) or cold (1-s tetrafluoroethane spray) stimuli to the snout. Heat and cold hyperalgesia were estimated as decreases in the latency to display head withdrawal or vigorous snout flicking, or increases in duration of bilateral facial grooming behavior, respectively. Additional groups of rats received the selective NK1 receptor antagonist SR140333 (10 lg/50 ll or 1-3 mg/kg) 30 min before the injection of carrageenan (50 lg/50 ll) or SP (1 lg/50 ll) or 1 h before the injection of formalin (2.5%, upper lip) and the heat hyperalgesia or formalin-induced nociception was assessed. SP (at 1 lg/50 ll) induced heat hyperalgesia, but failed to evoke significant facial grooming or cold hyperalgesia, even in rats pre-treated with captopril. Systemic treatment with SR140333 prevented SP and carrageenan-induced heat hyperalgesia and reduced by about 50% the first and the second phase of nociception induced by formalin. Our results suggest that NK1 receptors may represent a potential target for the control of orofacial inflammatory pain. Lower third molar extraction provides a clinical model for studying analgesic drugs such as non-steroidal anti-inflammatory drugs (NSA-IDs). In this double-blinded, randomized, crossover investigation, 47 patients received ketorolac sublingually and piroxicam sublingually during two separate appointments after lower third molar extraction of symmetrically positioned lower third molars. A surgeon evaluated objective parameters and patients documented subjective parameters comparing postoperative results for a total of 7 days after surgery. Patients treated with either sublingual ketorolac (40 mg) or piroxicam (20 mg) reported low pain scores during postoperative period. Similarly, the subset of patients who received ketorolac sublingually exhibited similar average amount of analgesic rescue medication (paracetamol) when compared to the subset of patients who received piroxicam sublingually (p [ 0.05). Additionally, values for mouth openings measured just prior to surgery and immediately following suture removal 7 days later were similar among patients (p [ 0.05), and the type of NSAID used in this study showed no significant differences between swellings on the second or seventh postoperative days (p [ 0.05). In summary, pain, trismus and swelling after lower third molar extraction, independent of surgical difficulty, could be successfully controlled by ketorolac administered sublingually (40 mg) or piroxicam administered sublingually (20 mg) and no significant differences were observed between the type of NSAID used in this study. Disclosure of interest: None declared. We have previously shown that i.art. injected CFA can cause secondary mechanical and thermal hyperalgesia in the mouse paw via distinct TRPV1-and TRPA1-dependent mechanisms (Keeble et al., 2005; Fernandes et al., 2010) . We are now trying to establish a link between primary mechanical hyperalgesia and blood flow in animals with arthritis. CD1 mice (12 weeks old) received a unilateral intraarticular injection of CFA (10 ll/joint; ipsilateral joint) in the knee. The contralateral joint received 10 ll of saline. Primary hyperalgesia was assessed by using a pressure application measurement device once a week, over 4 weeks. Animals developed primary mechanical hyperalgesia for 3 weeks after arthritis induction. We also assessed blood flow in the knee joint in these animals by using a Moor laser Doppler flowmeter. Two weeks after arthritis induction, baseline knee blood flow was recorded. At this time point, primary hyperalgesia was more accentuated in the ipsilateral joint. We found that blood flow was increased in both ipsilateral and contralateral knee joints (1.3and 1.7-fold increase, respectively). Interestingly, blood flow recordings were 25% higher in the contralateral when compared to the ipsilateral joint. We therefore, suggest a link between primary hyperalgesia and blood flow regulation. Further investigations are necessary to fully understand the link between blood flow and pain in arthritis. Work supported by the Arthritis Research UK and British Heart Foundation. Keeble, et al. Arthr Rheum. 2005; 52:3248-56. Fernandes, et al. Arthr Rheum. 2010 Recent evidence suggests a potential interaction between the TRPA1 and TRPV1 receptors as the TRPA1 receptor is known to be coexpressed in 50% of all TRPV1-positive sensory neurons [1] . Both receptors are activated or sensitised in inflammation and involved in cutaneous neurogenic vasoactive responses [2] . This study investigated (i) if in vivo deletion of TRPA1 affects TRPV1 (capsaicin)mediated responses and (ii) the role of TRPV1 in TRPA1 (mustardoil)-induced cutaneous blood flow in pharmacogenetic studies. Peripheral blood flow was measured, as a marker of in vivo activation in the ears of anaesthetised mice using laser Doppler techniques for 30 min following topical application of agents. Ear swelling was also determined as a measure of oedema formation. Topical application of capsaicin (10 mg/ml) induced a significant (p \ 0.05) and similar increase in blood flow and swelling in the ipsilateral ears of wild-type and TRPA1 knockout mice. Furthermore, mustard-oil (1%) induced vasodilatation was greater in TRPV1 knockout and in CD1 mice pretreated with TRPV1 antagonist SB366791 (5 mg/kg, i.p., 2 h) as compared to wild-type and CD1 mice pre-treated with vehicle (2% DMSO, i.p., 2 h), respectively. No significant change in oedema formation was observed. This study provided evidence that capsaicininduced vasodilatation and swelling do not involve TRPA1 receptor. Our previous findings showed that mustard-oil induced vasodilatation was dependent on TRPA1 and vasodilator neuropeptides [2, 3] . The present study shows that TRPV1 may also be involved in regulating TRPA1-mediated neurogenic responses in mouse vasculature. Our results highlight that genetic deletion or pharmacological blockade of TRPV1 may potentiate TRPA1-mediated responses in the mouse skin at least. This study was supported by a BBSRC-led IMB capacity building award & the BHF. 1. Andersson, et al. J Neurosci. 2008; 28:2485 -89. 2. Grant, et al. Eur J Pharmacol. 2005 507:273-80. 3. Pozsgai, et al. Cardiovasc Res. 2010; 87:760- Diterpenoids extracted from the red algae Laurencia sp. were isolated and tested for their anti-inflammatory and analgesic properties in models of visceral pain. The structure and the relative stereochemistry of this compound were established by means of spectral and data analyses. Methods: The analgesic effects of the diterpenoid extract were evaluated on two in vivo models of abdominal pain induced by the intraperitoneal injection of acetic acid (1%) or visceral pain induced by the intracolonic administration of mustard oil (0.001%). In vitro, calcium mobilization recordings in primary cultures of mouse dorsal root ganglia sensory neurons were performed. Results: Mouse treatment with the diterpenoid extract (intraperitoneal, 1 mg/kg per mouse) inhibited nociception induced by both acetic acid and mustard oil. Further, the pretreatment of sensory neuron with this compound (300 lM) inhibited significantly free intracellular calcium mobilization induced by PAR 2 agonist (200 lM) or histamine (100 lM) treatment. The effects of the Laurencia sp. extract treatment on histamine-induced calcium flux was blocked by naloxone, an antagonist of opioid receptors. The use of specific antagonist of opoids receptors delta, Kappa and mu (naltrindole, nor-binaltorphimine dihydrochloride and CTOP) confirmed the implication of mu and kappa opioid receptors. Conclusions: These results showed strong analgesic effects for a diterpenoid extracted from red algae, demonstrating opioid receptor activation properties for this natural product. This diterpenoid extract could be considered as a new natural product that could be use in the management of pain. Disclosure of interest: None declared. The California bay laurel (Umbellularia californica), has been known by native Americans as the headache tree', because the scent of its leaves produces violent headache crises. However, the mechanism by which U. californica causes headache is unknown. We recently described a case of cluster headache-like attacks triggered by the inhalation of the scent of U. californica. The major volatile constituent of the leaves of U. californica is umbellulone, a monoterpene ketone. The irritant properties of umbellulone are recapitulated by its ability to affect respiration, heartbeat, and blood circulation in laboratory animals, eventually causing death. Despite the b,b-dialkyl substitution, a structural feature known to suppresses Michael reactivity, umbellulone reacted quickly with thiols, suggesting that it could act as a trigeminovascular stimulator via TRPA1 activation. We found that umbellulone selectively stimulates TRPA1 expressing HEK293 cells and rat trigeminal ganglion neurons, but not untransfected cells or neurons after TRPA1 blockade. Umbellulone also released CGRP from trigeminal nerve terminals. In wild type mice, but not in TRPA1 deficient mice, umbellulone excited cultured trigeminal neurons. Finally, the increase in rat meningeal blood flow induced by umbellulone applied to the rat nasal mucosa was selectively inhibited by TRPA1 and CGRP receptor antagonists. The TRPA1-dependent ability of umbellulone to activate the trigeminovascular system, thereby causing nociceptive responses and CGRP release, represents a plausible mechanism for U. californica-induced headache. Disclosure of interest: None declared. Aim: The aim is to underline the importance of the paediatric dentist and orthodontist in the contribution to the early diagnosis of Psoriatic Arthritis (PA), avoiding and preventing the orofacial and systemic complications. Materials and methods: Psoriatic Arthritis is a chronic systemic disease that is difficult to detect. The diagnosis is made mainly on clinical grounds based on the findings of psoriasis and inflammatory arthritis of the joints. Many reports have described the damaging effects of PA on the temporomandibular joints (TMJs), but no study has clearly reported the TMJ as the first articulation to be involved in PA. This work reports a case of PA that was diagnosed several years after a TMJ onset because no other signs apart from psoriasis were present. Results: The missed early diagnosis resulted in severe TMJ damage. The TMJ can be the first joint involved in PA. It is often unilateral, with a sudden onset. Symptoms include pain and tenderness of the joint area and the muscles of mastication, morning stiffness, tiredness in the jaws, joint crepitation, occasional painful swelling of the TMJ capsule and painful mandibular movements associated with a progressive decrease in the interincisal opening. In severe cases, ankylosis of the TMJ may occur. Discussion: the diagnosis of PA of the TMJ is difficult and is made mainly on the basis of the systemic presentation of the disease. In general, the diagnosis is based on a triad of psoriasis, radiographic evidence of erosive polyarthritis, and a negative serologic test for RF. Conclusions: For a correct, early diagnosis of PA, collaboration between the dentist and rheumatologist it is very important. The dentist should recommend in addition to exercise and local pain treatment, an occlusal splint to help keep the TMJs working properly, improve function, relieve pain, reduce swelling, and prevent further severe TMJ damage. Disclosure of interest: None declared. Envenoming caused by snake from Bothrops sp. induces local effects in the victims, which are represented by intense edema formation, pain and necrosis. The most effective treatment for Bothrops snakebites is antivenom (AV) therapy. However, this procedure does not reverse the local reaction caused by the venom. Other alternatives are being investigated with the purpose of diminishing the local effect induced by bothropic snakebites that include the LED therapy. The aim of this study was to investigate the effectiveness of LED therapy to revert the edema formation and hyperalgesia caused by Bothrops moojeni venom (BmV). Also, the effect on edema formation and hyperalgesia induced by BmV when the LED treatment is combined with the use of AV was also evaluated. Male swiss mice were used. To measure the edema formation and hyperalgesia, 1 lg/paw (50 lL) of BmV was injected into the right paw of mice. The edematogenic effect was evaluated by pletismograph at 15, 30 min, 1, 3, 6 and 24 h after BmV injection or saline. The LED therapy on hypernociceptive effect induced by BmV was evaluated using a Von Frey test applied before, 1, 3, 6 and 24 h after venom injection. The animals had been treated with red LED (k635 nm, density of energy of 4 J/cm 2 , irradiation time of 41 s and area of 1.2 cm 2 ) and infrared LED (k945 nm, density of energy of 4 J/cm 2 , irradiation time of 38 s and area of 1.2 cm 2 ) applied: 30 min and 3 h after the venom injection. The results showed that paw edema and hyperalgesia caused by BmV was significantly reduced by red and infrared LED. Treatment with AV did not modify the edema formation or hypernociception caused by BmV. The combined therapy with LED and AV did not modify the LED effect. In conclusion, both LED therapy significantly reduced edema formation and hyperalgesia caused by BmV, suggesting that LED therapy should be considered as a potentially therapeutic approach for the local effects of Bothrops species. Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and, with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated following tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals. Disclosure of interest: None declared. The Transient Receptor Potential Vanilloid 1 (TRPV1) receptor is expressed predominantly on sensory nerve fibres and is activated by a variety of noxious stimuli including heat[43°C, protons and a variety of inflammatory mediators. Therein, TRPV1 plays an integral role in the pain response and, thus, has been highlighted as a therapeutic target for the treatment of pain. However, TRPV1 antagonists cause hyperthermia in humans and animals which is associated with increased thermogenesis and decreased cutaneous blood flow (Romanovsky et al., 2009) . The present study has investigated the effect of the TRPV1 antagonists SB366791 (Gunthorpe et al., 2004) on thermal nociception, body temperature and capsaicin-induced vascular responses in mice. Female, CD1 mice (Charles River, UK) were used for all experiments in accordance with the Scientific Procedures Act 1986. Mice were injected i.p. with SB366791 (5 mg/kg) or vehicle (10 ml/kg). Thermal nociceptive responses in both paws were measured 4 h after injection of SB366791 or vehicle using the Hargreaves test. Body temperature was measured using radiotelemetry via subcutaneously implanted transmitters (DSI). For measurement of vascular responses, capsaicin or vehicle was applied to the ears and exposed knee joint synovial membranes of urethane-anaesthetised mice and blood flow was measured by laser Doppler imagery (Moor instruments). SB366791 increased thermal nociceptive thresholds (P \ 0.05) without increasing core body temperature. Concomitantly, SB366791 had no significant effect on capsaicin-induced vascular responses. Thus, to conclude, SB366791 can increase thermal nociceptive thresholds in mice without increasing core temperature. This may be due, at least in part, to the lack of inhibition of vascular responses by SB366791. Gunthorpe, et al. Neuropharmacology. 2004; 46:133-49. Romanovsky, et al. Pharmacol Rev. 2009; 61:228- Introduction/aim: Riparin II (ripII) is a alkamide compound isolated from Aniba riparia, collected from the Amazonas. We decide evaluate the anti-inflammatory effect of riparin II in animals models. Methods: Male rats (180-240 g) were divided into groups: controls (tween 80 2%), ripII 25 mg/kg, ripII 50 mg/kg and indometacin 10 mg/kg, cyproheptadine 10 mg/kg or dexametasone 5 mg/kg. All drugs were injected orally. The anti-inflammatory activity of ripII were studied in animals models of inflammation: carrageenan-and dextraninduced edemas (Winter et al., 1962) , carrageenan-induced peritonitis (Souza and Ferreira, 1985) and MPO and Protein measurement (Bradley et al., 1982) and (Lowry et al., 1951) . Data were analyzed using one-way ANOVA and Newman-Keuls test. This work was approved by the local ethics committee (protocol number 40/10). Results: The previous administration of ripII reduced the paw edema (2 h: ripII25: 2.258 ± 0.284; ripII50: 2.161 ± 0.1769; Ctrl: 3.54 ± 0.2706; 3 h: ripII25: 2.683 ± 0.2308; ripII50: 2.31 ± 0.2151; Ctrl: 4.18 ± 0.2459; 4 h: ripII25: 2.615 ± 0.2683; ripII50: 2.549 ± 0.255; Ctrl: 4.271 ± 0.309). The pre-treatment with ripII 25 and 50 mg/kg had a reduction in edema formation by dextran (2 h: ripII25: 2.212 ± 0.1957; ripII50: 2.047 ± 0.3502; Ctrl: 3.371 ± 0.2737; 4 h: ripII25: 2.108 ± 0.2062; ripII50: 1.71 ± 0.3759; Ctrl: 3.356 ± 0.3457). The treatment with ripII 25 and 50 mg/kg had a decreased in migration of leucocytes to peritoneal cavities and the amount of total protein: ripII25: 8.750 9 10 6 ± 1.027 9 10 6 ; ripII50: 6.88 9 10 6 ± 854,342; Ctrl: 1.145 9 10 7 ± 1.042 9 10 6 ; ripII25: 59.2 ± 11.4; ripII50: 53.31 ± 12.12; Ctrl: 91.28 ± 5.534, respectively. RipII decreased the MPO activity (Ctrl: 240. 5 ± 26.86; ripII25: 173.8 ± 13.59; ripII50: 190 .0 ± 6.063).Conclusion: RipII in both doses was able to reduce the edemas induced by carrageenan and dextran. RipII reduced the influx of leukocyte and myeloperoxidase activity as well as the protein extravasations in response to carrageenan. Oxaliplatin (OXP), a platinum-based agent, plays a wide range of antitumor activities, limited due to a prominent peripheral sensory neuropathy (PSN). Amifostine (AMF) is an antioxidant agent used in management of some anticancer side effects. This study aimed to evaluate the protective effect of amifostine on OXP-induced PSN. The study was approved by the Ethics Committee (protocol 27/08). Swiss male mice (25-35 g) were divided into 3 groups, I: Saline (0.1 mL/kg, i.v.); II: OXP (1 mg/kg, i.v.) twice a week for 4.5 weeks; III: AMF (1, 5, 25, 50 or 100 mg/kg, s.c.) injected 30 min before OXP. Mechanical hyperalgesia, thermal allodynia and Rota-rod tests were performed weekly. Histopathology for dorsal horn of spinal cord and immunohistochemical assay for Fos, CASP3, IL-1b, nitrotyrosine, iNOS, nNOS and NMDA was performed. OXP significantly decreased mechanic and thermal nociceptive threshold from 14th day on (p \ 0.01) when compared to control group. AMF treatment inhibited these effects in all doses tested (p \ 0.001), and the best dose was 25 mg/kg, which increased mechanic (94.33% of AUC) and thermal (100.85% of AUC) nociceptive threshold compared to OXP group. Locomotor impairment was not evidenced through Rota-rod test. Furthermore, we observed edema and neurons atrophy in dorsal horn of OXP group, not showed in control and improved in AMF group. OXP group had overexpression of Fos, CASP3, nNOS, iNOS, and nitrotyrosine, but a reduced NMDA expression when compared to control group. AMF group had hypoexpression of Fos, nitrotyrosine and increased NMDA expression, but not altered CASP3, nNOS, iNOS expression when compared to OXP group. These results suggest that AMF protects tissue and sensory changes on OXP-induced NSP, probably through an antioxidant effect, since it inhibited c-Fos, nitrotyrosine expression, and through an antiapoptotic signaling mediated by NMDA receptor. Support: CNPq/CAPES/FUNCAP. Disclosure of interest: None declared. Peroxisome Proliferator Activated Receptors (PPARs) have been arisen as promising targets to treat chronic inflammatory diseases acting by mechanisms pro-resolution. Fasted swiss and BALBc mice (20-30 g) were used in in vivo tests of inflammation and pain (n = 7-10 animals/ group; *p \ 0.05 ANOVA one-way). Compounds were orally administered at a dose of 100 lmol/kg. All the studies have been approved by the UFRJ ethic committee. In vitro studies for TNF-a were carried out in LPS-stimulated murine macrophages (100 ng/ml) (Gallily, JPET:283, 1997). Among seven derivatives evaluated in the writhing test (Coolier, Br J Pharmacol Chem:32, 1968) we emphasize the antinociceptive effect displayed by LASSBio-1474 and LASSBio-1470 which inhibited the writhing behavior in 50 and 30%*, respectively. The PPAR-a agonist fenofibrate inhibited by 30%*. In order to better characterize the antinociceptive and anti-inflammatory profile of the compounds, we performed the formalin-induced nociception and carrageenan-induced peritonitis tests (Tjolsen, Pain:51, 1992; Vinegar, Proc SEBM:143, 1973) . LASSBio-1523 inhibited the neurogenic phase by 30% without interfering with inflammatory phase. LASSBio-331, LASSBio-1471 and LASSBio-1474 significantly inhibited the inflammatory phase in 72, 42 and 53%*, respectively. These compounds also inhibited the S206 Inflamm. Res. leukocyte migration in peritonitis such as Rosiglitazone, a PPARc agonist. Compounds were evaluated in TNF-a production given the ability of PPAR agonists modulate inflammatory cytokines. LASSBio-1473 and LASSBio-1474 (100 lM) inhibited the TNF-a in 83%*, being more effective than thalidomide (300 lM; 67% of inhibition), an anti-TNF-a drug. None of the compounds showed cytotoxicity. This study identified derivatives with anti-inflammatory and antinociceptive activities that can be useful in treating inflammatory conditions and chronic pain associated with diseases such as diabetes. Hymenoptera venoms are complex mixtures of biochemically and pharmacologically active components such as biogenic amines, peptides and proteins. Some of these peptides are involved with the activation of different basophiles types, chemotaxis of polymorphonucleated leukocytes, smooth muscle contraction and neurotoxicity. Stinging accidents produce acute pain, local tissue damage and occasional deaths in large vertebrates, including man. The goal of this work is to evaluate the hemolytic, mast cell degranulation, chemotaxis, antinociceptive/nociceptive, and inflammatory/antiinflammatory effects of small peptides such as Protonectin, Protonectin (1-5)-OH and Protonectin (1) (2) (3) (4) (5) (6) , isolated from the social wasp Agelaia pallipes pallipes venom. Also, some of the mechanisms involved in nociceptive and inflammation phenomena were evaluated for these peptides. Hyperalgesia and edema were determined, after application of peptides, by electronic von Frey apparatus and a paquimeter. Results showed that Protonectin, Protonectin (1-5)-OH, and Protonectin (1) (2) (3) (4) (5) (6) induced hemolytic, mast cell degranulation and chemotactic activity. Also, these peptides produced a hyperalgesic and edematogenic responses. Both phenomena were detected 2 h after peptides injection. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that indomethacin blocked protonectin-induced hiperalgesia and induced a decrease of the edematogenic response. Zileuton inhibited protonectin (1-5)-induced hyperalgesia and decreased oedema. On the other hand, zileuton and indomethacin did not interfere with these phenomena using Protonectin (1) (2) (3) (4) (5) (6) Introduction: Alcoholic neuropathy is one of the devastating complications of long term alcohol consumption which involves decreased nociceptive threshold characterized by spontaneous burning pain, hyperalgesia and allodynia. Alcoholic neuropathy has been associated with behavioral alterations, increase in oxidative-nitrosative stress and pro-inflammatory cytokines. Objectives: The present study was designed to explore the protective effect of epigallocatechin-3-gallate against alcoholic neuropathy in rats. Methods: Adult male Wistar rats (150-200 g) were administered alcohol (10 g/kg, 35%, v/v) for 10 weeks and pain was measured by using Tail immersion test, Randall-Sellito test and von-Frey hair test at 6th, 8th and 10th week. After 10 weeks, rats were sacrificed under deep anesthesia and sciatic nerves were isolated for various biochemical and molecular estimations. Results: Chronic alcohol (35%) treated rats developed neuropathy after 6 weeks, which was evident from decreased tail flick latency (thermal hyperalgesia), paw withdrawal threshold in Randall-Sellito test (mechanical hyperalgesia) and von-Frey hair test (mechanical allodynia) along with enhanced in oxidative-nitrosative stress and inflammatory mediators (TNFa, IL-1b and TGF-b1 levels). Coadministration of epigallocatechin-3-gallate significantly and dosedependently prevented behavioral, biochemical and molecular changes associated with alcoholic neuropathy. Conclusions: The current findings suggest the neuroprotective potential of epigallocatechin-3-gallate in attenuating the behavioral and biochemical alterations associated with alcoholic neuropathy through modulation of oxido-inflammatory cascade. Disclosure of interest: None declared. Antihistamines have been shown to produce analgesia in some situations, but also hypernociception in experimental models, which may explain why they are not always effective analgesics in clinic situations. Herein, we investigated the effect of antihistamine on the articular incapacitation, edema, and plasma leakage (PL) induced by formalin in the rat knee joint. Low and high doses of the brain permeant H1 antihistamine promethazine increased and decreased the incapacitation, respectively. However, both non-permeant H1 antihistamines loratadine and cetirizine only increased this formalin effect. None of the treatments changed the edema and PL. Loratadine given locally with formalin increased incapacitation, but had no effect when administered in the contralateral knee. Systemic loratadine was also without effect when formalin was co-injected with sodium chromoglycate. Histamine (0.2 and 2 nmol/knee), and the selective H1 agonist 2-pyridylethylamine (0.05; 0.5 and 5 nmol/knee) decreased incapacitation with no change in the edema or PL. Cetirizine prevented the antinociceptive effect of the H1 agonist. Serotonin (1, 10 and 100 nmol/knee) administered alone did not cause incapacitation, but increase the edema and the highest dose changed the PL. When co-injected with formalin, serotonin only enhanced incapacitation and the highest dose also increased the edema. NAN 190 (0.1 and 0.6 pmol/knee), cyproheptadine (0.16 and 0.5 pmol/ knee), and ondansetron (0.26 pmol/knee) decreased the incapacitation without changing the edema or PL. These results suggest that in the rat knee joint the histamine H1 receptor seems to play an antinociceptive role, while serotonine is important for the formalin-induced effect. Disclosure of interest: None declared. Menstrual-related migraine headaches occur in about half of all women migraineurs. The mechanisms that play a role in mediating the increased propensity of women to develop migraine headaches during this hormonal phase is unknown, but the abrupt decline (withdrawal) in estrogen levels during the premenstrual phase has been suggested to play a role. Mast cells (MCs) play an important role in inflammation and are found throughout the intracranial meninges where they are thought to play a key role in migraine headache triggering. We postulated that changes in meningeal MC density could serve as one factor that modulates the propensity to develop migraine headache such as during menstrual migraines. We therefore examined whether changes in estrogen levels modulate the density of meningeal MCs and whether this effect increases the propensity of meningeal sensory neurons to become activated by common migraine triggers. In cycling female rats, meningeal MCs exhibited fluctuations in their density with increases noted during the stages of reduced estrogen levels of estrous and diestrus 1. An increase in meningeal MC density was also noted in ovariectomized (OVX) females subjected to an experimental estrogen repletion and withdrawal paradigm. In electrophysiological experiments, systemic administration of the migraine triggers nitroglycerin or CGRP resulted in enhanced activation of meningeal sensory neurons during diestrous 1 compared to the high estrogen stage of proestrous. Migraine triggersevoked neuronal responses were also enhanced in OVX animals subjected to exogenous estrogen withdrawal paradigm when compared to untreated OVX rats. The MC stabilizing agent sodium cromolyn or depletion of meningeal MCs ameliorated the estrogenrelated enhanced responses of meningeal sensory neurons. We propose that estrogen-related recruitment of MCs to the meninges play an important role in the pathophysiology of menstrual migraines. Disclosure of interest: None declared. Hemorrhagic cystitis (HC) is a common side effect observed in patients under chemotherapy with cyclophosphamide (CYP). The urotoxic side effects of CYP are attributed to the metabolic compound acrolein and can be partially prevented by the uroprotector agent 2-mercaptoethene sulfate (Mesna). This study analyzed the anti-inflammatory and the antinociceptive effects of the selective CXCR2 and TRPV1 receptor antagonists-SB225002 and SB366791-in the rat model of CYPinduced HC. Male Wistar rats were used (200-220 g) and HC was induced by a single injection of CYP (200 mg/kg, ip). Breathing rate, closing of the eyes, and specific posture were scored at different time points after cystitis induction as nociception indexes. The mechanical hypernociception was measured with Von Frey filaments in the bladder area and in the rat paw. As inflammatory parameters, hemorrhage presence, edema formation, and bladder weight were determined macroscopically at 24 h after CYP administration. The neutrophil migration and inflammatory cytokines profile was assessed 4 h after cystitis induction by means of myeloperoxidase (MPO activity) and IL-1b and TNFa, respectively. As expected, Mesna treatment was able to reduce in a significant manner all the inflammatory and the nociceptive parameters induced by CYP. Of note, the pre-administration of SB225002 (1 mg/kg ip) and SB366791 (500 lg/kg ip) significantly attenuated the hemorrhage, the edema formation and the neutrophil recruitment, as well the increase in MPO activity and the elevation of IL-1b. Interestingly, the treatment with either SB225002 or SB366791 markedly reduced the bladder wet weight and the nociceptive responses. The present results indicate that CXCR2 and TRPV1 antagonists might represent important alternatives to prevent nociception following chemotherapy with CYP. Disclosure of interest: None declared. Background: Osteoarthritis (OA) is a degenerative joint disease causing pain and disability. Inflammatory mediators are responsible for synovitis and pain transmission, and contribute to the pathology development. Aim: This study aimed to evaluate the effect of intra-articular administration of the kinin B2 receptor antagonist MEN16132 and dexamethasone on pain response in the experimental model of OA induced by monosodium iodoacetate (MIA) in rats. Material and methods: MIA (1 mg) and 7 days later MEN16132 (1-3-10 lg), dexamethasone (10-30-100-300 lg) or their combination were injected in the right knee in a volume of 25 ll. Pain was measured with the incapacitance tester for the following 21 days. Results: MIA was able to produce pain and to increase the levels of bradykinin and prostaglandin metabolite in synovial fluid. MEN16132 or dexamethasone caused a dose-dependent and long lasting analgesia with a maximum effect of about 60% inhibition of pain at the doses of 3-10 and 100-300 lg/25 ll, respectively. In this model MEN16132 was about 30-fold more potent than dexamethasone. The combination S208 Inflamm. Res. of submaximal doses of MEN16132 (1 lg/25 ll) and dexamethasone (10-30 lg/25 ll), each producing a mean 20-25% inhibition of pain, caused analgesia of about 40-45%, ascribable to the sum of the respective antinociceptive effects. Conclusion: Intra-articular combination of low doses of MEN16132 and dexamethasone induces an additive increase of analgesia that might allow to reduce the doses of corticosteroids for the treatment of pain and inflammation associated to diseases such as osteoarthritis. Disclosure of interest: None declared. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the motor neurons of the spinal cord and brain. Degeneration of spinal motor neurons leads to muscular atrophy and weakness without accompanied sensory changes, whereas degeneration of motor neurons in the cerebral cortex leads to hyper-reflexia. Because of its rapid and progressive clinical course, ALS patients generally die within 2-5 years of onset. Matrix metallo proteinases (MMPs) are a relatively large family of more than 20 Zn 2+ -containing, Ca 2+ -requiring endopeptidases, characterized by their ability to digest components of the extracellular matrix. Excessive expression of MMPs may result in tissue damage. Therefore, precise control of the regulation of expression and activity of MMPs is important physiologically and pathologically. ALS patients were selected using the World Federation of Neurology (WFN) clinical criteria. Elisa kit for the estimation of MMP-9 was obtained from R&D Systems, Minneapolis, MN, USA and was measured as per the manufacturer's specifications. Standard curve was prepared as per the instructions and the MMP-9 levels were expressed as ng/ml. Results show that the neuroinflammatory marker, MMP-9 levels in the serum of ALS patients were significantly (P \ 0.01) increased with respect to control subjects and also showed significant correlation (R 2 = 0.9) with the duration of the disease. Neuroinflammation can both be a cause, and a consequence of chronic oxidative stress which is produced by glutamate excitotoxicity. Cytokine-stimulated microglia generate copious amounts of reactive oxygen and reactive nitrogen species, creating a stress upon ambient neurons. Conversely, oxidants can stimulate proinflammatory gene transcription in glia, leading to various inflammatory reactions including production of MMP-9. Disclosure of interest: None declared. A. Banani 1,* , S. Mostafalou 1 , A. Garjani 1 , N. Maleki-Dizaji 1 Introduction: N-Acetylaspartylglutamate (NAAG) is widely distributed peptide transmitter in the mammalian nervous system, activates mGlu3 receptors at presynaptic sites which results to inhibit glutamate release. Evidence suggests that glutamate receptors are involved in inflammatory response carried through the COX-2 up regulation. In present study we aimed to investigate the effect of NAAG (10 mg kg -1 ) on carrageenan-induced acute inflammation. Methods: Acute inflammation was induced by subplantar injection of carrageenan 1% (w/v), in the right hind paw of the rats. The animals received an intraperitoneal injection of NAAG 20 min before induction of inflammation. The severity of inflammation was assessed 4 h later using maximum paw edema volume, Area Under Curve(AUC), microscopic changes of inflamed paw, measurement of oxidative stress markers; myeloperoxidase(MPO), lipid peroxidation(MDA level), SOD and GPx activity in paw tissue. Results and conclusion: NAAG reduced maximum inflammatory response and AUC by 52.56 and 58% (P \ 0.001), respectively, compare to control. Microscopic studies indicated significant reduction in neutrophil infiltration. MPO activity in NAAG treated group was inhibited by 80.19% (P \ 0.01) and MDA level was decreased by 88.13% (P \ 0.01) compare to controls, but there were no significant reduction in SOD and GPx activity. Nitric oxide (NO) is an important signaling messenger with a broad range of functions in both the central and peripheral nervous systems. In the skin, NO is involved in vasodilation, melanogenesis, and immune/inflammatory processes. NO is synthesized from Larginine by 3 isoforms of NO synthase (NOS)-neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Keratinocytes constitutively express nNOS and can be induced to express iNOS by inflammatory cytokines. Preliminary studies using human immortalized keratinocytes (hTERT) demonstrates that iNOS is induced in a time-and dose-dependent manner by UVB and cytokines such as CGRP, substance P, and VIP. To extend these findings, we selected primary cortical neurons as a primary culture paradigm with cellular lineage distinct from keratinocytes. Expression of all NOS isoforms following neuropeptide treatment (24 vs. 48 h) was assessed by Western blot. Primary cortical neurons were treated in vitro with the nociceptive neurotransmitter substance P or bicyclic monoterpene diols (BMTd) reported to increase NO levels in neural crest-derived cells and compared to neurons that received brain-derived neurotrophic factor (BDNF, 100 ng/ll), a growth factor known to increase nNOS. BMTd is known to increase nitric oxide levels in both melanoma and neuronal cells. Compared to BDNF control, 0.1-3 mM BMTd and 1-50 nM substance P selectively upregulated iNOS at the protein level while have less of an effect on nNOS or eNOS. Our results provide additional support for the involvement of iNOS upregulation in the development and testing of agents of interest, which may specifically modulate NOrelated pathways. Disclosure of interest: None declared. Activation of microglia and astrocytes contributes to neuroinflammation observed in a number of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Such chronically activated glial cells secrete an array of cytokines and inflammatory mediators, some of which may be directly toxic to neurons and could therefore exacerbate neurodegeneration. Identifying the neurotoxins released by stimulated glial cells and searching for novel compounds that could be used to inhibit the neurotoxic activity of glia were the goals of this study. We used immortalized monocytic and astrocytoma cell lines to model microglia and astrocytes respectively. In addition, we used primary human astrocytes. Lipopolysaccharide and interferon-gamma, two potent proinflammatory mediators, were used to induce glial cell activation, which was accompanied by secretion of substances that were toxic to neuronal cells. We demonstrated that incubation of supernatants with phospholipase A2 group IIA-specific immunosorbents diminished their toxicity. We also prepared a series of four-coordinate cobalt compounds bearing beta-ketoaminato ligands which varied in the number of -CF3 ligand substituents. We identified a cobalt complex that inhibited the neurotoxic activity of glial cells at low micromolar concentrations without affecting their viability. The anti-neurotoxic activity of this cobalt complex was reduced by specific inhibitors of mitogen-activated protein kinases (MAPK). The corresponding ligand that did not contain cobalt was not effective. Our data indicate that phospholipase A2 group IIA could be one of the toxins responsible for glial cell-induced neurotoxicity. We also identified a novel cobalt-containing compound that inhibited the neurotoxic activity of glial cells in a MAPK-dependent manner. Cobalt-containing metallocomplexes may have potential as anti-inflammatory agents beneficial in neurodegenerative diseases. Supported by NSERC and Jack Brown and family AD foundation. Disclosure of interest: None declared. Introduction: Currently microglial cells have received great attention within the immune responses, due to the fact that its activation by inflammatory cytokines can promote the infiltration and destruction of the central nervous system (CNS) specially in the case of multiple sclerosis (MS). Besides, the it has been shown that they are also capable of expressing suppressive molecules such as indoleamine-2,3dioxygenase (IDO), able to suppress the proliferation of T cells. However, little is known about its true role in the pathogenesis of MS. Aims: This study aims to better understand the relationship between the secretion of cytokines by activated microglial cells and their connection with the pathogenic Th17 cells. Methods: C57Bl6 animals had their spinal cords and brains removed and their mononuclear cells extracted. Later, cells were stimulated or not with LPS or rm-IFN-g, for 24 h. IDO functional activity was also performed after co-cuture with MOG 35-55 specific 2D2 CD4 in the presence or absence of 1-methyl tryptophan, an IDO inhibitor. Results: Our results showed that, except for the TLR-4 and TLR-2, all the other molecules showed a significant increase in its expression after stimulation with LPS, which was not observed after IFN-g, except for TGF-beta and socs3. TLR-4 was decreased after IFN-g. Conclusions: Our data suggest that activation of microglial cells through the TLR-4 is able to induce the synthesis of a series of cytokines both pro-(IL-6, IL-23) as anti-inflammatory (IL-10, IL-27, TGF-b and IDO). The results obtained here shows the ability of inflammatory microglial cells to produce inflammatory cytokines which are important for th17 cells maintenance. On the other hand, it has also a regulatory role, due to its capacity to inhibit T CD4 specific response. In summary, our data show the importance of microglial cells during EAE, both as promoter or regulator of CNS inflammation. Disclosure of interest: None declared. Neuroinflammation occurs as a defensive response to pathogenic insult in brain and plays a crucial role in a variety of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's diseases S210 Inflamm. Res. (PD). Cyclooxygenase (COX), prostanoid-synthesizing enzyme, is well known to be involved in the regulation of inflammatory process. Two distinct COX isoforms, COX-1 and COX-2, are characterized as a constitutively expressed housekeeping enzyme and an inflammatory inducible enzyme, respectively. Recently, changes of COX-1 and COX-2 in the brain of patients with ischemic, traumatic brain injury and of several neurodegenerative diseases such as AD, PD and amyotrophic lateral sclerosis (ALS), have been reported, indicating that not only COX-2 but also COX-1 mediated neuroinflammation is a critical component in neuronal degeneration. In the present study, to determine the involvement of COX-1 in neuroinflammatory process, we performed positron emission tomography (PET) study with 11 C labeled ketoprofen-methyl ester (KTP-Me), its acid form rather specific to COX-1, during neuroinflammatory process of rats which were intracranially injected lipopolysaccharide (LPS) or quinolinic acid (QUIN). In addition, the cell type of COXs expression during neuroinflammation was determined by immunohistochemical studies. In the PET studies, both LPS and QUIN increased 11 C-KTP-Me accumulation in the inflamed hemisphere. It peaked at day 1 after the injury at which microglia/macrophages was activated with COX-1 but not with COX-2 expression. In addition, the increased accumulation of 11 C-KTP-Me was decreased rapidly from day 3 after the injury. This time-course of 11 C-KTP-Me was closely corresponded to that of changes in number of COX-1 expressing activated microglia/macrophages in the inflamed area. These results strongly indicate that COX-1 but not COX-2 expression in microglia/macrophages is critically involved in the neuroinflammatory process in the brain. Disclosure of interest: None declared. Oxysterols are oxidized cholesterol metabolites generated enzymatically or non-enzymatically during bodily metabolic processes. They function as ligands for liver X receptor (LXR), thus perform important roles during lipid metabolism and transport. Recently, anti-inflammatory actions of them attracted the interests of many researchers, but the precise mechanism of actions are largely unknown. In the present study, we unraveled that 22(R)-hydroxycholestrol (HC), a family member of oxysterol, increased MAPK phosphatase (MKP)-1 induction in brain astrocytes. Transcript levels of MKP-1 showed peak levels in 3 h, but protein levels were sustained up to 12 h and more prominent, suggesting that MKP-1 expressions are regulated at post-transcriptional levels. Moreover, experiments using actinomycin D revealed that 22(R)-HC increased MKP-1 mRNA stability in a LXR receptor-independent manner. mRNA stability is regulated by RNA-binding proteins (RBPs), of which HuR is a representative RBP that increase mRNA stability. As expected, 22(R)-HC increased cytoplasmic translocation of HuR in immunohistochemistry and nuclear/cytoplasmic fractionation experiments. Since nucleus/cytoplasm shuttling of HuR is regulated by its phosphorylation on Ser residues in the hinge region, we first tested the involvement of PKC, using its pharmacologic inhibitors. Immunoprecipitation assays using antibodies against phospho-serine and HuR revealed that 22(R)-HC increased cytoplasmic translocation of phosphor-HuR, whereas nuclear phospho-HuR was decreased. Much remains to be clarified to reveal in-depth mechanisms underlying HuR translocation, our data show that 22 ( Purified cultures of these glial cells appeared to proliferate more slowly than those cultured with neurons, therefore the aim of this study was to determine if neurons regulated proliferation and activation of glial cells. Three groups of DRG cells were prepared: mixed DRG cell cultures (neurons were 31 ± 3% total cells); neuron-enriched cell cultures (neurons were 73 ± 5% total cells); and glial cell cultures (glial cells were 97 ± 1% total cells). After 6 days in culture, there was a significant increase in the number of glial cells (as %Day 1) only in the mixed DRG cell and neuron-enriched cell cultures (P \ 0.05). Protein expression and viability (MTT assay) data was also significantly greater in the mixed DRG cell group compared with the glial cell preparation (P \ 0.05). The intensity of GFAP-immunoreactivity increased from Day 1 to Day 7, with no obvious difference between the three cell groups. In conclusion, our results suggest that DRG glial cells proliferate faster in the presence of DRG neurons when assayed in vitro, which may reflect the increased glial cell proliferation reported in intact DRG following nerve damage. Taken together, these results suggest that either intact DRG neurons release factor/s to prevent glial cell proliferation, or damaged DRG neurons release factor/s to stimulate glial cell proliferation. This study was supported by a direct grant from The Chinese University of Hong Kong (CUHK2041584). Disclosure of interest: None declared. Spinal cord injury (SCI) has a significant impact on quality of life, expectancy, and economic burden, with considerable costs associated with primary care and loss of income. The complex pathophysiology of SCI may explain the difficulty in finding a suitable therapy for limiting neuronal injury and promoting regeneration. Although innovative medical care, advances in pharmacotherapy have been limited. The aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective effect of PEA on inflammatory reaction associated with an experimental model of SCI. The compression model induced by applying an aneurysm clip to the spinal cord in mice is closer to the human situation, since it replicates the persistence of cord compression. Spinal cord trauma was induced in mice by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. Repeated PEA administration (10 mg/kg i.p., 6 and 12 h after SCI) significantly reduced the degree of the severity of spinal cord trauma through the reduction of mast cell infiltration and activation. Moreover, PEA treatment significantly reduced the activation of microglia and astrocytes expressing cannabinoid CB 2 receptor after SCI. Importantly, the protective effect of PEA involved changes in the expression of neurotrophic factors, and in spinal cord dopaminergic function. Our results enhance our understanding about mechanisms related to the anti-inflammatory property of the PEA suggesting that this N-acylethanolamine may represent a crucial therapeutic intervention both diminishing the immune/inflammatory response and promoting the initiation of neurotrophic substance after SCI. Disclosure of interest: None declared. Inflammation is involved in a large array of pathological conditions as a homeostatic response aimed to tissue repair and to defend organ functional integrity. Many signals and stimuli, including traumatic events, can be responsible of inflammatory process activation. An uncontrolled and excessive post-traumatic inflammatory reaction may play an important role in the secondary injury processes, which develop after spinal cord injury (SCI). Glucocorticoids (GC), among the most used drugs for SCI patients treatment, exert their antiinflammatory action through the inhibition of lymphocyte activation/ proliferation, production of proinflammatory cytokines and downregulation of specific adhesion molecules resulting in modification of lymphocyte migration and redistribution. Using GILZ transgenic mice, in which GILZ is selectively over-expressed in the T-cell lineage, we show that GILZ antagonizes the development of SCI in mice further suggesting that GILZ is an anti-inflammatory molecule that mediates the effect of GC and represents a potential pharmacological target for immune responses modulation in inflammatory disorders such as SCI. In particular, leukocyte infiltration, NF-jB activation, pro-inflammatory cytokine production and tissue damage were significantly reduced in GILZ-TG mice. These results indicate GILZ acts as an anti-inflammatory molecule in experimental SCI. Results could suggest for new therapeutic approaches alternative to treatment with GC. Disclosure of interest: None declared. Introduction: Scavenger receptor A (SR-A) is involved in recognition of endogenous molecules and pathogens, and inflammatory activation. It was considered to be expressed almost exclusively in myeloid cells. However, we described for the first time the presence of SR-A in astrocytes, which provide key functions for the modulation of the neuroinflammatory response. Results: Astrocytes derived from SR-A -/or SR-A +/+ mice were stimulated with LPS or Polyinositic acid (Poly I, a SR-family-A ligand). In contrast to microglial cells, which showed little dependence on SR-A mediated activation, we demonstrated the participation of SR-A on the activation of NF-jB pathways, and the modulation of nitric oxide (NO) and IL-1b production by astrocytes. MAPKs (JNK, ERK1/2) signaling pathways were evaluated. The absence of SR-A had no effect on the activation of ERK1/2 or JNK induced by inflammatory stimulation. In contrast, NF-jB transcriptional factor was unable to translocate into the nucleus of SR-A -/astrocytes, as it normally does in SR-A +/+ astrocytes exposed to LPS. At the functional level, astrocytes from SR-A -/mice exposed to proinflammatory ligands produced 50% less NO than SR-A +/+ astrocytes, whereas NO production by microglia was similar for both genotypes. On the other hand, LPS or Poly I stimulated SR-A -/astrocytes increased by 4-fold their production of IL-1b compared with SR-A +/+ counterparts. Discussion: Our results show that stimulation with proinflammatory molecules of astrocytes from SR-A -/animals activated MAPK but not NF-jB signaling, resulting in higher levels of IL-1b but lower NO production compared with SR-A +/+ astrocytes. Altogether, we propose that SR-A is involved in the modulation of the inflammatory response of astrocytes. In vertebrates microglial cells are a major glial cell element of the central nervous system (CNS). Reports in animal models underlined their involvement in various steps of neuron repair. The ability of nerve cells to survive damage is different across phyla. Higher vertebrates have a limited ability to regenerate efficiently their CNS. In contrast, some invertebrates as leeches can completely repair its CNS S212 Inflamm. Res. after injury. From a specific leech nervous system EST library, we characterized two specific factors. First, HmC1q (for H. medicinalis), homologous to vertebrate C1q, was detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were shown to respond to human C1q. The effect was reduced when microglia was preincubated with signalling pathway inhibitors (pertussis toxin or wortmannin) or a specific anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration. By using a recombinant HmC1q, the chemotactic effect was shown to be dependent on a specific C1q binding cell surface protein. Second, HmIL-16, a homologous of interleukin-16 (IL-16) human active form was detected in leech nervous system and was involved in microglia recruitment. By using either an anti-human IL-16 antibody or anti-HmIL-16 antibody, the cell migration was significantly reduced. In human, mature IL-16 might bind to CD4 (as receptor) to stimulate migration of immune cells. Interestingly HmIL-16 was shown to promote human CD4 + T cell migration. Indeed an anti-human IL-16 antibody, an IL-16 antagonist peptide or the soluble CD4 highly reduced the chemotactic effect. Thus HmIL-16 effect might be the result of binding to a protein homologous to human CD4. This study emphasizes the role of leech factors in the microglial cell recruitment known to be essential for neuron repair and, in invertebrates, highlights the presence of potential ''cytokines'' that are highly evolutionarily and functionally conserved in CNS. Disclosure of interest: None declared. Rationale: Chronic neuroinflammation has been suggested as a driving force for Alzheimer's disease (AD). AD is a neurodegenerative disease characterized by amyloid-b (Ab) deposition, neurofibrillary tangles, neuronal loss and glial cell (microglia and astrocytes) activation. Ab causes the phosphorylation of MAPKs and activation of NFjB pathway inducing the expression of inflammatory cytokines and reactive species like nitric oxide (NO) that have been shown to accelerate neurodegeneration and contribute to progression of pathology. Negative regulation of MAPK signaling is exerted by MAPK phosphatases (MKPs), being MKP-1 the archetype of the family. TGFb1 is an inflammation modulator whose level is increased in AD. We have previously shown that TGFb1 has a neuroprotective role against Ab toxicity, and recently it has been demonstrated that its signaling pathway is impaired in AD. These findings suggest that TGFb1 has a key role in the pathogenesis of AD, although the molecular mechanism underlying its neuroprotective effect has not been completely elucidated. Here we studied the potential role of MKP-1 in the modulatory effect of TGFb1 on cytokine-and Abstimulated glial cells. Results: Using rat primary glial cultures, we found that TGFb1 prevented the NO production in a time-and dosisdependent manner, being most effective after 48 h of pretreatment. a-Synuclein (a-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system and its dysfunction is related with several neurodegenerative diseases, including Parkinson's disease (PD). The microglial neuroinflammatory responses affect the onset and progression of PD. Nuclear-kB factor (NF-kB) is a transcription factor involved in cellular defense response, apoptosis and regulation of inflammatory genes expression. The aim of the present study is to evaluate the influence of reactive glia cells in the a-syn expression and NF-kB activation in SH-SY5Y cells infected with lentiviral (LV) vectors expressing wild-type (WT) or A30P a-syn. Glial cells were prepared from 1 day Wistar pups. Cortices were isolate and cells were dispersed with 0.25% trypsin. Cells were placed onto flasks and maintained in DMEM supplemented with 10% Fetal Bovine Serum. We challenged SH-SY5Y cells transduced with LV system with conditioned medium (CM) from reactive glia previously treated with LPS (1 mg/ml). The expression of a-syn was evaluated by Western blot assay. Cell nuclear extract was used to run EMSA to measure NF-kB binding activity. Exposures of SH-SY5Y cells transduced with LV vectors expressing wild-type or mutant (A30P) a-syn protein to CM from treated glia cells produced a pronounced increase in a-syn expression when compared to control treated cells. In addition, CM induced an increase in NF-kB activation in nuclear extracts from SH-SY5Y cells infected with LV vectors expressing WT protein when compared to control group. Our results suggest a putative interaction between a-syn expression and NF-kB signaling in response to an inflammatory stimulus. Therefore, a-syn can be converted to a toxic compound in the presence of a chronic inflammatory condition which might be linked to NF-kB signaling cascade. Technical support: LS Lima and DZ Andreotti. Introduction: There is a growing body of evidence indicating the involvement of immune and/or inflammatory dysfunction and impaired neuroplasticity in the physiopathology of bipolar disorder (BD), a condition that affects millions worldwide. Objective: The aim of this study was to evaluate circulating levels of a series of cytokines, chemokines and neurotrophic factors in BD patients in comparison with healthy controls. Secondary analysis was performed to evaluate predictors of BD diagnosis and state (euthymia or mania). Methods: Fifty-three BD patients (19 in euthymia and 34 in mania) and 30 healthy controls were comprehensively assessed with a structured psychiatric interview (Mini-International Neuropsychiatric Interview) and psychopathological scales (Young Mania Rating Scale and Hamilton Depression Rating Scale) . Plasma levels of BDNF, GDNF, NGF, TNF-alpha, sTNFR1, sTNFR2, CCL2, CCL3, CCL11, CCL24, CCXL8 and CCXL10 were determined by ELISA. Results: There were no significant differences in the proportion of gender, age and years of schooling between controls and BD patients. In univariate analysis, BD patients presented increased levels of BDNF, GDNF, sTNFR1, CCL24 and CXCL10, but decreased levels of NGF, CCL3 and CXCL8 in comparison with controls. In multivariate analysis, elevated plasma levels of sTNFR1 (p \ 0.001) and BDNF (p \ 0.001), and reduced plasma levels of CCL3 (p = 0.003) were associated with BD diagnosis. The logistic regression model presented good adjustment in Hosmer-Lemeshow test and a predictor power of 85.7%. No variable was associated with mania state in the multivariate analysis. Conclusion: The present study corroborates the view of involvement of inflammatory and neurotrophic mediators in BD physiopathology. Inflammatory mediators might be regarded as promising biomarkers of BD trait. Disclosure of interest: None declared. Introduction: The inflammatory process is involved in neurological disorders such as Parkinson's and Alzheimer's disease. The control and knowledge of these changes can help treat these and other diseases. The aim of this study was to investigate the neuroinflammatory dependence on pilocarpine-induced seizure model in mice. Methods: Mice (28-32 g) were pretreated with thalidomide (25, 100 and 200 mg/kg, ip), an inhibitor of the release of TNFa, Infliximab (2, 5 and 10 mg/kg, sc), an antibody anti-TNFa, Aminoguanidine (25 and 100 mg/kg, ip), an inhibitor of inducible nitric oxide synthase, or ODQ (2.5, 10 and 20 mg/kg, ip), an inhibitor of guanylate cyclase, and 30 min after was induced the seizure with pilocarpine (400 mg/ kg, sc). In the behavioral assessment was observed following parameters: Latency for a first seizure and latency to death. For statistical analysis, we used analysis of variance (ANOVA) and Student-Newman-Keuls as post hoc. This work was approved by the local ethics committee (protocol number 41/10). Results and discussion: Our results show the attenuation of the behavioral parameters (increased latency to first seizure and decreased of mortality) at higher doses of the groups pretreated with thalidomide and infliximab. In groups pretreated with the higher dose of Aminoguanidine and in a dose of 10 mg/kg of ODQ showed the increased latency and survival of animals death. Conclusion: We demonstrate the neuroprotective activity of thalidomide and infliximab, which may be related to the decreased amount of TNF, while, aminoguanidine and ODQ to increase the survival rate can be related to an aggressor effect of nitric oxide via cGMP. The adverse cardiovascular events associated with acute exposure to particulate air pollution are exacerbated among individuals with hypertension. This study was undertaken to determine the cardiovascular effect of diesel exhaust (DEP; 15 lg/mouse) on TO mice made hypertensive by implanting osmotic minipump infusing angiotensin II or vehicle (control). On day 13, the animals were intratracheally instilled with DEP (15 lg/mouse) or saline. 24 h later, pulmonary exposure to DEP insignificantly decreased systolic blood pressure (SBP) in normotensive (NT) mice. However, it caused a significant decrease of SBP in hypertensive (HT) mice (-16 %, p \ 0.005). The PaO 2 was decreased, and PaCO 2 was increased in DEP-treated HT mice compared to NT mice treated with DEP (P \ 0.05). Number of circulating platelets was significantly increased in DEP-treated HT versus saline-treated HT and DEP-treated NT mice. Moreover, DEP exposure of NT mice showed a prothrombotic effect in pial arterioles compared to saline-treated NT mice (P \ 0.05). Interestingly, in DEP-treated HT mice the prothrombotic events were significantly aggravated compared to saline-treated NT and DEP-treated HT mice. The direct addition of DEP (0.1-1 lg/ml) to untreated mouse blood significantly induced in vitro platelet aggregation in a dosedependent fashion, and these effects were potentiated in blood of HT mice. DEP caused the activation of intravascular coagulation which was confirmed by a shortening of activated partial thromboplastin time (aPTT) and the prothrombin time (PT) following in vitro exposure to DEP (0.25-1 lg/ml). The effect of DEP on aPTT was potentiated in the plasma of HTmice. It can be concluded that the thrombotic events caused by DEP are exacerbated in HT mice. Our findings, therefore, provide a plausible explanation for the cardiovascular morbidity and mortality accompanying urban air pollution. Background: C-reactive protein (CRP) and pentraxin 3 (PTX3) are members of a highly conserved pentraxin superfamily. CRP is synthesized in the liver and may represent a systemic response to local inflammation, whereas PTX3 is synthesized locally at the inflammatory sites by endothelial and smooth muscle cells or by monocytes/ macrophages and may represent a rapid marker for local inflammation at sites of vessel injury. We compared plasma high-sensitivity CRP (hsCRP) and PTX3 concentrations after bare-metal stent (BMS) and drug-eluting stent (DES) implantation. 33 patients with stable coronary artery disease who underwent percutaneous coronary intervention (PCI) were divided into two groups: 1-16 patients (BMS group); and 2-17 patients (DES group). All patients were scheduled for an elective, 6-month clinical follow-up. Major adverse cardiovascular events (MACEs) (death, re-hospitalization for acute coronary syndrome, target vessel revascularization) were assessed. Results: Baseline demographics and clinical characteristics were similar in both groups. Patients after BMS implantation had a higher median PTX3 concentration 1.19 ng/mL (0.72-1.33) compared to patients after DES implantation 0.85 ng/mL (0.55-1.02, p = 0.04). Median hsCRP concentrations were similar in both groups: 0.9 mg/L (0.78-1.1) versus 0.89 mg/L (0.7-1.0), respectively (p = 0.7). Six out of 33 patients had MACEs during follow-up. The cut-off value to predict MACEs for PTX-3 was [1.291 ng/mL (AUC 0.907, sensitivity 83%, specificity 96%, p \ 0.0001) and for hsCRP was [0.9 mg/L (AUC 0.92, sensitivity 83%, specificity 81%, p \ 0.0001). Conclusions: DES showed significantly lower plasma PTX3 levels after PCI compared with BMS. hsCRP levels were comparable between the two groups. Thus, PTX3 may be a more sensitive marker of local inflammatory response due to vessel injury than hsCRP. Both PTX3 and hsCRP had a high predictive value for MACE during follow-up. Disclosure of interest: None declared. Acute viral myocarditis is a serious condition, which can cause sudden cardiac death in previously healthy adults. Despite intense research to unravel the mechanisms underlying the severe inflammation by cardiotropic viruses, treatment for acute myocarditis is primarily supportive, and specific therapies targeting inflammation are needed. Our laboratory has previously demonstrated a role for extracellular matrix proteins such as Osteonectin (SPARC) and Thrombospondin-2 in the wound healing process after myocardial infarction and in pressure overload. Here we investigate the role of matricellular protein SPARC in the coxsackie B3 (CVB3) murine model of viral myocarditis. Exposure to the CVB3 virus resulted in a biphasic response of SPARC cardiac transcript levels, with a twofold decrease after 24 h and a fourfold increase at 7 days. This was also observed in protein levels at 5 and 11 days post infection, respectively. Immunohistochemical analysis showed low SPARC expression under normal conditions with an abundant presence after 11 days of viral presence. Immunofluorescence studies confirmed SPARC expression on CD-3 positive lymphocytes and V-CAM positive cells, but not on a-smooth muscle actin-positive capillaries, CD45-positive leukocytes or F4/80-positive macrophages. SPARC WT and null mice were exposed to the CVB3 virus, and an increased mortality was observed in the SPARC null mice, associated with increased cardiac inflammation. To further demonstrate the role of SPARC in myocarditis, adenoviral over-expression of SPARC improved the mortality in both SPARC WT and null mice. These data demonstrate the protective role of SPARC in viral myocarditis and suggesting that SPARC may be a novel target for the treatment of viral myocarditis. Disclosure of interest: None declared. Atherosclerosis is characterized by a chronic immune response. An increased Th1 activity has been associated with plaque instability and risk of acute cardiac events. Lymphocyte analyses in blood are widely used to evaluate the immune status in patients with coronary artery disease. We investigated whether thoracic lymph nodes (LN) from patients with stable angina (SA) and acute coronary syndrome (ACS) revealed changes in the immune response that were not detectable in peripheral blood (PB). PB and thoracic LN were collected in 13 patients with SA and 14 patients with ACS during coronary by-pass surgery. PB and LN lymphocytes were assessed by flow cytometry using antibodies to CD3, CD4, CD8, CD19, CD16/56, CD25, Foxp3, CD69, HLA-DR, IL-18R and CCR4. LN showed a cell composition that was different from PB with a higher proportion of B cells, lower proportions of CD8+ T cells and NK cells and a twofold higher CD4/ CD8 ratio. Introduction: Epidemiological studies have shown that periodontitis, an inflammatory disease from an infection of the oral cavity, is a risk factor for cardiovascular diseases (CVDs). Endothelial dysfunction is the initial step in the development of CVD. The aim of the present study was evaluated endothelial function in an experimental model of periodontitis. Methods: Procedures were approved by ethic committee. A ligature was placed around rat mandibular and maxilar molars to induce periodontitis. A simulated procedure was performed in the Sham (Sh) group. Seven, 14 or 28 days after procedure, effects of intravenous injection of acetylcholine (Ach), sodium nitroprusside (SNP) or phenylephrine (Phe) on blood pressure (BP) were evaluated. Acetylcholine and phenylephrine response were also assessed in constantly perfused mesenteric bed. The blood was obtained for analyze serum lipid profile. Results and discussion: There was no difference in BP reduction to SNP in either group and in none of the evaluated times. Fourteen days after ligation, Ach response in BP was significantly reduced in ligature-induced periodontitis when compared tom Sh group (Ach 30 nmol/kg: Sh 50.6 ± 4.1; Ligature 31.5 ± 3.3 mmHg, n = 8; p \ 0.05; expressed as change in MAP). There was no difference in Ach response in other times (7 and 28 days). In mesenteric beds removed 28 days after ligature procedure, the response to Phe was significantly increased while the response to Ach was decreased. Seven and 14 days after ligature no changes in response to Ach or Phe were observed. The ligature resulted in plasma LDL increase 14 days after procedure. Plasma cholesterol, HDL, and triglycerides were not changed in any time. Periodontitis-induced endothelial dysfunction and increase in LDL levels may be involved in the early development of CVD. Background: It has been reported that the better outcome after rt-PA therapy could relate to the reduction of inflammatory response. The rt-PA administration was reported to decrease the activation of astrocytes after ischemic insult. In this study, we explored whether the inflammatory response is altered by rt-PA therapy and whether the free radical scavenger administration can reduce the inflammatory response providing better outcome or not. Methods: Acute ischemic stroke patients were consecutively enrolled in this study (n = 57, 72 ± 12.2 yo). Thrombolysis with rt-PA intravenous administration was performed on 11 patients. IL-6, oxidative LDL (OxLDL) and the radical productivity were measured as inflammatory biomarkers at 3 time points: before medication, 4 and 24 h following medication. All patients were classified into cardiogenic embolism (n = 39), atherothrombotic infarction (n = 18) and lacunar infarction (n = 10) by clinical examinations and records. Neurological alteration was assessed by comparing NIH Stroke Scale (NIHSS) on admission and at 1 month later. Results: IL-6, OxLDL and the radical productivity were significantly increased at 4 and 24 h following medication in rt-PA administration compared with conventional therapy (p \ 0.02). Whereas, patients with both rt-PA and free radical scavenger administration showed 32% reduction in IL-6 and 18% reduction in OxLDL at 24 h following medication as compared to patients with only rt-PA. Moreover, rt-PA and free radical scavenger treated patients exhibited better outcome compared with only rt-PA treated patients (NIHSS reduction: 9.4 and 1.7, respectively), although NIHSS was not different on admission (15. 3 and 19.8, respectively) . Conclusion: The free radical scavenger could decrease the inflammatory biomarkers which were amplified by rt-PA administration. Reduction of inflammation may provide better outcome. Disclosure of interest: None declared. Background and objectives: Thrombin is a procoagulant and proinflammatory molecule and is also a potential mediator of bone resorption and vascular healing. Osteoprotegerin (OPG) is a key molecule that binds to the receptor activator of the nuclear factor-kB (NF-kB) ligand (RANKL), and it inhibits osteoclast differentiation. In addition, OPG protects against vascular calcification. This study aimed to evaluate the biological effects of thrombin on OPG production in human dermal microvascular endothelial cells (HuDMECs). Materials and methods: Cells were treated with various concentrations (0, 0.1, 2.4, and 4.8 U/mL) of thrombin for 24 h. Thrombininduced OPG production in HuDMECs was determined using an enzyme-linked immunosorbent assay (ELISA). RANKL expression in HuDMECs was estimated using a reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of thrombin on OPG production in HuDMECs and the thrombin signaling pathway were investigated using inhibitors. Results: Thrombin induced OPG production in the HuDMECs in a dose-dependent manner. RANKL mRNA expression was also upregulated by thrombin. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and the Src kinase inhibitor, PP1, exerted an inhibitory effect on thrombin-induced OPG expression. In addition, thrombin-induced OPG production was inhibited by the proteaseactivated receptor (PAR)-1 antagonist. Conclusion: Thrombin induces OPG expression in HuDMECs, possibly through PAR-1. Thrombin-induced OPG production is regulated by the PI3K and Src pathways. This finding suggests that thrombinactivated HuDMECs may contribute to bone metabolism and vascular healing, partly through the OPG/RANKL system. This research was supported by a Grant-in-Aid for Scientific Research, Japan The aim of this study was to evaluate the levels of CINC-2 and neutrophil migration (NM) in the gingival tissue (GT) from spontaneously hypertensive rats (SHR) and Wistar (W) (180-200 g) (Protocol 52/06) submitted to periodontal disease (PD) and the role of mast cells (MAST) on this process. Thus, a group of animals was depleted of MAST by treatment with compound 48/80 (48/80), i.p. After 5 days, the animals were performed ligation of the first molars counterparts with silk thread to induce PD. The induction was also performed in animals of the groups that did not receive treatment with the 48/80 and after 7 and 14 days, GT were collected for the determination of CINC-2 production by ELISA, the MN was evaluated by the level of myeloperoxidase (MPO), and the mandibles were dissected and radiographed to evaluate the level of the bone resorption (BR) using Digora software. We observed that the levels of CINC-2 tends to increase in W and SHR animals in the presence of PD. CINC-2 is increased in the absence of MAST in SHR with 7 days after induction of PD and tends to increase in animals W without MAST 7 days after induction of PD. In animals with 14 days of induction, the SHR with PD showed a significant increase over its control. NM observed into the GT in SHR with PD is higher when compared with W animals with PD, both 7 and 14 days after disease induction. This migration was decreased in MAST-depleted SHR animals but not in animals W. The induction of PD, since we observed higher BR in all animals with PD. The presence of MAST increases BR only in SHR 14 days after induction of PD. In conclusion, we observed that the presence of MAST enhances the inflammatory process in SHR and BR with PD but not in animals W. In contrast, the presence of MAST decreases the production of CINC-2 suggesting that this chemokine is not related to bone resorption and neither MN in this model (SHR animals). Serum amyloid A (SAA) is an acute-phase reactant. Levels are elevated in coronary artery disease (CAD) patients and deposits are localised in macrophages in atheroma. SAA is an important mediator of inflammation and coagulation. It increases monocyte/macrophage cytokine production and tissue factor (TF). Similarly, the calcium-binding protein, S100A12, is expressed in foam cells and macrophages in vessels with atherosclerotic lesions, and circulating levels are elevated in CAD patients. S100A12 has pro-inflammatory properties, but our recent studies indicate its pleiotropic activities. Immunoaffinity experiments using human serum showed that SAA bound S100A12, suggesting functional interaction. Western blotting of sera from CAD patients confirmed low (10 kDa) and high mass SAA (200 kDa) and S100A12 (200 kDa), suggesting complex formation with components of HDL. We assessed whether S100A12 influenced the pro-inflammatory and pro-thrombotic functions of SAA in vitro. S100A12 did not directly induce cytokines in human PBMC, monocytes/macrophages but significantly suppressed SAA-induced cytokine production. IL-8 staining suggested that the secretory pathway was unaffected by S100A12. Conversely, S100A12 did not suppress LPS-induced cytokines or alter SAA-induced TF pro-coagulant activity, indicating selective effects. S100A12 and SAA share similar putative receptors, but competition studies indicated that this was unlikely. SAA activates the NFjB and MAPK pathways. S100A12 suppressed SAA-induced phosphorylation of ERK1/2; no effects on NFjB, p38 and JNK MAPK pathways were observed. S100A12 and SAA mediate inflammatory processes. Since S100A12 downregulated SAA-induced cytokine production, and bound SAA in plasma, it may protect circulating monocytes from activation when SAA and S100A12 levels are elevated during inflammatory episodes. However, S100A12 is unlikely to moderate the reported SAA-induced prothrombotic tendencies of circulating monocytes in patients with CAD. Disclosure of interest: None declared. Objectives: To estimate point prevalence of asymptomatic PAH and determine the associated factors for PAH in a cohort of SLE patients Methods: A prospective cross-sectional study of 101 patients with SLE were recruited in a single tertiary centre. Transthoracic echocardiography was performed to estimate the pulmonary arterial pressures. PAH was defined as resting systolic pulmonary artery pressure (sPAP) C 40 mmHg, in the absence of left heart disease. The patients were also evaluated with respect to their clinical and serologic features, and disease treatment. Results: PAH was identified in eight patients (7.9%) who had no history of interstitial lung disease. Fourteen patients (13.8%) had sPAP of 30-40 mmHg. Higher SLEDAI score (13.14 ± 3.4 vs. 5.8 ± 0.5, p = 0.001) and lower positivity of anti-Ro/SSA antibody (12.5 vs. 66.3%, p = 0.03) was observed in patients with PAH than patients without PAH. Of note, serum uric acid (UA) was significantly higher in patients with PAH than in those without PAH (7.8 ± 2.2 vs. 5.4 ± 1.9 mg/dl, p = 0.002). However, no significant differences in disease duration, Raynaud's phenomenon, antiphospholipid antibodies were found between these groups. In multivariate analysis, higher serum UA level was independently associated with the presence of PAH. Interestingly, serum UA level correlated significantly with plasma B-type natriuretic peptide level (r = 0.453, p \ 0.001 Objectives: In the present study, effects of preischemic administration of natural honey on cardiac arrhythmias and myocardial infarction size during zero flow global ischemia were investigated in isolated rat heart. Materials and methods: The isolated hearts were subjected to 30 min zero flow global ischemia followed by 120 min reperfusion then perfused by a modified drug free Krebs-Henseleit solution throughout the experiment (control) or the solution containing 0.25, 0.5, 1 and 2% of natural honey for 15 min before induction of global ischemia (treated groups), respectively. Cardiac arrhythmias were determined based on the Lambeth conventions and the infarct size was measured by computerized planimetry. Results: Myocardial infarction size was 55.8 ± 7.8% in the control group, while preischemic perfusion of honey (0.25, 0.5, 1 and 2%) reduced it to 39.3 ± 11, 30.6 ± 5.5 (P \ 0.01), 17.9 ± 5.6 (P \ 0.001) and 8.7 ± 1.1% (P \ 0.001), respectively. A direct linear correlation between honey concentrations and infarction size reduction was observed (R 2 = 0.9948). In addition, total number of ventricular ectopic beats were significantly decreased by all used concentrations of honey (P \ 0.05) during reperfusion time. Honey (0.25, 0.5 and 1%) also lowered incidence of irreversible ventricular fibrillation (P \ 0.05). Moreover, number and duration of ventricular tachycardia were reduced in all honey treated groups. Conclusion: Preischemic administration of natural honey before zero flow global ischemia can protect isolated rat heart against ischemia/ reperfusion injuries as reduction of infarction size and arrhythmias. Maybe, antioxidant and free radical scavenging activities of honey, reduction of necrotized tissue and providing energy sources may involve in these cardioprotective effects of honey. Keywords: Natural honey, global ischemia, preischemic administration, arrhythmia, isolated rat heart. Disclosure of interest: None declared. There are some data on beneficial effects of mesenchymal stem cells (MSCs) for the myocardial infarction. One mechanism is ability of MSCs to secrete cytokines. It is important assess changes of growth factors in serum after therapy. Purpose: Assess TGF b, VEGF-C and GM-CSF concentrations in the serum of rats after postinfarct intramyocardial injection of mesenchymal stem cells. Materials and methods: The experiment were conducted on 32 adult male Wistar rats. Myocardial infarction was simulated by the cryodestruction of the left ventricular anterior wall. Mesenchymal stem cells were derived from rat bone marrow and injected into myocardium 21 days after cryoinjury. In the control group, normal saline was infused. The blood levels of TGF b, VEGF-C and GM-CSF were measured at 3, 7 and 14 days. Results: The mean serum TGF b in group with MSCs was 40.53 ± 7.9 ng/ml on day 3, and this significant (p \ 0.05) increase a compared with control group (15.99 ± 6.9 ng/ml). The maximum TGF b concentration observed on day 7 after transplantation (62.35 ± 33.02 ng/ml). On day 14 concentration of TGF b made up 20.8 ± 10.02 ng/ml. The maximum VEGF-C concentration in the group with MSCs was noted on day 3 after transplantation 132.2 ± 18.94 pg/ml. By 7 days VEGF-C levels reduced to 72.88 ± 29.48 pg/ml. There was an increase the content of VEGF-C by day 14. No statistically significant differences between control and groups with MSCs were observed. GM-CSF concentration was 14.05 ± 6.08 pg/ml and there were no significant changes on 7 and 14 days. No statistically significant differences between control and treated groups were observed. Conclusions: Introduction of mesenchymal cells into the infarcted myocardium stimulated increase TGF b in the peripheral blood of rats. MSCs do not demonstrate a significant effect on the level of VEGF-C and GM-CSF in the peripheral blood. Disclosure of interest: None declared. Introduction: Hypertension is a multifactorial trait resulting from the combined influence of environmental and genetic determinants. It remains a major risk factor for cardiovascular diseases and is therefore one of the most important causes of premature morbidity and mortality in developed and developing countries. Very recent studies have focused on the relationship between inflammation and arterial stiffness in essential hypertension. Furthermore, according to the latest research, Vitamin D deficiency may increase the risk of developing hypertension. Because of lingering uncertainty of these associations, we examined in this study, the link between serum concentration of vitamin D and inflammation in blood samples of Algerian patients with hypertension. The Inflammation status was evaluated with serums levels of ferritin and c-reactive protein. Patients and methods: The study was conducted on 53 Algerian patients suffering from hypertension: 34 women and 19 men. The middle age was: 49 ± 7.5 years. For each patient, we measured serum levels of The CDKN2A locus, which contains the tumor suppressor gene p16 INK4a , is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize towards classically (CAMU) or alternatively (AAMU) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16 INK4adeficiency (p16 -/-) skews macrophages towards an alternative phenotype. Transcriptome analysis revealed that p16 -/bone marrowderived macrophages (BMDM) exhibit a phenotype resembling alternatively activated macrophages. In line with this observation, p16 -/-BMDM displayed a decreased response to classically polarizing IFNg and LPS and an increased sensitivity to alternatively polarizing IL-4. Furthermore, mice transplanted with p16 -/bone marrow displayed higher hepatic AAMU marker expression levels upon Schistosoma mansoni infection, an in vivo model of AAMU phenotype-skewing. Surprisingly, p16 -/-BMDM did not display increased IL-4-induced STAT6 signaling, but rather a decrease in STAT1 and IKKa,b phosphorylation, which correlated with higher levels of inhibitory acetylation of STAT1 Background: The CGRP family of peptides, signal via unique receptors that comprise of a G-protein-linked calcitonin-like receptor (CLR) with a single transmembrane protein receptor associated modifying protein (RAMP) that forms 3 distinct types, RAMP1, RAMP2 and RAMP3. Of these, CLR linked with RAMP1 leads to a high affinity CGRP receptor, CLR with RAMP2 or RAMP3 leads to ADM receptors (McLatchie et al., 1999) . We and others have good evidence that ADM mediates vascular protection through the CLR-RAMP2 receptor (Tam et al., 2006; Liang et al., 2009 ). Material and methods: RAMP1, 2 & 3, CLR, ADM and CGRP gene expressions were monitored in human carotid endarterectomy specimens in stable and unstable lesions (Collot- Teixeira et al., 2009) . Total RNA was extracted from unstable (n = 15) and stable (n = 12) atherosclerotic lesions, retrotranscribed and gene expression was assayed using quantitative real time PCR. Gene expression data normalisation was carried out against the geometric mean of three reference genes (18S, GAPDH and PPIA) and analysed using the DDCt method. Statistical analyses were performed using unpaired t-test with Welch's correction. Results: Results show a significant decrease of RAMP1 gene expression and increase in RAMP2, CLR and ADM gene expressions in ''unstable'' atherosclerotic lesions compared to ''stable'' parts of the lesions. RAMP3 and CGRP did not show any change in expression. Conclusion: CGRP is a potent vasodilator with vascular protective properties and structurally related to the vascular protective peptide ADM, with which it shares a unique family of receptors. This study suggests that the CGRP receptor pathway may be downregulated as the atherosclerotic plaque develops, but conversely the ADM pathway may be upregulated. The switch in the signalling activities of this peptide family as the plaque develops is of potential interest to understanding possibly lack of adverse effects for CGRP antagonists, currently under development for migraine. Disclosure of interest: None declared. Introduction: NSAIDs are the most frequently prescribed drugs in the world. There are a lot of controversial information published during recent years about NSAID cardiosafety. It is still unclear do NSAIDs develop cardioprotective or negative effects in acute coronary patients. Aim of the study was to investigate safety and efficacy of lornoxicam, nonselective COX-inhibitor, in patients with acute coronary syndrome (NSTEACS) and to evaluate the influence of lornoxicam on the inflammatory markers. Methods: Overall 85 patients with NSTEACS were randomized to the prospective single-blind trial to receive lornoxicam (8/12 mg/day) plus aspirin (100 mg) or aspirin alone for 15 days. Primary end points were: cardiovascular death, non-fatal myocardial infarct, nonfatal stroke, unstable angina. Results: According to the 6 months follow-up there were a significant reduction of primary end points in patients receiving lornoxicam compared with placebo (8.5 vs. 33.3%, OR = 0.18, P = 0.006). There was also significantly lower unstable angina event rate in lornoxicam group ( Center for Transdisciplinary Research, Niigata University, Niigata, Japan Objectives: Atherosclerosis is an inflammatory disease which is accelerated by chronic infection such as periodontitis. NKT cells have been shown to accelerate atherosclerosis in mouse model and human. NKT cells can recognize lipid antigens derived from bacterial pathogens presented by CD1d, and have a potent ability to produce IFN-c, a potentially pro-atherogenic cytokine. The aim of the present study is to elucidate the role of NKT cells in infection-related atherogenic response in mouse model. Materials and methods: CD1d-/-mice with C57BL/6 background and C57BL/6 mice which were administered a-galactosylceramide (aGalCer) were orally infected with Porphyromonas gingivalis, a representative pathogen of periodontitis, 10 times at 3-day intervals. After completion of infection alveolar bone loss and atherogenesisrelated gene expression in liver and artery were examined. Results: CD1d-/-mice demonstrated less alveolar bone resorption whereas aGalCer -administered mice showed increased resorption compared with C57BL/6 mice. Serum IL-6 level was increased in both CD1d-/-and aGalCer-administered mice. In liver, gene expressions of IFN-c, IL-4 and TNF-a were down-regulated in CD1d-/-mice, whereas up-regulated in aGalCer-administered mice. In artery, CCL2 and early growth response 1 gene expression was down-regulated and natriuretic peptide precursor B was up-regulated in CD1d-/-mice compared to wild type mice, however, opposite tendency was seen in aGalCer-administered mice. Hematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodeling and pulmonary hypertension. Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilization of hematopoietic c-kit+ progenitor cells in the perivascular niche. We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in pulmonary hypertension. CXCL12, CXCR4 and CXCR7 protein expression, hemodynamic parameters, right ventricular mass, extent of vascular remodeling and perivascular progenitor cell accumulation were studied. Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of pulmonary hypertension. This was associated with significantly increased right ventricular systolic pressure, and evidence of right ventricular hypertrophy, vascular remodeling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodeling, pulmonary hypertension and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents. This study offers important pathophysiological insights into the role of S220 Background: Highly active antiretroviral therapy (HAART) has been linked to the development of cardiovascular diseases such as myocardial infarction and atherosclerosis. Efavirenz (EFV) is one of the most widely used antiretroviral agents and, thus, cannot be ruled out as a causal agent of these side effects. The present study was designed to assess the role of clinically relevant doses of EFV on human leukocyte-endothelial cell interactions in vitro, which are a hallmark of the early stages of these pathological states. Methods: The interaction between human leukocytes-peripheral blood polymorphonuclear (PMN) and mononuclear (PBMC) cells-and human umbilical vein endothelial cells (HUVEC) was analysed using the parallel plate flow chamber system. Leukocyte b 2 integrin and endothelial expression of adhesion molecules was evaluated by flow cytometry. A one-way ANOVA followed by Newman-Keuls post hoc test were performed and statistical significance was set at *p \ 0.01 (vs. vehicle). The ubiquitin-proteasome system (UPS) mediates the main proteolytic pathway of targeted protein degradation in the cell. UPS dysfunction has been observed in animal models of pressure-overload cardiac hypertrophy and failure. UPS dysfunction has also been implicated in failing human hearts. Both clinical observation and some animal experiments have suggested that proteasome functional insufficiency may play an important role in the development of congestive heart failure. It has been well elucidated that IFN-c results in replacement of the constitutive catalytic subunits b1, b2, and b5 with the immunosubunits b1i, b2i, and b5i, respectively. The different proteasome complexes, such as the regular proteasome and immunoproteasome, exhibit different catalytic efficiencies. These catalytic alternations in the immunoproteasome produce well-defined effects on the antigen processing for major histocompatibility complex class I. However, a precise function of immunoproteasome in a pressure overload-induced heart remodeling has not been identified. To address this, we examined mouse model of pressure overload cardiac hypertrophy induced by transverse aortic banding (TAB) using Balb/c mice (WT) and IFN-c deficient mice. Three weeks after TAB, substantial hypertrophic changes of the hearts were observed in WT mice with mild fibrotic changes of the left ventricle. On the other hand, IFN-c deficient mice exhibited more significant hypertrophic change with exaggerated fibrosis in the hearts, compared with WT mice. After TAB, both mRNA and protein levels of immunosubunit b1i (LMP2) significantly increased in left ventricles of WT mice but not in those of IFN-c deficient mice. Furthermore, more significant polyubiquitinated peptides accumulated in the hearts of IFN-c deficient mice, compared with WT mice. These observations suggest that IFN-c plays a protective role in pressure overload-induced cardiac hypertrophy though the induction of immunoproteasome activation. Disclosure of interest: None declared. Rationale: Progress on the isolation and culture of various endothelial cells (EC) has allowed comparison of their biochemical and physiologic properties. However, very few studies compared the proteomes of EC from different sources. Objective: To compare proteomes of macro-vascular and micro-vascular EC. Methods: Proteomes of human umbilical vein EC (HUVEC) and 2 sources of micro-vascular EC, human pulmonary (HMVEC-P) and dermal micro-vascular EC (HMVEC-D) from healthy caucasian donors (4 in each group) were compared using two-dimension differential in gel electrophoresis (2D-DIGE) at pH ranges of 3-11 and 4-7 and mass spectrometry. Results: Among the protein spots detected in pH 4-7 and in pH 3-11 gels, we identified 17 and 64 proteins which were differentially expressed between HUVEC and HMVEC-P and between HUVEC and HMVEC-D with a ratio C2 and a T-test B0.01. Between HMVEC-P and HMVEC-D, 6 and 3 different proteins were differentially expressed in pH 4-7 and in pH 3-11 gels respectively with a ratio C2 and a T-test B0.01. Among identified proteins are cytoskeleton proteins and enzymes implicated in glycolysis. Ingenuity software analysis interestingly showed that numerous proteins overexpressed in HMVEC-D compared to HUVEC are implicated in the retinoic acid pathway. Conclusion: Major differences were observed between proteomes of macro-vascular and micro-vascular EC. Some of the differentially expressed proteins might be of great importance in the immune rec- Background: Elevated serum amyloid A (SAA), a systemic inflammatory marker, strongly associates with increased cardiovascular (CV) risk, endothelial dysfunction, future CV events and worse coronary artery disease (CAD) prognosis. Increased plasma endothelial microparticles (EMPs) are biomarkers of endothelial dysfunction in CV diseases, associate with high-risk angiographic lesions and predict future CV events in CAD. Aim: To investigate the role of SAA in EMP generation in cultured primary human coronary artery endothelial cells (HCAEC) and determine whether EMPs could reflect SAA-induced procoagulant and/or proinflammatory responses in HCAEC. Methods: HCAEC were stimulated with human recombinant SAA (1,000 nM; 4, 8, 16, 24 h) . EMPs were isolated from culture supernatants by differential centrifugation. Flow cytometry was used to measure EMP number, Annexin V binding, CD31, CD62E and tissue factor (TF) surface exposure. Gene expression (TF, CD62E, IL-6, IL-8) was measured by Real-Time PCR; TF activity by Actichrome TF Activity Assay, IL-6 and IL-8 levels by ELISA. Results: SAA-stimulated HCAEC released significantly increased numbers of Annexin V+ and CD31+ EMPs. Time-dependent rise in SAA-induced Annexin V+ EMPs highly correlated with IL-6 and IL-8 levels (p \ 0.01). CD62E antigen was expressed only on SAAinduced EMPs and reflected time-dependent changes in CD62E mRNA levels, indicating cell activation. TF mRNA expression, TF surface exposure and activity in HCAEC peaked at 4 h following SAA stimulation and were accompanied by TF surface exposure and activity on EMPs. Later during SAA stimulation TF expression and activity progressively decreased in SAA-induced HCAEC and EMPs, suggesting tight TF regulation. Cardiovascular diseases represent the major cause of mortality in western countries. Among these conditions, atherosclerosis is the most prominent one. A hallmark of atherosclerosis is the atheromatous plaque formation, characterized by different disturbances, like vascular smooth muscle cell (VSMC) accumulation. Under the effect of different pathological stimuli, VSMC are able to migrate and proliferate, contributing to the fibrous cap formation. Notably, these events occur mainly in sinuous vessels, and are associated to turbulent blood flow, what may lead to hemolysis and consequent free heme accumulation. Our group has characterized heme as a proinflammatory molecule that mediates its effect via NADPH oxidase complex activation and reactive oxygen species (ROS) production. In this work we aim to elucidate the putative role of free heme in VSMC physiology and the molecular mechanisms underlying the affected processes. We observed that free heme induces VSMC migration and proliferation in a ROS-dependent manner. Heme-induced ROS production relies on NADPH oxidase activation, once diphenyleneiodonium (DPI), a NADPH oxidase inhibitor completely abolished this effect, and also inhibited heme chemotactic and proliferate effect. Additionally, heme activates proliferation-relationed and redox-sensitive signaling pathway, such as MAP kinases and NFkB. It was also observed a critical crosstalk between NADPH oxidase and heme oxygenase (HO-1) system. Heme induces HO-1 expression, and the pretreatment of VSMC with HO inhibitors potentiated heme proliferative effect. In summary we show that free heme induces VSMC proliferation via NADPH oxidase activation, which is elegantly counter-regulated by HO-1 activity, and the depiction of the signaling events underlying this process may lead to the development of novel and more effective therapeutic interventions in cardiovascular diseases (Financial Support: FAPERJ, CAPES, CNPq). Disclosure of interest: None declared. Biochemistry, Eskisehir, Turkey, 2 Endocrinology, Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey, 3 Statins which inhibit the enzyme HMG CoA reductase are used in the treatment of hypercholesterolemia. These HMG-CoA reductase inhibitors have antiinflammatory properties, but their effect on neopterin which are produced by interferon-g stimulated macrophages largely unknown. Also chitotriosidase and myeloperoxidase (MPO) are produced during the inflammatory process in pro-atherogenic state within the vasculature. In this regard we aimed to search effects of atorvastatin terapies on inflammation which induce cardiovascular disease risk in hypercholesterolemic patients. Material and methods: The study included 30 patients who were diagnosed as hypercholesterolemic (0 month) ([200 mg/dl). All patients were treated by atorvastatin (10 mg/day) for 3 months (3 months). Control group was composed of healthy individuals (n = 30). Bloods samples were taken before and 3 months after treatment. Neopterin levels were measured by HPLC method, MPO activity was measured by spectrophotometric method and chitotriosidase activity were measured by fluorometric method in both groups. Results: Chitotriosidase activity was higher in hypercholesterolemic patients compared to control. (37.8 ± 12.3 vs. 21.5 ± 8.4 nmol/L, p \ 0.001). Chitotriosidase activity was decreased after 3 months in atorvastatin treatment in hypercholesterolemic patients (32.3 ± 10.6 vs. 37.8 ± 12.3 nmol/L, p \ 0.05). Both MPO and neopterin levels decreased after 3 months atorvastatin therapy in hypercholesterolemic patients (p \ 0.01 U/L for both). Conclusions: Atorvastatin therapy is effective therapeutic strategy in decreasing inflammation which increases the incidence of cardio vascular diseases in hypercholesterolemic patients beyond decreasing serum cholesterol levels. Disclosure of interest: None declared. Introduction: Hypercholesterolemia usually occurs prior to the onset of atherosclerosis and renders tissue more susceptible to inflammation. In this process some markers of activated macrophages like chitotriosidase and myeloperoxidase are released into circulation. In this regard we aimed to assess the antiinflammatory effects of atorvastatin and glitazone using an in vivo hypercholesterolemic rat model. Material and methods: The control group (group I) fed basal diet. All other animals fed with hypercholesterolemic diet which contains 2% (w/w) cholesterol and 1% cholic acid in basal diet for 8 weeks. The hypercholesterolemic group (group II) was continued taking hypercholesterolemic diet while the group III, IV and V, VI, VII, VIII, IX received 1 mg rosiglitazone, 4 mg rosiglitazone, 10 mg atorvastatin, 1 mg rosiglitazone plus 10 mg atorvastatin, 4 mg rosiglitazone plus 10 mg atorvastatin, 3 mg pioglitazone and 3 mg pioglitazone plus 10 mg atorvastatin, respectively, by oral gavage in addition to hypercholesterolemic diet for 3 weeks. The plasma concentrations of Myeloperoxidase were measured by spectrophotometric method. Chitotriosidase enzyme activity was measured by fluorometric method. Results: Serum Myeloperoxidase levels of control group, hypercholesterolemic group and group III, IV, V, VI, VII, VIII, IX were 80 ± 40 a , 440 ± 180, 290 ± 70, 200 ± 40 a , 290 ± 60, 190 ± 50 a , 170 ± 40 a , 102 ± 20 a , 104 ± 30 a mU/L, respectively. Also serum chitotriosidase levels of these groups were 5.97 ± 1.48 a , 16.25 ± 2.5, 9.67 ± 1.68 a , 7.20 ± 1.55 a , 8.57 ± 1.20 a , 8.74 ± 1.26 a , 8.24 ± 2.63 a , 9.41 ± 1.33 a , 8.61 ± 1.58 a nmol/mL, respectively. a Significant different from hypercholesterolemic group p \ 0.05. Conclusions: Glitazones decreased inflammatory process but combined atorvastatin and glitazone therapy was more effective on inflammation. Also pioglitazon is better than rosiglitazone for decreasing inflammation. Glitazones may be strategical target for decreasing inflammation in hypercholesterolemia. Atherosclerosis is considered a Th1-dominated inflammatory disease, but there is a growing interest for the role of CD8 + T cells. The cytotoxicity of CD8 + T cells has been reported to be influenced by CD56. We hypothesized that the distribution and cytokine-producing capacity of CD56 + CD8 + T cells was altered in patients with coronary artery disease compared with healthy individuals. We also examined if the expression of Annexin 1 in CD56 + CD8 + T cells was related to a Type 1 response. Blood was collected from 30 patients with acute coronary syndrome (ACS), 34 patients with stable angina (SA) and 36 healthy controls. Blood sampling was repeated in patients at 3 and 12 months. T cells were analyzed by flow cytometry using antibodies to CD3, CD8 and CD56. Whole blood from 9 ACS, 14 SA and 10 controls was stimulated ex vivo and intracellular staining for IFN-c, TNF, IL-10, IL-13 or IL-17, as well as double-staining for IFN-c and Annexin 1 was determined by flow cytometry. Data are given as mean (SD). Total numbers of CD56 + CD8 + T cells were similar in ACS and SA but higher compared with controls, 184 (160) versus 109 (83) cells/ll, p = 0.003. The proportions of CD56 + CD8 + T cells were expanded in patients, 30 (19) versus 23 (11)%, p = 0.05, not changing over time. Upon mitogenic stimulation, the majority of CD56 + CD8 + T cells produced IFN-c and TNF, but no IL-10, IL-13 or IL-17, with no difference between ACS, SA and controls. After stimulation, an increased expression of Annexin1 was seen in IFN-c + cells, p \ 0.001. However in ACS, the Annexin 1 MFI expression was markedly lower compared with SA and controls, 45735 (8381), 65581 (9369), and 70806 (20372), p = 0.001. In both ACS and SA patients, accumulations of peripheral CD56 + CD8 + T cells seem to be a constitutive phenomenon independent of clinical manifestation. The cells are high producers of IFN-c and TNF which may indicate an increased Type 1 activity in the patients. The differential expression of Annexin 1 highlights its potential role in ACS. Disclosure of interest: None declared. Human embryonic stem cell-derived endothelial cells (hESC-EC) are being investigated as research tools and cell therapy for vascular inflammation. It is known that hESC or hESC-EC do not respond to LPS [1] . Thus, the full extent of how these hESC-EC function is not known. Endothelial cells release the vasoconstrictor hormone endothelin-1 (ET-1), elevated levels of which are associated with vascular inflammation. In the current study we have compared the ability of unstimulated human endothelial cells from umbilical vein (HUVEC), human aorta (HAEC) and the microvasculature of the lung (HMVEC) with hESC-EC to release ET-1. ET-1 is produced by endothelial cells under basal culture conditions, but can be increased further by agonists including thrombin. hESC-EC were derived as described previously [1] . All cells were cultured for 24 h in endothelial cell specific medium (Lonza-EGM2). ET-1 release in conditioned media was measured by ELISA. For hESC-EC and HUVEC, experiments were run in parallel and cell number was assessed by nuclear staining with DAPI and automated cell counting. HAEC and HMVEC were run in separate experiments but under similar conditions. HUVEC (215.2 ± 70.0 pg/ ml), HAEC (248.7 ± 14.2 pg/ml) and HMVEC (339.8 ± 19.5 pg/ml) all released detectable levels of ET-1 (data are mean ± SEM). However, hESC-EC failed to release detectable levels of ET-1 in this time period (\42 pg/ml). There was no significant difference (p [ 0.05; Student's t test) in cell number between HUVEC (151.5 ± 30.7 cells/ field) and hESC-EC (256.0 ± 82.5 cells/field). Compared with other types of human endothelial cells, hESC-EC released relatively low levels of ET-1. As ET-1 can contribute to cardiovascular disease this could represent a benefit of hESC-EC over native endothelium in a damaged organ. However, the findings remain preliminary and a detailed examination of the ET-1 pathway in these cells remains the subject of investigation. Vascular inflammation plays an important role in the development of myocardial infarction. Lymphotoxin-alpha (LTA), a member of the tumor necrosis factor family, is a cytokine produced by lymphocytes that mediates a wide variety of inflammatory, immunostimulatory, and antiviral responses. Acute myocardial infarction (AMI) is the clinical manifestation of the chronic development of coronary artery atheroma, with the final process of plaque rupture and coronary thrombosis. The aim of this study sought to assess the association of LTA Thr26Asn polymorphism with AMI. One hundred and sixty eight AMI patients were compared to 169 healthy controls. The Frequencies of the LTA Thr/Thr, Thr/Asn, and Asn Asn genotypes among AMI patients were 45. 8, 46.4, and 7.8%, respectively, and in controls were 43.1, 45, and 10 .9%, respectively. There was no significant difference in the distribution of LTA Thr26Asn genotypes or in the allelic frequencies between AMI patients and controls (P = 0.098). The frequency of Asn/ Asn genotype not increased with the number of stenoses in coronary vessels in AMI patients. In conclusions, the LTA gene is not associated with susceptibility to AMI and not influences the severity. Disclosure of interest: None declared. Introduction and objectives: Arterial peripheral disease is a condition caused by the blocking of blood flow as a result of cholesterol deposit in the arteries of the arms, legs and aorta. Studies have shown that macrophages in the atherosclerotic plaque are highly activated, and this process makes these cells important antigen presenting cells, with the development of a specific immune response, having LDLox as a possible inducing antigen. As functional changes of cells which participate in the atherogenesis process may occur in the peripheral blood, the objective of the present study was to evaluate plasma levels of anti-inflammatory and inflammatory cytokines such as, interleukin-6 (IL-6), IFNc, IL-10 and TGF-b in patients with peripheral arteriosclerosis obliterans, as well as the level of monocyte activation in the peripheral blood through the ability of these cells to release hydrogen peroxide (H 2 O 2 ) and to develop fungicidal activity (FA) against Candida albicans (C. albicans). Methods: IL-6, IFN-c, IL-10 and TGF-b from plasma of patients were detected by ELISA. Monocyte cultures activated with TNF-a and IFN-c were evaluated by FA against C. albicans using plating and CFU recovery and by H 2 O 2 production. Results: Plasma levels of all cytokines were significantly higher in patients compared to those detected in control subjects. Monocytes from control group did not release substantial levels of H 2 O 2 , however, these levels were significantly increased after activation with IFN-cand TNF-a. We found that patient's monocytes, before and after the activation process, showed lower responses than control subjects. Similar results were found when FA was evaluated. The results of the patients were always significantly smaller than those detected for controls. Conclusions: The results revealed an unresponsiveness state of patient's monocytes in vitro probably due to the high activation process occurring in vivo as evidenced by high plasma levels of cytokines. Disclosure of interest: None declared. Inflamm. Res. Background: IVS-I-6 (T-C), IVS-I-110 (G-A), IVS-II-1 (G-A) and IVS-II-745 (C-G) B globin gene mutations are common in the Mediterranean population and were reported to be common in Egyptians. In dilated cardiomyopathy, immune related disorders show preferential associations with HLA genes. It was known that the mutations occurred with bthalassemia major have a strong relation to severity and complication accompanying the disease. We aimed to study the frequency of the previous mentioned mutations and HLA-DR in Upper Egyptian bthalassemia patients. Patients and methods: The study included 68 Egyptian patients with bthalassemia major classified into two groups; the first group was 35 patients suffering from left sided heart failure, while the second group formed from 33 patients without heart failure. PCR amplification using five oligonucleotides primers to the concerned mutations. Dotblot analysis was carried out with allele-specific probes and detection was done by using streptavidin-peroxidase and Chemiluminescent liquid detection. HLA class II-DR was investigated for all patients and controls using sequence specific oligonucleotide (SSO). Results: The following frequencies of the investigated mutations were noticed IVS-I-110 (G-A): 41%, IVS-II-1 (G-A): 18%, IVS-I-6 (T-C): 14%, and lastly IVS-II-745: 12%. DR4 and DR7 were significantly increased in patients with heart failure with a P value of.02, Odds Ratio (OR) = 3.3 and p \ 0.01, OR = 2.1, respectively. A statistical significant difference was found between patients who are positive for IVS-I-1 and patients with heart failure (p \ 0.02). Conclusions: The recorded frequencies of tested mutations are very close to the previous studies which were performed in Egyptian bthalassemia patients. Immunogenetic pathogenesis mediated mechanism attributed to myocarditis must be considered in addition to other risky factors as iron overload. Disclosure of interest: None declared. Introduction: Atherosclerosis involves the cellular infiltration in the arterial intima of several cell types, including monocytes. Then, monocytes differentiate into macrophages and become foam cells after oxidized Low Density Lipoprotein (oxLDL) uptake. The aim of this work was to develop a model of primary human macrophagederived foam cells and to study their inflammatory profile. We have focused our attention on the capacity of macrophage-derived foam cells to metabolize polyunsaturated free fatty acids into inflammatory and pro-resolving molecules. Methods: For this purpose, monocytes from human donor were differentiated into macrophages, which were then incubated in the presence of oxLDL. The uptake of oxLDL was associated with an increase of total cholesterol and a higher level of cholesterol ester into the cells. We have then incubated those cells in presence of arachidonic, eicosapentaenoic and docosahexaenoic acids to evaluate the capacity of macrophage-derived foam cells to synthetize lipid mediators after their stimulation by a phorbol myristate and a calcium ionophore. Supernatant were then collected and mediators analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/ MS) methodology to quantitatively evaluate bioactive lipids production. Results: We showed that in presence of oxLDL, macrophage-derived foam cells produce more inflammatory (TXB2, PGE2) and oxidative stress mediators The search of new anti-inflammatory agents is target of many researches. Natural products and their analogues modified synthetically are molecules of great interest. The aim of our investigation is to study the anti-inflammatory effect of the natural product extracted from Tabebuia, lapachol, and its synthetic derivative, a new pterocarpanquinone. LQB 118, in the inhaled lipopolysaccharide (LPS)induced lung inflammation model, using C57J/BL6 mice. Our results demonstrated that intraperitonial treatment with 1 mg/kg of lapachol or LQB 118 reduced neutrophil influx to lungs in mice submitted to inhaled LPS (0.5 mg/mL). Additionally, LQB 118 reduced the concentration of the inflammatory mediators TNF-a and KC in the bronchoalveolar lavage fluid (BALF). These effects of LQB 118 are comparable to those of dexamethasone or aspirin, two well-established anti-inflammatory drugs. The reduction of mediators concentration by LQB 118 is partially due to reduction of NF-kB activation in the cell lungs. In vitro, LQB 118 also inhibited TNF-a production in LPS-stimulated mononuclear cells of human peripheral blood (PBMC). The highest concentration tested (100 mM) was able to inhibit almost 100% of TNF-a liberation. These data confirm the anti-inflammatory action of lapachol observed in a paw edema model (Almeida de et al., 1990) , and reveal the lapachol derivative, LQB 118, as a potent modulator of inflammation, through the reduction of inflammatory mediators. Disclosure of interest: None declared. The airway epithelium acts as a barrier protecting the lung from inhaled substances notably airborne pollutants. In some respiratory inflammatory disease such as asthma, these substances can either induce and/or exacerbate the disease. Situated beside airways epithelial cells and at the junction of innate and adaptative immunity, dendritic cells (DCs) have an important role in setting up how immune responses are initiated and perpetuated. The aim of our study is to better understand how bronchial epithelial cells (BECs) could interact with DCs particularly when epithelium is submitted to environmental risk factor such as particulate matter (PM). For this purpose, we used a coculture system composed of BECs differentiated in air-liquid interface and monocyte-derived immature dendritic cells (iDC), placed on basolateral side of BECs. Particulate matter are then added on apical surface of BECs. The expression of different cytokines/chemokines (IL-8, CCL-20, TSLP for example), which are notably involved in T cell activation and DCs chemoattraction, was studied in BECs by RT-PCR or ELISA. We have shown that coculture of BECs with iDC could regulate release of some molecules involved in inflammation process or DCs chemoattraction. However, addition of PM on this system seems to have little effect on these expressions. Devising of BECs/DCs coculture system could allow imitating first step of airways epithelium responses to inhaled substances and notably better understanding the modulation of antigenic presentation and immune system activation. Disclosure of interest: None declared. Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. Using the murine model of bleomycin-induced acute lung injury resulting in pulmonary fibrosis we reported before that neutrophil airway influx, remodeling and lung fibrosis depend on the activation of the NLRP3 inflammasome leading to mature IL-1b production. Since interleukin-17 (IL-17) is a major pro-inflammatory cytokine involved in neutrophil recruitment and chronic lung pathologies, we asked whether IL-17 plays a role in lung injury-induced inflammation and fibrosis. We find that IL-17A pulmonary expression is upregulated after bleomycin or rmIL-1b administration in the airways. Lung inflammation, remodeling and fibrosis are diminished in IL-17RA deficient mice or after IL-17A antibody neutralization. Early IL-17A producing cells in response to lung injury are mostly gdT cells and in a lesser extent CD4abT cells. Finally local bleomycin or IL-1b administration enhanced pulmonary inflammation, remodeling and fibrosis which are IL-23p19 dependent. In conclusion, we highlighted the existence of an IL-1b-IL-23-IL-17A axis. IL-17A and IL-17RA signaling play a pivotal role in bleomycin-induced acute lung inflammation and fibrosis, IL-1b and IL-23p19 expression being critically involved in IL-17A dependent lung pathology. Disclosure of interest: None declared. Introduction: Experimental and clinical studies have reported that intestinal ischemia/reperfusion (I/R) induces acute lung inflammation (ALI), which is characterized by the release of many inflammatory mediators, neutrophil infiltration and increased vascular permeability. It is known that inflammatory mediators, generated at the site of intestinal I/R, are transported by lymphatic system and, reaching the lungs, contributes to ALI. Nevertheless, data on late effects of intestinal I/R in ALI are still fragmented. Material and methods: Male Wistar rats were subjected to 45 min of ischemia of the superior mesenteric artery and then to 2, 24, 72 or 120 h of reperfusion. After these times of reperfusion, the activity of myeloperoxidase (MPO) and the extravasation of Evans blue dye (EB) in lungs were determined. Some inflammatory mediators in lymph and in lung explants were quantified. Results: The MPO activity and the EB extravasation in lung after 2 h of reperfusion increased, but have not changed from basal after 24 and 72 h. However, after 120 h of reperfusion they increased again. Lymph collected from intestinal I/R animals contains significant amounts of IL-1b, IL-6, VEGF and LTB 4 compared to basal. Lung explants from reperfused animals revealed that while the levels of IL-1b increased during the hours of reperfusion, the levels of IL-10 decreased. The amount of IL-6 is higher after 2 h of reperfusion than in the others periods studied, whereas the levels of VEGF increased only after 120 h of reperfusion. Proinflammatory cytokine generation and neutrophil infiltration into the lung during mechanical ventilation are thought to play important roles in the development of ventilator-induced lung injury, although the exact cellular and molecular mechanisms which trigger lung inflammation are still not clear. Calpains, a family of Ca 2+ -dependent intracellular cysteine proteases, have been shown to be involved in the inflammatory response and multiple organ failure. The aim of the present study was to determine the role of calpain in the pathogenesis of lung inflammation due to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of pharmacological calpain inhibitor. Additionally, depletion of calpain expression in pulmonary microvascular endothelial cells in vivo using siRNA was performed at 48 h before ventilation. Mechanical ventilation caused a rapid (within minutes), persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of the cytokines TNF-a and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition as well as depletion of calpain-1 or calpain-2 significantly attenuated these inflammatory responses caused by lung hyperinflation. Inhibition of calpain activation also reduced eNOS-mediated NO production and subsequent ICAM-1 phosphorylation induced by mechanical stretch. This study demonstrates that calpain activation during mechanical ventilation may trigger lung inflammation via eNOS/NO-mediated ICAM-1 phosphorylation and neutrophil recruitment. Thus, inhibition of calpain activation may be a novel therapeutic strategy for the The aim of this study is to investigate the effects of 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), which is a specific inhibitor of the activation of mitogen-activated protein kinase kinase (MAPK3/MAPK1), on the development of acute lung inflammation caused by carrageenan injection or pulmonary fibrosis induced by instillation of bleomycin (BLEO). Mice were injected with carrageenan into the pleural cavity or were subjected to intratracheal instillation of BLEO (1 mg/kg), a glycopeptide produced by the bacterium Streptomyces verticillus. Mice subjected to injury developed significant inflammatory response characterized by marked neutrophil infiltration and tissue edema. An increase in immunoreactivity to nitrotyrosine, iNOS, TNF-alpha and IL-1b were also observed in the lungs from injured mice. In contrast, administration of PD98059 (10 mg/kg, 10% DMSO, i.p.) 1 h after carrageenan injection or BLM instillation significantly reduced: (1) cytokines production, (2) IjB-a degradation and NF-jB nuclear expression (3) iNOS expression (4) nitrotyrosine and PARP localization (5) Smoking and air pollutants are recognized as key pathogenic factors for chronic obstructive pulmonary disease (COPD), leading to gradual loss of lung function. GLPG0259, a potent inhibitor of MAPKAPK5 currently in Phase II trials for rheumatoid arthritis, was characterized in various in vivo and in vitro models of inflammation and showed strong capacity to inhibit cytokine-induced MMP expression as well as innate immune response. In view of similar processes in COPD pathogenesis, we investigated the efficacy of GLPG0259 in models of COPD. GLPG0259 was profiled in normal human bronchial epithelial cells (NHBECs) with the addition of poly(dI-dC). IL-6 and CCL5 RNA levels were determined by qPCR. The in vivo efficacy was characterized in the mouse LPS model (serum TNFa levels) and in a 4-day tobacco smoke mouse model of COPD (inflammatory cell counts and cytokine levels in bronchoalveloar lavage fluid (BALF), inflammatory gene expression in lung tissue). In NHBECs, GLPG0259 concentration-dependently inhibited both IL-6 and CCL5. In the LPS model, GLPG0259 strongly reduced the TNFa release (ED 50 3 mg/kg). In the COPD model, GLPG0259 dose-dependently (0.1-10 mg/kg b.i.d.) inhibited the cell influx into BALF. At 1 mg/kg, GLPG0259 reduced the total cell number (40%), neutrophils (51%), lymphocytes (61%), macrophages (29%) and epithelial cells (50%), comparable to the p38 inhibitor BIRB796. Disease and compound effects were also assessed by cytokine measurements as well as gene expression profiling. GLPG0259 is active on pathways involved in the COPD pathogenesis both in vitro and in vivo, indicating that this compound might have therapeutic benefit in COPD. Disclosure of interest: None declared. Background: Interleukin-32 (IL-32) is a newly described proinflammatory cytokine which is expected to have an important role in autoimmune disorders. Recently, it was shown that chronic obstructive pulmonary diseases have a component of autoimmunity, though the role of IL-32 in those diseases is not known. The aim of this study was to investigate IL-32 concentration in different tissue compartments from patients with COPD, asthma and compare with healthy subjects. Methods: Outpatients aging from 40 to 80 years with stable COPD (n = 51), asthma (n = 31), and healthy subjects (n = 9) were studied. IL-32 concentration in serum, induced sputum and BAL was analyzed by enzyme-linked immunosorbent assay. Results: IL-32 concentration was significantly increased in all studied tissue compartments from patients with COPD in comparison to asthmatics and healthy subjects (serum: 26.77 ± 2.56 vs. 6.09 ± 1.16 and 4.63 ± 1.03 pg/ml; induced sputum: 19.66 ± 1.69 vs. 5.82 ± 1.15 and 3.59 ± 0.66 pg/ml; BAL: 22.46 ± 2.48 vs. 6.25 ± 1.08 and 4.21 ± 1.13 pg/ml, respectively), and significantly correlated with smoking history (pack years). There was no statistically significant difference of IL-32 concentration between asthmatics and healthy subjects. Conclusion: The fact that IL-32 concentration is increased in patients with COPD and related to the smoking history let us suggest that IL-32 may play an important role in the pathogenesis of COPD. Disclosure of interest: None declared. Rationale: PDE4 inhibitors increase cAMP levels, and as a consequence have shown anti-inflammatory effect in various animal models of inflammation. PDE4 inhibitors are in various stages of development with roflumilast in particular being approved in Europe for treatment of COPD. OCID 2987 a novel PDE4 inhibitor was evaluated in murine models of asthma and COPD. Methods: In LPS induced acute lung injury model Sprague-Dawley rats were exposed to LPS (100 mg/ml) for 40 min. For evaluation of neutrophil influx BALF (broncho-alveolar lavage fluid) was collected at 6 h and for TNF-a estimation at 1.5 h. In COPD model C57 mice were exposed to the tobacco smoke of 10 cigarettes/day for 20 days. 24 h after last cigarette smoke exposure BALF was collected and differential cell counts conducted. In guinea pig model of asthma, animals were sensitized and challenged with aerosolized ovalbumin. BALF was collected 48 h after last ovalbumin challenge for total cell count and differential cell count. Results: In LPS induced acute lung injury model OCID 2987 produced inhibition of neutrophil influx in BALF with ED 50 value of 0.81 mg/kg, p.o. and inhibition of TNF-a in BALF with ED 50 value of 1.9 mg/kg, p.o. OCID 2987 at 1 mg/kg, p.o. produced 66.96% inhibition of neutrophil influx in BALF of cigarette smoke exposed mice. Methodology: Male Wistar rats (n = 12) were breastfed by rats whose diet contained 17 and 8% of protein during lactation (N and MN group). After weaning, both groups were administered a normoproteic diet. Following the macrophage isolation, four systems were formed: negative control composed by macrophages (C-), positive control added with lipopolysaccharide (C?) and two systems, MSSA and MRSA, stimulated by the strains. Cytokine release was evaluated after 24 h of incubation. Results: Malnutrition led to the decrease of body mass as well as reduction in the production of IFN-c in the systems C? (p = 0.001) and MRSA (p = 0.001). However, it was not observed significant difference among systems C-(p = 0.707) and MSSA (p = 0.065). Pharmacological modulation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is emerging as a means to control inflammatory disorders. The SH2-containing inositol-5 0 -phosphatase 1 (SHIP1) metabolizes PI [3, 4, 5] P3 to PI [3, 4] P2. SHIP1-deficient mice exhibit a marked degree of inflammatory cell recruitment into the lung, leading to pulmonary inflammation. Pharmacological, allosteric activation of SHIP1 exerts anti-inflammatory effects (Ong et al., Blood, 2008 The bleomycin-induced pulmonary fibrosis model in the mouse is the most common model to investigate potential new therapies for idiopathic pulmonary fibrosis (IPF). The main read-outs used to quantify the severity of fibrosis are based on the amount of collagen deposition (either measured using histological sections or by biochemical assay). However, this becomes a problem when the model is run using a therapeutic treatment protocol in which treatment is only started after fibrosis has been allowed to establish. Since the production of collagen (the main component of fibrous tissue) already starts during in the initial inflammatory phase, the window for measuring effects of new compounds is much smaller in the therapeutic protocol versus preventive treatment. In this study, we investigated the kinetics of collagen deposition during bleomycin-induced lung fibrosis in C57Bl/6 mice using deuterated water (D 2 O) to label newly formed collagen synthesis. Furthermore, we used whole gene array analysis in combination with the ToxProfiler software to differentiate between the different pathways induced during fibrosis. Array analysis showed that inflammatory processes were maximal during the first week of disease, while extracellular matrix related pathways were maximal during the second and third week after induction, after which they slowly returned to base line levels. The new collagen assay showed that during the first week already substantial collagen formation took place, despite strong upregulation of inflammatory processes. The production of collagen was further upregulated during the second and third week, after which collagen synthesis returned to pre-induction levels. This high level of collagen synthesis during the first week explains the difficulties in obtaining sufficient window during therapeutic treatment. Disclosure of interest: None declared. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, which encodes an epithelial anion channel. Morbidity is mainly due to the lung disease, characterized by a chronic neutrophilic inflammation. Deregulation of inflammatory pathways is observed in the airways of CF patients, as evidenced by an increased AP-1 activity and NF-jB response. Consequently, proinflammatory cytokines such as IL-8 are increased. CSN5 and COM-MD1 are two proteins involved in the trafficking of CFTR but also in inflammatory processes. We observed a larger proportion of COM-MD1 and CSN5 in the nucleus of CF cells. We studied the influence of COMMD1 or CSN5 overexpression/extinction on NF-jB pathway in CF and non-CF bronchial epithelial cells (IB3-1 and S9, respectively). To decipher the underlying mechanisms, we have first performed a set of luciferase assays and NF-jB binding assays in order to test NF-jB transcriptional activity in the presence or absence of each protein. TNFa stimulation produced a higher increase of NF-jB activity in CF versus non CF cells. Overexpression of COMMD1 and CSN5 in the same experimental conditions decreased NF-jB activity by half in both cell lines. As IL-8 promoter contains AP-1 and NF-jB responsive elements, we performed such experiments with the wild-type and mutant constructs. Our preliminary results did not show a clear-cut effect on IL-8 promoter. However, new experimental conditions such as IL-1b stimulation are currently investigated. Our results show for the first time the anti-inflammatory properties of COMMD1 and CSN5 in bronchial cells. These data could expand investigations to other chronic inflammatory diseases such as asthma or chronic obstructive pulmonary disease. This work was supported by public grants from Institut National de la Santé Cyclooxygenase (COX) is the first enzyme in the conversion of arachidonic acid to prostanoids. There are two isoforms of COX; COX-1, which is constitutively expressed with a homeostatic role in most tissues, and COX-2, which while constitutively expressed in some discreet sites is generally inducible by growth factors and during inflammation. In the current study, we have used tissues and cells from knock-out mice to investigate the relative contributions of COX-1 and COX-2 to PGE 2 production by lung tissue ex vivo and by proliferating lung fibroblasts in vitro. Lung tissues from WT (C57Bl6), COX-1 -/and COX-2 -/mice were immediately dissected (\15 min after death) and incubated (37°C) for 30 min in DMEM containing 50 lM calcium ionophore (A23187). Release of PGE 2 was determined by competitive immunoassay. In parallel studies, murine lung fibroblasts from COX-1 -/and COX-2 -/mice were explanted and cultured before being seeded in 96-well plates at sub-confluence (5,000-8,000/well) and incubated for 24-48 h in the presence of 10% FCS. Accumulated release of PGE 2 was then measured as above. Over 30 min PGE 2 was released by lung pieces from wild type (1,117 ± 55 pg/ml) and COX-2 -/-(2,013 ± 255 pg/ ml) but not from COX-1 -/-(\61 pg/ml) mice (n = 4). In contrast, proliferating lung fibroblasts from COX-1 -/-(4,978.9 ± 1,392 pg/ml) mice released higher levels of PGE 2 than cells from COX-2 -/- (1, 194 ± 617 ng/ml) mice (n = 4 using cells from 2-3 separate mice for each genotype). These results show that COX-1 activity underpins the stimulated release of PGE 2 in healthy mouse lung tissue. Conversely, COX-2 activity predominates in proliferating lung fibroblasts, which may be important as COX-derived PGE 2 mediates proliferation of lung fibroblasts [1] . Our results suggest a switch in COX isoform in lung cells during proliferation which could be relevant to our understanding of conditions such as idiopathic pulmonary fibrosis [1] Trends Immunol. 2004; 25 (1) Introduction: Pulmonary fibrosis has high rates of mortality and morbidity, but there is no established therapy at present. Oxidative stress plays an important role in the pathogenesis of and pulmonary fibrosis. The aim of the present study was to determine whether there is any significant genetic influence on the development of lung fibrosis related to oxidative stress after bleomycin stimulus. Methods and Results: C57BL/6, DBA/2 and BALB/c male mice were divided in control group (50 lL saline; i.t., n = 10 each strain) and bleomycin (Bleo) group (0.1 U/kg i.t., n = 10 each strain). After 21 days the mice were sacrificed. The lungs of 20 animals were homogenized for oxidative stress marker determinations and compared with the respective control groups by with Tukey post-test. Lung ventilation was performed in 10 animals of each group and elastance and resistance determined. Malondialdehyde equivalent levels were increased in the C57BL/6 Bleo group (p \ 0.01), and were decreased in DBA/2 and BALB/c (p \ 0.0001) when compared with the respective control strain group. Both activity and expression of catalase were increased in C57BL/6 Bleo group (p \ 0.01), decreased in DBA/2 and BALB/c Bleo groups when compared with control groups (p \ 0.01). Superoxide dismutase activity increased in C57BL/6 and BALB/c Bleo groups (p \ 0.01). Nitrite levels were increased in C57BL/6 and BALB/c (p \ 0.01) Bleo groups. Lung resistance was increased in C57BL/6 Bleo group (p \ 0.01) and in DBA/2 (p \ 0.05). Conclusion: Our results shown that C57BL/6 mice are more susceptible to redox imbalance and oxidative damage and bleomycin response in DBA/2 and BALB/c strains we not associated with oxidative stress injury. Disclosure of interest: None declared. Inflamm. Res. Intestinal ischemia and reperfusion (intestinal I/R) frequently occurs in human pathological conditions, and it has been considered as a significant cause of Acute Respiratory Distress Syndrome (ARDS). It is thought that polymorphonuclear neutrophils and mediators generated in the setting of oxidative stress, such as reactive oxygen species (ROS), have important roles in its pathophysiology. In this sense, we hypothesize that heme oxygenase-1 (HO-1) up regulation, an enzyme with antiinflammatory and antioxidant activities, could modulate the expression of antioxidant enzymes and neutrophils recruitment in the lung after an intestinal I/R, therefore inducing cytoprotection. Upon anesthesia male Wistar rats were subjected to 45 min occlusion of the superior mesenteric artery (SMA) followed by 2 h of reperfusion. Before ischemia, one group of rats had the lymphatic thoracic duct sectioned and another group of rats was treated 48 and 24 h before induction of SMA occlusion with an HO-1 inductor, Hemin (10 mg/kg, i.p.). Neutrophils recruitment to the tissues was indirectly measured by myeloperoxidase (MPO) activity. HO-1, superoxide dismutase (SOD) 1 and 2, catalase (CAT) mRNA were determined in the lung tissue. Intestinal I/R determined a significant increase of MPO activity in lung that was reduced in rats upon lymphatic duct sectioned. Hemin treatment of rats increased the activity of HO-1, SOD-1 and SOD-2, but did not modify the expression the CAT in lung tissue. In addition, the MPO activity in lung tissue was significantly reduced in hemin-treated group as compared with non treated rats upon intestinal I/R. Introduction: The incidence of rheumatoid arthritis (RA), an autoimmune disease characterized by persistent synovial inflammation as well as articular cartilage and bone destruction, is greater in women compared with men and can be related to menopause. Estrogen deficiency has demonstrated to increase incidence and progression of RA. We have shown previously that CO-releasing molecule, CORM-3 exerts anti-inflammatory effects in vitro and in vivo. The aim of this study was to investigate the effects of CORM-3 on the inflammatory response as well as on cartilage and bone metabolism in comparison with alendronate (ALN). Methods: Two weeks after ovariectomy (OVX), collagen-induced arthritis (CIA) was induced in DBA/1J mice. ALN (100 lg/kg po once a day) and CORM-3 (10 mg/kg ip once a day) were administered from day 22. The clinical score was studied using a scale of 0-2 in each paw. Mice were killed at day 36 or 50. Histological analyses were performed on hind paws. Trabecular microarchitecture was analyzed by lCT. Results: CORM-3 treatment resulted in a significant reduction of the clinical score with respect to the control animals (OVX + CIA). The bone parameters of tibia of animals treated with CORM-3 were similar to naïve group at day 36. At day 50, CORM-3 reduced chondrocyte death compared with control group. ALN administration did not improve the clinical score nor the articular cartilage damage. At day 50, ALN administration showed to be an effective treatment to reduce bone resorption. Conclusions: These data corroborate the anti-inflammatory role of CORM-3 and demonstrate protective effects against cartilage destruction. In addition, CORM-3 is able to reduce the bone loss in the early phase of the disease. ALN confirms its antiresorptive action in this animal model of arthritis + osteoporosis but it fails to protect joint cartilage damage. Therefore, these data suggest that a combination of both drugs could be an effective strategy in postmenopausal arthritis. Disclosure of interest: None declared. Tendinitis and tenosynovitis were detected in 32 patients (40%) and 12 patients (15%), respectively. These abnormalities were observed more frequently in selected SLE patients with arthralgia (n = 11). Moreover, synovial thickness in joints and tendon thickness were greater in patients with arthralgia compared to those without (P \ 0.006 and P \ 0.03). However, only alteration found in healthy subjects was synovitis in MCP joint (5.9%). Among patients without arthralgia, patients with synovitis had shorter disease duration (P = 0.022), while patients who showed tendinitis had higher mean age of disease onset (P = 0.002). Importantly, tendon thickness was positively correlated with mean daily prednisolone dosage (r = 0.23, P = 0.037). Conclusions: Asymptomatic joint and tendon involvement of hand and wrist is common in SLE patients. Longitudinal study is needed to determine if silent sonographic abnormalities will develop relevant musculoskeletal symptoms in those patients and progress to more serious pathology leading to deformity. Disclosure of interest: None declared. Nuclear factor-kappaB (NF-kB) plays a major role in arthritic diseases by promoting the expression of inflammatory and catabolic mediators, namely nitric oxide (NO). Our previous study showed that a fraction (named Falpha-p) of the essential oil from the leaves of Juniperus oxycedrus, mainly composed of a racemic mixture of alpha-pinene [2,6,6-trimethyl-bicyclo(3.1.1) hept-2-ene] decreases IL-1-induced NF-kB activation and NO production in human chondrocytes. This study aimed at further elucidating the mechanism of action of alpha-pinene. NF-kB activation was evaluated in the human chondrocytic cell line, C-28/I 2 , by western blot for cytoplasmic phospho-IkB-a levels and by ELISA for DNA binding activity. To determine the ability of the test compounds to directly inhibit NO production, regardless of their effect on NF-kB-dependent iNOS expression, nitrite levels were measured by the Griess reaction in human chondrocytes treated with the test compounds after pretreatment with IL-1 for 18 h to induce iNOS expression. Falpha-p decreased phospho-IkB-a, NF-kB DNA binding and nitrite concentration to 22.1% ± 7.8, 46.4% ± 13.2 and 11.3% ± 0.4 of the levels induced by IL-1 alone, respectively. When added 18 h after IL-1, Falpha-p decreased nitrite levels to 42.1% ± 7.3. The commercial racemic and (+) enantiomer of alpha-pinene were less effective while the (-) enantiomer had no significant effects. (+) alpha-pinene probably accounts for most of the ability of Falpha-p to inhibit IL-1induced NF-kB activation and NO production. The later seems to involve NF-kB dependent and independent mechanisms, probably through direct inhibition of iNOS activity. Aim: The Notch family is involved in cell differentiation during embryogenesis. Osteoarthritic chondrocytes undergo morphological and biochemical changes leading to the de-differentiation process. In this study, we were interested in the involvement of the Notch pathway in human articular chondrocyte de-differentiation. Methods: Human articular cartilage was obtained from cadavers after informed consent of the family. Chondrocytes were isolated then subjected to several cell culture passages and treated with or without the [N-[N-(3,5-difluorophenacetyl-L-alanyl) ]-S-phenylglycine t-butyl ester] (DAPT): a Notch inhibitor. Chondrocyte morphology was studied using optical microscopy. Immunoblot was performed to study the expression of differentiated chondrocyte markers (Collagen II, aggrecan) and dedifferentiated chondrocyte markers (collagen I, metalloproteinases and nitric oxide synthases) as well as Notch family members. Results: Without DAPT treatment, chondrocyte de-differentiation resulted in fibroblast-like morphology. This was confirmed by immunoblot analysis, which showed an increase in collagen type I, metalloproteinases, nitric oxide synthases and a decrease in collagen type II and aggrecan expression. With DAPT treatment, de-differentiation was delayed. Immunoblot analysis showed during the first passages inhibition of collagen type II and aggrecan expression, which then was re-expressed during the last passage, suggesting chondrocyte re-differentiation. Conclusion: In this study, we showed that inhibition of the Notch receptor not only delayed the de-differentiation process, but also leaded to chondrocyte re-differentiation, which confirms the involvement of the Notch pathway in chondrocyte de-differentiation. This suggests that modulation of this pathway could constitute a new therapeutic target for the treatment of osteoarthritis. Disclosure of interest: None declared. Duchenne muscular dystrophy (DMD) is a severe X-linked muscle disease characterized by progressive skeletal muscle atrophy and weakness. DMD is caused by mutations in the dystrophin gene, which encodes the cytoskeletal protein dystrophin. We have found that hematopoietic prostaglandin (PG) D synthase (HPGDS) was induced in grouped necrotic muscle fibers in DMD patients. Cytosolic form of phospholipase A 2 and cyclooxygenase-2, the upstream enzymes of the arachnoid acid cascade, were similarly observed in the HPGDSpositive fibers. We performed functional analysis of prostaglandin D 2 produced by HPGDS in DMD and developed a novel therapy for DMD based on the inhibition of PGD 2 signaling. HPGDS was induced in the necrotic muscle fibers of chemically induced rat and mouse models of DMD and in genetically dystrophin-deficient mdx mice and DMD dogs. The necrotic muscle in DMD models constitutively expressed HPGDS. Oral administration of HPGDS inhibitor for 5 days prevented the expansion of muscular necrosis in mdx mouse, as measured by X-ray computed tomography (CT) imaging. mdx mice were treated with HPGDS inhibitor for 30 days, and then the recovery of muscle strength, necrotic muscle volume evaluated by X-ray CT, locomotor activities and muscle histology were examined. The HPGDS inhibitor decreased the volume of necrotic muscle, improved locomotor activity and the histological analysis revealed that the inhibitor prevented the expansion of muscle damage. These data demonstrate the pathological role of PGD 2 produced by HPGDS in the expansion of muscle necrosis. These results indicate that PGD 2 produced by H-PGDS is involved in the expansion of muscle necrosis in DMD and that inhibition of PGD 2 signaling is a novel therapy for DMD. Disclosure of interest: None declared. Objective: Oleocanthal has been shown to mimic the actions of a nonsteroidal anti-inflammatory drug (NSAID) and acts as a natural NSAID through the inhibition of cyclooxygenase (COX). NSAIDs attenuate proliferation in C2C12 myoblasts through COX inhibition. COX-2 dependent prostaglandin synthesis is required in muscle regeneration and is implicated in myoblast proliferation. Therefore the use of anti-inflammatory COX-2 inhibitors may be detrimental to muscle growth and regeneration. The objective of the present study is to determine if a natural NSAID such as oleocanthal has adverse effects on myoblast proliferation in vitro. Methods: Mouse C2C12 myoblasts were grown in 96 well plates. Cell viability was determined by fluorescent assay (CytoTox-Fluor, Promega). Myoblasts were treated with 0.1, 0.5, 1, 5, 10 lM oleocanthal for 24 h. DMSO (0.24%) was used as a vehicle control. Myoblast proliferation was determined by the incorporation of 25 lM 5-bromo-2 0 -deoxyuridine (BrdU) for 24 h in the presence of oleocanthal. BrdU levels were measured a 405 nm with a reference wavelength of 690 nm. Results and discussion: Concentrations of oleocanthal between 0.1-5 lM were not toxic to myoblasts over 24 h. Additionally, these concentrations did not affect myoblast proliferation. NSAIDs are prescribed for the treatment of pain associated with inflammatory related disease states such as arthritis leaving patients susceptible to potential adverse effects on skeletal muscle. Long term ingestion of a naturally occurring NSAID such as oleocanthal at low concentrations may have pharmacological benefits without compromising muscle growth or regeneration. Ongoing studies are presently determining the anti-inflammatory effects of oleocanthal in attenuation of inflammatory markers and proteins associated with proteolysis including p38-MAPK, NFjB, antrogin-1 (MFAbx) and MuRF-1 in C2C12 cells and Background: Blocking TNF alpha (TNFa) with monoclonal antibodies (mAbs) has been very successful in the treatment of severe rheumatoid arthritis. However, nearly 30% of the patients failed to respond and most of them escape mAb efficiency during time. Active immunization with a TNF-Kinoid induces self polyclonal anti-TNFa antibodies and effectively treats TNFa mediated arthritis in transgenic mice. This novel therapeutic strategy could be an attractive alternative to anti-TNFa mAbs. Objectives: We evaluate the safety, immunogenicity, clinical response to TNF-K in patients with rheumatoid arthritis who develop secondary resistance to one TNFa mAb. Material and methods: TNFa-Kinoid (TNF-K, Neovacs SA, Paris, France) is an immunotherapeutic composed of recombinant human TNFa conjugated to KLH as a carrier protein, inactivated and adjuvanted with ISA-51 emulsion. Patients with active rheumatoid arthritis (DAS28 C 3.2) with history of positive clinical response to one anti-TNFamAb followed by secondary failure are being enrolled in a double-blind, placebo-controlled, phase 2, dose escalation study to evaluate three different doses of TNF-K. Immune responses are being evaluated through titration of anti-TNFa and anti-KLH antibodies with isotyping, evaluation of neutralizing capacity and of cellular responses. Clinical response is being assessed by regular evaluation of DAS28, ACR and EULAR scores and titration of C reactive protein Results: Up to now, no related serious adverse event has been reported. Objectives: Adipocytokine adiponectin is involved in the regulation of energy metabolism and appetite, and possibly in the development of insulin resistance. Recently, it has also been shown to regulate inflammatory responses. The aim of the present study was to investigate the association and effects of adiponectin on inflammation and cartilage destruction in osteoarthritic joints. Methods: Blood and cartilage samples were collected from 35 male patients with OA, undergoing total knee replacement surgery. Plasma concentrations of adiponectin and biomarkers of cartilage degradation, i.e. COMP (cartilage oligometric matrix protein) and MMP-3 (matrix metalloproteinase 3) were measured by immunoassay. Cartilage samples were cultured for 48 h, and adiponectin released in the culture medium was measured and correlated to nitric oxide (NO), interleukin-6 (IL-6), MMP-1 and MMP-3 production. Finally, the effects of adiponectin on NO, IL-6, and MMP production in cartilage were studied in tissue culture experiments. Results: Plasma adiponectin concentrations correlated positively with biomarkers of cartilage degradation, i.e. with COMP (Pearson r = 0.54, p \ 0.001, ln transformation) and with MMP-3 (r = 0.34, p = 0.046). In addition, OA cartilage was found to produce adiponectin, and the adiponectin amounts released from cartilage in tissue culture correlated positively with the production of NO (r = 0.34, p = 0.049), IL-6 (r = 0.39, p = 0.021, ln transformation) and MMP-3 (r = 0.36, p = 0.037, ln transformation). Further, adiponectin was found to enhance production of NO, IL-6, MMP-1 and MMP-3 in OA cartilage in vitro by a p38 kinase dependent manner. Conclusions: These findings suggest that adiponectin is involved in the pathogenesis of joint inflammation and cartilage destruction in osteoarthritis. Disclosure of interest: None declared. Background: For monitoring the drug resistance among Campylobacter populations which has become a serious concern worldwide, there is a need to reliable and reproducible laboratory techniques. There are several methods; including disk diffusion, broth micro-dilution, agar dilution and E-test to apply to determine in vitro susceptibility profiles of Campylobacter to a range of antimicrobial agents. Objectives: The aim of present study was isolation of Campylobacter jejuni and Campylobacter coli from children with diarrhea and determination of antimicrobial susceptibilities of the isolates to clinically relevant antimicrobials. Methods: In total 220 stool samples of children with diarrhea were cultured on Preston agar and the isolated Campylobacter spp. were identified by further standard identification test. Susceptibility testing was carried out using Kirby-Bauer disk diffusion method and E-test. Results: In this study, 14 Campylobacter strains were isolated (6.36%), of which 9 cases (64.3%) were identified as C. jejuni and 5 cases (35.7%) as C. coli. Using disk diffusion, all the Campylobacter isolates were fully resistant to cephalothin, oxacillin and ampicillin followed by ceftazidime with resistance rate of 71.42%. Gentamicin and ciprofloxacin were the most effective antibiotics against both isolated Campylobacter species. According to E-test results, Campylobacter isolates demonstrated the greatest resistance to cephalothin (92.85%), oxacillin (92.85%) and ampicillin (78.57%). Conclusions: Our study reveals a high-level correlation between the E-test and agar disk diffusion method in evaluating the resistance of Campylobacter spp. to tested antimicrobial agents. Intrinsic contractile activity of lymphatic vessels is critical in tissue fluid homeostasis, immune cell trafficking and nutrient uptake. Correlation between inflammatory bowel disease (IBD) and dilated and/or obstructed mesenteric lymphatics is described in clinical settings and animal models. In the guinea pig model of TNBS-ileitis, mesenteric lymphatic vessels are enlarged and their contractile function is impaired. This dysfunction can be caused by the action of inflammatory mediators. Among them, nitric oxide (NO), an inflammatory mediator upregulated in intestinal inflammation can decrease lymphatic muscle contractility through activation of ATP-sensitive potassium channels (K ATP ). Our objective is to understand the role of K ATP in inflammation-induced lymphatic dysfunction and their possible dysregulation by NO using the guinea pig model of TNBS ileitis. Following initiation of the inflammation, animals were sacrificed at day 1 for PCR and day 3 for in vitro studies. Lymphatic tissues were microdissected out and real time PCR was performed for K ATP subunits Kir6.1 and SUR2B, as well as iNOS. Pharmacological studies were performed in vitro on isolated, cannulated mesenteric lymphatic vessels. The contractile activity was assessed in the presence of 10 lM of the K ATP antagonist glibenclamide, the iNOS inhibitor 1400 W, or the sGC inhibitor ODQ. Our data show that expression of Kir6.1, SUR2B and iNOS mRNAs was significantly upregulated in TNBS versus sham animals (p \ 0.05). Administration of glibenclamide induced a large contractile response in the quiescent lymphatic vessels from TNBS animals (p \ 0.0001). Administration of 1400 W and ODQ also improved lymphatic contractile activity in TNBS animals (p \ 0.05). Our findings suggest that K ATP and iNOS play a major role in the lymphatic contractile dysfunction seen in TNBS animals. Identifying molecular targets to reverse lymphatic dysfunction can lead to pharmacological tools to treat intestinal inflammation. Disclosure of interest: None declared. Acute enteric infections may initiate post-infectious irritable bowel syndrome (PI-IBS) via unknown mechanisms. Giardia duodenalis (G. intestinalis, G. lamblia) is the most common cause of parasitic diarrheal disease worldwide. Follow-up after recent outbreaks have implicated giardiasis in the development of PI-IBS in a number of patients. Whether disruptions in the resident intestinal microbiota structure and/or species distribution by enteropathogens may be implicated in the pathogenesis of PI-IBS has yet to be determined. This study examined (1) whether Giardia trophozoites modify human intestinal mucosal biofilms retrieved from intestinal biopsies, and (2) whether these biofilm changes may disrupt human epithelial monolayers. Methods: Experiments in vitro used Caco-2 and HCT-8 intestinal monolayers. Representative, multi-species biofilms were cultured anaerobically (72 h) on the Calgary Biofilm Device from human intestinal mucosal biopsies collected during routine colonoscopies. Results: Cell counts reveal Giardia promotes the planktonic growth phase of bacteria over that associated with the biofilm structure. Scanning electron microscopy indicates that Giardia inhibits the secretion of the normal extracellular matrix covering the biofilm. ELISA revealed that monolayers co-incubated for 3 h with Giardiaexposed biofilms (24 h) exhibited greater levels of apoptosis, compared to those incubated with biofilms not exposed to Giardia. Conclusion: In this study, multi-species anaerobic biofilms, representative of the endogenous mucosal microbiota, were successfully grown from human intestinal biopsies. Our findings indicate that Giardia modifies the structure and phenotype of these communities. Lastly, Giardia-exposed biofilms disrupt the homeostasis of intestinal monolayers by enhancing their apopotic decay. These results pave the way towards a better understanding of the ramifications that complex host-polymicrobial interactions have in chronic disease manifestation. Glucocorticoids exert anti-inflammatory and immunomodulatory effects that are regulated in part by activity of the enzyme 11b-hydroxysteroid dehydrogenase type 1 (11HSD1). This enzyme is responsible for the interconversion of inactive glucocorticoids (cortisone, 11-dehydrocorticosterone) into biologically active steroids (cortisol, corticosterone) and plays an important role in various diseases. We have shown recently that 11HSD1 is upregulated in bioptic samples taken from patients with ulcerative colitis. The aim of the present study was (1) to characterize the role of proinflammatory cytokines in upregulation of colonic 11HSD1 and (2) to identify the effect of inflammation associated with colitis on 11HSD1 in spleen and mesenteric lymph nodes (MLN). Experimental colitis was induced in rats by the intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid. Our results showed that colitis upregulated in the large intestine not only 11HSD1 mRNA and 11-reductase activity but also mRNA levels of proinflammatory cytokines tumor necrosis factor a (TNFa) and interleukin 1b (IL-1b). Similarly, treatment of colon tissue explants with TNFa significantly increased 11HSD1 mRNA expression. In contrast, IL-1b was without any effect even if both cytokines upregulated cyclooxygenase-2 (COX-2) mRNA. Inflammation also increased mRNA levels of 11HSD1, TNFa, IL-1b and COX-2 in intraepithelial (IEL) and lamina propria lymphocytes (LPL). Similar increase of 11HSD1 mRNA expression and 11-reductase activity was found also in mesenteric lymph nodes and spleen, particularly in T cells. Comparison of 11HSD1 levels showed that enzyme expression was lower in MLN than in IEL and particularly in LPL. Thus, inflammation stimulates the reactivation of glucocorticoids in lymphoid organs and in gut-associated lymphoid tissue. This reactivation system seems to exhibit a higher activation in the cells of the effector regions of mucosal immune system. Aims: The water channel protein aquaporin 1 (AQP1) is widely expressed on epithelial cells with the primary purpose of regulating transmembrane water flux. Chronic inflammatory states like inflammatory bowel disease (IBD) are characterized by high levels of interferon (IFN)c and exhibit abnormal transepithelial fluid flux. We sought to determine the effect of IFNc on AQP1 expression in the intestinal epithelium. Methods: (1) C57Bl6 mice were given 4% dextran sodium sulfate (DSS) in their drinking water to induce acute colitis and were euthanized at 3 days. Colons were removed and AQP1 expression was assessed by immunofluorescence. (2) . Confluent CMT93 cells (mouse cecal epithelial cell line that constitutively expresses AQP1) on Transwell membranes were pre-treated with either siRNA to knock down STAT1 (signal transducer and activator of transcription 1) or a JAK2 (Janus Kinase 2) inhibitor and then treated with IFNc (10 ng/ mL, 24 h) in serum free media. Cells were then lysed and protein levels for AQP1 and STAT1 were assessed by Western blot. Results: (1) In C57Bl6 mice, AQP1 immunoreactivity was observed in vascular endothelial cells and on the apical membrane of colonic crypt epithelial cells. Acute colitis induced by DSS was associated with a loss of crypt epithelial, but not vascular endothelial, AQP1 immunoreactivity. (2) . CMT93 cells demonstrated a significant suppression in AQP1 protein expression after treatment with IFNc at 24 h. Pre-treatment with JAK2 inhibitor, but not STAT1 siRNA, prior to IFNc administration resulted in the amelioration of the IFNc-induced suppression of AQP1 protein expression. Conclusions: Epithelial AQP1 expression is reduced by DSS colitis in mice and by IFNc in CMT93 cells through a unique JAK2-dependent, STAT1-independent mechanism. Thus, inflammatory cytokines present during IBD are able to influence AQP1 levels and may contribute to the water transport deficiencies observed in these diseases. Disclosure of interest: None declared. The NAD-dependent deacetylase Sirtuin-1 (SIRT1) has been implicated in a number of critical physiological functions including control of inflammation. Genetic and biochemical studies show a key role of SIRT1-mediated deacetylation in the inflammatory response. Most notably, SIRT1 mediated deacetylation of p65/RelA directly reduces NF-kB transcriptional activity thus suppressing proinflammatory responses. SRT2104 is a novel small molecule that activates SIRT1 in biochemical and cell-based assays and has demonstrated selectivity against over 200 GPCRs, nuclear hormone receptors, enzymes and ion channels. In the present study the efficacy of SRT2104 to attenuate inflammation in DSS-induced colitis was compared to the corticosteroid prednisolone. Colitis was induced by administering 3% DSS in drinking water for 5 days following which the severity of the inflammatory response was assessed by endoscopy. Treatment was then begun with SRT2104 (1-30 mg/kg), prednisolone (3 mg/kg) or vehicle administered orally for 9 days followed by endoscopic analysis on days 7 and 13 S240 Inflamm. Res. with mice sacrificed on day 14 for histology. Prednisolone reduced colitis severity but worsened the body weight loss seen in this model. By contrast, SRT2104 produced a greater reduction in the colitis score compared to prednisolone while improving the loss in body weight. On histological examination, SRT2104 also produced a greater effect than did prednisolone. The ability of SRT2104 and prednisolone to reduce colitis was then examined in mice in which SIRT1 had been genetically knocked out in the gastrointestinal tract. Prednisolone was equally efficacious in wild-type and SIRT1 KO mice whereas the anti-inflammatory effect of SRT2104 was absent in the SIRT1 KO mice demonstrating a SIRT1-dependent efficacy for SRT2104. These data demonstrate that small molecule activators of SIRT1, like SRT2104, may have therapeutic value in inflammatory disorders such as ulcerative colitis. In addition, multiplex analysis reveals elevated serum levels of pro-inflammatory cytokines, including IL-17, IFN-c, IL-1b, and MIP-1a, suggesting the involvement of Th1 and Th17 cells. The development of these aspects of colitis is sensitive to suppression by intrarectally or orally administered budesonide. Prophylactic treatment by oral administration of L. plantarum or the VSL#3 probiotic mixture suppressed the development of recurrent TNBS colitis, evident from less macroscopic alterations, reduced mucosal infiltration by inflammatory cells and serum levels of IL-17, IFN-c, IL-1b, MCP-1 and MIP-1a to concentrations comparable to those found in healthy mice. Transcriptome analysis of colon showed increased expression of mast cell proteases and a-defensins in mice with colitis, but significantly less in mice treated with probiotics. Our data indicate that the recurrent TNBS colitis model shows several aspects of inflammatory bowel disease, which are sensitive both to corticosteroids and even to subtle modulation by probiotics. Disclosure of interest: None declared. Giardia duodenalis is a non-invasive protozoan parasite of the small intestine and common cause of diarrheal disease. Despite a parasite load that may exceed one million trophozoites per centimetre of gut, the small intestinal mucosa of Giardia-infected individuals is devoid of overt inflammation, for reasons that remain obscure. In the small intestine, acute inflammatory responses include the secretion of the potent neutrophil chemoattractant interleukin-8 (CXCL8) by intestinal epithelial cells. Following exposure to enterocytes, G. duodenalis trophozoites express several cathepsin-like proteases of unknown function. We hypothesized that G. duodenalis trophozoites attenuate pro-inflammatory responses in intestinal epithelial cells by releasing cathepsin-like proteases. Methods: Experiments were performed in vitro using Caco-2 and HCT-8 human intestinal epithelial monolayers, and ex vivo, using human small intestinal biopsy tissues. Cells were co-incubated with assemblage A or B G. duodenalis trophozoite isolates and subsequently stimulated with pro-inflammatory IL-1b. Results: Co-incubation of Giardia trophozoites (both assemblage A and B genotypes) with cell monolayers or small intestinal mucosal biopsy tissues results in the secretion of an unknown, heat-sensitive factor that attenuates IL-1b-induced CXCL8 secretion. Recombinant CXCL8 administered to cell monolayers co-incubated with Giardia trophozoites was significantly reduced versus control groups. Cathepsin B/L activity was increased in cell supernatants and monolayers co-incubated with Giardia trophozoites. Pre-treatment with a cysteine protease inhibitor, E-64, abolished cathepsin B/L activity in these groups and reversed Giardia-mediated attenuation of IL-1binduced CXCL8 secretion. Conclusion: The interaction between Giardia trophozoites and IECs results in the secretion of a cysteine protease that cleaves CXCL8. This factor may represent a potent anti-inflammatory compound. Disclosure of interest: None declared. It was demonstrated that NO, CO and H 2 S are gastric defense mediators. However, the interactions of these three endogenous gases in the gastric protection mechanisms are not totally defined. In this study, we evaluated the participation of NOS in CO and H 2 S gastroprotective effect against ethanol-induced gastric injury in mice. Swiss mice, (20-30 g), previously fasted for 24 h, were pre-treated with L-NAME (NOS inhibitor, 3 mg/kg, i.p.) or saline. After 30 min., received saline, hemin (HO-1 inducer, 3 mg/kg, i.p.), dimanganese decacarbonyl (DMDC) (CO donor, 12.5 lmol/kg) or NaHS (H 2 S donor, 150 lmol/kg, i.p). One hour after, ethanol 50% (0.5 ml/25 g, by gavage) was administrated. At the end of 1 h, the mice were sacrificed and the stomachs removed. Mucosal lesion area was measured by planimetry using an Image J program. Gastric corpus fragments were removed and stored until assayed for malondialdehyde (MDA) and glutathione (GSH) concentration. Data were analyzed using One-Way ANOVA and Newman-Keuls test. All animal treatments and surgical procedures were approved by the local ethics committee (protocol No 63/07). CO inducer/donor (hemin: 35.0 ± 5.7 mm 2 , MDA = 55.3 ± 6.9 nmol/g; GSH = 523.3 ± 43.9 mg/g; DMDC: 13.2 ± 2.7 mm 2 , MDA = 45.5 ± 5.7 nmol/g; GSH = 501.8 ± 25.2 mg/g) or H2S donor (NaHS: 22.3 ± 7.1 mm 2 , MDA = 50.2 ± 2.4 nmol/g, GSH = 621.1 ± 58.1 mg/g) significantly (p \ 0.05) decreased the gastropathy induced by ethanol (103.9 ± 5.9 mm 2 , MDA = 89.1 ± 5.6 nmol/g, GSH = 323.6 ± 23.9 mg/g). The pre-treatment with L-NAME reversed the gastroprotective effect induced by hemin (80. Methods: Twelve-to-fifteen week-old, Fyn-deficient and wild-type control mice were either supplied tap water or water supplemented with 2.5% (w/v) DSS for a ten day period (n = 5-6/treatment group). Throughout the study, animals were monitored for weight loss, stool appearance and rectal bleeding. Upon autopsy animals were assessed for colonic macroscopic damage, colonic length, hematocrit levels and myeloperoxidase activity. Further, colonic tissue was dissected and subjected to microscopic assessment of histopathology (0-12 scale) based upon epithelial structure, cell infiltrate, ulceration and changes to intestinal smooth muscle. Results: Wild-type mice treated with DSS lost significantly more body weight relative to Fyn-deficient mice and had significantly greater decreases in hematocrit. Wild-type mice also had significantly shorter colon lengths following DSS treatment relative to Fyn -/mice. Finally microscopic analysis indicated wild-type animals had substantially greater histopathology than Fyn -/mice, typified by marked loss of epithelial architecture, cell infiltrate, edema and smooth muscle thickening. Conclusions: Fyn-deficient mice exhibit reduced inflammatory disease following exposure to DSS. Further examination of whether this phenotype is due to improved intestinal barrier function or a modulation of the pro-inflammatory response will define the protective, anti-colitic effect of Fyn kinase deficiency. Disclosure of interest: None declared. Aims: Inflammatory bowel diseases (IBD) are characterized by altered water transport leading to the development of diarrhea. However, the status of aquaporin (AQP) 3 expression, localization and its role in the barrier dysfunction that characterizes IBD remains unknown. We hypothesized that AQP3 expression and localization were altered in IBD. Methods: C57Bl/6 mice were treated with 2.5% dextran sodium sulfate (DSS) in their drinking water for up to 7 days to induce colonic inflammation. The establishment of colitis was verified morphologically and biochemically. AQP3 expression was assessed using real-time RT-PCR and localization was assessed by immunofluorescence of formalin-fixed paraffin-embedded colonic tissue. Furthermore, AQP3 expression was assessed both in human biopsy samples and in serum-starved HT29 colorectal adenocarcinoma cells exposed to human recombinant tumour necrosis factor (TNF) a and/or interferon (IFN) c. Results: DSS-treated mice had significantly increased granulocyte infiltration at 5, 7 and 14 days, but not at 3 days following commencement of DSS. AQP3 mRNA expression in mucosal scrapings was unaltered at both 3 and 7 days following the start of treatment. However, immunofluorescence studies revealed overall downregulated expression of AQP3 in 3 day DSS-treated tissues, with diminished basolateral staining in cells lining colonic crypts. Human biopsy sample analysis revealed no significant difference in AQP3 mRNA expression between healthy control, active and quiescent ulcerative colitis biopsy samples. Time-course experiments with a single treatment of TNFa (25 ng/mL) or IFNc (500 U/mL) resulted in significantly reduced expression of AQP3 mRNA at 6-12 h posttreatment, although total protein expression levels in these cells remained unaltered at 24-96 h post-treatment. We recently demonstrated that cyclooxygenase metabolite prostaglandin E 2 induces the disruption of intestinal epithelial barrier function through the interaction with EP 1 and EP 4 receptors. In the present work, our objective was to study the effect on epithelial barrier function of eicosanoids produced by the lipoxygenase pathway: leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) and cytochrome P-450 monooxygenase pathway: 20-HETE and epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs). Moreover, we further investigated the receptors and the signaling pathways involved in this event. Paracellular permeability (PP) was estimated from fluorescein isothiocyanate-dextran fluxes and transepithelial electrical resistance in differentiated Caco-2 cells. Our results suggest that 20-HETE and EETs/DHETs have no effect on PP whereas LTD 4 , 5-HETE, 12-(R)-HETE, 12-(S)-HETE and 15-HETE increased this parameter. Moreover, our findings indicate that cysteinyl-leukotriene receptor 1 participates in the effects induced by LTD 4 while no candidate was found for 5-HETE. For both eicosanoids, these effects were mediated through the activation of the phospholipase C/Ca 2+ /protein kinase C pathway in addition to cAMPindependent PKA activation. Furthermore, we observed a correlation between increased PP and the redistribution of TJ proteins. In conclusion, on the basis of our results, we propose that lipoxygenase pathway metabolites could be contributed to the disruption of epithelial barrier function characteristic of inflammatory bowel disease. Vanin molecules are pantetheinases which release cysteamine in tissues. Based on the use of Vanin-1 deficient mice, we showed that Vanin-1 is a major regulator of intestinal inflammation. A Vanin-1 deficiency protects mice from acute infectious or toxic stresses by dampening inflammation and ensuing lesions. Mechanistically, Vanin-1 behaves as a proinflammatory molecule by controlling glutathione levels and PPARgamma anti-inflammatory potential in gut mucosa. However, Vanin-1 role is probably more complex. Vanin-1 exerts a cytoprotective role on islet beta cells and consequently contributes to the progression of type 1 diabetes. We have investigated the contribution of human VNN1 to human gut inflammatory diseases. We sequenced the Vanin-1 gene in 400 IBD patients and controls and identified novel polymorphisms and mutations which might contribute to the progression of the disease by modulation of the enzymatic activity in gut epithelium. In parallel we developed novel tools to quantify cysteamine activity in tissues and show that VNN1 is highly expressed in epithelial cells of patients with ulcerative colitis. We also document the presence of a seric form of the enzyme which may serve as a marker in various disease conditions. Preliminary results indicate that seric pantetheinase might be involved in the control of systemic inflammation and blood homeostasis. Disclosure of interest: None declared. Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is partly caused by dysfunction of immune system and overproduction of inflammatory cytokines. Our previous studies identified an anti-inflammatory peptide; pyroglutamyl-leucine (pyro-Glu-Leu) in Japanese rice wine (sake). The objective of the present study was to prepare pyroGlu-Leu rich fraction from food and check its therapeutic potency to IBD. Constituents in Japanese rice wine were fractionated into 10 fractions on the basis of isoelectric point and charge by an ampholyte-free preparative isoelectric focusing (autofocusing) apparatus. Fr. [1] [2] [3] [4] [5] Fr. 8 (pH 3.40), Fr. 9 (pH 8.66) and Fr. 10 (pH 12.14) were prepared. Then, Fr. 1-5 was fractionated by solid phase extraction using Sep Pak C18. Pyroglutamic acid and pyroGlu-Leu were rich in Fr. [1] [2] [3] [4] [5] and Fr. 8 and Fr. 9 also contained some amount of pyroGlu-Leu. Acute colitis was induced in mice by oral administration of 2.5% dextran sulfate sodium (DSS) in drinking water for 7 days. Mice were treated with each fraction at 30 mg peptide/kg body weight/day or various concentration of synthetic pyroGlu-Leu during entire colitis induction period. Fr. 9 treated group significantly improved the colon length (p = 0.05) and percent loss in weight (p = 0.04) compared with control group on DSSinduced acute colitis. 0.1 mg/kg of pyroGlu-Leu treated group also significantly improved percent loss in weight (p = 0.05). On the other hand, Fr. 1-5 treated group significantly reduced the colon length (p = 0.02). The group treated with Sep Pak C18-unabsorbed fraction of Fr. 1-5 showed high mortality while all in control group survived. This result indicates that Japanese rice wine-derived components have a potent of therapeutic agent on IBD and pyroGlu-Leu might be one of these components, though Japanese rice wine contains low amount of detrimental substance within acidic and hydrophilic properties. Disclosure of interest: None declared. Background: Ulcerative colitis (UC) is a chronic inflammatory disease of the rectal and colonic mucosa and seems to result from a complex series of interactions between susceptibility genes, the environment and the immune system. There is a paucity of data on the positive effect of vitamin A on intestinal mucosal immunity. We aimed to evaluate the effect of vitamin A supplementation on proinflammatory cytokine interleukin 12 (IL12) and acute phase protein, high sensitive-C-reactive protein(hs-CRP) and anti-inflammatory cytokines, IL4 and IL10 in ulcerative colitis. Methods: Immunological assessment was done in 43 outpatients with ulcerative colitis participating in a before and after interventional survey in Sheikh al rais, a public clinic of Tabriz. All participants were at clinical remission stages. The severity of disease activity was assessed by Truelove-Witt Index. Vitamin A supplement was injected twice (50000 IU) with 2 weeks interval. Study period was considered 45 days. Serum interleukins and hs-CRP levels were measured by ELISA and turbidimetric methods, respectively. Results: Vitamin A supplementation had significant effect on decrease of IL12 (P = 0.003) and clinical symptoms severity (P = 0.001). IL4 increased considerably(P = 0.001). While hs-CRP and IL10 changes were not significant. Conclusion: Vitamin A supplementation could suppress serum proinflammatory cytokines and be beneficial in reduction of clinical symptoms severity of disease in ulcerative colitis in remission and should be explored in a larger study. Disclosure of interest: None declared. Inflammatory bowel disease (IBD) is a disease of chronic intestinal inflammation, which causes severe diarrhea, abdominal pain, bleeding and finally development of colon cancer. Serotonin is a neurotransmitter and gastrointestinal hormone, which regulates intestinal movements in the gut. In human IBD patients, serotonin level is increased in inflamed gut. In a previous study, we found that serotonin induces adhesion of monocytes to HT-29 colon epithelial cells, similar to the action of TNF-a. Since the presence of high levels of bacteria creates a biofilm on the surface of the gut mucosa in patients with IBD, we evaluated whether serotonin and its precursor indole inhibit bacterial biofilm formation in vitro. Indole and its derivative 3,3 0 -methylene-bisindole (MBI) reduced enterohemorrhagic Escherichia coli O157:H7 biofilm formation, whereas serotonin did not. In terms of adhesion of monocytes to HT-29 cells, indole and MBI significantly inhibited TNF-a as well as serotonininduced adhesion of monocytes to HT-29 cells. In addition, indoles were more potent at inhibiting serotonin-induced than TNF-ainduced adhesion. Taken together, serotonin acts as proinflammatory factor in IBD by inducing inflammatory response without affecting bacterial biofilm formation. In contrast, indoles inhibited serotonin and TNF-a-induced monocytes adhesion to HT-29 cells as well as bacterial biofilm formation. Disclosure of interest: None declared. induced monocyte adhesion to HT-29 colon epithelial cells similar to TNF-a. No additive effect was seen in case of co-treatment of the cells with serotonin and TNF-a. Serotonin-induced inflammatory response was subsided well by the treatment with different polyphenols compared to TNF-a-induced inflammation. In vivo, intra-colonic administration of serotonin in rats through rectum increased myeloperoxidase activity and expression of IL-8, IL-6 and VCAM-1. Since several lines of evidence have implicated that IL-8 induces abnormal angiogenesis in the inflamed site, we evaluated whether serotonin induces angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. In the CAM assay, serotonin (0.1 and 1.0 lg) dosedependently induced the formation of new blood vessels. In conclusion, our results indicate that serotonin acts as a proinflammatory factor in the colon. Background: Ulcerative colitis (UC) is a disease of unknown etiology caused by an inappropriate mucosal immune response to antigens from the intestinal flora. Its etiology has not yet been resolved, but immunological and genetic factors are known to play a very important role. Active UC is often associated with elevated peripheral blood granulocytes and imbalanced inflammatory cells (CD4, CD8 Th cells). As these cells are major sources of inflammatory cytokines, selective changes of these cells in patients with UC should alleviate inflammation. According to the anti inflammatory role's of vitamin A, we decided to evaluate the effect of vitamin A supplementation on circulatory white blood cell (WBC) subtypes and inflammatory cells in this study. Methods: Forty-three patients with ulcerative colitis were included in a before and after, interventional survey in Sheikh al rais, a public health clinic of Tabriz. All participants were at clinical remission stages. The severity of disease activity was assessed by colitis activity index (CAI).Vitamin A supplement was injected twice (50,000 IU) with 2 weeks interval. Study period was considered 45 days. Total WBC count was performed by using an automatic blood cell counter. The serum levels of hs-CRP were evaluated by immunoturbidimetry. CD4 and CD8 T cells were analysed by flow cytometry. Results: In UC, neutrophil lymphocyte ratio (NLR) and hs-CRP levels positively correlated with CAI (P \ 0.05). There was a negative correlation between CD4 T cell and CAI (P = 0.01).We found no significant effect of vitamin A supplementation on CD4 and CD8 T cell subsets, neutrophil and monocyte count after supplementation. NLR and hs-CRP levels decreased not considerably, while platelet count decreased significantly (P = 0.004 Crohn's disease is an inflammatory bowel disease (IBD) with a Th1 inflammation of unknown etiopathogenesis. Accumulating evidence points to breakdown of the anti-inflammatory cytokines IL-10 and/or TGFb production/signaling in subpopulations of Crohn's disease. However, as to whether these defects can be the driving force of the inflammatory process remains to be defined. To address the issue of the consequences of depleting a normal human intestinal mucosa from IL-10 or TGFb, we have recently setup an ex vivo model system of explant cultures of human normal colonic mucosa, microdissected from surgical resections, in combination with immunoneutralization of IL-10 or TGFb. This model maintains cellular interactions between mucosa resident cells and is amenable to a multiparametric methodological approach (QPCR, ELISA, in situ immunofluorescence, flow cytometry after collagenase digestion). We previously demonstrated that immunoneutralization of IL-10, endogenously produced by resident mucosa cells including epithelial cells, leads to a Th1 response, mainly IFNc, triggered by LPS from the microflora, and responsible for epithelial barrier damage. In this work, aimed at elucidating how resident mucosa cells are converted into ''colitogenic'' pro-inflammatory cells, we demonstrate that the IFNc response elicited by IL-10 breakdown in explant cultures is preceded by active caspase-1 production and early secretion of mature IL-18 by epithelial cells and lamina propria macrophages. Furthermore, the IFNc response is blocked by a caspase-1 inhibitor and by IL-18-binding protein. These results show that the Th1 response upon IL-10 breakdown depends on IL-18/caspase-1, part of the inflammasome pathway. In addition, we also show that immunoneutralization of another anti-inflammatory cytokine, TGFb, also elicits a Th1 (IFNc) response, but via a distinct, non redundant pathway. These distinct signalling pathways we identified can therefore represent targets in some forms of IBD. Disclosure of interest: None declared. Physiology and Pharmacology, University of Calgary, Calgary, Canada The lymphatic system is involved in many disease pathologies, including inflammatory bowel diseases (IBD). Lymphatic vessel abnormalities reported in IBD include vessel dilation, hypocontractility and flow obstruction. These may lead to inefficient removal and transport of excess interstitial fluid, antigens, and immune cells. Inflammatory mediators, particularly prostaglandins can potently modulate lymphatic vessel contractile function. Our hypothesis is that key mediators released during the inflammatory process can signal through the lymphatic vessels to modulate lymphatic function and phenotype. Inflammation was assessed in the rat ileum 24 h after surgical instillation of the hapten TNBS using a macroscopic damage scoring system and a myeloperoxidase activity assay. Mesenteric lymphatic vessels were assessed for diameter and collected for quantitative real time PCR analysis of the following mRNAs: Cyclooxygenase 1 and 2 (COX-1, COX-2), TNFa receptor 1 (TNFR1), interleukin 1-b receptor (IL1R1), interleukin 6 receptor (IL6R), interferon gamma receptor (IFNR1), prostacyclin synthase (PTGIS), prostacyclin receptor (IP), prostaglandin E2 synthase 1/2 (PGES 1/2), EP2, and EP4 receptors. Ileum from TNBS-treated rats shows an increased granulocyte infiltration, and higher macroscopic S246 Inflamm. Res. damage scores compared to sham counterparts. Lymphatic vessels from TNBS-treated animals exhibited an increase in diameter, suggesting lymphatic contractile dysfunction. Quantitative real-time PCR shows a significant upregulation of COX-2, TNFR1, IL1R1, PTGIS and IP mRNAs in lymphatic vessels from TNBS-treated animals. Our findings suggest an increased responsiveness of the lymphatic vessels to IL-1b and TNFa, as well as an increase in lymphatic prostacyclin production and signalling. These changes could lead to the dilated lymphatic phenotype we observed in this study. Lymphatic dilation could have potential implications in modulating the course of intestinal inflammation. Disclosure of interest: None declared. Arup Institute for Clinical And Experimental Patho, 2 Pathology, University of Utah, Salt Lake City, United States TGF-b1 is the important mediator of immune and anti-inflammatory responses; it is involved in normal renal function and in the development of different nephropathies. Present research was dedicated to the validation of urine TGF-b1 measurement by Quantikine Human TGF-b1 ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). Human recombinant TGF-b1 was spiked into normal urine at different concentrations, and frozen in aliquots. Collection of 100 frozen urine samples from healthy donors were used to investigate distribution of urine TGF-b1 in normal population. Creatinine concentration in urine was determined at ARUP Laboratory. After the thawing, considerable amount of precipitate was found in some samples. Such samples were centrifuged, and supernatants or un-spun specimens were run with TGF-b1 assay in duplicates. TGF-b1 activation procedure (which precedes ELISA steps) clarified all samples no matter how much precipitation they contained. It was shown that supernatants contained only fraction of the whole sample TGF-b1 activity (77-29%, even less for the normal low concentration samples). Validation involving un-centrifuged urine samples demonstrated following TGF-b1 assay characteristics: -linearity: from 23 to 1286 pg/mL, recovery from 95-116%; -precision: \17%; -analytical specificity-Limit-Of-Blank: 3 pg/mL; -reference interval: from 0 to 39 pg/mg creatinine -freeze-thaw stability: after first thawing, urine samples could loose from 5 to 40% of TGF-b1 activity on each following freeze-thaw circle. The prostate is an accessory reproductive gland that is found in all male mammals. This gland is prone to infection, inflammation, and cancer, making it one of the most diseased tissues in the body. Specifically, the role of protease-activated receptor 1 (PAR1) has not been addressed in the context of prostatitis. Therefore, we have developed a murine model of prostatitis to investigate the inflammatory effects of PAR1. We hypothesized that administration of 50% ethanol (EtOH) and dinitrobenzenesulfonic acid (DNBS) into the mouse prostate would incite an inflammatory response, and that treatment with the PAR1 activating peptide TFLLR-NH 2 would modulate this reaction. EtOH + DNBS were administered alone or in conjunction with TFLLR-NH 2 via catheter and mice were sacrificed 48 h following treatment. Prostates treated with EtOH + DNBS alone showed a 4-fold increase in weight and myeloperoxidase (MPO) specific activity and a respective 3-fold and 16-fold increase in CXCL-1 and IL-10 production over controls (p \ 0.05). Furthermore, histological analyses of these prostates revealed edema, extensive leukocyte infiltration, and destruction of the prostatic acini. Compared with EtOH + DNBS treatment alone, prostates given EtOH + DNBS in conjunction with TFLLR-NH 2 showed a 4-fold decrease in weight and MPO specific activity, a 3-fold decrease in CXCL-1, and a 3-fold increase in IL-10 production (p \ 0.05). Histological analyses of these prostates revealed minor edema, minimal leukocyte recruitment, and minor tissue damage. In conclusion, acute inflammation of the prostate can be induced in mice using a combination of EtOH and DNBS. Furthermore, activation of PAR1 modulates this inflammatory reaction by increasing IL-10 production, decreasing MPO and CXCL-1 production, and reducing tissue damage. These findings support an anti-inflammatory role for PAR1 in the mouse prostate. Disclosure of interest: None declared. Cyclophosphamide (CYP)-induced bladder inflammation is widely used as an in vivo model for bladder pain syndrome. We previously showed that CYP-induced bladder pain was reversed by ibuprofen and aspirin, 2 non steroidal anti-inflammatory drugs (NSAIDs), and morphine, an opioid agonist. We studied the modulation of different inflammatory mediators by these pharmacological treatments in CYPinduced bladder inflammation. Female rats were dosed with aspirin, ibuprofen, morphine or vehicle prior a single injection of CYP. Urines were then collected over 4 h and rats sacrificed. Bladders were assessed for weight, wall thickness and macroscopic damage. Inflammatory mediators were quantified in bladders and urines using enzyme immunoassays or Multiplex technology. After CYP injection, an increase in bladder weight and wall thickness was observed associated with strong edema and erythema. Bladder levels for interleukin-1b (IL-1b), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion molecule (VCAM) were significantly increased as well as urinary levels for prostaglandin E 2 (PGE 2 ), lipoxin A 4 (LXA 4 ) and 15-epi lipoxin. After NSAID treatment, macroscopic parameters were not affected except for a decreased wall thickness. Urinary PGE 2 levels were strongly reduced by both treatments, while IL-1b and IL-6 tissue levels were decreased by ibuprofen treatment only. In contrast, morphine-treated rats presented a strong increase in wall thickness and macroscopic scores, associated with increased bladder levels for IL-6 and MCP-1, and increased urinary 15-epi lipoxin. Other mediators like LXA 4 were not significantly affected by these drugs. In summary, antinociceptive effects of NSAIDs are associated with a decrease in key inflammatory mediators such as IL-1b, IL- Control mice were fed with standard food. Another group received adenine chow and allopurinol diluted in drinking water. At sacrifice time, blood and renal tissue were collected for analysis. Serum creatinine was measured and histopathologic changes were evaluated. Also, RT-PCR was performed for IL-18, TNF, IL-1b, and IL-6. Furthermore, uric acid, ox-LDL, TGFb and pro-inflammatory cytokines were measured on renal tissue and immunoblotting was done for IKK. Results: WT animals presented higher levels of serum creatinine and enhanced cellular infiltration and collagen deposition. They also showed higher gene and protein expression of inflammatory cytokines. In contrast, KO mice showed renoprotection, with serum creatinine levels, histology evaluation, and inflammatory molecules expression comparable to control. This protection was also observed on allopurinol group, which also presented decreased levels of uric acid, ox-LDL, TGFb and IKK expression. Conclusions: TLR2/4 and Myd88 seem to participate on the development of TIN. Inhibition of XD also seems to be a promising therapeutic target to combat the developing inflammatory process. Financial support: FAPESP/INCT Complex Fluids/CNPq. Disclosure of interest: None declared. Acute kidney injury (AKI) presents close association with immune system including the participation of Toll-like receptors (TLRs). TLRs may interact with potential ligands released by cell death leading the production of several inflammatory cytokines, which can amplify the tissue damage. Thus, this study was designed to evaluate the influence of TLR-2 and TLR-4 receptors on prevention of cisplatin-induced AKI. So, we used wild type (WT), TLR-2 -/and TLR-4 -/mice to evaluate the morphological and functional renal parameters, besides cytokines profile and gene expression analysis. Firstly, it was observed that both KO animals showed a significant reduction of TNF-a (3.6-and 2.2-fold decreased), IL-6 (1.9-and 4.9fold decreased) and IL-1b mRNAs (13.6-and 16.3-fold decreased) transcripts. Additionally, the production of pro-inflammatory cytokines (IL-1a, IL-1b, IL-3, IL-6, TNF-a, IFN-g, CCL-5, KC, MIP-1a and G-CSF) was considerably diminished in TLR-2 -/and TLR-4 -/mice. Although, only TLR-4 -/mice have a trend toward expressed anti-inflammatory molecules as Th1-and Th17-related cytokines (IL-4, IL-10, IL-12 and IL-17). Likewise, the absence of TLR-4 but not TLR-2 receptor protects mice from severe weight loss, acute tubular necrosis (ATN) and high levels of serum creatinine (SC) at 96 hs of injury (C4 to\1 mg/dL, respectively). In order to evaluate the mechanisms behind of TLRs activation, we also investigated the cytoprotective effect of Heme oxygenase-1 (HO-1). Surprisingly, the treatment with COPPIX (5 mg/kg), an intracellular activator of HO-1, decreased ATN and SC levels in WT and TLR-4 -/mice, but not in TLR-2 -/animals. All together, these results suggest that TLR-2 and -4 responded to injury signals of kidney milieu, intensifying the renal failure. However, TLR-4 have a fundamental role as pro-inflammatory agent and their cross-talking with HO-1 and TNF-a can help in better understanding of process that govern the AKI. Results: Of 50 patients who had acute allograft rejection, nine patients had combined treatment of more than one anti-lymphocyte. In group 1 had more syndrome and invasive disease and lower incidence of asymptomatic. The viral load of these patients, showed that patients with invasive disease had high viral load in compared with others groups. When analysed viral load inside groups, the group with used in induction of anti-lymphocyte had increased of viral load. Patients that used antilymphocyte had more disease (syndrome and invasive disease). All patients with invasive disease had involvement primarily in the gastrointestinal system. Conclusion: Patients who used anti-lymphocyte had more CMV disease (syndrome and invasive disease) with involvement of the gastrointestinal system and showed higher viral load in relation asymptomatic patients. An excessive oral intake of adenine in mice leads to the accumulation of 2,8-dihydroxyadenine (DHA) in renal tubules and that causes progressive renal dysfunction accompanied by interstitial fibrosis. Physiopathology of renal diseases involves several elements in the immune response. Since NKT cells are a unique T lymphocyte sublineage that has been implicated in either up-or downregulating immune responses, the aim of our work was to study the role of NKT cells in an adenine-induced tubulointerstitial nephritis. Materials and methods: Male C57BL/6 wild-type and Ja18 and CD1d KO mice were used in this experiment. They were allowed food and water ad libitum. A 0.25% adenine-containing diet was used in all adenine groups during 10 days. Control group received standard diet. Mice were sacrificed at day 10, after the start of adenine diet, for collecting blood and kidney. Serum urea and creatinine were measured and kidney samples were used to isolate RNA. RT-PCR assay and Western Blotting was performed as described in standard protocols. Results: Serum urea and creatinine values were significant higher in adenine-fed WT mice compared with the control group. Although urea and creatinine were higher in adenine-fed WT than in KO groups, there was no significant difference between then. There was a significant increase in gene expression of IL-6, IL-1b, TNF-a, TLR-2, Myd88 and XDH (xanthine dehydrogenase) in adenine-fed WT mice compared with the control group. IKK protein expression was increased in adenine-fed WT mice than in control group. Our findings suggest that NKT cells may play a role on tubulointerstitial nephritis. DHA accumulation seems to be involved in the inflammation process in kidney injury since there was an increase in genes and protein expression that implicate it. Disclosure of interest: None declared. The role of the cytokine tumour necrosis factor-a in the pathophysiology of renal ischemia/reperfusion (I/R) injury is unclear. Here we investigate the effects of on the degree of renal injury induced by I/R, dysfunction and inflammation in (1) tumour necrosis factor-R1 knock out (TNF-aR1KO) mice and in (2) wild-type mice administered with infliximab, a chimeric antibody which neutralise the biological activity of TNF. All mice were subjected to bilateral renal artery occlusion (45 min) and reperfusion (6 h) . In addition, wild-type mice were pretreated (30 min before ischemia) with a TNF-a soluble receptor construct, infliximab, to mimic the effects that would be seen in TNF-aR1KO mice. At the end of experiments, indicators and markers of renal dysfunction, injury and inflammation were measured. Kidneys were used for histological evaluation of renal injury, for the localization of the intercellular adhesion molecules and selectins, as well as apoptosis (TUNEL, Bax, Bcl-2 and Fas-L expression) by immunohistochemistry and western blot analysis. TNF-aR1KO mice and wild-type mice administered with infliximab demonstrated significantly reduced plasma urea and creatinine levels in comparison with I/R-mice, indicating reduction of renal dysfunction. Neutrophil infiltration was also significantly reduced in TNF-aR1KO mice and wild-type mice administered with infliximab subjected to renal I/R. TNF-aR1KO mice demonstrated reduced histological tubular injury and markedly reduced immunohistochemical evidence of ICAM-1, P-selectin, and apoptosis when subjected to renal I/R. Our results clearly demonstrate that TNF-a play an important role in the renal ischemia and reperfusion injury and put forward the hypothesis that modulation of TNF-a expression may represent a novel and possible therapeutic option to attenuate the ischemia-reperfusion injury process that leads to acute kidney injury, a frequent complication with renal transplantation, also associated with graft function. Disclosure of interest: None declared. Retinol binding protein (RBP) is a small tubular protein, freely filtered by the glomeruli barrier and totally reabsorbed by the proximal tubular cells. High levels of uRBP indicate proximal tubular dysfunction (PTD) and closely correlate with long term renal and heart transplant outcomes. However, at this stage of PTD, histology or renal function are not good screening tests. Therefore, we were interested in identify the gene profile of those patients, with PTD assessed by uRBP, in order to discover new biomarkers. Renal transplanted patients with good (serum creatinine \1.5 mg/dL) and stable (for at least 3 months) were screened for PTD. Sixteen patients with high levels ([1.000 mg/L) and 11 patients with normal uRBP levels were consecutive selected a graft biopsied of performed after uRBP measurement. Agilent (GE) platform of 44,000 genes were used. Each slide quality was assessed and those considered good and discovered were selected for plotting the spots. We calculated gene fold difference between groups and those above and below 2 were selected. 135 genes were differentially expressed; 123 down regulated and 12 up regulated in patients with higher uRBP levels. Among those genes, we observed difference in cell adhesion molecules, cell communication, cytosolic calcium ion concentration and ribosome pathways. PTD is associated with disturbance in cell physiology reflecting the impairment in the endocytic machinery. uRBP can identify the patients with higher risk for the development of chronic allograft dysfunction that have no abnormalities in renal function or no outstanding changes in histology but a modification in renal gene profile. Our data demonstrated that although PTD is associated with normal renal function and no specific histological abnormalities, it is characterized by a set of genes that might unveil its link with development of chronic allograft dysfunction. Immunology, University Of Sao Paulo, 2 Nephrology, Federal University of Sao Paulo, Sao Paulo, 3 Pathology, Federal University of Triangulo Mineiro, Uberaba, 4 Cellular Biology, University Of Sao Paulo, Ribeira˜o Preto, Brazil Introduction: Acute kidney injury (AKI) constitutes important clinical problem, leading to high rates of morbidity and mortality. The most important etiology of AKI involves ischemia/reperfusion injury (IRI), an inflammatory response related to IL1-b participation. These data prompted us to evaluate the participation of the inflammasome complex, which is responsible for secretion of active IL-1b, in the development of IRI. Methods: C57BL6 WT animals and KO for caspase-1, ASC and MyD88 were subjected to 45 min of ischemia and 24 h of reperfusion. Blood samples and kidney tissues were collected for analysis. Results: Serum creatinine and histological data showed that KO animals for caspase-1, ASC and mainly MyD88 had a significant protection of the renal function compared to WT animals 24 h after IRI. The protected animals showed lower areas of hypoxia and gene expression of HIF-1a. In addition, protected animals had higher expression of anti-apoptotic gene BCL-2. Regarding gene and protein expression of pro-inflammatory cytokines, we found that WT animals had higher expression of these inflammatory factors. Some of these results are presented here: IL-6 (WT: 6.78, casp- Glomerular endothelial cell damages are a hallmark for atypical hemolytic uremic syndrome (aHUS) and involve an uncontrolled activation of complement alternative pathway (AP), as suggested by the association with mutations of complement proteins. Starting from the hypothesis that endothelial cells themselves trigger such complement activation, we investigated conditions which could transform quiescent cells into AP activators, and the effect of a poorly regulated complement on these cells. Human quiescent umbilical vein HUVEC and glomerular GEnC endothelial cells were treated with hemolysisderived or inflammation stimuli (free heme, neuraminidase, E. coli LPS, TNFa/IFNc/IL1b). Cells were, then, incubated with human serum and analyzed for complement membrane deposition, by flow cytometry after cells detachment. Blocking anti-MCP mAb, factor H depleted serum or aHUS sera with genetic complement abnormalities were used as models for poorly regulated complement. With normal human serum, minimal C3 deposits were detected on quiescent adherent cells. Among tested stimuli, maximal levels of cell-bound C3 were observed on cells pre-incubated with heme or TNFa/IFNc/ IL1b, but these levels remained limited. In vitro functional inhibition of MCP or aHUS sera resulted in a modest increase of C3 on quiescent adherent cells. This C3 deposition was, however, further enhanced on cells pre-treated with heme or pro-inflammatory cytokines and became significantly higher than that observed with normal serum, emphasizing the amplifying effect of defective complement control. In conclusion, quiescent endothelial cells are well protected against autologous complement activation on their surface. Common agonists released during inflammation or hemolysis trigger low level complement activation on endothelial cell membrane. This is amplified by uncontrolled or over-reactive AP components and could result in the release of C5a and C5b-9, with known pro-inflammatory and pro-coagulant effects. Disclosure of interest: None declared. Introduction: Adriamycin (ADM) nephropathy (AN) is a disease characterized by glomerular sclerosis, proteinuria and up regulation of proinflammatory cytokines. Many works pointed for a possible role of kinin receptors in inflammatory studies. On renal diseases models the blockage of B1 receptor (B1RBK) seems to diminish the inflammatory markers on the other hand the role of B2 receptor (B2RBK) has not been unveiled yet. In this way we studied the role of these kinin receptors in the inflammatory reaction induced by ADM injection using specific antagonists for these receptors. Material and methods: The AN was induced by a single injection of ADM in the tail vein of Balb/c mice. The mice were treated with the kinin antagonists DALBK (B1RBK antagonist) in for 3 days, DABK (B1RBK agonist) for 3 days and with HOE-140 B2RBK (antagonist) for 3 or 10 days. The renal function parameters were analyzed by levels of albuminuria, Western Blot and PCR real time expression of podocyte proteins. The levels of cytokines like TGFb1 were evaluated by ELISA, the macrophage infiltration by Flow cytometry and the levels of proinflammatory cytokines were analyzed by a mouse cytokine Bioplex assay kit. Results: The use of DALBK protected and revert the signs of mice albuminuria and podocyte dysfunction. This result was associated with the downregulation of proinflammatory markers such as TNF-a, TGF-b, IL-1b, MCP-1 and microphage infiltration, a mirrored result was observed with the Treatment with DABK (B1RBK antagonist) showing a positive effect of B1RBK blockage in AN. Differently the treatment with HOE-140 (B2RBK antagonist) did not protected animals from AN signs at the beginning of the AN on the other hand in an established disease downregulated the levels of proinflammatory cytokines, suggesting that the role of this receptor depends of the stage of AN progression. Differently the B1RBK blockade that at all phases of the disease was showed to be an important target to AN. Disclosure of interest: None declared. Ischemia and reperfusion injury (IRI) is an acute inflammatory response considered to be the main cause of acute renal injury in kidneys. Dendritic cells (DC) have been recently associated to have a role in IRI, although a few is known about their functionality and their possible role in T cell activation in this model. In this way, we investigated whether DC are involved in IRI and their participation in T cell modulation in this model. We used C57Bl/6 and C57Bl/6-CD11c-DTR mice to perform the ischemia and sham group as control. We observed that after 24 h of reperfusion, urea ([250 mg/dl) and creatinine ([1 mg/dl) levels were increased in ischemic group, indicating the lesion. No difference of DC numbers were observed in the kidney and draining lymph nodes, however, DC from ischemic group were more activated, presenting more pronounced expression of CD86. We then used two models of depletion of dendritic cells: clodronate depletion of phagocytes and C57B/l6-CD11c-DTR mice with diphtheric toxin (4 ng/g of mice). Both protocols resulted in increased levels of urea ([300 mg/dl) and creatinine ([2 mg/dl) in the serum of DC depleted mice. In order to investigate the influence of DC in CD4+ T cell modulation in IRI, we analysed the phenotype of CD4+ T cells in DC depleted mice after ischemia and reperfusion induction and we observed that the population of CD4+ CD69+ (activated) T cells was increased, while no differences were observed in CD4+ CD25+ FOXP3+ T cells (Tregs), indicating that DC might be important for the regulation of T cell response in IRI. We conclude that DC are involved in IRI and they modulate CD4+ T cells in order to control the injury. Support: FAPESP. Disclosure of interest: None declared. Introduction: The objective of this study was to highlight the effects of aspirin, ibuprofen and morphine on the production of pro-resolving lipid mediators during a cyclophosphamide (CYP)-induced bladder inflammation. Methods: For this, female rats were dosed with aspirin, ibuprofen, morphine or vehicle prior to a single injection of CYP. Urines were then collected over 4 h and mediators analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology to quantitatively evaluate bioactive lipids production in urine. Results: During these experiments, we showed that CYP induced the production of lipid mediators derived from the cyclooxygenase (COX) and lipoxygenases (LOX) pathways. Moreover, CYP treatment induced bladder pain, which was inhibited by aspirin, ibuprofen and morphine treatments. The use of aspirin and ibuprofen completely abrogated the mediators depending on COX pathways while morphine had no effects on those. On the other hand, aspirin boosted the LOX pathways and the production of the precursors of resolvins, maresin and protectins. In contrast, the use of ibuprofen and morphine inhibited the production of these same precursors. Conclusions: Taking together the results suggest that aspirin may act on bladder pain through the induction of key pro-resolving mediators, while ibuprofen and morphine may promote the desensitization of bladder nociceptors by other pathways. Disclosure of interest: None declared. Background: Skin infection by Leishmania braziliensis results in cutaneous and mucosal leishmaniasis. In between this clinical spectrum, disseminated leishmaniasis (DL) aside primary ulcer and ulcerated secondary lesions differs from others by clinical manifestation of multiple papules and non ulcerated lesions. In this study, we describe the histopathology of DL papular lesions, correlating them with inflamed area, CD4, CD20, CD68, CD31 cells and von Willebrand factor (vW) amount characterizing immune inflammatory aspects. Methods: Eighteen biopsies from patients diagnosed by clinical criteria, positive skin test and fine needle aspiration in vitro cultivation were analyzed. Histopathological aspects were obtained by analysis of Hematoxylin and Eosin (H&E) stained slides. To quantify the inflamed area and cells positivity for CD4, CD20, CD 68, CD31 and vW the Image-Pro (Media Cybernetics) software was used. Results: Diffuse lymphoplasmocytic perivascular infiltrate and granulomas not well organized were found in dermal skin. Histopathological patterns were graded from I to III [39% (I), 11% (II) and 50% (III)]. Inflammation extent varied from 3 to 73%, presenting a significant linear correlation with vW+ vessels and with graded patterns. The most frequent inflammatory cells were macrophages (CD68+) followed by B cells (CD20+) and T cells (CD4+). Significant linear correlation between CD4+ and CD20+ cells and inflamed area was also found. In more inflamed lesions we found grater amount of CD31+ cells. Amastigotes were visualized or immunostained in 8 biopsies. Conclusions: Our findings suggest that chronic inflammation was present in all papules predominantly formed by macrophages plasmocytes, T and B cells. As inflammation area expands, granulomas and vessel framework increases proportionally. Thus, the present study highlights the possible role small vessels may have in clinical evolution and spectrum from acneiform papules to larger nodular skin lesions. Disclosure of interest: None declared. Background: Studies have shown the association between psoriasis and celiac disease (CD), an immune-mediated gluten-dependent enteropathy due to inappropriate T cell mediated response against ingested gluten, and also improvement of psoriasis after gluten-free diet. However, the exact relationship between psoriasis and CD remains controversial. Aim: We aimed to assess the prevalence of IgA anti gliadin (AGA) antibodies and other celiac disease associated antibodies as IgA tissue transglutaminase (tTG) antibodies and IgA antiendomysium antibodies (EMA) in psoriatic patients, and their possible role in the pathogenesis of psoriasis. Patients and methods: Forty-one psoriasis patients and 41 healthy controls were included in this study. Blood samples were taken from all subjects and screened for AGA, tTG, measured by ELISA, and EMA assessed by standard immunofluorescence with cryo section to monkey oesophagus. Results: There was a significant difference between patients and controls as regards AGA but no significant difference between the 2 groups as regards tTG and EMA was detected. Conclusion: Finally, the significant increase in the level of some CD antibodies detected in this study indicates that their serological evaluation should be considered in psoriasis patients. Also, glutenfree diet could lead to better management and decreased morbidity of psoriasis. Disclosure of interest: None declared. Dermatology, 2 Clinical Immunology, University of Naples Federico II, Naples, Italy IL-33 is a pro-inflammatory cytokine recently identified as a ligand for the orphan receptor ST2. IL-33 constitutive nuclear expression has been found in cells of tissues exposed to the environment. Moreover, endothelial cells have been shown to abundantly express IL-33. Many of the inflammatory effects of IL-33 define its ability to induce Th2-type mediated responses but recently IL-33 was found to have a pro-inflammatory role in arthritis, which is Th1 and/or Th17 mediated. It can be hypothesized that IL-33 has a proinflammatory role in psoriasis too. In the present study we aimed to assess the role of IL-33 in psoriasis, investigating its property to promote inflammation via mast cell (MC) and keratinocyte (KC) activation. Here we report that IL-33 is elevated in the skin of psoriasis patients. Furthermore, IL-33 upregulates IL-4 and IL-13 and is able to induce a higher increase of MCP-1 and VEGF respect to TNF-a in MCs. In presence of TNF-a, IL-33 induces MCP-1 and IL-6, whereas in association with IL-17, is able to induce IL-20 in KCs. In conclusion, our study provides evidence that IL-33 is involved in psoriasis biology. Furthermore, our results reinforce the IL-33 activity in driving Th1 response too, contributing to the maintenance of psoriasis pathogenesis. Disclosure of interest: None declared. Objective: Dimethylfumarate (DMF) is used in the treatment of psoriasis. Macrophage migration inhibitory factor (MIF) is elevated in patients with severe psoriasis. We studied the effect of DMF on the MIF induced activation of the Mitogen and Stress-Activated Kinase 1 (MSK1) and p90 kDa Ribosomal S6 kinase (RSK1) signaling pathways which regulate the proliferation of human keratinocytes via transcription factors. Methods: The effect of DMF on the MIF induced activation of MSK1, RSK1, CREB, Cox-2 and c-Jun, JunB and p53 were studied by Western Blotting using phospho-specific antibodies. Results: DMF inhibited the MIF induced phosphorylation of MSK1, RSK1, CREB and JunB, and reduced Cox-2 expression and the proliferation of cultured human keratinocytes. The expression of p-p53 (S15) was induced, simultaneously with the inhibition of Cox-2. Addition of DMF before MIF induced nuclear expression of p-c-Jun (S63) and c-Jun. Transfection with small interfering MSK1 and RSK1 RNA before MIF incubation, stimulated p-p53 (S15) and nuclear p-c-Jun (S63) similar to DMF. Conclusion: Our results indicate that the specific inhibitory effects of DMF on RSK1 and MSK1 activation together with the induction of p-c-Jun (S63) and p-p53 (S15) lead to the inhibition of keratinocyte proliferation, partly explaining the anti-psoriatic effect of DMF. Disclosure of interest: None declared. Many authors reported a significant involvement of nitric oxide (NO) in the process of keratinocytes proliferation with many divergences. In order to determine the involvement of NO in hyperproliferative process of epidermis in vivo, chronic inflammatory process was induced by multiple applications of croton oil (0.4 mg/ear) for 9 days on alternate days. Mice were topically treated with the NG-nitro-Larginine-methyl ester (L-NAME, 10 lmol/ear), Aminoguanidine (AG, 10 lmol/ear), Sodium nitroprusside (SNP, 2.5 lmol/ear), 7-nitroindazole (7-NI, 10 lmol/ear), or Dexamethasone (0.1 mg/ear), starting on the 5th day. Oedema was measured daily as increase in ear thickness and on the last day 6 mm sample of ear tissue was collected, weighed and analyzed by histology and immunohistochemistry (PCNA). Procedures were approved by Institutional Ethics Committee (130). L-NAME, 7-NI, and dexamethasone treatments were able to decrease, while SNP increased ear oedema during treatment. However, the weight of ears at the end of experiment was reduced in the groups that received L-NAME (16.9%) and 7-NI (44.3%), while the AG (24.9%) and SNP (22.0%) groups presented an increment in the ear weight compared to control (24.3 ± 1.4 mg). The evaluation of epidermis thickness show that L-NAME (28.2%), AG (20.7%) and 7-NI (42.0%) reduce, while SNP application contributed to enlarge in 35.6% when compared with control group (133.8 ± 8.1 lm). Similar results were observed in the PCNA immunohistochemical staining, where treatments with L-NAME, AG and 7-NI caused reduction of 34.2, 15.9 and 70.2%, respectively, while SNP enhanced in 15.6% when compared with control group (87.2 ± 6.9 cell/field). Therefore, in the skin NO must be produced by iNOS and nNOS, and is probably involved in the control of keratinocytes hyperproliferation in the epidermis, also with a contribution to the oedema formation. Support: CAPES, CNPq, REUNI. Disclosure of interest: None declared. Statins represent a class of drugs that effectively lowers cholesterol, but have shown to possess also pleiotropic effects, like promotion of vasculogenesis, prevention of bone loss, immunomodulating and antiinflammatory effects. Thus, the aim of this study was to investigate the activity of simvastatin in acute and chronic inflammatory models through topical treatment. Skin inflammation was induced in the ear of female Swiss mice (25-30 g) by topical application of 12-O-tetradecanoylphorbol acetate (TPA). In the acute model, ear oedema was measured by the increase of ear thickness 6 h after TPA (2.5 lg/ear). The chronic inflammatory process was induced by multiple applications of TPA (2 lg/ear) for 9 days on alternate days and the oedema was measured daily as the increase in ear thickness. Topical treatment with simvastatin (0.24-2.40 lmol) or dexamethasone (0.13 lmol) started on the 5th day of experiment. Samples of ear tissue (6 mm) from acute and chronic model were collected, weighed and analyzed: ear weigh, histology, and myeloperoxidase (MPO) enzymatic activity. Procedures were approved by Institutional Ethics Committee (390). Simvastatin application was able to reduce the ear oedema induced by a single TPA treatment in a concentration-dependent manner [ID 50 of 0.48 (0.22-1.13) lmol], with maximal inhibition of 70 ± 4% (2.4 lmol). The activity of MPO was reduced by simvastatin with maximum inhibition of 53 ± 4%. In the chronic model, simvastatin (1%) was able to reduce ear oedema in 16 ± 3% and ear weight in 11 ± 2%, at the end of experiment. Histological analysis showed a reduction of swelling and decrease in leukocyte infiltration. The results confirm the anti-inflammatory activity of simvastatin when applied topically in acute and chronic models of skin inflammation. Support: CAPES, CNPq and REUNI. Disclosure of interest: None declared. Inflamm. Res. Pusan National University, Yangsan-si, Gyeongsangnam-do, The effects of cilostazol on stimulating heme oxygenase (HO)-1 expression including signal pathways and suppression of inflammatory cytokines and molecules, were studied. Cilostazol stimulation time-and concentration-dependently increased the HO-1 mRNA and protein expression associated with increased HO-1 activity, as did cobalt protoporphyrin IX in J774A.1 macrophages. In addition, cilostazol concentration-dependently reduced lipopolysaccharide (LPS)mediated nitrite and TNF-a production, in accordance with the inhibition of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in the J774 macrophages, as did CoPP. In parallel with these results, LPS-induced IkBa degradation and NF-jB nuclear translocation were significantly decreased after treatment with cilostazol as well as with CoPP. These effects of cilostazol and CoPP were significantly reversed by Zn protoporphyrin IX. The effects of cilostazol on IkBa expression and nitrite production were not manifested in the cells transfected with HO-1 small interfering RNA. In the J774 macrophages, cilostazol time-and concentration-dependently increased the nuclear expression of NF-E2 related factor (Nrf2) and antioxidant response element (ARE) activity. PI3-kinase and Akt play a role in the major signal pathways with cilostazol-induced HO-1 expression. In summary, cilostazol suppressed production of anti-inflammatory cytokines and molecules via inhibition of NF-jB activation, through a mechanism involving up-regulation of cyclic AMP-dependent protein kinase activation-coupled Nrf2-linked HO-1 expression in J774A. In obesity, white adipose tissue (WAT) is considered a site of production of inflammatory factors and is characterized by macrophage infiltration. The contribution of WAT secretion and macrophages to weight loss-induced amelioration of systemic inflammation in obese subjects is not clear. Subcutaneous WAT was obtained from 20 morbidly obese patients before, 3 and 6 months after gastric bypass (GP). Luminex technology was used to measure the release of 27 factors in WAT explants over 24 h incubation. Adipocyte diameters and CD68+ macrophages content were determined in WAT slides obtained at the same time points. Following GP, BMI and adipocyte diameters regularly decreased, accompanied by an overall amelioration of patients' systemic inflammation. In WAT explants, leptin release steadily dropped while adiponectin remained roughly stable. The secretion rate of the anti-inflammatory cytokines IL-10 and IL-13 increased up to 2-fold the initial values after 3 months and remained stable thereafter. Unexpectedly, a similar kinetic profile was observed for pro-inflammatory factors, including TNFa, IL-12 and RANTES. WAT release of three major angiogenic factors, bFGF, PDGF-BB and VEGF, increased regularly, reaching 5-fold the pre-surgical values at month 6. Different patient's kinetic profiles were found for WAT CD68+ macrophages, with increased, stable or decreased counts. Early variations in WAT macrophage content were positively correlated with changes in the release of pro-inflammatory factors in paired explants. These variable secretion profiles in WAT do not directly account for amelioration of systemic inflammation but might be related to variations in macrophage content. Secretion of angiogenic factors suggests a local vascular response to sustained weight reduction. Change in WAT macrophage phenotype is under study to further explore their contribution to the dynamics of WAT secretion, tissue remodeling and bioclinical phenotypes during GP-induced weight loss. Disclosure of interest: None declared. Obesity is associated to a systemic inflammatory process characterized by proinflammatory cytokine production and macrophage infiltration in adipose tissue (AT). Methotrexate (MTX) is an anti-TNF-a drug and it could be a role in inflammation associated to obesity. The aim of this project was to evaluate MTX effects in inflammatory alterations induced by obesity. Swiss mice were feed with high-fat diet (HFD) during 12 weeks and received MTX 2 (MTX2) or 4 (MTX4) mg/kg/week during the last 2 weeks. Body weight, food intake, glucose blood levels and insulin tolerance were evaluated. Visceral AT depots were quantified and biopsies were obtained for protein expression analysis, immunohistochemistry, adipokines production and basal lipolysis measurement. MTX actions were evaluated in vitro using 3T3-L1 adipocytes or Raw 264.7 macrophage/3T3-L1 adipocytes co-cultures. There was no reduction in body weight, food intake or adiposity in obese mice after MTX treatment. However, glucose blood levels were reduced and insulin sensitivity was increased in MTX2 and MTX4. Ex vivo, the basal lipolysis, leptin, TNF-a and IL-6 production were reduced while adiponectin and IL-10 were increased in AT from MTX4 mice. The MTX also reduced iNOS expression, JNK phosphorylation, macrophage infiltration (F4/80 + cells) and MCP-1 expression in visceral AT. In vitro, MTX inhibited lipolysis through adenosine receptor A1 activation in 3T3-L1 adipocytes. IL-6 in LPS-stimulated co-cultures was also inhibited by MTX. Our results demonstrate that MTX improves glucose homeostasis in obese mice. This improvement seems to be associated with a reduced production of proinflammatory and increased production of anti-inflammatory adipokines by adipose tissue. The effect anti-lipolysis could also contribute to glucose homeostasis, suggesting that drugs with anti-inflammatory associated to anti-lipolytic action could be a tool for controlling inflammatory alterations associated to obesity. Oleic acid (OA) is a nonesterified fatty acid (NEFA) released during lipomobilization around calving in cows or following traumatic bone injury in humans. OA induces well known effects on pancreatic cells via binding to GPR40 receptor, however, recent evidence suggests a possible role on innate immune response. In this study, we assessed the effect of oleic acid on bovine neutrophils activation and the role of calcium on reactive oxygen species (ROS) production, degranulation, CD11b expression and MAPK phosphorylation. Neutrophils isolated from blood samples of healthy heifers were incubated with 100-500 lM OA, in presence or absence of calcium chelating agents. Intracellular calcium mobilization was measured by spectrofluorimetry using Fura-2AM. Superoxide production and CD11b expression, using hydroethidine and CD11b-APC antibody, respectively, were assessed by flow cytometry. Metalloproteinase-9 (MMP-9) release was assessed by zymography gel and MAPK phosphorylation by immunoblot. Oleic acid, in a dose-dependent manner, raised the intracellular calcium, superoxide release and CD11b expression. The intracellular calcium mobilization was partially reduced by EGTA, an extracellular calcium chelating, and inhibited by BAPTA-AM, an intracellular calcium chelating. Superoxide release and CD11b expression induced by 250 lM OA were reduced by BAPTA-AM. OA induced degranulation of neutrophils, as was demonstrated by MMP-9 release. A dosedependent increase in MMP-9 activity was observed. OA also induced, in a dose-dependent manner, the ERK1/2 phosphorylation between 2 and 5 min of stimulation. In conclusion, we show that oleic acid induces CD11b expression, superoxide production, degranulation and ERK1/2 phosphorylation, dependent of intracellular calcium release. Thus, these results provide evidence about the role of OA in blood neutrophils activation that could affect the innate immune response. Supported by grant from IFS (B/4720-1) and Fondecyt 11100413 and 1090401. DID-UACH. Disclosure of interest: None declared. Background: The obese state is characterized by inflammation of low intensity and this increased production of pro-inflammatory molecules by adipose tissue has been associated with the accumulation of macrophages in this tissue. The molecular signals that initiate macrophages activation in obese adipose tissue are not yet known, but there are several candidates. Objective: In order to determine the role of adipocyte released from IL-6 in this activation process, we used a model of co-cultivation between adipocytes and monocytes. Procedure: J774 cells were maintained in DMEM containing 10% fetal calf serum. We extract the adipocytes of epididymal adipose tissue of mice C57 black using collagenase type II and incubated with J774 cells for different periods of time (1 and 3 h) in the presence or not of IL-6 neutralizing antibody. We then evaluated J774 cells gene expression of TNF-a, IL-1b, IL-6, IL-10, MRS-1 and MIP-1 by realtime PCR. Total RNA was extracted using Trizol reagent. The expression of genes was compared with that of the constitutive Hprt1 gene whose expression was not modulated by treatment. Results: The co-culture with adipocytes stimulated the expression of TNF-a, IL-1b, IL-6, IL-10 and MRS-1 cells by J774, at specific periods of incubation. The expression of TNF-a increased about 4 times after 1 h of incubation. All other genes had their expression increased after longer periods of co-cultivation besides MIP-1. In cells incubated with adipocytes and anti-IL-6 antibody we noticed a further increase in mRNA expression of these inflammatory mediators. Conclusion: The results suggest that, at least locally, IL-6 has an antiinflammatory effect in monocytes exposed to adipocytes. When periodontal disease develops, many inflammatory cells, mainly lymphocytes, can be observed infiltrating the lesion. It is believed that chemokines like MCP-1 and IL-8, which are produced by gingival epithelial cells undergoing stimulation by bacterial cell components from plaque bacteria through toll-like receptor-2, are involved in the migration of these immunocompetent cells to the lesion site. It has been confirmed that the expression of these inflammatory cytokines is elevated in the gingival tissue and gingival sulcus exudates of periodontal disease patients. When we investigated the dynamics of the inflammatory cytokines IL-1b, IL-6, TNF-a, IFN-c, and MCP-1 and antiinflammatory cytokine IL-10 in rats with spontaneous periodontal disease (ODUS/Odu), we identified them to be an animal model of periodontal disease, and maintained them in our laboratory. In this study, we measured the plasma levels of the PGE 2 and Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) and the antiinflammatory cytokine IL-4 in ODUS/Odu. Plasma cytokine levels were measured in these and control rats (Res) at 0 (5 weeks old), 1,3,6,9, and 12 months after the start of the experiment using an enzyme-linked immunosorbent assay (ELISA) kit. The plasma levels of the PGE 2 and CINC-1 were greater in ODUS/Odu than in Res throughout the experimental period. However, the plasma level of the anti-inflammatory cytokine IL-4 was lower in ODUS/Odu than in Res. These results, similar to those obtained in patients with periodontal disease, suggest that ODUS/Odu is a useful animal model of human periodontal disease. Disclosure of interest: None declared. In obesity, adipocyte hypertrophy and hyperplasia might generate hypoxic areas within the tissue. In a close relationship with hypoxia, the adipose tissue produces inflammatory molecules involved in obesity-related complications, such as cardiovascular disease and metabolic disorders. Besides the hypoxia-inducible factor 1a (HIF-1a), several proteins have their stability or expression modulated by hypoxia. It was also recognized that adipocyte size was correlated with the expression and production of the serum amyloid A (SAA). This protein acts as a potent stimulus for the production of cytokines in immune cells. Thus, the aim of the present study was to assess the influence of hypoxia on the expression of adipocyte-derived protein SAA isoforms, and investigate, in normoxia, the effect of SAA on the production of proinflammatory cytokines by 3T3-L1 adipocytes and human adipocytes. Western blotting was used to measure HIF-1a and SAA; for the last RT-PCR analyses was also performed. The production of the cytokines TNF-a, IL-6, IL-1b and IL-8 in cell culture supernatants was determined by ELISA assays. Ambient hypoxia (1% O 2 ) caused a two and threefold increase in SAA3 mRNA expression in differentiated 3T3-L1 adipocytes under 12 and 24 h of induction, respectively. For mature human adipocytes, the low oxygen pressure caused an approximately twofold increase in SAA1, SAA2 and SAA4 mRNA expression on 12, 6 and 24 h, respectively. During the differentiation of 3T3-L1, the presence of SAA markedly increased the release of TNF-a and IL-6. The same effect was observed only for TNF-a when human adipocytes were stimulated with SAA. At 24 h, it was observed a sevenfold increase in the TNF-a production. Our data show that the SAA gene is responsive to hypoxia, and that SAA possibly favors the improvement of the inflammatory profile of the adipose tissue that may contribute to the overall health complications related to obesity. During acute ruminal acidosis, the animals show a clinical pattern similar to septic shock. The ingestion of excessive amounts of highly fermentable concentrates is followed by an increase in propionic acid production, in this environment, cellulolytic and lactate-using bacteria and protozoa are destroyed, while the growth of lactobacilli is promoted, increasing D-lactic acid production. During overload of oligofructose, we observed an increase of metalloproteinase-9 (MMP-9) release and L-selectin shedding in bovine neutrophils. We assessed if neutrophil response increase during ruminal acidosis by effect of propionate or D-lactic acid. Neutrophils isolated from venous blood of healthy heifers were incubated with 0.3-30 mM propionate or 0.2-10 mM D-lactic acid. The intracellular calcium release was measured by spectrofluorimetry with Fluo-4AM. Reactive oxygen species (ROS) production was assessed by chemiluminescence. CD11b and L-selectin expression was detected by flow cytometry. Metalloproteinase-9 (MMP-9) release was assessed by zymography and the myeloperoxidase enzyme activity with a microplate reader. The presence of GPR41 and GPR43 was evaluated using RT-PCR, western blot and confocal microscopy. 0.3 mM propionate induced MMP-9 secretion, meanwhile 30 mM increased the myeloperoxidase release. Also we demonstrated that bovine neutrophils expressed the putative receptors of propionate GPR41 and GPR43. On the other hand, D-lactic acid induced MMP-9 release, and increased the CD11b expression and L-selectin shedding. Additionally, we observed that Dlactic acid interfered with the ROS production, CD11b expression, Lselectin shedding and MMP-9 release induced by platelet activating factor. We concluded that propionic and D-lactic acid active the blood neutrophils and could contribute with the septic shock signs observed during acute ruminal acidosis. Inflamm. Res. The long chain x-3 polyunsaturated fatty acid of fish oil, docosahexanoic acid (DHA) and polyphenols like procyanidins have been found to inhibit the inflammatory process. Our study investigated the anti-inflammatory effects of pure DHA, B 1 , B 2 , C 1 procyanidins and the combination between procyanidins-DHA, on interleukin-6 (IL-6) and prostaglandin E 2 (PGE 2 ) secretion, cyclooxygenase activity and in the nuclear factor NF-jB signaling in lipopolysaccharide (LPS)stimulated THP-1 monocyte-derived macrophages. Pretreatment with 10 lg/mL of B 1 , B 2 and C 1 procyanidins with or without 25 lM of DHA for 48 h, significantly decreased proinflammatory IL-6 secretion, nevertheless any of the pretreatments had a significantly effect on PGE 2 secretion in (LPS)-stimulated THP-1 macrophages. The pretreatments DHA, B 1 , B 2 and C 1 , decreased cyclooxygenases inducible (COX 2 ) and housekeeping (COX 1 ) in vitro activity. The pretreatment with B 2 were significantly more selective in inhibition of COX 2 than COX 1 activity. The nuclear factor NF-jB activation was significantly reduced by DHA, B 1 , B 2 , C 1 procyanidins and the combination procyanidins-DHA pretreatments. The IkB kinase activity was decreased through the block of the qIjB-a phosphorylation by the combination of DHA-procyanidin pretreatments. B 1 , B 2 , C 1 with or without DHA pretreatments blocks, both p65 and p50, nuclear translocation and the retention of both subunits in the cytoplasm. The nuclear factor NF-jB binding activity was dramatically decreased as a response of the pretreatments with DHA with or without B 1 , B 2 and C 1 procyanidins. The results indicate that the antiinflammatory effects of DHA polyunsaturated fatty acid and B 1 , B 2 , C 1 procyanidins, alone or together are due to inhibition of IL-6 secretion and cyclooxygenase activity, as well as the inhibition of NF-jB activation by blocking NF-jB p65 translocation and nuclear binding. In the last years, studies have clearly demonstrated that non-microbial molecules released upon cellular damage have the ability to alert immune system through activation of macrophages and dendritic cells (DC). Among these signals, monosodium urate (MSU), the etiological agent of gout, has been found to trigger inflammation through the activation of NLRP3 inflammasome in macrophages, leading to secretion of the proinflammatory cytokines IL-1b and IL-18. Although the inflammasome is currently under investigation for its role in the activation of innate cells, less is known about its ability to program DC to prime a specific T cell response. In the present study, we investigated the response elicited by MSU in DC and how this could promote adaptive immune response. Bone marrow-derived DC (BMDC) stimulated with MSU combined with the NOD2 agonist muramyldipeptide (MDP), strongly produced IL-1b, IL-6, TNFa, and IL-23. Co-culture of BMDC with naïve CD4 + T cells in the presence of MDP/MSU revealed a specific T cell polarization. We show that MDP/MSU stimulation induced the maturation of DC into stably committed Th17-promoting effector DC, as assessed by the presence of IL-17-secreting T-cells both in vitro and in vivo. Similar effects were seen using MSU-activated BMDCs primed with non-microbial stimuli, such as CD40L, GM-CSF or TNFa. In vitro, the MSU-driven skewing of CD4 + T cell polarization towards the Th17 lineage was dependent on IL-1, IL-18 and NLRP3. In vivo antigen-specific Th17 polarization was similarly dependent on IL-1 and IL-18 signaling, but not on NLRP3 activation. Collectively these data show for the first time that a sterile danger signal, acting in synergy with pro-inflammatory priming signals, induces Th17 cell differentiation in vitro and in vivo through an IL-1-and IL-18-dependent mechanism. Disclosure of interest: None declared. Obesity is often described as low-grade systemic inflammation and several proinflammatory cytokines are involved in this complex process. Due to the limited studies performed on children, the relationships between obesity and some inflammatory parameters as well as acute phase reactants were investigated in children with normal blood count and iron status parameters. 153 children aged 6.0-17.9 years were recruited for this study. They were divided into two groups; 63 constituted the control group (BMI 16.1 ± 1.6 kg/ m 2 ). Based on WHO criteria, those defined as having a BMI above the gender-specific 95th percentile were included in obese group (BMI 25.6 ± 3.9 kg/m 2 ). Complete blood count, serum iron, total iron binding capacity (TIBC), ferritin, C-reactive protein (CRP), fibrinogen analyses were performed. Data were analyzed by SPSS. p B 0.05 was accepted as the degree of statistical significance. There was not any statistically significant difference between the groups in terms of mean age, male to female ratio, serum iron, TIBC, white blood cell (WBC), red blood cell, platelet counts as well as hemoglobin, hematocrit, mean corpuscular volume values. No gender difference was noted for these parameters, either. In comparison with those of the control group, CRP values were significantly higher (p B 0.001) in obese children with normal WBC count. Furthermore, in obese group, the concentrations of the other acute phase proteins such as fibrinogen and ferritin were also higher (p B 0.01) than those obtained for non-obese children, which confirms the hypothesis of low-grade systemic inflammation in childhood obesity. Even in the absence of elevated WBC count as well as anemia indicators, inflammatory parameters are elevated in obesity during childhood. These may be the pre-indicators of the development of atherosclerosis in the future. Therefore, the importance of the understanding of early life-style interventions is essential for the prevention of cardiovascular diseases. Disclosure of interest: None declared. The consequences of maternal malnutrition in early life on thymocyte responses and the role of leptin were investigated in rats. The young progeny (PD) of dams submitted to protein deprivation during lactation showed at 30 days of age lower body and thymus weights, with no alteration in the thymocyte number or any difference in the profile of T cell subsets, or in their proliferative response. Similar circulating levels of corticosterone and of GR nuclear contents were detected in thymic cells of PD or control groups. In contrast, despite the rats from PD group did not present alterations in leptin circulating levels, the expression of leptin receptor ObRb was enhanced in their thymocytes. This change was accompanied by amplification in leptin signaling response of thymocytes from PD rats, which showed an increase in JAK2 and STAT3 phosphorylation after stimulation with leptin. Moreover, the thymic cells from PD rats presented a decreased rate of spontaneous apoptosis when compared to controls. Accordingly, higher expression of anti-apoptotic protein Bcl-2, and lower of proapoptotic protein Bax, with no change of pro-apoptotic Bad, and higher pro-caspase 3 content were detected in PD thymocytes. Moreover, thymocytes from PD group exhibited a constitutive higher nuclear content of p65 NF-kB associated to a lower IkB content in the cytoplasm. Finally, although there was no change in ObR genic expression in PD thymocytes, a higher expression of mRNA for Ob gene was observed in the thymic microenvironment from PD animals. Taken together, the results show that maternal protein deprivation during lactation affects thymic homeostasis, inducing leptin activity, which protect thymocytes from apoptosis in young progeny and, perhaps, may prone these animals for alterations in immune response in adult life. Disclosure of interest: None declared. Obesity is associated with chronic inflammation that increases the risk of type II diabetes and cardiovascular disease. Accumulation of visceral adipose tissue (VSAT) is more proinflammatory and carries a higher risk for disease compared with an increase in subcutaneous adipose tissue (SCAT). Annexin A1 (AnxA1) is an antiinflammatory proresolving mediator whose regulation has not been investigated in obesity. Expression of AnxA1 was evaluated by RT-PCR in VSAT and SCAT of male C57BL6 mice fed either a 60% kcal/fat diet (HFD) or chow diet. A significant upregulation of AnxA1 was observed in VSAT (p \ 0.006), but not SCAT, of mice fed a HFD for 5 weeks compared with chow groups. Upregulation of AnxA1 by HFD was an early event that preceded both significant weight gain and macrophage infiltration in VSAT, as measured by expression of CD68. When HFD was prolonged for 13 weeks, mice developed obesity and insulin resistance. At this time point, a significant induction of AnxA1 was observed in both VSAT and SCAT (p \ 0.0001) compared with chow groups. Despite equivalent induction of AnxA1 in VSAT and SCAT, expression of CD68 was significantly lower in SCAT (2-fold increase compared to chow) vs VSAT (100-fold increase) of obese mice, indicating a dissociation between upregulation of AnxA1 and degree of adipose tissue inflammation. Comparable results were obtained in ob/ob mice, a model of genetic obesity. A significant induction of AnxA1 was observed in both VSAT (p \ 0.001) and SCAT (p \ 0.007) of ob/ob mice compared to their lean littermates, even though upregulation of CD68 was significantly higher in VSAT vs SCAT. These results indicate that modulation of AnxA1 in adipose tissue is an early event and is dissociated from the process of macrophage infiltration. Upregulation of AnxA1 likely represents a frustrated attempt at controlling adipose tissue inflammation, a response that might be exploited therapeutically to reduce the risk of obesity-associated pathologies. Disclosure of interest: None declared. (2) Ty-IRS and SP-IRS status, in white adipose tissue (WAT). Therefore, 20 female Wistar rats (210 g bw) were divided into two groups: (1) with PD, that was induced by a suture ligature placed around teeth; (2) control rats (CN). After 7 days of the placement of the ligature in the PD group, the rats of both groups were placed for mating. After the offspring birth, male offspring were divided into two groups: (1) PDO group, offspring from female rats with PD; (2) Nitrodative stress and inflammation it is implicated as a final common pathway in the development of diabetic neuropathy. The study was designed to evaluate the impact of epigallocatechin-3-gallate alone and in combination with insulin on neuropathic pain and neuroinflammatory cascade in streptozotocin-induced diabetic rats. After 4 weeks, streptozotocin induced diabetic rats developed neuropathy which was evident from decreased tail-flick latency in tail-immersion test (thermal hyperalgesia) and decreased paw withdrawal threshold both in Randall sellitto (mechanical hyperalgesia) and von-Frey hair test (mechanical allodynia). Decrease in nociceptive threshold was accompanied by significant increase in lipid peroxidation and nitrite levels in sciatic nerve of diabetic rats along with marked decrease in reduced glutathione and superoxide dismutase activities. Decrease in pain threshold was coupled with marked increase in TNF-a, IL-1b, caspase-3 activities in cytoplasmic lysate and active p65 subunit of NFjb in sciatic nerves of diabetic rats. Administration of epigallocatechin-3-gallate (25, 50, 100 mg/kg) significantly and dosedependently attenuated all the behavioral and biochemical alterations. Insulin alone corrected the hyperglycemia and partially reversed the pain response in diabetic rats. However, combination with epigallocatechin-3-gallate not only attenuated the diabetic condition but also reversed neuropathic pain through modulation of functional, biochemical and molecular deficits associated with diabetic neuropathy. Collectively, the data reveal that activation of nitrodative stress and inflammation induced NFjb signaling pathway is associated with diabetes induced neuropathic pain and point towards the therapeutic potential of epigallocatechin-3-gallate in diabetic neuropathy. Disclosure of interest: None declared. The etiology and pathogenesis of Behçet's disease (BD) have remained unclear, however, it has been assumed that genetic factors, infectious, and immune mechanisms are involved in the onset of this disease. A recent study has shown elevated production of IL-17, IL-23, and IFN-c by peripheral blood mononuclear cells (PBMCs) besides increased frequencies of IL-17 and IFN-c producing T cells in BD patients with active uveitis. It was also reported that increased expression of IL-23 p19 mRNA in erythema nodosum-like lesions in patients with active BD. In this study, we investigated the expression Th17 and related cytokines according to clinical activity of BD. A total of 22 patients with active and inactive BD were enrolled in this study. The disease control and healthy control (HC) group consisted of age-and sex-matched 10 recurrent aphthous ulcer (RAU) patients without any other evident disease and 10 healthy volunteers, respectively. PBMCs of subjects were cultured and stained with the appropriate fluorescent antibody for analysis by flow cytometry. To measure the IL-17, IL-23 and IFN-c concentrations in serum and in culture supernatants, ELISA was performed. To investigate the mRNA expression level of IL-12p35, IL-12/23p40 and IL-23p19 in PBMCs, real time polymerase chain reaction (RT-PCR) was performed. In the active BD group, CD4+ and CD8+ T cells from patients expressed significantly higher level of IL-17 and IFN-c, compared with the cells from the control group. High serum and supernatant concentration of IL-17 and IFN-c was in active BD group compared with HC and RAU groups. In addition, mRNA expression of IL-23p19, IL-12p35 and IL-12/23p40 in PBMCs was also upregulated in BD group. Expression of IL-23p19 mRNA was significantly higher in the active BD group compared with HC group. Increased expression of the IL-17 and IFN-c was observed in this study and these results suggest that Th17 cells have an important implication in immune modulation of BD. Disclosure of interest: None declared. Objectives: Graft versus host disease (GVHD) is a major complication that limits the use of allogeneic hematopoietic stem cell transplantation. In the present study we investigated the gene expression patterns among different organs (target and non target organs) in GVHD mouse model based on chemotherapy conditioning regimen. Methods: Female BALB/c (H-2K d ) mice were conditioned with busulfan (80 mg/kg) for 4 days followed by cyclophosphamide (200 mg/kg) for 2 days. Allogeneic and syngeneic bone marrow transplantation (BMT) were carried out at day 0 by infusing bone marrow and spleen cells from C57BL/6 (H-2K b ) or BALB/c (H-2K d ) mice, respectively. Snap frozen samples of liver, intestine, kidney and muscle were collected from control (day -7), conditioned (day 0), allogeneic and syngeneic (day +7) transplanted mice. After extraction of total RNA, samples were analyzed using Affymetrix Mouse Genome 430 2.0 GeneChip micro array, data analyzed by Gene Spring TM software version GX 10. Results: Gene expression pattern after conditioning (day 0) compared to control mice (day -7) showed down regulation of gene expression in all tissues. After BMT gene expression pattern were extremely different in allogeneic transplanted mice compared to syngeneic. Moreover, we found a unique pattern of CD274 or programmed cell death ligand-1 that is known to be a negative regulator of T cell activation in GVHD mice. The expression of CD274 was significantly up regulated in the muscle and kidney by 66 and 52-fold, respectively, while much lower increment was observed in the liver (15-fold) and intestine (threefold). This could be a potential protective mechanism that renders muscle and kidney less affected organs by acute GVHD. Two mouse lines were phenotype-selected for maximum (AIRmax) or minimum (AIRmin) acute inflammation responses to polyacrylamide bead (Biogel) subcutaneous injection. These lines differ in terms of bone marrow granulopoiesis, neutrophil resistance to apoptosis, and inflammatory cytokine production. Genome wide scan using SNP markers detected two significant quantitative trait loci (QTL) on chromosomes 5 and 7 (p \ 10 -6 ), as well as suggestive ones on chromosomes 2, 4, 6 11, 12 and 17 (p \ 10 -3 ). We compared gene expression profiles of epidermal tissues of AIRmax and AIRmin mice submitted to acute inflammatory reactions, in order to correlate with QTL found. Epidermal tissues were recovered 48 h after Biogel subcutaneous injections. Global gene expression analysis was performed on Affymetrix bioarrays (27 k genes) using individual RNA from both control and Biogel-treated AIRmax and AIRmin mice. Differentially expressed genes were statistically established and the over-represented gene ontology biological process categories were identified. Up-regulations of about 479 and 121 genes were observed in Biogel-treated AIRmax and AIRmin mice, respectively, but only 1 gene were found to be down-regulated in AIRmin, as compared to 167 genes in AIRmax mice. The over-represented biological themes of the differently expressed genes among AIRmax and AIRmin mice represent inflammatory response, signal transduction and cell adhesion. We were able to demonstrate a distinct gene usage between both lines during acute inflammation, and significant differentially expressed genes co-localized with mapped regions on chromosomes 2, 4, 5, 6, 7, 11, 12 Inflammation and impaired function of endothelium are crucial factors of initiation, progression and clinical implications of essential hypertension (EH). Cytokines are signaling molecules, modulating cellular cooperation during inflammation process. The aim of the present study was to investigate the expression pattern of cytokine genes and their association with cardiovascular disease, and to study possible interactions between genes involved in the blood pressure maintenance. Study group consisted of 355 EH patients and was divided into three subgroups: first included 219 patients with noncomplicated EH, second-95 hypertensive patients with myocardial infarction, third-41 hypertensive patients with ischemic stroke. Control group included 273 unrelated healthy subjects. In the experiment, we used SNPs in 5 cytokine genes and 5 genes encoding for renin-angiotensin system (RAS) and endothelial relaxation factor synthesis components, and performed an association study between EH and control subjects. 84 cytokines and cytokine receptor genes were screened for their expression profile with the RT 2 Profiler TM PCR Array (SuperArray Bioscience Corporation, USA). An association was detected between IL1B T511C (P = 0.029) and IL10 C627A (P = 0.016) SNPs and EH. IL1B, IL6, IL10, IL12B and TNFA gene variants were found to be associated with cardiovascular complications of EH. Evidence for association with stroke was observed for IL1B T511C (P = 0.022), IL-6 G572C (P = 0.03), IL12B A1159C (P = 0.017) and TNFA G308A. VNTR-polymorphism in 4-th intron of endothelial nitric oxide synthase gene ( Background: Passenger cells, normal constituents of whole organs, migrate from the graft create microchimerism which is suggested to be essential for sustained survival of allografts. The process of DNA transfer between mammalian cells remains not well understood. In our previous study we found donor Sry-PCR product in recipient tissues. This product was detected in splenic macrophages and DC's, but was absent from hepatocytes and parenchymal cells. It has been suggested that donor DNA may play a role in rejection having influence on tolerance. Aim: In our study we tried to assess the amount of Sry gene in different recipient tissues after allogeneic heart transplantation with and without immunosuppression (FK506). Material/methods: In allogeneic combination male rats BN served as donors and female rats LEW as recipients. Genomic DNA was extracted from the recipient blood and tissues at different times after grafting. Sry was detected using Real-time PCR. Relative amount levels in the different samples were calculated by using the comparative C T method with Gapdh as internal control. Results: The relative amount of Sry gene was high in all female tissues 30 days after heart transplantation with immunosuppression, with highest values in blood, liver, spleen and lymph node. Thirty days after heart transplantation without immunosuppression the relative amount of Sry gene was in all tissues lower than with immunosuppression. Conclusion: Detection of donor male DNA isolated from female blood and tissues suggests its spontaneous transport into recipients. Its higher levels in immunosuppressed recipients point to cytotoxicity of anti-rejection drugs. The question remains open whether long-lasting presence of donor DNA may have any relevance to the rejection or tolerance process. Disclosure of interest: None declared. the passenger cells and debris of damaged organ endothelial and parenchymal cells. Immunosuppression may cause additional release of genetic material due to its cytotoxic effects. Aim: The question arises whether the level of released donor DNA may be helpful in assessment of the intensity of the rejection process. In our previous studies, we looked for the presence of donor-specific STR loci (phospholipase A2-HUMPLA2A1(AAT) n , cytochrome P-450 A2-HUM-CYARO(AAAT) n and locus D1S80) in the recipient blood. Material/methods: In this study we have examined the plasma and blood mononuclear cells of recipients to assess the amount of SRY gene in sex-mismatched combinations and phospholipase A2-HUMPLA2A1(AAT) n in the same sex recipients after kidney transplantation, in relation to the patients probes before grafting by the use of the Real-time PCR method. Recipient's blood and donors spleen samples were collected before kidney transplantation and at different times after grafting. Genomic DNA was isolated from plasma and mononuclear cells. The amount of DNA in different samples after tx was calculated by using the comparative Ct method with GAPDH as internal control. Results: We observed increase in donor DNA level in recipient's blood mononuclear cells already on day 1 after grafting. The relative amount of SRY gene was much higher in female's lymphocytes 21 days after transplantation than in plasma. The amount of HUM-PLA gene was also higher in blood lymphocytes than in plasma 14 days after kidney transplantation. Objectives: Most published studies on infections and genetic polymorphisms are dealing with sepsis. Aim: We studied polymorphisms of selected allele of cytokines and TLRs at 9 polymorphic sites in patients with sepsis, acute tissue infections and prolonged wound suppuration as we hypothesize with same genetic predilection. Results: (1) in entire group of patients with systemic and local infections, higher frequency of TNFaG308A GG, TNFb G525A mutated homozygote AA, and CCR2 G190A mutated homozygote AA than in controls (all p \ 0.0001) was found. At TGFb G25C site there was a low expression of GG compared with controls (p \ 0.001). (2) comparison of sepsis, acute tissue infections and delayed infected wound revealed more of CD14 C-159T CT, TLR1,2 C2259A GA and C2029T CT in sepsis than other infections but differences were not significant. There was lack of differences in subgroups in expression of Acute anterior uveitis (AAU) is a rare disease but the most common form of uveitis with proposed autoinflammatory rather than autoimmune background. At the moment, defined genetic component responsible for this sight threatening disease manifestation is unknown. There is, however, growing number of evidence that single nucleotide polymorphisms (SNPs) in cytokine genes can influence the development of ocular inflammation through their effect on cytokine level production. The gene encoding IL-10, an anti-inflammatory cytokine, was broadly investigated and several SNPs were proved to be implicated in various forms of uveitis and outcome (Stanford et al. 2005; Mizuki et al. 2010; Remmers et al. 2010; Atan et al. 2010 ). Data obtained with idiopathic intermediate uveitis patients revealed that homozygotes for rs1800896A (-1082) in the IL10 promoter region were strongly associated with bad outcome. Functional studies also reported that the -1082A, -819T and -592A haplotype is associated with low IL-10 production whereas GCC haplotype formed by the same set of SNPs is linked with IL-10 upregulation and is protective against disease recurrence. For this study, DNA from 137 patients with AAU and 92 healthy controls recruited through Oregon Health and Science University were analyzed for polymorphisms in the promoter region of the IL10 gene at the established -1082A/G, -819C/T and -592A/C positions by SSP-PCR. Data analysis revealed no significant association of haplotypes with AAU. Four additional markers of interest were analyzed by TaqMan SNP genotyping assays: rs1518111, rs3024490, rs2222202 and rs3024505. Gender influences the severity and evolution of various inflammatory conditions. Recent clinical studies showed significant gender differences in inflammatory markers of prepubertal children with very low sex steroid levels, suggesting a genetic contribution. We studied in 24 healthy adults of both sexes some main leukocyte functions of the innate immunity depending on X-linked genes. The respiratory burst measured by chemiluminescence of purified neutrophils stimulated with fMLP was not different in males and females (p [ 0.05) although the main NADPH oxidase subunit gp91phox is linked to the X chromosome. Percentage of monocytes expressing CD99 was higher in men than in women in the basal state and after stimulation with LPS and fMLP in whole blood (p \ 0.05). This confirms the higher CD99 protein expression reported in males using RT-PCR and probably due to the partial silencing of the CD99 gene in the inactivated X chromosome of female cells. But using flow cytometry, we were unable to observe any difference in the level of CD99 expression (p [ 0.05). Using Cytometric Bead Array, we measured inflammatory cytokines (IL-1b, IL-6, IL-8, IL-10, IL-12p70, TNF-a) produced in whole blood after a 24 h incubation with and without LPS at 1 ng/ml and 1 lg/ml. Only TNF-a showed a significant difference with a higher production in men than in women, either with LPS at 1 ng/ml (p \ 0.05) or with LPS at 1 lg/ml (p \ 0.05). TNF-a production was significantly correlated with the monocyte count (r = 0.57, p \ 0.01) and men had a higher monocyte count than women (p \ 0.01). Normalization of cytokine levels to monocyte counts suppressed differences in TNF-a production but showed a higher IL-8 production in women than in men after stimulation with both LPS concentrations (p \ 0.05). IL-8 being the main neutrophil chemoattractant, this difference could explain the higher neutrophil count observed in girls with acute inflammatory disease. Disclosure of interest: None declared. Nakajo-Nishimura syndrome (ORPHA2615, MIM256040), also designated as familial Japanese fever, is a particular autosomal recessivelyinherited inflammatory and wasting disease which onsets with perniolike eruptions in early infancy and gradually develops emaciation and long-clubbed fingers resulting in joint contracture, occasionally accompanied with periodic spiking fever and nodular erythema-like eruptions. This syndrome had originally been reported as ''secondary hypertrophic periostosis with pernio'' by Japanese dermatologists Nakajo (Tohoku University) and Nishimura (Wakayama Medical University) in 1939 and 1950, respectively. Afterwards, a group of neurologists described the disease as ''hereditary lipo-muscular atrophy with joint contracture, skin eruptions and hyper-gammaglobulinemia'' in 1993. So far about 20 cases have been reported as this syndrome from Japan, most of which arise in consanguineous parents and are concentrated in Tohoku and Kansai areas. Therefore, the concept is being accepted that this syndrome represents a new hereditary autoinflammatory disorder accompanied with lipodystrophy. Here we report the summary of Japanese cases, including one infant case, as the result of the national surveillance of this syndrome. Furthermore, comparison to the recently-reported related disorders, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome and an autosomal recessive syndrome of joint contractures, muscular atrophy, microcytic anemia, and panniculitis-associated lipodystrophy (JMP syndrome), is discussed. Identification of the responsible gene mutation will be presented in another poster. Disclosure of interest: None declared. A new class of auto-inflammatory syndromes called NLRP12-associated disorders (NLRP12AD) has been associated with mutations in NLRP12. Conflicting data have been generated in vitro on the putative role of NLRP12 on IL-1B signaling. This prospective study assesses the secretion of IL-1B and of three IL-1B-induced cytokines (IL-1Ra, IL-6 and TNFa) in patients' PBMC cultured ex vivo and evaluates the patients' response to IL-1 receptor antagonist (IL-1Ra, anakinra), a major drug in the treatment of auto-inflammatory disorders. Recording of patients' manifestations and cytokine measurements were performed before onset of anakinra treatment, during 14 months of therapy, and after anakinra withdrawal. Spontaneous secretion of IL-1B by patients' PBMC was found to be dramatically increased (80-to 175-fold) compared to controls. Consistently, anakinra initially led to a marked clinical improvement and to a rapid near-normalization of IL-1B secretion. However, a progressive clinical relapse occurred secondarily, associated with an increase in TNFa secretion, a persistence of elevated levels of IL-1Ra and IL-6 and a reincrease in IL-1B secretion. Anakinra was withdrawn after 14 months of therapy. This study, which provides in vivo evidence for the crucial role of IL-1B in the pathophysiology of NLRP12AD, reports the first use of anakinra in this disorder and one of the few examples of resistance to anti-IL1 therapy in autoinflammatory syndromes. Our data also point to the potential interest of cytokine ex vivo measurements in predicting response to treatment and provide new insights into the cellular mechanisms underlying resistance to anti-IL1 therapy. Disclosure of interest: None declared. tumor suppressor and is required for the anti-proliferative effect exerted by retinoids and its expression is frequently lost in many neoplastic tissues like cervical cancer. Conditional mice generated by floxing RARb gene and expressing Cre recombinase (RARb L-/L-) represent a useful model to analyze the function of this receptor. This work examined the role of RARb on DCs and macrophages in spleen as well as the effect of RARb ablation in cervix of RARb L-/Lconditional mice. Our results showed that RARb is expressed mainly in the splenic white pulp zone of wild type mice. In addition, low levels of RARb expression were detected in the spleen of RARb L-/Lmice, as determined by immunohistochemistry and Western blot analyses. These results are consistent with a decrease in the population of splenic CD11c+MHC-II+ white cells. Histopathology analyses of conditional mice spleen showed a reduction of macrophage-like cells and defects in cell organization and structure. These results correlated with RARb ablation, histological alterations and a neutrophil-morphology-cell inflammatory infiltrate in cervix of RARb L-/ Lmice as detected by Western Blot and histopathology analyses. Our results suggest that RARb is involved in splenic and cervical cell organization as well as the homeostatic maintenance of cells implicated in inflammation like macrophages and DCs. Disclosure of interest: None declared. Introduction: Human Leukocyte Antigen (HLA) is significantly variable in the inflammatory process of the burn trauma. HLA-Dr antigens expressed several days after stimulation by burn injury and remains expressed for several weeks. This work aims to detect if there is a relation between the inflammatory process of burned patients and HLA as an important immunological marker, and HLA-Dr expression on peripheral T lymphocytes. Patients and Methods: Twenty six patients suffering from major burn versus 100 apparently healthy controls were included. Five milliliters of whole blood were collected from control and patient groups at 24 h, 1 week and 2 weeks after burn injury. HLA-DRB1 was tested using sequence specific oligonucleotide (SSO) technique. HLA-Dr expression in T lymphocytes was also tested using flowcytometry. Results: Comparisons between patients and controls in the frequency of HLA-DRB1 different alleles were performed revealing that HLA-DR4, DR11 and DR13 were significantly decreased in patients group with the P values of (.046, .04 and .04, respectively). In addition, DR13 frequency was significantly increased in patients with gram negative bacilli infection (p \ 0.05). The absolute number of HLA-Dr + T lymphocyte subsets all over the time of the study are lower than controls except that of HLA-Dr + T lymphocytes after 2 weeks (p = 0.009) in patient compared to control groups. No correlations were found between the HLA-DRB1 different alleles and the expression level of HLA-Dr. Conclusions: Significant associations of the polymorphism of HLA-DR13 with gram negative bacilli occurring in severe burn inflammation must be considered. Therefore, it seems possible that DR13 may influence the occurrence of severe infection in burned patients and explained that as an evidence of natural selection by microorganisms. HLA-Dr expression on T lymphocytes occurred independently with no relation to different inflammatory conditions accompanying burn injury. Disclosure of interest: None declared. Introduction: Studies on the effects of low level laser therapy (LLLT) on fracture bone repair have shown different results: some of them have reported positive effects of LLLT on bone healing, whereas others did not obtain any significant results. The aim of this study was evaluation of therapeutic effect of LLLT on the rate of callus formation and bone healing time in the radius of rabbit. Methods: 30 white male rabbits of Dutch race (2 ± 0.2 kg weight) were used. After applying anesthesia, 3 mm in diameter lesion was induced in the craniomedial aspect of radius bone by orthopedic saw. Then rabbits were randomly divided into control and experimental groups. In the experimental group a laser (k = 830 nm, F = 1500 Hz, P = 50 mW, D = 4) was applied transcutaneously on 3 points at 9 sessions during 21 days. The first irradiation was performed 24 h after surgery. The control defects were allowed to heal spontaneously. To assess the callus formed, 4 series of radiographic images from 2 views (lat and AP) with 1 week interval were taken. Data were analyzed using independent sample t-test and repeated measurement. Results: Statistical analysis of X-ray data show that LLLT can increase the average amount of callus in comparison to control group, but the difference is not significant (P [ 0.5 Hypertension is an unfavorable condition for dental treatments that require bone healing. The purpose of this study was to evaluate the alveolar wound healing in spontaneously hypertensive rats (SHR) and atenolol effect on this process. Wistar rats and SHR treated with atenolol (100 mg/kg/day) were submitted to the extraction of the upper right incisive tooth and sacrificed at different days after surgery. The hemi-jaws were extracted and the radiographic images were obtained and analysed. Histological, histomorphometric and immunohistochemical reactions were done in histological sections, 5 lm thick, stained with hematoxylin and eosin or subjected to immunolabeling to RANK, RANKL, OPG and MMP-9 proteins. The results were analyzed by two-way ANOVA. In immunohistochemical analysis, scores were assigned to the images and the results were analyzed by Kruskal-Wallis and Mann-Whitney statistical tests (p \ 0.05). Reduced bone mineral density (BMD), lower bone percentage and less thickness of trabecular bone was observed in the final periods of alveolar bone healing in SHR. Increased RANKL, RANK and MMP-9 immunolabeling were observed at 28 days after surgery in SHR alveolus. Atenolol increased the BMD observed in most of the periods analyzed and increased trabecular bone thickness at 28 and 42 days in SHR alveolus. Increased OPG immunolabeling at 7 and 42 days of alveolar healing were observed in treated SHR. Increased expression of RANKL and MMP-9 at 21 and 42 days, and increased RANK expression at 21 day was also observed in alveolus of SHR treated with atenolol. Our results suggested that the delay in alveolar healing observed in SHR was associated with increased peripheral sympathetic tone described in these animals. Changes in alveolar bone formation and remodeling would be associated with differential expression of proteins in bone metabolism OPG, RANKL, RANK and MMP-9, which is modulated by the effect of atenolol. Disclosure of interest: None declared. Some food-derived peptides have inhibitory activities against various peptidases such as ACE (Angiotensin I-converting enzyme), which are involved in processing of cytokines and hormones. TACE (tumor necrosis factor-a converting enzyme) is currently being explored as a good target for anti-inflammatory drugs. Our previous studies demonstrated that the occurrence of food-derived di-peptides in human blood after oral ingestion of some peptides. Pro-Hyp, Hyp-Gly, Gly-Pro, Pro-Gly, pGlu-Leu, and Val-Ala have been identified in human blood at relatively higher level ([lM) after ingestion of food peptides. The objective of the present study was to investigate the inhibitory activity against TACE of the food-derived peptides (Pro-Hyp, Hyp-Gly, Gly-Pro, Pro-Gly, pGlu-Leu, and Val-Ala) occurring in human peripheral blood. The enzymatic activity of TACE was measured by using the quenched fluorogenic peptide substrate Mac-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH 2 . TAPI-2 (TNF-a processing inhibitor) was used as a positive control. Assays were performed in 100 ll of reaction buffer (50 mM Tris, 5 lM ZnCl 2 , 0.01% Brij35, pH 9.0) containing 112 lM substrate at 37°C in the absence or presence of test compounds. Substrate and products were separated by HPLC using C18 reversed phase column and detected by fluorescence detector at 320 nm excitation and 405 nm emission, respectively. Tissue reaction after transplantation of engineered tissues could hamper their regeneration. The understanding of those biological events provides valuable information to inhibit severe inflammation and could improve the clinical results of tissue engineering. This study was conducted to elucidate the interaction between tissueengineered cartilage using biodegradable polymer scaffolds and host cells. Tissue-engineered cartilage consisting of C57BL/6 mice chondrocytes and poly-L-lactic acid (PLLA) scaffolds were transplanted in EGFP transgenic mice of the same genetic background. Host-derived EGFP-positive cells were mostly F4/80-positive macrophages, which were drastically decreased and excluded to noncartilage areas after transplantation. Meanwhile, IL-1b was significantly lower in the tissue-engineered cartilage than PLLA scaffolds alone. Fas ligand (FasL) was immunolocalized on the chondrocytes in tissue-engineered cartilage, indicating that immune privilege was formed in tissue-engineered cartilage, suppressing tissue reactions. We then used FasL-hypomorphic mice (gld) to prove the function of FasL in the formation of immune privilege. Chondrocytes of wildtype induced apoptosis of macrophage with higher incidence, than those of gld. In the transplantation of tissue-engineered cartilage, the constructs of wild-type underwent more accumulation of cartilage matrix than those of gld. To examine the trigger of FasL expression on chondrocytes interacted with macrophages, the co-culture medium of both cells were exhaustively analyzed, which detected G-CSF. The G-CSF-treated constructs underwent less infiltration of macrophages with increased formation of cartilage after transplantation. The G-CSF secreted from macrophages could increase the expression of FasL on chondrocytes and render immune privilege, which may in turn suppress the viability and localization of macrophages, promoting the maturation of tissue-engineered cartilage. Disclosure of interest: None declared. Inflammation is a defensive response of the body against pathogens and injury, which is tightly controlled by endogenous pro-resolving factors (such as lipids, e.g. lipoxins and resolvins, and peptides, e.g. Annexin A1) operative in restoring homeostasis after tissue injury. Melanocortin (MC) peptides are emerging as another group of anti-inflammatory mediators, though their ability to activate resolving circuits is yet not investigated. Here we studied an analogue of aMSH, compound AP214, currently in clinical development for cardiac surgery (http://clinicaltrials.gov). In acute peritonitis, AP214 (400 lg/kg) afforded *30% reduction of 4 h neutrophil infiltration promoted by zymosan. In vitro, zymosan-stimulated cytokine (IL-1b, IL-6 and TNFa) release from macrophages was inhibited by AP214 (1-100 nM) . At these concentrations, a potent pro-resolving action of the phagocytosis of human apoptotic neutrophils (process of efferocytosis) was observed: this occurred in vitro as well as in vivo. To identify the receptor target for the anti-inflammatory and pro-resolving properties of AP214, cells and mice lacking the MC 3 receptor were employed observing that inhibitory effects of the compound on neutrophil infiltration, IL-1b release and efferocytosis were no longer measured. Similar observations were obtained with another compound, AP1189: when AP1189 was given at the peak of inflammation (12 h after injection with zymosan) in the peritonitis model, it was able to increase resolution compared to vehicle treated mice, an effect independent on increased leukocyte apoptosis. In summary, MCrelated drugs are anti-inflammatory therapeutics endowed with genuine pro-resolving properties, thus adding this family of compounds (peptides or small molecules) to the successful exploitation of the resolution of inflammation paradigm for innovative drug discovery programmes. Skeletal muscle regeneration is associated with macrophage infiltration. We have previously shown that macrophages exert dual and beneficial role during skeletal muscle repair. Soon after injury, infiltrating monocytes/macrophages are classically activated and phagocyte tissue debris while preventing too early myogenic differentiation. Then macrophages switch their phenotype to resolve inflammation: the second phase of muscle repair is characterized by the presence of so called M2 macrophages that, while dampening environmental inflammatory signals, directly support myogenesis and myofibre growth. We aimed at exploring the effects of these different types of macrophages at the cellular level on the sequential steps of in vitro human myogenesis, namely expression of the differentiation (myogenic) programme, prefusional migration of the cells, and eventually fusion of the cells to form myotubes. We found that pro-inflammatory (M1), alternatively activated (M2a) and anti-inflammatory (M2c) macrophages exert distinct, sometimes opposite effects on the steps of in vitro myogenesis, essentially through secreted factors. Vegetables oils for wound treatment have been used in Brazil currently, but do not have scientific endorsement. Therefore, our aim was to describe the mechanism of action of Curatec AGE Ò (commercial product rich with oleic acid, linoleic acid and medium chain triglycerides) in skin wound healing in mice. Methods: The wound was surgically induced and treated daily with Curatec AGE Ò . Mineral oil was used as control. Results: The Curatec AGE Ò induced an increase of neutrophil accumulation in the wound at days 1, 4, 7 and 14, which has a peak at day 4. A high level of IL-13 in wound homogenate was observed at day 7 but no difference was found in IL-10, IL-6, TNFa and TGFb levels. Curatec AGE Ò showed an increase of new blood vessels and predominance of fibroblasts in newly formed tissue at day 7 after wound. However the wound closure in treated and control groups was similar throughout the 14 days of evaluation. Conclusions: Taken together, Curatec AGE Ò promoted pro-inflammatory effects only in early days after injury. The presence of new blood vessels and fibroblasts indicate granulation tissue formation, which was not evident in control group. Data also suggest that fatty acids from Curatec AGE Ò affect the wound microenvironments leading to an improvement of new tissue formation after injury. We intend to evaluated the Curatec AGE Ò influence on the total collagen production at the wound, as well as parameters of oxidative stress and deposit of matrix proteins in the wound bed. Cutaneous wound healing is a complex regenerative and immunological process. Its disturbance leads to chronic wounds and represents a great medical problem. In the past, we demonstrated that the novel IL-10 cytokine family members IL-20, IL-22, and IL-24 play a major role in inflammatory skin diseases like psoriasis. Here, we studied the significance of these cytokines in cutaneous tissue repair. Surprisingly, using an in vivo mouse model, IL-20 was almost constantly expressed upon wounding and following healing process, whereas IL-22 was not expressed at all. In contrast, IL-24 was highly upregulated in the inflammatory phase of wound repair. The major sources of IL-24 appeared to be T cells and keratinocytes. IL-24 acts through a transmembrane receptor complex, which is composed of IL-22R1 and IL-20R2. Importantly, mice lacking IL-22R1 (IL-22R1 -/-) compared to corresponding wild-type mice (WT) showed a delayed wound closure starting in the early phase of the healing process. Further studies identified keratinocytes, but not dermal fibroblasts, endothelial cells, melanocytes, or subcutaneous adipocytes as being targets of IL-24 action. The IL-24 treatment of human keratinocytes regulates the expression of antimicrobial proteins, chemokines and keratinocyte differentiation-associated genes. This study suggests that the IL-24/IL-22R1 system plays a key role in the inflammatory phase of cutaneous wound healing. Disclosure of interest: None declared. The induction of neutrophil apoptosis could be of potential benefit in the control of inflammatory diseases. Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate several GC functions, such as, apoptosis, anti-proliferative and anti-inflammatory activities. In this study, we investigated the role of GILZ on the spontaneous and pharmacologically-induced resolution of neutrophilic inflammation in the pleural cavity of LPS-challenged mice. The injection of LPS induced a time-dependent influx of neutrophils into the pleural cavity of mice which was maximal at 8-24 h, diminished at 48 h, with complete resolution occurring at 72 h. The spontaneous resolution of neutrophilic inflammation was accompanied by an increase of mononuclear cells at the pleural cavity and was associated with an increase in the number of apoptotic cells at 8-48 h, interval of time that precedes complete resolution, as demonstrated by morphological and biochemical criteria (increase of Bax and caspase-3 cleavage). The expression of GILZ was detected in PBS challenged mice and its levels decreased markedly at the peak of inflammation and increased during the resolution phase of the response. Pharmacological treatment of mice with drugs that cause resolution of inflammation, including rolipram, wortmannin and dexamethasone, reduced the number of neutrophil in the pleural cavity and increased expression of GILZ, which was associated with activation of a proapoptotic program (increase of Bax and caspase-3 cleavage and decrease of Bcl-xL). In conclusion, our results showed a positive correlation between expression of GILZ, increased expression of proapoptotic pathway and resolution of inflammation, suggesting GILZ plays an important role in the signaling events leading to resolution of neutrophilic inflammation. Matrix metalloproteinase-9 (MMP-9) degrades extracellular matrix (ECM) substrates and plays an important role in neutrophil infiltration, e.g. during zymosan peritonitis. In other inflammatory models, similar roles were documented for heparanase that degrades ECM heparan sulphate proteoglycans. We report here that synthesis of heparanase is, surprisingly, increased only during the late phase of zymosan peritonitis (at 24 h). An anti-inflammatory role of heparanase is revealed by finding that specific inhibition of heparanase does not decrease neutrophil influx, and in contrast, neutrophil numbers are increased but without affecting neutrophil apoptosis. For MMP-9, we showed previously that during zymosan peritonitis, early neutrophil infiltration in MMP-9-deficient (MMP-9-/-) mice was significantly weaker during maximal neutrophil influx (6 h) than in wild-type mice. However, during the late stages of peritonitis (24 h) an extended accumulation of neutrophils was observed in MMP-9-/-mice. Recently, we reported that this was due to increased release of COX-1-derived PGE 2 that inhibited apoptosis of inflammatory neutrophils. The aim of the current study was to investigate the production of mediators/enzymes in the resolution of inflammation and evaluate whether these are altered in mice lacking MMP-9. mRNA expression and release of IL-10 and TGF-b were significantly decreased at 6 h in MMP-9-/-mice, whereas in late inflammation (24 h) they were significantly augmented. Moreover, we detected that intracellular and extracellular heparanase contents were increased in leukocytes from MMP-9-/-mice. This phenomenon was also observed in the early stage of peritonitis. Overall, we reveal that the resolution of zymosan peritonitis is enhanced in MMP-9-/-mice as the production of IL-10, TGF-b and heparanase is increased. This suggests that MMP-9 has unexpected impacts on the termination of inflammation. Disclosure of interest: None declared. Aim: Two different graft materials, Beta-tricalcium phosphate (Beta-TCP) and autogenous bone, were used in the same patient. The aim of this study was to determine whether donor site morbidity could be avoided by using pure-phase Beta-TCP. Materials and methods: Bilateral sinus grafting was performed on 20 selected patients; Beta-tricalcium phosphate was used on the experimental side, and autogenous bone was used on the control side. In addition to routine panoramic radiographs, 2-and 3-dimensional computerized tomographic examinations were performed pre-and postoperatively and after implantation. Eighty bone biopsy specimens were taken at the time of implant placement. Results: Histologically and histomorphometrically, there was no significant difference between the experimental and control grafts in terms of the quantity and rate of ossification. For each histologic sample, the total surface area, the surface area that consisted of bone, and the surface area that consisted of graft material were measured in mm 2 , and bone and graft material were analyzed as percentages of the total. The mean percentage bone areas were 36.47 ± 6.9 and 38.34 ± 7.4%, respectively; the difference was not significant (P = .25). The autogenous bone was initially less visible than the Beta-TCP, but new bone formation was clearly observed for both materials. The rate of graft resorption was lower on the experimental side than on the control side. The mean graft area percentages were 13.95 ± 5.38 and 8.47 ± 3.17%, respectively, and the difference was highly significant (P \ .001). The mean areas of the biopsy samples taken from the 2 sides were quite similar: 9.18 ± 2.42 mm 2 on the experimental side and 8 Cytotherapeutic approaches have been investigated to overcome the limitation of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences of cell source in a canine severe defect model (one-wall infrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from 4 beagle dogs, respectively. Three layered cell sheets of each cell source supported with woven polyglycolic acid were transplanted to the denuded root surface autologously. One-wall intrabony defects were filled with the mixture of b-tricalcium phosphate (b-TCP)/collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers in the PDLCs group compared to other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLCs group. Alveolar bone regeneration was observed in all groups. These results indicate that PDLCs combined with b-TCP/collagen scaffold is the promising tool for periodontal regeneration. Disclosure of interest: None declared. Glutamine is an non-essential amino acid, but it becomes essential under surgical trauma and some pathological conditions. Gln has been supplemented to patients suffering from hepatitis and other illness. However, Gln is easily degraded into pyroglutamic acid in solution and lost its beneficial nutritional value. Wheat gluten is rich in glutaminyl residue. Then, the enzymatic hydrolysate of wheat gluten has been considered as good glutamine source. There is an episode suggesting that ingestion of wheat gluten hydrolysate (WHG) could moderate symptom of hepatitis (Horiguchi et al. 2004) . The objective of the present study was to identify the active compound in WHG. Ingestion of WHG can moderate galactosamin-induced acute hepatitis in rat model. To identify the active component in WHG, in vivo activity-guided fractionation was carried out. Peptides in WHG were fractionated on the basis of amphoteric nature of sample peptide by the method of Hashimoto et al. (2005) . The acidic peptide fractions showed significant moderation of the hepatitis. Peptides in active fraction predominantly consisted of free pyroglutamic acid (pyroGlu), pyroGlu-Leu, pyroGlu-Ile, pyroGlu-Gln, and pyroGlu-Gln-Gln. Among them, pyroGlu-Leu significantly suppressed the hepatitis. On the basis of the present results, pyroGlu-Leu can at least partially be responsible for the beneficial effects of WHG. The pyroGlu-Leu is absorbed into portal blood of rat after oral ingestion and suppress induction of proinflammatory cytokines and NO. Then it could be concluded that pyroGlu-Leu moderates acute hepatitis by suppressing inflammatory response. Disclosure of interest: None declared. The possibility that homeostatic mediators, effectors of the resolution of inflammation, might impact on chondrocyte biology has been poorly explored. Here we tested the potential effects of anti-inflammatory and tissue-protective peptides discovered in the melanocortin biology arena. The human chondrocytic cell line C-28/I2 was cultured in high-density micromass (MM) cultures. Peptides AP214 (10-1,000 nM; under clinical development http://clinicaltrials.gov) and NDP-MSH (1-100 nM) were tested both alone and/or upon IL-1b stimulated cells. qRT-PCR allowed quantification of anabolic (COL2A1, SOX9, ACAN) and catabolic (MMP-1, ADAMTS5) gene products, to reveal the chondrocyte phenotype. Production of matrix glycosaminoglycans (GAG) was quantified by staining the MM with Alcian blue (AB) dye. Data are from 3 to 4 independent experiments. In resting cells, AP214 produced a concentration dependent anabolic response (+250% on COL2A1). Upon IL-1b stimulation, AP214 was predominantly effective in offsetting the catabolic response, with 30-50% reduction in IL-1b-induced metalloproteinase MMP1 and ADAMTS5. This effect was not restricted to AP214, and NDP-MSH also potently inhibited the IL-1 response. Detection of melanocortin receptor MC1 and MC5 mRNA in these cells confirmed recently published data (Grassel S et al A&R 10:3017, 2009 ). In summary, applying a novel predictive protocol using C-28/I2 chondrocyte cell line cultured in MM, we report the chondroprotective properties of AP214. These findings may impact on the development of melanocortin receptor agonists and, more in general, may become paradigmatic for exploiting the resolution of inflammation area of research for novel chondroprotective therapeutics. We thank Dr M Goldring (Cornell Medical College, New York) for the C-28/I2 cells. Disclosure of interest: None declared. Background: Periodontitis is a bacterial infection and can lead to tooth loss. Periodontal ligament (PDL) is a connective tissue that exists between tooth and alveolar bone, and considered to contain mesenchymal stromal cells. So PDL is suggested to be useful for periodontal regeneration. When PDL cells are cultured with osteoinductive medium (OIM), alkaline phosphatase (ALP) activity and osteogenic gene expressions are enhanced and PDL cells form mineralized nodules after the long-term cultivation. The aim of this study was to investigate the involvement of Wnt signaling during osteogenic differentiation in human PDL cells. Methods: To investigate the multipotency of hPDL cells, colony forming assay (CFA) and differentiation assay were performed. hPDL cells were cultured in OIM, and ALP activity was measured with various reagents. To analyze the Wnt-related genes during osteoblastic differentiation, mRNAs were quantitatively analyzed using real-time RT-PCR. About interested genes, loss and gain of function experiments were performed. Results: hPDL cells in this study showed mesenchymal stromal cell-like properties. hPDL cells grown in OIM showed high ALP activity, however it was decreased by Wnt inhibitor, XAV939. From the analysis of PCR array, secreted Frizzled-related protein 3 (SFRP3) was up-regulated, on the other hand, that of SFRP4 was down-regulated during the osteoinduction. TaqMan PCR assay also confirm the gene expressions, and time course studies revealed the expression of SFRP3 was gradually increased, whereas that of SFRP4 was decreased. SFRP3 siRNA and recombinant human SFRP4 decreased ALP activity. Obesity exacerbates severity in acute pancreatitis (AP) and is associated with elevated levels of IL-6 in both humans and experimental animals, including the murine model of AP induced by co-administration of IL-12 + IL-18. To study the role of IL-6 in AP exacerbation by obesity, WT and IL-6 KO C57BL6 male mice were fed either a high-fat diet (HFD) or chow diet (lean) for 13 weeks. IL-6 deficiency did not affect the degree of obesity or insulin resistance induced by HFD. Lean and HFD WT and IL-6 KO mice received two injections of IL-12 + IL-18 and were euthanized at 1, 3, 7 or 15 days. Acinar cell and fat necrosis were both significantly more severe and prolonged in HFD compared with lean groups, irrespective of genotype. The degree of inflammatory infiltrate was comparable in each group at day 1. However, a significant delay in the resolution of inflammation was observed in the pancreas of HFD WT mice compared with both HFD IL-6 KO and lean mice. Specifically, resolution of the neutrophil and lymphocyte infiltrate followed a comparable kinetics in HFD IL-6 KO and lean mice, whereas a significant delayed resolution was observed in HFD WT mice. In contrast, no effect of genotype was observed for resolution of macrophage infiltration, which was delayed in both HFD groups compared with lean mice. The pattern of chemokine production was in agreement with histologic data, with significantly higher levels of CXCL1 and CXL2 in HFD WT compared with HFD IL-6 KO mice, whereas levels of CCL1 were not significantly different between the two groups. Despite the important role of IL-6 in augmenting the inflammatory infiltrate, overall the exacerbated pathological response and associated lethality was not affected by IL-6 deficiency in HFD mice. Therefore, elevated production of IL-6 cannot by itself explain the increased AP severity of obese mice receiving IL-12 + IL-18, although IL-6 mediates the increased pancreatic inflammatory infiltrate of obese mice. Disclosure of interest: None declared. Introduction and aims: The tubule-interstitial fibrosis is a major event of chronic kidney disease and is also an important predictor of renal dysfunction. The fibrous tissue is caused by the accumulation of fibroblasts, myofibroblasts and extracellular matrix. Recently it has been demonstrated that fibroblasts can also be derived from endothelial cells by the process of endothelial to mesenchymal transition (EndMT). Transforming growth factor-beta protein (TGF-b1), a master cytokine acts as a stimulator of EndMT. Our aim was to demonstrate EndMT in vitro using human umbilical vein endothelial cells (HUVECs) stimulated with TGF-b1 and verify the expression of inflammatory mediators. Methods: The HUVECS were stimulated TGF-b1 (10 ng/mL) at 72 h. The phenotypical changes of HUVECs were analyzed by confocal imaging, protein expression (western blot) and gene expression (real time PCR) of endothelial cells related molecules such as Von Willebrand factor (VWF) and VE-cadherin, and mesenchymal cells markers such as a-smooth muscle actin (a-SMA) and vimetin protein. Results: TGF-b1 stimulated EndMT in HUVECs showed expression of a-SMA and vimentin protein, decrease in the expression of VEcadherin, VWF and TGF-b1 protein by western blot. It was demonstrated that mesenchymal cells markers appear after 72 h when HUVECs are stimulated with TGF-b1 (10 ng/mL). It was also positive to antibodies specifics from mesenchymal cells, capture confocal imaging indicate that the cells were undergo to EndMT. Conclusions: TGF-b1 was able to induce EndMT in HUVECs being a suitable model to study in vitro and these new findings suggest that targeting EndMT may be a novel therapeutic strategy, which is broadly applicable not only to chronic kidney disease but also to various other disease states. Disclosure of interest: None declared. Macrophage phagocytosis and clearance of apoptotic inflammatory cells is a key mechanism in resolution of inflammation. The interaction of macrophages with apoptotic cells has also been demonstrated to suppress pro-inflammatory cytokine release by macrophages. In disease states such as severe asthma and COPD resolution is defective, resulting in chronic inflammation. It has been demonstrated that macrophage phagocytosis is impaired in these diseases. Glucocorticosteroids are powerful anti-inflammatory drugs used in the treatment of these respiratory diseases and have been shown to augment phagocytosis of apoptotic cells by macrophages. Here, different phenotypes of human macrophage were characterised and the effects of corticosteroids on phagocytosis of apoptotic neutrophils examined. Human blood monocyte derived macrophages were cultured by differentiating with GM-CSF or M-CSF. Responses were contrasted with those of macrophages from human lung. Conditions were identified that mimic the phagocytic defects observed in chronic inflammation and the effect of corticosteroids examined. GM-CSF differentiated macrophages demonstrated poor phagocytosis which was greatly enhanced by corticosteroids. In contrast, M-CSF differentiated macrophages were found to be efficient at phagocytosis and corticosteroids showed little enhancement. Lung macrophages showed similar phagocytic levels to the M-CSF differentiated phenotype, with good basal phagocytosis and little enhancement with corticosteroids. Pre-treatment of all subsets of macrophages with TNFa or LPS/IFNc was found to induce a phagocytic-defective state. Under these conditions, the enhancing effect of corticosteroid treatment was also impaired. The efficacy of corticosteroids in clearance of apoptotic cells by lung macrophages under inflammatory conditions was found to be suppressed, thus providing an opportunity for alternative therapeutic intervention in the resolution of respiratory inflammation. Introduction: The biological response to skin injury can be subdivided into two distinct categories, regenerative and non-regenerative types of wound healing. Homozygous AIRmax and AIRmin sublines for Slc11a1 S alleles, produced by genotype-assisted breeding, differ in ability to completely repair ear holes. AIRmax SS mice showed fast ear tissue regeneration while AIRmin SS mice did not show regeneration after ear punch. Objectives: Our aim in this work was to evaluate the influence of the early inflammatory response in the determination of those distinct phenotypes. Methods: Two-millimeter ear holes were punches in mice of each subline and the inflammatory reaction was characterized by histomorphometric analysis and MPO activity. Global gene expression analysis was used to identify sets of differentially expressed genes during the inflammatory stage of regeneration and quantitative PCR experiments were performed to validate microarray results. Results: The local inflammatory response was slightly more intense in AIRmin SS than AIRmax SS mice 24 and 48 h after ear punch, which was demonstrated by histomorphometric analysis and MPO levels. Global gene expression analysis demonstrated over-represented distinct biological themes between AIRmax SS and AIRmin SS control mice. Inflammatory response biological theme was observed only in AIRmin SS . At 24 h after punch, both AIRmax SS and AIRmin SS mice showed significant (P \ 0.001) up-regulated genes related to inflammation. However, angiogenesis, regulation of myeloid cell differentiation and epidermal growth factor receptor signaling pathway related genes were expressed only in AIRmax SS . Microarray results were validated by quantitative PCR. Conclusions: These results suggest that distinct group of genes related to inflammatory response in the early events after injury modulates the quality of regeneration or wound healing. Introduction: Previous work by our group has investigated the mechanisms of resolution of innate inflammation. Recently, we identified a unique phenotype of macrophage during resolution. Termed resolution phase macrophage (rM), they possess a phenotype that is a hybrid of both classically activated M1 and alternatively activated M2 macrophages, expressing both iNOS and COX-2. This unique phenotype is controlled by cAMP and mediates the repopulation of protective lymphocytes (rLØ) post inflammation and the restoration of tissue homeostasis. Having identified the unexpected expression of iNOS in rM, we sought to further investigate its role during resolution using iNOS knockout (iNOS-/-) mice. Methods: Wild type mice (WT) and iNOS-/-were injected intraperitoneally with 0.1 mg zymosan. At various time points throughout inflammation cells were analysed and sorted by FACS, as well as protein expression of iNOS, and cytospins of sorted samples. Cell free inflammatory exudates were analysed for NOx, cytokines and lipids. Results: In iNOS-/-mice we identified a population of cells, which occupied a discreet region on size versus granularity plot. These were identified as ring cells by their characteristic nuclear morphology following sorting and rapid Romanowsky staining of cytospins. We have further characterised the phenotype and potential function of ring cells during resolution. FACS analysis showed that ring cells can be subdivided into ring neutrophils (RPMN) and ring monocytes (RMon). RPMNs are F480low/Gr1low, whilst RMons are F480hi/ Gr1low. These cells have a morphological phenotype similar to myeloid suppressor cells. There is a higher occurrence of ring cells in iNOS-/-compared to WT. This coincides with a decrease of 12-Lipoxygenase (12-LOX) protein expression, as well as 12-LOX derived lipids in iNOS-/-inflammatory exudates. Conclusions: We propose that these 12-LOX derived lipid mediators are produced by rM, at least in part under the control of iNOS, may have a role to play in the appearance of ring cells. Disclosure of interest: None declared. Introduction: Depending on the activating stimuli, macrophages (Mu) can develop into different subsets, broadly referred as either M1 or M2. So far, however, most of the characterisation of subsets has been carried out using mu in vitro. As the environment in which mu develop in vivo is complex, it is likely that mu phenotype is tissue-, stimuli-and inflammatory phase-specific. Therefore it was the aim of this work to catalogue the inflammatory status of mu isolated from the resolution-phase of a murine zymosan-induced peritonitis model and determine their post-inflammation tissue homeostatic role. Methods: Resolution-phase Mu (rMu) were isolated from the peritoneum 72 h after intra-peritoneal injection of a very mild dose of zymosan (0.1 mg). Its transcriptome was analysed using Affymetrix microarray technology and genes that were found to be exclusively upregulated in the rMu compared to 'inflammatory' and/or naive resident peritoneal mu were validated at the transcriptional level by quantitative RT-PCR. Results: We have found that the transcriptome of rMu has a distinct molecular signature to that of both 'inflammatory' and naïve resident mu. Some of the genes exclusively upregulated have been previously shown to be important for resolution functions such as efferocytosis (Alox15, Tgfb2 and Timd4), and in initiating the adaptive response, i.e. antigen presentation (5 subunits of the class II, major histocompatibility complex) and chemoattraction, activation and proliferation of B-and T-lymphocytes (Cxcl13, Ccr2, Ccl5, Tnfsf4, Clec2i and Clcf1). Conclusions: Transcriptome profiling not only reveals novel molecules and signatures associated with murine rMu but has also helped to determine their role post-inflammation. We find that in addition to having a crucial resolution role, these Mu function to develop the S276 Inflamm. Res. adaptive immune response by initiating the repopulation of B and T cells into the cavity and by presenting antigen to these cells. Disclosure of interest: None declared. New targets for drug development P-581 CARDAMONIN ( In anti-arthritic drug development, non-steroidal anti-inflammatory drug (NSAID), disease modifying anti-rheumatic drug (DMARD), steroidal agents, immunosuppressants have been widely used to control and suppress the syndrome of arthritis patient. However, these drugs are known to produce various adverse effects including gastrointestinal disorders, immunodeficiency and humoral disturbances. On the natural end of the treatment spectrum, our group has shown that cardamonin (2 0 ,4 0 -dihydroxy-6 0 -methoxychalcone) isolated from Alpinia rafflesiana exert promising anti-inflammatory activities in activated macrophage cells. Therefore, we further explored the antiarthritic effects of cardamonin as well as investigated its underlying mechanism action on PMA-stimulated synovial fibroblast (SF) in vitro. Zymography results reveal that cardamoin treatment significantly inhibited the enzymatic activities of gelatinase B (MMP-9) in PMA-stimulated SF. In addition, cardamonin suppressed collagenase activities of PMA-stimulated SF which mediated by inhibition of MMP-1. Beside, cardamonin strongly suppressed MMP-3 and IL-6 gene expression as well as inhibited COX-2 gene expression albeit in moderate level. We also demonstrated that the cardamonin abolished the p65 subunit of NF-jB translocation from cytosol to nucleus and DNA binding activity of NF-jB in dose-dependent manner. Collectively, these findings suggested that cardamonin exerts an antiarthritic effect through immunomodulatory action on MMP production/activity and pro-inflammatory genes at least partly via NF-jB pathway in PMA-stimulated SF. This study provides a new evidence of cardamonin used as potent inhibitor of joint destruction in rheumatoid arthritis patient. Luteolin, the flavonoids that were isolated from the active fraction of Vitex negundo leaf. The purpose of this study is to clarify the role of luteolin as anti-inflammatory agent in V. negundo. Anti-inflammatory actions were assessed using 4 different in vitro bioassays techniques. Nitric oxide (NO) synthesis by inducible nitric oxide synthase (iNOS) is increased in inflammatory diseases and leads to cellular injury. Luteolin concentration-dependently (1.95-125 lg/ml) inhibited NO production upon stimulation by lipopolysaccharide/interferon-g (IFNg) in RAW 264.7, a murine macrophage cell line. 5-LOX is the initial key enzyme for the production of leukotrienes (LTs) from arachidonic acid, inhibitors of LTs biosynthesis are potential candidates for further development into novel therapies for inflammation, in particular for asthma and allergic diseases. Luteolin also concentration-dependently (1.56-100 lg/ml) inhibited activity of soybean lipoxygenase (LOX) and the IC 50 value is lower than standard control for LOX inhibitor Nordihydroguaiaretic acid (NDGA). It was also shown to contain anti-gout activity by reducing the activity of xanthine oxidase, the key enzyme that catalyses the oxidation of hypoxanthine to xanthine and then uric acid, which plays a crucial role in gout. This study also showed that luteolin are relatively strong platelet activating factor (PAF) receptor binding inhibitor. PAF is known to be a biologically active phospolipid, involved in many pathological conditions such as allergy, inflammation, asthma, cardiac anaphylaxis, gastrointestinal ulceration, endotoxin shock, transplanted organ rejection and thrombosis. In summary, we demonstrated that luteolin possesses anti-inflammatory properties in various mechanism of action. It is crucial to study one of mechanism in deep in order to further validate the actual mechanism of action. Methotrexate (MTX) remains the first choice in the management of rheumatoid arthritis (RA). Resistance to this drug is largely associated to MTX entry into the cell. Lipidic nanoemulsion has emerged as a new strategy since it carries drugs directed to affected tissue. Our aim was to evaluate the anti-inflammatory efficacy of a cholesterol-rich nanoemulsion coupled MTX (MTX-LDE) in antigen induced arthritis (AIA). Toxicity, pharmacokinetics and biodistribution assays were performed. The dose-effect curve of the complex was also determined. Arthritis was induced in the knee joint of methylated serum albumin sensitized NZW rabbits. Twenty four hours after AIA induction animals were intravenously (I.V.) or intraarticularly (I.A.) injected with a single dose of commercial MTX (0.25 mg/kg), LDE or saline. To give an equivalent dose of MTX-LDE, the amount of drug was evaluated by HPLC. Animals were sacrificed 48 h after AIA induction when synovial fluid (SF) and membrane (SM) were collected for total and differential leukocyte count, protein leakage and histopathological evaluation. Our data shows that MTX-LDE did not promote any hematological toxicity. The uptake of this complex was preferentially to liver, spleen and inflamed SM. MTX-LDE I.V. promoted a reduction of 65% of leukocyte infiltrate, 41% in protein leakage and a significant reduction in the SM cell infiltrate. Likewise, the treatment with MTX-LDE I.A. also reduced the inflammatory parameters. In contrast, a single dose of commercial MTX, a DMARDs that requires several weeks to have a clinical effect, did not alter any evaluated parameter. LDE alone or saline were also ineffective. Our study suggests that MTX-LDE is effective, well tolerated and exhibit a rapid effect, probably as a result of their good uptake thus representing a novel therapeutic strategy to treat RA patients. Non-steroidal anti-inflammatory drugs (NSAID) function to reduce pain, fever, and inflammation, and are commonly used in people and animals for this purpose. Currently there are no NSAIDs approved for the control of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. A swine model based on inflammation biomarkers could prove very useful in the drug approval process. The study was designed to accomplish the following objectives: to verify expression patterns of in vitro candidate markers in an in vivo model and to determine the utility of the biomarkers through simultaneous administration of LPS and flunixin meglumine in swine. A previous in vitro study investigated the differential expression of genes altered in response to endotoxin-induced inflammation. Based on that study five of the genes previously identified were chosen for in vivo verification. Two groups of healthy swine were tested: group one was stimulated with only LPS while group two received both flunixin meglumine and LPS. Five genes (MCP-1, Serum Amyloid A2 (SAA2), CD4, CD1, and Caspase 1) were investigated in this system. After LPS stimulation, blood was retrieved at four time points (0, 3, 24, and 48 h). Total RNA was extracted from the blood and analyzed via quantitative RT-PCR (qRT-PCR). Following the analysis, two genes (SAA2 and CD1) were found to have statistically significant (p-value .05) temporal differences in gene expression. In addition to these findings, the animals that received both flunixin meglumine and LPS demonstrated reduced mRNA expression relative to the group stimulated with LPS alone. These results demonstrate that two out of five genes (SAA2 and CD1) may serve as biomarkers of inflammation as well as indicators of NSAID efficacy. As swine are also used for human drug development, the results of this study represent the development of an alternative model for NSAID drug development. Disclosure of interest: None declared. [1, 2] oxaborol-5-yloxy)-2-(2-isopropoxyethoxy) nicotinonitrile)is a novel benzoxaborole under investigation for the treatment of inflammatory diseases. AN6414 inhibits PDE4 with an IC 50 of 5.2 nM, and inhibits TNFa secretion from human leukocytes with an IC 50 of 2.5 nM. AN6414 binds to the active site of PDE4 in a different mode from catechol PDE4 inhibitors making contact with the activated water in the bimetal center. In an in vivo mouse model measuring inhibition of LPS-induced TNFa levels in mouse plasma, AN6414 demonstrates an ED 50 of 0.15 mg/kg, Roflumilast exhibits an ED 50 of 7.5 mg/kg. In a mouse collagen induced arthritis (CIA) model AN6414 reduces disease burden by 90% when dosed orally BID for 21 days at 10 mg/kg. At the same dose and duration reduction by Roflumilast is 80%. Once a day dosing of AN6414 also demonstrates activity in the mouse CIA model, with doses as low as 5 mg/kg showing significant arthritis suppressive activity. Histologic examination of the feet and ankles of AN6414 and Roflumilast treated animals show a strong suppression of inflammation, bone erosion, cartilage erosion and extra pannus formation. In a mouse 5-day toxicity study, AN6414 demonstrates lesser GI toxicity compared to Roflumilast, (NOAEL of 20 mg/kg/day compared to 10 mg/kg/day). In the monkey AN6414 is less emetic than Roflumilast, since it requires a higher plasma concentration of AN6414 to cause emesis. The in vivo pharmacological effect in both a 4 h monkey LPS-induced TNFa secretion test and a 21 day mouse CIA joint damage test can be well explained for both AN6414 and Roflumilast by free plasma drug level and enzyme affinity. AN6414 shows good in vivo efficacy, safety, and pharmacokinetics, and thus provides a new candidate therapeutic for autoimmune diseases. Disclosure of interest: K. Jarnagin: None declared, T. Hydrogen sulfide (H 2 S) is synthesized endogenously from L-cysteine by two pyridoxal-5 0 -phosphate-dependent enzymes, cystathionine bsynthase (CBS) and cystathionine c-lyase (CSE). S-propargyl-cysteine (SPRC), an H 2 S releasing drug, may modulate the endogenous H 2 S production. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 lg/ kg) for 10 h. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H 2 S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC treatment. Furthermore, the antiinflammatory effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. Plasma H 2 S concentration showed significant difference in H 2 S levels between control, vehicle and SPRC treatment groups. In conclusion, these data provide evidence for antiinflammatory effects of SPRC by modulating endogenous H 2 S production. Disclosure of interest: None declared. Idiopathic pulmonary fibrosis (IPF) is a fatal disease manifested by a gradual loss of lung function. Jun N-terminal kinase (JNK) has been implicated in multiple processes that promote fibrosis pathology including the production of cytokines such as TNF-alpha, TGF-beta, and CTGF, myofibroblast transformation, and matrix deposition. In an accompanying abstract, we describe the identification of potent aminopurine-based JNK inhibitors which showed efficacy in animal models of ischemia reperfusion injury following i.v. administration. In this paper, we present the chemical optimization of CC-930, a potent, selective, and orally available aminopurine JNK inhibitor, and its pharmacological profile in animal models of inflammation and fibrosis. Disclosure of interest: None declared. Polymorphonuclear neutrophils (PMN) play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in case of excessive or misdirected responses. Release of the alarmin HMGB1 by cells that die at an inflammatory site may act as an alert signal for the immune system. We studied the effect of HMGB1 on human PMN migration, using whole-blood samples to avoid cell activation associated with isolation procedures. HMGB1 50-100 ng/ml reduced baseline PMN migration as well as fMLP-and IL-8-induced PMN chemotaxis. This inhibitory effect was mediated by the RAGE receptor and was reversed by preincubation with ERK1/2 inhibitor. It was also associated with a decrease in PMN phospho-p38MAPK content and a concomitant increase in phospho-ERK1/2. In contrast, a higher HMGB1 concentration (5,000 ng/ml) had a chemoattractant effect on PMN, through IL-8 production. This effect required the engagement of TLR2 and TLR4 in addition to the RAGE receptor. Finally, the A Box component of HMGB1, which antagonizes the endogenous protein, reduced chemotaxis and also strongly inhibited the enhancement of PMN migration observed with the highest HMGB1 concentration. These results strongly point to a key regulatory role of HMGB1 in PMN recruitment to inflammatory tissues. The A Box component could potentially serve to inhibit inappropriate PMN recruitment during chronic inflammatory disorders associated with excessive HMGB1 release. Disclosure of interest: None declared. Melanocortin peptides exert anti-inflammatory effects via activation of melanocortin receptors (MC) of which five have been identified-MC 1 and 3 bring about the anti-inflammatory effect of melanocortins (Getting et al. 2006) . Here, the effect of melanocortins was determined by utilizing stimulated primary bovine articular chondrocytes. Western blotting was used to identify expression of MC on chondrocytes. Chondrocytes were treated with TNFa (0-80 pg/ml). In separate experiments cells were treated with PBS, aMSH or selective MC 3 agonist DTRP 8 -c-MSH (0-10 mg/ml), or in combination with the MC 3/4 antagonist SHU9119 for 30 min prior to 6 h stimulation with TNFa (60 pg/ml). Cell-free supernatants were collected and analysed by cytokine ELISAs. Data is presented as mean ± SEM of n = 4 determinations vs. control. Western blot showed expression of MC 1,3,5 on chondrocytes; TNFa caused concentration-dependent increase in IL1b, IL6, IL8, whereas IL10 levels were not altered. Maximal release of cytokines was caused by 60 pg/ml TNFa with 27and 2-fold increase in IL1b and IL8, respectively (p \ 0.05) compared to controls, whilst maximal 48-fold increase of IL6 occurred at 80 pg/ml. aMSH and DTRP 8 -c-MSH suppressed TNFa-stimulated production of proinflammatory cytokines causing significant increase in IL10 secretion. aMSH (1 lg/ml) caused 42% decrease in IL1b and DTRP 8 -c-MSH (10 lg/ml) caused 76%, p \ 0.05. Similar reductions in IL6 and IL8 were observed following peptide treatment. SHU9119 failed to inhibit aMSH, but attenuated the effect of DTRP 8c-MSH. TNFa upregulates pro-inflammatory cytokine release in bovine articular chondrocytes and this was abrogated by aMSH and DTRP 8 -c-MSH, whilst treatment of cells with these peptides led to increase in anti-inflammatory cytokine IL10. The MC 3/4 antagonist SHU9119 blocked the effects of DTRP 8c-MSH but had no effect on aMSH activity. These data suggest that both MC 1/3 mediate the anti-inflammatory effect of melanocortins in bovine chondrocytes. Disclosure of interest: None declared. University of Naples ''Federico II'', Naples, Italy Suppressor of cytokine signalling (SOCS)1 negatively regulates the molecular cascades triggered by IFN-c by disabling Jak2 and STAT1 phosphorylation and, thus, impeding the activation of IFN-c-dependent genes. In human keratinocytes, the inhibitory activity of SOCS1 inhibits the IFN-c signalling by binding Jak2 through its kinase inhibitory region (KIR) and, thus, inhibiting the IFN-c receptor (IFN-cR) and STAT1 phosphorylation. As consequence of STAT1 lack, keratinocytes overexpressing SOCS1 cannot longer express ICAM1 and MHC class II as well as CXCL10, CXCL9, and CCL2 molecules. Aimed to potentiate the inhibitory function of SOCS1 in IFN-c-activated keratinocytes, we developed a sets of peptides mimicking the SOCS1 KIR domain. The effects of two cell-permeable mimetic peptides, termed TAT-KIR and TAT-P5, were evaluated on cultured keratinocytes and in a mouse model of allergic contact dermatitis. We found that IFN-c-activated keratinocytes treated with TAT-KIR and, to a lesser extent with TAT-P5, exhibited a reduced Jak2, IFN-cRa and STAT1 phosphorylation, compared to cells treated with a control peptide. The expression of IRF-1, a late IFN-c-inducible transcription factor, and of STAT1dependent genes, such as ICAM-1, HLA-DR, CXCL10 and CCL2, were quite totally abrogated. Also, the reduced expression of ICAM-1 determined by treatment with peptides, resulted in an impaired adhesiveness of keratinocytes to autologous T lymphocytes. Consistently, the migratory responses of T helper (Th)1 lymphocytes towards supernatants from keratinocytes treated with peptides were drastically reduced due to their impaired release of the CXCL10 and CCL2. Finally, we found that the topical administration of peptides on mouse ears during skin challenge with the hapten TNCB led to a significant reduction of the contact hypersensitivity responses. Our data suggest that TAT-KIR and TAT-P5 could be effective for the treatment of Th1dependent inflammatory skin diseases in humans. Disclosure of interest: None declared. D. Schwanzer-Pfeiffer*, E. Roßmanith and D. Falkenhagen. Objectives: MicroRNAs (miRNAs) are under intensive research in the last years. Nevertheless we are still at a very early stage in understanding regulation mechanisms and functions of miRNAs in sepsis. Methods: In a first step a conditioned medium was produced by the stimulation of THP-1 monocytes with 10 ng/ml LSP [4 h ]. This conditioned medium was used in a second step for the stimulation of HUVECs or HPMEC [16 h] and changes in miRNA expression were analyzed by miRNA array analysis. MiR-146a, miR-146b, and miR-155 were selected for further analysis due to their different expression pattern after stimulation. Altered expression of proinflammatory cytokines were investigated after transfection of HUVECs with inhibitory miRNAs. Results: The expression of up to 1,900 microRNAs was investigated after stimulation of HUVECs and HPMECs by a conditioned medium. MiRNA array analysis identified among others a twofold upregulation of miR-146a, miR-146b, and miR-155. MiRNA inhibitors for the selected miRNAs were used in order to investigate the functional properties of miR-146a, miR-146b, and miR-155. Inhibition of miR-146a resulted in a 68% diminished expression of Interleukin (IL)6 and a 64% reduced expression of IL8. Further, expression of IL6 and IL8 was reduced by inhibition of miRNA-146b at 49 and 26%, respectively. Additionally, inhibition of miR-155 resulted in a reduced expression of IL6 of 31% after stimulation of HUVEC miR-155 inhibited cells. Conclusions: In conclusion, miR-146a, miR-146b, and miR-155 as well, seem to be highly involved in the regulation of inflammatory cytokines, especially IL6 and IL8. Disclosure of interest: None declared. Toll like receptors (TLRs) have emerged as key players in the detection of pathogens and the induction of anti-microbial and antiviral immune response, shaping both innate and adaptive immune response. To date, 10 TLRs have been identified in humans. Toll-like receptor (TLR) agonists have attracted a lot of attention from the pharmaceutical industry. Two TLR agonist are approved either for treatment of cancer or as vaccine adjuvant, while numerous clinical trials are currently evaluating the efficacy of TLR agonists in vaccine, cancer and viral infection, as well as in allergy/asthma. An emerging field in TLR targeting drug development is the use of TLR antagonists for use in inflammation, including nucleic acid, phospholipids but few monoclonal antibodies. Indeed, there is accumulating evidences that while TLR activate the immune systems to fight infections or cancer, TLR may also respond to endogenous ligands and maintain unwanted inflammation, leading to tissue damage and inflammatory pathologies. TLR3, a receptor for dsRNA, is widely expressed by immune cells but also epithelial, endothelial and fibroblast, and TLR3 stimulation induces IFN type I and inflammatory cytokines. TLR3 over-expression has been demonstrated in several inflammatory pathologies, and TLR3 ligands can mimic inflammatory diseases in vivo in mice. In the present study we generated and selected antibodies targeting S280 Inflamm. Res. specifically the human TLR3 receptor and demonstrating antagonist activity in vitro. We further characterized their specificity and bioactivity in vitro. Using a surrogate anti-mouse TLR3 antagonist antibody, we established proof of concept of inhibiting TLR3 in vivo, in mice models of inflammatory pathologies. Chronic obstructive pulmonary disease (COPD) is characterized by incompletely reversible airway obstruction associated with inflammation in which macrophages are the predominant inflammatory cells. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is the ligand of the chemokine receptor 2 (CCR2) and a mediator of inflammation in COPD. In order to assess the therapeutic potential of MCP-1 inhibition in murine models we generated a polyethylene glycol conjugated, mirror image oligonucleotide (Spiegelmer Ò ), mNOX-E36, with high specific binding affinity to mouse MCP-1. The mNOX-E36 was characterized in the LPS induced neutrophilia rat model and the sub chronic (11 days) tobacco smoke induced inflammation mouse model. Anti-inflammatory effects were determined based on differential cell counts of the broncheo-alveolar lavage fluid (BALF). In the acute rat model intravenous administration of mNOX-E36 prior to LPS challenge significantly reduced the number of infiltrating neutrophils in the BALF at a dose of 2 mg/kg body weight (bw). In the sub-chronic tobacco smoke mouse model mNOX-E36 (0.2 mg/kg bw), given subcutaneously every other day, starting just prior to first smoke exposure, resulted in a significant decrease of macrophages (41%), lymphocytes (52%), eosinophils (86%), and neutrophils (57%) in the BALF compared to vehicle treated control animals. The effects were comparable to those obtained in the positive control arm with the reference compound roflumilast. These preliminary results demonstrate potent effects of MCP-1 inhibition in an animal model of COPD. Spiegelmer mNOX-E36 administration significantly prevented the recruitment of different inflammatory cells, involved in both the innate and the adaptive immune response, into the broncheo-alveolar space. Preventing the recruitment of these inflammatory cells with the human MCP-1 specific NOX-E36 into the lung might translate into a benefit for patients with COPD. Disclosure of interest: None declared. Hepcidin is a central regulator of systemic iron homeostasis. Increased expression results in functional iron deficiency and is a potential reason for the genesis of anemia of inflammation. With the objective to neutralise the biological activity of hepcidin, the Spiegelmer NOX-H94 was developed. This compound has a high affinity and specificity for human and cynomolgus hepcidin. The aim of this study was to further characterize NOX-H94 in in vitro and in vivo models. In vitro the expression of the iron exporter and hepcidin receptor ferroportin was assessed in the cell line J774A.1 after stimulation with hepcidin. Western blot analysis was used to analyse ferroportin expression. After binding to its receptor the ferroportin-hepcidin complex is degraded and intracellular iron cannot be exported. Acute models of serum iron reduction induced by human hepcidin (mouse) and interleukin-6 (cynomolgus monkey) were established to characterize the efficacy of NOX-H94 in in vivo models. NOX-H94 inhibited hepcidin-induced ferroportin degradation in J774A.1 cells and also a decrease of the serum iron concentration in two in vivo models. These results demonstrate the efficacy of NOX-H94 in in vitro and in acute in vivo models. This ferroportin protecting and iron liberating strategy may supply sufficient iron for erythropoiesis and avoid treatment with intravenous iron preparations. The potential harmful increase of intracellular iron is prevented. Based on these preliminary data, NOX-H94 may be used in patients to influence one reason for the genesis of anemia of inflammation. Disclosure of interest: None declared. Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. The present study investigated the involvement of ETs in arthritis using the most reasonable experimental model of this disease, collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice, and on the day that CIA became clinically detectable, the mice were treated with a dual ETA/ETB receptor antagonist (Bosentan). Disease progression was assessed by measurements of visual clinic score, paw swelling and hypernociception. Gene expression in lymph nodes was evaluated by microarray technology. Validation of microarray genes and ET1-prepro expression in sham and immunized mice and in control and AR human patients treated with Methotrexate or anti-TNF, were evaluated by real time PCR. We observed that the oral administration of Bosentan markedly ameliorated CIA. Gene expression in the lymph nodes of ill mice showed a characteristic profile during Bosentan administration that allowed us to distinguish the treatment from disease state. Prepro-ETs mRNA expression was increased in the lymph nodes of arthritic mice compared with that of sham mice. From a clinical perspective, prepro-ETs mRNA expression was also increased in the PBMCs of RA patients during conventional treatment when compared with anti-TNF therapy and healthy individuals. The findings suggest a critical role of ETs in CIA and RA, through the modulation of important genes. This study offers new advances concerning the role of endothelin in arthritis mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation. Disclosure of interest: None declared. 87.7% for Apis (from bee venom, without mellitin) and 88.1% for Naja (from N. naja venom) secretory phospholipases A 2. Edema induced by exogenous sPLA 2 administration performed in mice paw by harpalycin 2 showed significant inhibition of the edema initial step induced by all sPLA2. In addition, harpalycin 2 also inhibited the myotoxic activity of these sPLA2. In order to understand how harpalycin 2 interacts with these enzymes, docking calculation were performed, showing strong interaction (hydrogen bond) with His48 and Asp49 residues in the active-site of these enzymes. These results pointed to a possible new anti-inflammatory drug. Disclosure of interest: None declared. Availability of rapid and robust assays to determine chondrocyte reactivity can help in the identification of novel treatments and mechanisms for osteoarthritis. We tested here the C-28/I2 chondrocytic cell line developing its culture in micromasses (MM). MM using C-28/I2 cells were obtained by pipetting 20 ll droplets of a cell suspension (density of 2.5 9 10 7 cells/ml) into 48-well plates. Medium was changed after 24 h and stimuli [IL-1b (20 ng/ml), TGFb1 (10 ng/ml) and/or Naproxen (0.1-10 lM)] added 24 h later for further 48 h culture. Human fibroblasts were used as negative control. MM were harvested for staining with Alcian blue (AB; 1% in HCl 0.1 N). Anabolic (COL2A1, SOX9, ACAN) and catabolic genes (MMP1, ADAMTS5) mRNA levels were analyzed by qRT-PCR. Data are from 3 to 4 independent experiments in triplicate. IL-1b promoted a catabolic response characterized by *40% induction in MMP1 and ADAMTS5 mRNA associated with reduction (-55%) in the production of matrix glycosaminoglycan (GAG) measured by AB staining. TGFb1 (10 ng/mL) had an important anabolic effect on gene expression complemented by +47% in GAG production. As a proof of concept for therapeutic development, we tested Naproxen; this compound abolished the catabolic effect of IL-1b. Such pharmacological actions were paralleled by inhibition of PGE 2 release in the MM supernatant. We describe a novel medium-throughput protocol for studying inflammatory processes, candidate compounds and gene expression in chondrocytes. The system is also amenable to different levels of analysis, from simple and rapid quantitative measurement of matrix GAG, to more complex investigations including multiple gene expression analysis at the mRNA level. We thank Dr M Goldring (Cornell Medical College, New York) for the C-28/I2 cells. Disclosure of interest: None declared. Hyperforin (HF) is a natural phloroglucinol isolated from St John's wort (Hypericum perforatum) with antibiotic and antidepressive activities. Acute myeloid leukemia (AML) is a deadly disease which results from the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. AML cells are characterized by abnormal proliferation and resistance to apoptotic signals, and Akt and NF-kB pathways are activated in AML. We studied here the potential antitumor efficacy of HF in AML cell lines and AML patients' cells ex vivo. HF inhibited the growth of AML cell lines U937, NB4, HL-60 and OCI-AML3 differing in phenotype and p53 status. The antiproliferative effects were associated with apoptosis induction as indicated by sub-G1 cell-cycle accumulation, phosphatidylserine externalization and DNA fragmentation. HF also triggered mitochondrial membrane depolarization, down-regulation of Bcl-2 anti-apoptotic protein that controls mitochondrial permeability, activation of caspases-3, -8, -9 and cleavage of the caspase substrate PARP-1. Pan-caspase inhibitor Z-VAD-fmk and caspase-3 inhibitor, but not caspase-8 inhibitor, prevented apoptosis indicating a mitochondrial caspase-dependent cell death (intrinsic pathway). HFelicited apoptosis was also associated with Akt inactivation and activation of pro-apoptotic dephosphorylated Bad which is downstream target of Akt. In contrast, using a specific IKK inhibitor and a specific repressor of NF-kB activation, we showed that NF-kB signaling pathway did not interfere with the proapoptotic effect of HF. Furthermore, HF induced apoptosis of cultured AML patients' cells while normal peripheral blood mononuclear cells were not affected. These results indicate that HF may be of interest in the development of new therapies for AML based on the induction of apoptosis warranting further in vivo investigation. Disclosure of interest: None declared. oxide-NO) of macrophages co-cultivated with LLC WRC 256 tumor cells was evaluated. Macrophages were obtained from peritoneal cavity and cells (2 9 10 5 ) were incubated with CTX (0.3 lg/mL) for 2 h at 37°C. After this time, the macrophages were co-cultivated in presence of LLC WRC 256 tumor cells (2 9 10 4 ), previously plated in 96-well culture dishes. The results showed that macrophages previously incubated in the presence of CTX and co-cultivated with tumor cells generated a greater quantity of NO (35%) than control cells. Tumor cells co-cultivated with macrophages pre-incubated with CTX showed reduction (25%) of proliferation. Boc-2 reversed the stimulatory effect of CTX on secretory activity of macrophages and the inhibitory effect these macrophages on tumor cells proliferation. Taken together, the results indicate that CTX modifies the secretory activity of M2 cells, which may contribute to the inhibitory action of the toxin on tumor growth and activation of formyl peptide receptors seems to play a major role in this effect. These data reinforce the actions of CTX on defence mechanisms and bring new perspectives for the development of a new substance with therapeutic properties. Prostaglandin D 2 (PGD 2 ) is a potent prostanoid released from mast cells in response to allergens. The subsequent actions of PGD 2 are not controlled by existing therapies. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2, also known as DP2) is a G-protein coupled receptor specific for PGD 2 and related metabolites. Activation of CRTh2 mediates chemotaxis and activation of basophils, eosinophils and Th2 lymphocytes. It is also involved in the production of IL-4, IL-5 and IL-13 from this subset of lymphocytes. Selective antagonism of CRTh2 offers a therapeutic approach for the treatment of allergic disease and, accordingly, drug discovery efforts were initiated. A set of substituted phenyl acetic acids were prepared and evaluated for binding affinity to the CRTh2 receptor. Selectivity over related prostanoid receptors was also evaluated. In the event, compound 2x (2-(4-(4-(4-chlorophenethylcarbamoyl)phenoxy)-3cyanophenyl)acetic acid) was identified as a potent, selective, functionally active CRTh2 antagonist. Moreover, compound 2x demonstrated good plasma exposure in rodents upon oral dosing and provided protection from inflammation in a murine model of delayedtype hypersensitivity. Disclosure of interest: None declared. Prostate cancer is one of most prevalent cancers among men in western world. Despite several hundred clinical studies of both experimental and approved anti-tumor agent, chemotherapy has limited activity due to chemo-resistance and up-regulated anti-apoptotic mechanisms. Therefore, development of new drugs for prostate cancer is urgently needed. It was reported that PI3K/AKT signaling plays an important role in the maintenance and viability of prostate cancer progenitors, suggesting that inhibition of the PI3K/AKT and mTOR pathways in prostate cancer cells together with conventional chemotherapy may provide improved therapeutic outcomes. Cucurbitacins are a group of triterpenoids plant families such as cucurbitaceae and cruciferae, which have been shown to have anti-inflammatory activities and anti-cancer effects on various tumor types. Recently, we reported that cucurbitacin D, which is isolated from Trichosanthes kirilowii, has anti-tumor activity through suppression of proteasome activity. Here, we demonstrate that cucurbitacin D leads to inhibit cell proliferation of prostate cancer cells with inhibition of mTOR activation. Western blotting showed that cucurbitacin D induced suppression of p62/SQSTM1 protein level and mTOR phosphorylation which are known as markers for autophagy in PC3 cells, DU145 cells and LNCaP cells. However, cucurbitacin D did not induced typical autophagic event such as LC-3II fragment. In addition, apoptosis inhibitor, z-VAD did not affect cucurbitacin D-induced cell death. These indicate cucurbitacin D induced new type cell death but neither typical apoptotic nor autophagic cell death in prostate cancer cells. Taken together, our results provide a new insight of cell death induced by cucurbitacin D, which was targeted to mTOR. It emerged as a promising candidate for cancer therapy. Disclosure of interest: None declared. The Immunopharmacology Research Group, University of Tampere, Tampere University Hospital, Tampere, Finland Mitogen-activated protein kinases (MAPKs), namely ERK, p38 MAPK and JNK, are important signaling pathways regulating several cellular functions, including inflammatory response. MAPK phosphatase-1 (MKP-1) dephosphorylates and thereby negatively regulates the activity of p38 MAPK and JNK. In the present study, the effect of MKP-1 on inflammatory gene expression in human A549 cells, mouse J774 macrophages and primary mouse bone marrow-derived macrophages (BMMs) from MKP-1 deficient and wild type mice were investigated. Stimulation with cytokines or LPS activated p38 MAPK and JNK in human and mouse cells and induced IL-6, TNF, IL-8 and COX-2 mRNA and protein expression. IL-6, IL-8 and COX-2 mRNA and protein expression were inhibited by p38 inhibitors SB202190 and BIRB 796 in a dose-dependent manner but not with a JNK inhibitor VIII. Down-regulation of MKP-1 with siRNA decreased MKP-1 expression and increased p38 phosphorylation in A549 cells and J774 macrophages. MKP-1 siRNA increased the expression of IL-6, IL-8, and COX-2 in A549 cells and J774 macrophages. Also, LPS-induced expression of IL-6, TNF and COX-2 was further enhanced in BMMs from MKP-1 deficient mice as compared to that seen in wild-type mice. In conclusion, our result suggests that MKP-1 regulates the expression of inflammatory genes by limiting the activity of p38 MAPK. Compounds that increase MKP-1 expression or modulate its function are potential candidates as anti-inflammatory drugs. Disclosure of interest: None declared. Rheumatoid arthritis (RA) is a chronic inflammatory disease with unclear etiology that affects approximately 1% of the world population. It is a systemic disease but mainly targets freely moveable joints, such as the wrists or the knees, and can cause severe disability. Resident synovial cells, such as fibroblast-like synoviocytes (FLS), acquire an altered phenotype during RA and present characteristic of transformed cells. They play an important role in the disease by directly destroying the cartilage, but also by inducing angiogenesis, inflammation, bone damage, and by transferring disease from affected to healthy joints. We have found that FLS from patients with RA (RA-FLS) express large numbers of functional KCa1.1 (BK, Maxi-K, Slo1) channels at their plasma membrane. Blocking KCa1.1 channels inhibited the proliferation, the motility, and the invasive properties of RA-FLS, and may therefore prevent disease progression. Blockers of KCa1.1 channels also inhibited the production of vascular endothelial growth factor (VEGF), interleukin-8, and matrix metalloproteinase-2 by RA-FLS, without affecting the production of interleukin-6 or inducing cell toxicity. These blockers therefore have the potential of reducing angiogenesis, inflammation, and joint destruction. KCa1 Although biologics have demonstrated utility for treatment of rheumatoid arthritis, a significant proportion of patients fail to respond or develop dose-limiting side effects. This, together with the cost and inconvenient parenteral mode of administration, has prompted renewed efforts to develop a broader armamentarium to include safe, efficacious and orally available small-molecule drugs. Inflammationassociated protein kinases fulfill these requirements and remain an attractive target for therapeutic development. However, despite promising performance in the discovery phase, several compounds have failed to deliver on efficacy and/or safety criteria in the clinical setting. BioSeek has pioneered the development of an in vitro human cell-based technology to integrate human biology into drug discovery. BioMAP Ò Systems model the complex signals and phenotypic biology of specific therapeutic areas including inflammation/auto-immunity. BioMAP Ò profiling generates a unique signature of human biological responses for every active compound. We have profiled a number of pathway-associated kinase inhibitors, JAK, p38MAPK or PI3K family members. BioMAP Ò profilingreveals both common and unique antiinflammatory/mechanism of action effects (efficacy), secondary and off-target activities (safety), and clinical insights (therapeutic guidance) for each of these target classes. Comparison of the resulting profiles using proprietary algorithms to BioSeek's database containing profiles of thousands of experimental compounds and approved therapeutics provides an enhanced understanding of the mechanism of action of these target classes. Such actionable data can guide lead selection, lead optimization (SAR), mechanistic understanding of compound action. BioMAP Ò profiling yields critical human cell-based information to guide nomination of candidates for preclinical development in the areas of inflammatory and autoimmune disease and enable develop safe and effective therapies. Disclosure of interest: None declared. Non-invasive imaging of inflammation to measure the progression of autoimmune diseases such as rheumatoid arthritis (RA) and to monitor responses to therapy is critically needed. V-Sense, a perfluorocarbon tracer agent that preferentially labels inflammatory cells, which are then recruited out of systemic circulation to sites of inflammation, enables detection by 19F MRI. With no 19F background in the host, detection is highly specific, yielding a quantitative marker of the degree of inflammation present. Collagen-induced arthritis in rats, a model with many similarities to human RA, was used to study the ability of V-Sense to measure the accumulation of inflammation over time. Disease progression in the rat hind limbs was monitored by caliper measurements and 19F MRI on days 14, 21 and 28, including the height of clinically symptomatic disease. Naïve rats served as controls. The capacity of V-Sense to assess the effectiveness of therapy was studied in a cohort of rats administered oral prednisolone on days 14-28. This study may support the use of V-Sense to clinically quantify and monitor the severity of inflammation, and to assess the effectiveness of treatments in RA and other diseases with an inflammatory component. Objective: Systemic Inflammation (SI) is considered as a key pathogenic base of critical states including sepsis. In our opinion, the main problem consists in absence of both of fundamental image of SI as the typical pathological process and it's specialities as compared to ''Classical'' (local) inflammation. It is significant because of one or another signs of SIR are observed under classical inflammation. In 2001 we defined SI as ''Typical multi syndrome phase-specific pathological process caused by systemic alteration and is characterized by total inflammatory responsiveness of endotheliocytes, plasma and cell factors, and connective tissue as well as microcirculation disorders in essential organs and tissues''. The main methodological approaches to study SI were suggested. Methods: Rates of IL-6, IL-8, IL-10, TNFa, CRP, troponin I, myoglobin, cortisol; and D-dimer were determined with immunochemiluminous assay (''Immulite'', Siemens Medical Solutions Diagnostics, USA). Methods of calculation of Reactivity Level (RL) to estimate intensity of SIR as well as SI scale score have been created. Integral SI scale registers the values of RL (maximum is 5 points) and occurrence of other phenomena of SI: systemic alteration, MODS, micro clottage, distress of neuroendocrine system [Patent No. 233577/Rus.] . Conclusion: SI scale intends to describe pathogenesis of shock and other critical states both of septic and aseptic origin. This scale makes it possible to define the SI phase, which could be applicable for monitoring and prognosis sever complications. Advantages of this way of estimation SIR and SI are the following: replaceability of parameters and the calculation has been based on overshooting of normal level by cytokines and CRP that makes it applicable in different method assay. Disclosure of interest: None declared. The behaviors of individual cells are thought to be influenced with many integrated factors, such as the humoral factors from adjacent and even distant cells, the attached extracellular matrix and the mechanical stimulation. Because the environment of the cells is not two dimensional (2D) but 3D in vivo, the cells receive those stimuli not from 2D but from 3D. Therefore, the engineered 3D tissue models in vitro have great possibilities in the study of such 3D complexities of physiology and 3D pathological processes of diseases in vivo. In addition, the use of such 3D tissue models with human cells is expected much more efficient in the screening and discovery of new therapies and new drugs, more than 2D culture cells and animal experiments. Then, we have addressed to develop some effective technologies to fabricate such 3D tissue models efficiently and accurately using living cells and biological materials. Then, we have developed 3D bioprinter applying inkjet and gelation technique, which enables to position living cells at the designed 3D position arbitrary. We have succeeded to fabricate simple 3D gel structures containing living cells. Next, we have improved our 3D bioprinter with a new printing mode based on the bitmap images to fabricate more complex 2D and 3D structures. We have succeeded to fabricate some 3D structures by laminating the 2D structures based on bitmap images, however, we could not fabricate them with sufficient precision. In this study, we developed our printer by adding with Z-axis control system. Using the new version system, we successfully improved its performance, and could fabricate more complex 3D structures, such as branched tubes and the multi-layered 3D pyramid structures. In addition, we also recognized that the living fibroblasts were extended in the fabricated 3D tube with fibrin gel. We confirmed that the approach of 3D biofabrication with our 3D bioprinter has a great potential to fabricate complex 3D tissue models in vitro. Disclosure of interest: None declared. E. Gusev 1 , L. Solomatina 1, *, J. Zhuravleva 1 1 Institute Immunology and Physiology, RAS, Ekaterinburg, Russian Federation Objective: Chronic renal allograft dysfunction is a progressive nephrosclerosis which arises under immune and nonimmune mechanisms. There is a supposition today that one of the reasons of chronic renal allograft dysfunction may be process of chronic systemic inflammation in recipients. Study Goal: To analyze signs of the systemic inflammation as a risk factor of chronic renal allograft dysfunction in patients with chronic glomerulonephritis. Patients and methods: There were studied three groups of patients with chronic glomerulonephritis: the group 1 is a ''patients with endstage renal disease which receive renal replacement therapy in the form of programmed hemodialysis'' (n = 22); the group 2 is a ''patients with chronic renal allograft dysfunction'' (n = 23); the group 3 is a ''patients with normal functions of allograft'' (n = 24). The control group was a cohort of healthy persons, age 18-55 (n = 50, mean age 34.1 ± 10.4 years). There were measured levels of systemic inflammation markers in blood plasma such as interleukins (IL-6, IL-8, IL-10), tumor necrosis factor (TNF)a, C-reactivity protein (CRP), cortisol, myoglobin, troponin I, D-dimer. On the basis of these markers we calculated integrated criterionscale of chronic systemic inflammation, ranging 0-8. Point One of the vital barriers in the human body is the air-blood interface in the lung. With an area of approximately 100-140 m 2 the alveoli of the human lung are functionally the most important element of the lung. Therefore, we developed an in vitro model that mimics the alveolo-capillary barrier in order to assess the toxicity of nanoparticles (NP). The aim of the model is to explore the toxicity, the passage and the inflammatory response of the air-blood barrier after a treatment by SiO 2 (20 and 100 nm) and TiO 2 (20 nm) NP. In order to better mimic the reality, the model of cell coculture is composed of 3 cell types which are, respectively: THP-1 macrophages, A549 or NCI-H441 epithelial cells and HUVEC-CS endothelial cells. Epithelial cells and macrophages are cultivated on the apical membrane of an insert, and endothelial cells on the basal side. Therefore, we studied toxicity, cellular inflammation and intercellular communication between the cells of the model. After 24 h of treatment with NP, IL-1b, IL-6, IL-8 and TNFa were measured in cell supernatants with a multiplex technology. SiO 2 and TiO 2 nanoparticles do not induce the same inflammatory response and the smallest nanoparticles (20 nm SiO 2 and TiO 2 NP) seem to induce more cytotoxic effects. Moreover, we demonstrate that a treatment of epithelial cells by NP-exposed-macrophage's conditioned medium does not trigger the same inflammatory response compared to direct treatment of epithelial cells by NP. Therefore, a model of air-blood barrier including macrophages is essential to assess inflammatory process close to reality. Otherwise, contrary to A549 epithelial cells, the inflammatory process in NCI-441 cells may link to the activation of the P2X 7 cell death receptor, functional, and known for his involvement in the inflammasome activation. Therefore, NCI-H441 cells better mimic the reality and is a better cellular model than A549 to assess the inflammatory process and damages caused by NP in the air-blood barrier. Disclosure of interest: None declared. Aim: The aim of this study is to show the importance of Cone Beam Computerized Tomography to volumetrically quantify TMJ damage in patients with Juvenile Idiopathic Arthritis (JIA), measuring condylar and mandibular real volumes. Material and methods: 30 children with temporomandibular involvement by JIA were observed by Cone Beam Computerized Tomography. The mandible was isolated from others craniofacial structures; the whole mandibular volume and its components' volumes (condyle, ramus, emibody, emisymphysis on right side and on left side) has been calculated by a 3D volume rendering technique. Results: The results show a highly significant statistical difference between affected side volumetric values versus normal side volumetric values above all on condyle region (P \ 0.01), while they does not show any statistical differences between right side versus left side. Conclusion: The Cone Beam Computerized Tomography represents a huge improvement in understanding of the condyle and mandibular morphological changes, even in the early stages of the Juvenile Idiopathic Arthritis. Early initiation and optimal adjustment of aggressive therapy with disease-modifying anti-rheumatic drugs have been extremely successful in preventing irreversible joint damage. Therefore, the accurate and early diagnosis of JIA and the sensitive monitoring of the disease process are essential. Advanced imaging technology capable of identifying even the slightest trace of erosive joint damage may enable the prediction of future structural and functional deterioration. Disclosure of interest: None declared. M. Saito 1, *, T. Tanaka 1 , K. Takahashi 1 , S. Yamanaka 1 , T. Nakahata 1 1 Kyoto university, Kyoto, Japan Background: In vitro studies for some of immunological disorders using human samples are sometimes difficult because (1) therapeutic medicines can affect the phenotype of primary immune cells, and (2) existence of somatic mosaisism interfares identifying responsible inflammatory cells. To overcome this, here we constructed an in vitro disease model for a NLRP3-drived hereditary autoinflammatory syndrome with human induced pluripotent cells (iPSCs). Chronic Infantile Neurologic Cutaneous and Articular (CINCA) syndrome is a severe autoinflammatory disease caused by gain-of-function mutations of NLRP3 gene. Mutant NLPR3 protein in macrophages causes overproduction of IL-1 beta, which leads to systemic inflammation. Results: We obtained fibroblasts from two male patients of CINCA syndrome with somatic mosaicism. Fibroblasts were transduced with lentiviral vector encoding ecotropic receptor Slc7a1 and with retroviral vector encoding OCT4, SOX2, KLF4 and cMYC. Compact embryonic stem (ES)-cell like colonies emerged after 2 weeks and we obtained more than 20 iPSC lines. We obtained both wild-type and mutant clones from each patient's fibroblasts. After evaluating transgene silencing by quantitative RT-PCR, we selected 3 mutant and wild type iPSC clones, respectively, for further analysis. All the clones expressed human ES-cell markers. iPSCs are differentiated into CD14+ macrophages via CD34+ KDR+ hematopoietic progenitors. Mutant iPSC-derived macrophages tend to secrete higher amount of IL-1 beta when stimulated with lipopolysaccharides or infected with Listeria monocytogenes, while wild-type clones not. Future directions: We have succeeded to recapitulate in vitro phenotypes of macrophages with NLRP3 mutations. In vitro disease modeling provides us a platform for drug screening. Now we are planning to look for novel NLRP3 inhibitors that can modulate inflammasome signaling more precisely. Disclosure of interest: None declared. The unique properties of Quantum Dots (QDs) such as broad excitation spectra, narrow emission spectra, high resistance to photobleaching, higher photo-stability and stronger fluorescence makes them one of the most attractive fluorescent reporter molecules for a wide range of biological applications including biosensing and assay development applications. However, the high cost of QDs has limited their application in assay development where at least 50-100 lL reagents are required for performing an assay. This research capitalizes on the superior properties of QDs by utilizing just 1-2 lL of sample and antibodies conjugated to QDs for detection of biomarkers. In this work we developed a simple and inexpensive microcapillary immunosensor based on quantum dot assay for detecting myeloperoxidase (MPO) in an animal model of intestinal inflammatory disease. The biomarker MPO was detected in the stool samples collected from animal at various stages of inflammatory disease. The method utilizes commercially available polymethylmethaacrylate (PMMA) micro-capillaries (ID 250 lm, OD 500 lm) as substrates for performing a sandwich assay. The results obtained indicate that the limit of detection (LOD) of the device and the assay is 100 picomolar of MPO (*15 ng/mL) in 1-2 lL of sample. Furthermore, the assay and device robustness were tested by detecting MPO in 1-2 ml of animal stool sample and comparing the results obtained with a commercially available MPO ELISA kit that uses 100 lL of sample. In conclusion, we are reporting here a low cost microcapillary immunosensor platform for detecting inflammatory markers. Disclosure of interest: None declared. Diglucopyranosylamines and the oligo-SO3 salts have a confusing array of immunological properties and suppress antigen and collagen arthritis. Also, in testimonial reports, dibbgluco-pyransylamine (dibbGA) resolves herpetic lesions and pain. In silico models of active enzyme and receptor binding sites are used in ligand design for drug candidates. A novel application of this technology is in silico screening of active sites for drug binding to a wide variety of binding sites. dibbGA, monoglucosylamine (MGA), dibbGA-oligo-SO3 were screened against the Pharmacophore Database HypoDB (Inte:Ligand) with 2500 chemical feature-based 3D pharmacophore models encoding 277 targets: mammalian, bacterial and viral enzymes, GPCRs, transduction factor receptors, signalling molecules, structural proteins, toxins, and transport proteins, from protein-ligand 3D complex structures via LigandScout software (Inte:ligand). An algorithmic score of 0.7 to a maximum of 1.0 is considered highly relevant. Glucose was included to exclude its monomer binding. dibbGA, a bacterial glucosidase inhibitor, fitted to 0.689, and MGA to 0.741. N-acetylation and oligo-SO3 removed activity. Herpes thymidine kinase fitted to 0.7. Viral DNA polymerase and influenza neuraminidases were negative. Surprisingly, adenosine A1 receptor (A1R) fit was 0.787 for dibbGA, 0,571 on N-acetylation, and all others negative. The model predicted the activity of dibbGA and revealed potential herpes viral enzyme interference, notably herpetic thymidine kinase, relevant to DNA synthesis and cell division. Unexpected was a high A1R fit. A1R limits inflammatory pain and A1 agonists are antinociceptive in inflammation. This may explain reports that dibbGA limits herpes lesions and peripheral pain. Verification of A1R and inflammatory nociceptive activity is needed. Lack of oligo-SO3derivative binding is unsurprising since heparinoid-binding receptors likely to be involved in the anti-rheumatic activity of these agents is excluded. Two leukocyte adsorbing apheresis techniques are available now for treatment of chronic inflammatory conditions: granulocyte and monocyte apheresis (GMA) (ADA Ò -column) using heparin as anticoagulant and leukocytapheresis (LCAP) (device Cellsorba TM ) using nafamostat mesilate or ACD-A as anticoagulant. We found recently that antimicrobial and immunoregulatory peptide LL-37 was released from neutrophil granulocytes within the plastic apheresis lines during LCAP using ACD-A as anticoagulant and may perform its regulatory functions when returned back into the patient. However, heparin is also known for its anti-inflammatory properties. Eight patients with chronic inflammatory diseases were treated by GMA using heparin as anticoagulant: three patients with inflammatory bowel disease, three-with sarcoidosis, one-with chronic dimyeliniserande disease and onewith pyoderma gangrenosum. Venous blood samples and samples from the apheresis lines (before and after the apheresis device) were analyzed in the same manner as we done previously in our LCAP study (ACD-A) by commercial ELISA. There was no increase of LL-37 in the lines in difference to our LCAP-study. Some slight increase of LL-37 concentration was observed over the apheresis device. We report hereby that heparin used as anticoagulant inhibits generation of LL-37. Thus, the anticoagulants have different impact on immunoregulatory molecules as LL-37 and choice of the anticoagulant influences antiinflammatory profile of respective apheresis treatment. Disclosure of interest: None declared. Heart failure in WT AngII mice, as quantified by percentage fractional shortening (FS, a) was accompanied by significantly increased cardiac weight (b), wall thickness (c) and larger cardiac myocytes (d), while these parameters were significantly lower in protected KO AngII mice. Levels of cardiac stress markers atrial natriuretic factor (ANF, e) and alpha skeletal actin (aska, f) remained low in miR-155 KO AngII as compared to WT AngII. Using antagomiR-155, we obtained a cardiac knockdown of 81.1 ± 3.8% in miR-155 antagomiR-treated mice as compared to mice treated with scrambled control (g). MiR-155 inhibition was accompanied by preserved FS (h), decreased cardiac weights (i) and wall thicknesses (not shown), and decreased induction of ANF (j) and aska (k). *P \ 0.05; **P \ 0.01; ****P \ 0.001 Leukocytic miR-155 drives cardiac hypertrophy Cardiac myocytes express minor amounts of miR-155 whereas cardiac interstitial leukocytes have high miR-155 levels (Fig. 2a) . Could miR-155 drive leukocyte mobilization and infiltration upon AngII? Indeed, while baseline numbers of circulating monocytes are equal in WT and KO mice, AngII mobilizes these cells in WT but not in KO mice (Fig. 2b) . Also, AngII-treated WT hearts experienced an influx of CD68+ macrophages and-to a lesser extent-CD3+ T cells, but absence of miR-155 abrogated this (Fig. 2c-e) . These data point towards a crucial role of miR-155 in cardiac leukocyte mobilization and infiltration during pressure overload. Fig. 2 In situ hybridization shows preferential miR-155 expression in cardiac inflammatory foci (a). AngII mobilizes CD11b+/Ly6Gmonocytes in WT but not in KO mice (b) but does not affect CD3+ T cells (not shown). In hearts of AngII-treated WTs, there is an influx of CD68+ macrophages (c) and CD3+ T cells (d), while these cell populations remain low in KO mice. AntagomiR-155 treatment similarly cardiac abrogated leukocyte infiltration, as shown by CD45 staining (e). *P \ 0.05; **P \ 0.01;*** P \ 0.005 We then wondered whether leukocyte miR-155 drives the cardiac hypertrophic response. Indeed, adoptive transfer of WT leukocytes in miR-155 KO hearts completely restored the cardiac hypertrophic response (Fig. 3a) . Vice versa, the removal of leukocyte miR-155 from WT hearts compromised hypertrophic growth following AngII. Cardiac myocytes cultured in miR-155 KO media also displayed a limited hypertrophic response (Fig. 3b, c) . Fig. 3 a Adoptive transfer of miR-155 KO bone marrow into WT mice (KO to WT) and vice versa (WT to KO). Heart weights are determined by genotype (WT or KO) of the bone marrow, and not by mouse origin (WT or KO). b, c nRCMs cultured in the presence of WT + LPS or KO + LPS media and stimulated with pro-hypertrophic endothelin-1 (ET-1) show abrogated hypertrophic response in the presence of KO + LPS media, as demonstrated by decreased myocyte areas (b) and blunted aska-expression (c). d, e AAV9-mediated overexpression of has-miR-155 introduces human miR-155 in all mice (d), but does not influence the cardiac hypertrophic response (e). f Et-1 treatment increases miR-155 expression 1.6 fold in nRCMs (P \ 0.05 versus control). However, inhibition of miR-155 in nRCMs does not influence the cardiac myocyte hypertrophic response, as shown by normal aska and BNP expression. *P \ 0.05; **P \ 0.01; ***P \ 0.005 We excluded a role for myocytic miR-155 in the heart. In the presence of AAV9-miR-155 or -scrambled expression (Fig. 3d) , hearts weighed equal at baseline and had similar degrees of hypertrophy and hypertrophic gene expression following AngII (Fig. 3e and not shown). Similarly, in vitro, cardiomyocyte-knockdown of miR-155 had no effect on hypertrophic markers (Fig. 3f) . Taken together, these data show that leukocytes impose hypertrophic growth on cardiac myocytes via miR-155. Leukocytic miR-155 drives cardiac hypertrophy via IL-6 MiR-155 stimulates production of inflammatory cytokines including IL-6, IL-12 and IL-236. IL-6 activates STAT3 in macrophages as well as cardiac myocytes, evoking hypertrophy and pro-inflammatory signaling, respectively. We found that AngII increased cardiac IL-6 levels in WT but not in KO mice (Fig. 4a) , and LPS had similar effects in BMMs (not shown). Following-up on this, we measured STAT3 activation in miR-155 KO and WT BMMs, in nRCMs cultured in miR-155 KO and WT BMM-media, and in hearts, and confirmed the cardiac IL-6/STAT3 axis to be regulated by miR-155 ( Fig. 4b-d) . The miR-155 target Suppressor of Cytokine Signaling 1, SOCS1, which is expressed by leukocytes but not cardiac myocytes, may mediate this effect. Indeed, we found that in the absence of miR-155, SOCS1 protein expression is elevated in unstimulated BMMs as well as in LPS-activated BMMs as compared to WT (Fig. 4e) . AngII-treatment increases cardiac interleukin-6 (IL-6) levels (a) and P-STAT3/total STAT3 levels (b) in WT but not in KO mice. BMMs also show blunted STAT3 activation upon LPSstimulation in the absence of miR-155 (c). Finally, nRCMs cultured in the presence of KO + LPS media fail to activate STAT3 (d). On the other hand, the miR-155 target SOCS1 is strongly increased in the absence of miR-155, at baseline and following LPS-treatment (e) 1 William Harvey Research Institute, Barts and The London SMD, QMUL, London, United Kingdom, 2 Dip Farmacologia Sperimentale, Universita' di Napoli Federico II, Naples, Italy, 3 Departamento de Farmacologia, Farmanguinhos-Fiocruz, Rio de Janeiro, Brazil Hydrogen sulphide (H2S), a gaseous mediator synthesized in several mammalian tissues by two main enzymes CBS and CSE, increases under inflammatory conditions or sepsis. Since H2S and H2Sreleasing molecules afford potent inhibitory properties on the process of leukocyte trafficking, we tested whether endogenous Annexin A1 (AnxA1) could display intermediary functions. Treatment of human PMNs the H2S donor NaHS (10-100 mM) provoked prompted and intense mobilization ([50%) of AnxA1 from the cytosolic pool to the cell surface, supporting the inhibitory effects of the gas in the flow chamber assay. Such in vitro actions could be translated in analyses of the inflamed microcirculation, where NaHS (100 mmol/kg s.c., -1 h) afforded marked inhibition of IL-1-induced cell adhesion and emigration in the mesenteric vessels of wild type, but not AnxA1-/-, mice. Next, we investigated whether endogenous AnxA1 could modulate H2S synthesis, indicating existence of a positive circuit. To this end, a marked increase in CBS and/or CSE expression in a variety of tissues (aorta, kidney, spleen) tested from AnxA1-/-mice, as compared to wild type animals, was quantified by qPCR. Moreover, NaHS was able to counteract the increase in expression in iNOS and COX2 (four-and sevenfold reduction, respectively) upon LPS-stimulation of bone marrow derived macrophages (BMDM), though it was totally inactive in cells prepared from AnxA1-/-mice. Taken together, these data strongly suggest-for the first time-the existence of a positive interlink between AnxA1 and the H2S pathway, providing a novel mechanistic explanation for the exquisite properties of H2S in the control of experimental inflammation. These finding may be relevant to innovative discovery programmes aiming at harnessing the biological properties of H2S. Supported by the Wellcome Trust (programme grant 086867/Z/08/Z). Introduction: H2S is a gaseous mediator synthesized by two pyridoxal-5-phosphatedependent enzymes, such as CBS and CSE. L-Cysteine represents the physiological precursor, which is metabolized leading to the H2S production. Recent literature has supported the involvement of H2S in cardiovascular homoeostasis (Zhong et al. 2003; Qu et al. 2006) . In particular, it has been demonstrated that H2S (a) exhibits vasodilator activity in vitro and in vivo (Ali et al. 2006) , (b) is pro-apoptotic on vascular smooth muscle cells (Zhao et al. 2001; Yang et al. 2004 ) and (c) is involved in leukocyte-endothelium interaction (Zanardo et al. 2006) . Furthermore, there is emerging evidence for a key role for H2S in cell trafficking and inflammation (Zhang et al., J Leukoc Biol, 2007) . AnxA1 is also a key mediator in the inflammation and it shows prominent features in activation of the resolution process, exerting anti-inflammatory activity. Since H2S and H2S-releasing molecules potently inhibit the process of leukocyte trafficking, we tested whether endogenous AnxA1 could display intermediary functions. Materials and methods: Human PMN isolation and membrane and cytosolic fractions preparation. Peripheral blood was collected from healthy volunteers and PMNs isolated. All healthy volunteers gave oral and written consent and cell separation was covered by ethical approval 05/Q0603/34 (East London and The City Research Ethics Committee 1). After treatment, cells (4 9 106) were re-suspended in lysis buffer and processed in order to get cytosolic and membrane fraction. Animals and treatment: C57Bl/6 mice (Charles River, UK) were treated with LPS (10 mg/kg) and then sacrificed after 6 h. Blood, liver, aorta, kidney, lymph nodes and spleen were collected for further analysis. Bone Marrow Derived Macrophages (BMDM) from AnxA1-/-and wild type mice: BMDM were obtained from femurs and tibias of 4-6 week old. The marrow was flushed from the bone, washed, resuspended (2-3 9 106 cells/ml) in culture medium (30% L929 conditioned medium) and incubated at 37°C for 5 days. BMDM were then treated with LPS (100 ng/ml, 6 h) alone or in presence of NaHS (100 mM, -1 h). Quantitative real-time PCR: Quantization of the expression level of selected genes (CBS, CSE, COX2 and iNOS) was performed by quantitative real-time PCR. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For realtime PCR, 10 ng template was used in combination with each primer solution (Qiagen, UK) and Fast SYBR Green Master Mix solution (Applied Biosystem, UK). All reactions were performed in a_ 7900HT Fast Real-Time PCR System instrument (Applied Biosystem). Relative expression (vs GAPDH) ±SEM was then expressed in the graphs and showed as fold-increase or decrease (n = 3). Intravital microscopy of the mesenteric microcirculation: Following treatment with IL1b (10 ng/mouse i.p., 2 h) alone or in combination with NaHS (100 mmol/kg s.c., -1 h) or PAG (10 mg/kg i.p., -30 min) AnxA1-/-or wild type mice were mesenteries were exposed and one to three randomly selected postcapillary venules (diameter between 20 and 40 mm) were observed for each mouse (n = 5). Adherent and emigrated leukocytes were then quantified by counting the number of cells (100 9 50 mm 2 ). All of the animal studies were conducted in accordance with current UK. Home Office regulations and complied with local ethical and operational guidelines. Results: Resting human PMNs retain the majority of AnxA1 in the cytosol as granular or free stores. NaHS treatment (10-100 mM, 30 min) induces mobilization of intracellular AnxA1 from cytosol towards the membrane and its further release in the culture medium (Fig. 1) . As LPS stimulation has bee reported to increase circulating H2S levels, we treated AnxA1-/-and wild type mice with LPS and monitored plasma levels for H2S. As shown in Fig. 2a , H2S levels are increased in LPS treated mice; furthermore, the gaseous mediator levels are higher in AnxA1-/-mice vs wild type even in non-treated mice. We then focused on the clear difference in H2S amount between the two animal genotypes and real time PCR data confirmed that in AnxA1-/-mice we have an increase in expression for CSE and/or CBS in all tissues analyzed ( Fig. 2b-f ). On the basis of these results we decided to investigate on possible antiinflammatory effect for H2S in a different setting such as BMDM. Preincubation with NaHS for 1 h before LPS administration could significantly prevent the increase in expression of COX2 and iNOS associated to LPS treatment. Surprisingly, this effect was occurring only in BMDM from wild type but not from AnxA1-/-mice (Fig. 3) . In order to better establish the physiological relevance for AnxA1 mobilization in human PMN we performed intravital microcopy study in a IL1b-induced inflammation model. Pre-treatment of mice with S292 Inflamm. Res. NaHS was able to reduce the number of adherent and emigrated cells and, once again, this effect was only evident in wild type but not in wild type mice. Furthermore, pretreatment with CSE inhibitor PAG significantly exacerbated the inflammatory pattern (Fig. 4 ). Discussion: The evidence that H2S is a new gaseous physiological modulator rather that a toxic and pollutant gas is now widely accepted, as many studies have shown that impairment in its production could lead to or are involved in many different pathological conditions (Brancaleone et al., Br J Pharmacol, 2008; Szabo C, Nat Rev Drug Discov 2007) . The data here reported represent an interesting novelty and clearly demonstrate, for the first time, the existence of a crosslink between H2S and AnxA1 pathways, where it appears to be an evident ''one-need-the other'' system. However, the data this study showed are preliminary and the aspects highlighted need to be confirmed and further investigated with more targeted experiments in order to get better understanding of the mechanisms behind this circuit. In addition to its enzymatic properties, the serine proteinase, Cathepsin G is known to activate proteinase-activated receptor 4 (PAR4). In joints, PAR4 activation leads to inflammation and pain. Since cathepsin G is found in high concentration in arthritic joints, we hypothesised that Cathepsin G would have a pro-nociceptive effect in the joint by cleaving and activating PAR4. Electrophysiological recordings were made from knee joint afferent fibres in male Wistar rats during normal and noxious joint rotations. Fibre firing rate was recorded in naive rats during 10 s rotations before and over 15 min after local administration of Cathepsin G (1 ng/100 ll-10 lg/100 ll). Responses were compared with animals pre-treated with the PAR4 antagonist, pepducin P4pal-10 (100 lg i.p.). Cathepsin G dose-dependently decreased the firing rate of joint fibres during rotation of the knee. At doses of 1 and 10 ng, where Cathepsin G alone had no effect, PAR4 antagonism caused a significant decrease in firing rate. Thus, Cathepsin G has an anti-nociceptive effect on joint fibres in normal rats that is enhanced when PAR4 is inhibited. This suggests that Cathepsin G can activate PAR4 but the anti-nociceptive effect of Cathepsin G is via an unknown mechanism. Introduction: Cathepsin G is a pro-inflammatory serine proteinase released from activated neutrophils that occurs in high levels in rheumatoid arthritic joints [1, 2] . This proteolytic enzyme is thought to play an important role in inflammation as evidenced by the reduced leukocyte accumulation and cytokine release observed in arthritic joints of Cathepsin G knockout mice [3] . As yet, a role for Cathepsin G in joint pain has not been examined. The intracellular signalling effects of Cathepsin G are thought to be mediated in part by the ability of this enzyme to cleave and activate members of the proteinase-activated receptor (PAR) family [2] . The PAR family currently consists of four G-protein coupled receptors. Cleavage of these receptors by serine proteinases at a specific site within the extracellular N terminus, reveals a tethered ligand sequence which can then bind to the active site and cause receptor activation [4] . PARs can also be selectively activated, without proteolytic cleavage, by synthetic activating peptides that mimic the tethered ligand sequence. PAR4 was the 4th member of the family to be discovered and we have previously shown that a PAR4 activating peptide had both pro-inflammatory and pronociceptive effects in the joint [5] [6] [7] . In addition, treatment with the selective PAR4 antagonist, pepducin P4pal-10 significantly reduced disease severity in a model of acute joint inflammation [5] . It is not yet known which particular serine proteinase activates PAR4 in vivo in the joint but Cathepsin G can activate PAR4 in platelets [2] . In this study we examined the effect of Cathepsin G on neuronal activity in knee joints of normal rats and determined whether this effect is mediated via PAR4. Methods: Experiments were performed on male Wistar rats (250-400 g; Charles River Laboratories, Quebec, Canada) in accordance with the Canadian Council for Animal Care guidelines for the care and use of experimental animals. Single unit extracellular recordings were made from knee joint primary afferents as previously described [6, 7] . Briefly, small nerve fibre bundles from the saphenous nerve were placed over a platinum electrode to permit extracellular recordings. Afferent nerve fibres originating from the knee joint were identified by the elicitation of a firing response to probing of the joint with a glass rod. Conduction velocities, electrical and mechanical thresholds of fibres were determined. Four movement cycles, each consisting of 10 s of normal rotation and 10 s of noxious rotation of the knee to discrete torque levels, were performed at the beginning of the experiment. The mean afferent nerve fibre firing rate associated with these movements was the control baseline level, which was set at 100%. Then, either Cathepsin G (1 ng-10 lg in 100 ll) or boiled denatured Cathepsin G (inactive control) were administered locally to the knee via a saphenous arterial cannulation. Movement cycles were repeated 1 min post-drug administration and then every 2 min until 15 min postdrug. Rats were either naive or pre-treated with the selective PAR4 antagonist, pepducin P4pal-10 (100 lg i.p.) 30 min prior to recordings. Neuronal activity was digitized using a data acquisition system (CED1401; Cambridge Electronic Design, Cambridge, UK) and stored on a microcomputer for offline analysis. The number of action potentials per movement was determined using Spike 2 software (Cambridge Electronic Design), and the % change in afferent nerve fibre activity from baseline was calculated. Results: One afferent joint fibre was recorded from each rat with a total of 34 fibres examined overall in this study. Table 1 summarises the electrophysiological characteristics of each fibre. All fibres tested responded to close intra-arterial administration of potassium chloride (KCl; 0.4 mM, 0.1 ml) at the conclusion of the experiment. Cathepsin G dose-dependently decreased the firing rate of joint afferent fibres during both normal and noxious rotations of the joint (Figs. 1, 2; p \ 0.05 compared to boiled enzyme using two-way ANOVA). Boiled inactive Cathepsin G had no effect on firing rate at any time point and at any dose (Figs. 1, 2) . At doses of 1 and 10 ng where Cathepsin G alone had no effect, pre-treatment with the PAR4 antagonist, pepducin, caused Cathepsin G to significantly decrease firing rate during noxious joint rotation ( Fig. 3 ; p \ 0.05 pepducin treated group compared to Cathepsin G alone, using two-way ANOVA.). Pepducin alone had no effect on firing rate (data not shown). Results are mean ± SEM, n = 9-14. *p \ 0.05 pepducin treated group compared to Cathepsin G alone using two-way ANOVA Discussion: We have shown that in normal rat knee joints, Cathepsin G reduces the mechanosensitivity of slowly conducting afferent fibres. This is in contrast to the pro-nociceptive effect of synthetic PAR4 activating peptide in the joint [6, 7] , suggesting that Cathepsin G acts via a PAR4independent pathway. Interestingly, inhibition of PAR4 enhances this antinociceptive effect of Cathepsn G, i.e. when PAR4 activation is blocked, low doses of Cathepsin G that do not normally have an effect, now cause a decreased firing rate. Therefore, PAR4 activation by Cathepsin G may be causing a pro-nociceptive effect but this is normally masked by a stronger anti-nociceptive effect through a PAR4-independent pathway. Cathepsin G can act on other PAR members including PAR1, which has previously been shown to be anti-nociceptive [8] . Thus, further work is required to investigate the mechanisms behind this antinociceptive effect of Cathepsin G so perhaps this effect can be enhanced in arthritic joints where high levels of this enzyme are observed [1] . Intestinal ischemia-reperfusion injury is associated with increased Arginin-cleavage proteolytic activity: After the ischemic period, Arginin-cleavage proteolytic activity levels were significantly increased both in plasma and in intestinal tissues, compared to the activity measured in sham-operated mice (Fig. 2) . This increase was maintained in tissues, or further increased in plasma, for 2 h (Fig. 2 ) and up to 5-h (data not shown) after reperfusion. Fig. 2 Plasma and tissue Arginin-cleavage proteolytic activity levels after 90-min of ischemia only (0 h), or after 90-min of ischemia followed by 2-h reperfusion (2 h). Data represents mean ± SEM of 8-10 animals per group. *Significant difference from the corresponding Sham-operated group (*P B 0.05, **P B 0.01, ***P B 0.001) Intestinal ischemia-reperfusion injury is associated with increased MMP-2 and MMP-9: As demonstrated by gelatin-zymography, intestinal ischemia followed by 2 h reperfusion induced in intestinal tissues the activation of MMP-2 with its conversion to the 66-kDa activated enzyme, but also the activation of MMP-9 with the conversion of the pro-form into the active form of 97 kDa (Fig. 3a) . Quantification of this activity showed that in I/R mice, MMP-2 and MMP-9 activities were up-regulated both in intestinal tissues and in plasma, compared to Sham-operated mice (Fig. 3b) . The nature of MMP-2 and MMP-9 was confirmed by Western-blot analysis (data not shown). Results are expressed as arbitrary activity units related to an internal standard (n = 6 in each group). * Significant difference from the corresponding Sham-operated group (** P B 0.01, *** P B 0.001) Correlation of proteolytic activities with granulocyte recruitment: Significant correlation between the Arginin-cleavage proteolytic activity and the granulocyte infiltration marker MPO activity was found in intestinal tissues (F = 21.13 for p = 0.0003) (Fig. 4a) , while similar correlation was not observed for the Arginin-cleavage proteolytic activity in the plasma of mice submitted to ischemiareperfusion (F = 0.91 for p = 0.35) (Fig. 4b) . Discussion-Conclusion: The present study shows that 90 min of ischemia followed by 2 h reperfusion causes intestinal inflammatory injury demonstrated by macroscopic inflammatory damage and an increased granulocyte recruitment. This inflammatory response is associated with the release of proteases both in tissues affected by ischemia and reperfusion (intestinal tissues), and systemically in plasma. The nature of those proteases appears to be diverse, with some proteases clearly identified as MMP-2 and MMP-9 and having a gelatinic activity, and others cleaving at an arginin site, similar to the proteases that are capable of activating PARs. The release of Arginincleaving proteases in intestinal tissues correlates with inflammatory parameters, but the systemic release of Arginin-cleaving proteases in the plasma does not correlate with inflammation. Considering the well known pro-inflammatory effects of a number of proteases [2] , this could suggest that the proteases released in the intestinal tissues, but not the ones released in the plasma, could participate to the generation of the massive inflammatory response associated with ischemia-reperfusion. The role of those different proteases in ischemia-reperfusion associated injury still has to be demonstrated. Contrasting roles can be suggested for MMP-2 and MMP-9. MMP-2 is constitutively expressed in almost all tissues, where it seems to play a protective role, particularly in gut diseases like colitis [5] . MMP-2 overexpression and over-activity in this model of intestinal ischemiareperfusion could serve to limit the deleterious effects of this massive inflammatory reaction. In contrast, MMP-9 is absent from most adult tissues and was found to be mostly neutrophil-associated [4, 6] . Therefore, in our study, the increased expression of MMP-9 could be due to the recruitment of neutrophils to ischemic tissues. As for the Arginin-cleaving proteases, it is tempting to suggest that they might exert a pro-inflammatory role through the activation of PARs in the intestine, since the activation of 3 of those receptors is considered as a major pro-inflammatory signal in acute models of colitis [3] . However, those Arginin-cleaving proteases could also exert proinflammatory effects through other mechanisms. One of them could be the activation of MMP-9, which proform is known to be transformed into an active form by cleavage at an Arginin site [7] . The correlation between Arginin-cleavage proteolytic activity in intestinal tissues and parameters of inflammation suggests that those proteases are active players of the tissue inflammation. This increased activity could be due to the massive recruitment of inflammatory cells, which express in their granules, a number of proteases, including elastase, proteinase-3 and cathepsins. However, the lack of correlation between MPO activity and proteolytic activity in plasma demonstrates that those plasmatic proteases do not influence and are not influenced by inflammatory cell recruitment. Taken together, these results showed the massive release of different proteases upon intestinal ischemiareperfusion and suggest that those proteases could actively participate to the associated inflammatory response. Enterohemorrhagic Escherichia coli (EHEC) induces elevated levels of IL-1b in infected patients. However, the molecular mechanisms involved in the activation of IL-1b by EHEC are unknown. Inflammasomes are multiprotein complexes in the cytosol that process proIL-1b to bioactive IL-1b. To identify the inflammasomes involved in IL-1b induction by EHEC, we infected macrophages derived from wild type and mice deficient in inflammasome components with EHEC and analyzed the processing and release of mature IL-1b. EHEC-induced IL-1b production was dependent on caspase-1 and the adaptor ASC. Notably, both NLRP3, a cytosolic sensor for microbial and endogenous products, and AIM2, a cytosolic sensor for DNA, were absolutely essential for EHECinduced IL-1b production. Though EHEC is a noncytosolic pathogen, bacterial products such as DNA were observed in the cytosol of infected cells, providing a potential means by which cytosolic inflammasomes might be activated. This also indicated that phagosomal degradation of bacteria is a prerequisite for inflammasome activation by EHEC. Supporting this hypothesis, blocking lysosomal acidification and type I interferon signaling that are essential for bacterial lysis in phagosome, led to a marked decrease in EHEC-induced IL-1b secretion. Thus, our results demonstrate that EHEC potently activates multiple inflammasomes as a result of release of bacterial products from phagosomes into the cytosol. Introduction: Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen linked to hemorrhagic colitis and a fatal renal complication, hemolytic uremic syndrome (HUS) [1] . An important marker for HUS is IL-1b [2] , a potent proinflammatory cytokine that orchestrates antimicrobial host defense. Dysregulation of IL-1b production is linked to several autoimmune and inflammatory diseases, but pathways regulating its production are only recently becoming understood. The synthesis of pro-IL-1b is triggered by the stimulation of pattern recognition receptors (PRRs; signal 1), and its activation is mediated by cytosolic multiprotein complexes called inflammasomes (signal 2). Inflammasomes are formed when cytosolic PRRs, such as NLRP3 and AIM2 sense specific microbial or endogenous danger signals, triggering recruitment of the adaptor. ASC and procaspase-1. The autocatalytic activation of procaspase-1 generates caspase-1, which converts proIL-1b to IL-1b [3] . In this study, we investigated the mechanism by which EHEC induces IL-1b secretion and found, surprisingly, that this extracellular pathogen activates NLRP3 and AIM2 inflammasomes to elicit IL-1b production. Materials and methods: Bone marrow-derived macrophages (BMDM) from wild type (WT) C57BL/6, ASC-/-, Caspase-1-/-, NLRP3-/-, IFNR-/-, AIM2+/+, and AIM2-/-mice were infected with EHEC. IL-1b and cleaved caspase-1 in supernatants were analyzed by ELISA and immunoblotting as described [4] . Statistical analysis was conducted by two-way ANOVA and Bonferroni's post-test. Results: EHEC infection of murine BMDM induced a robust production of pro-IL-1b and its cleavage into IL-1b by caspase-1, indicating that EHEC provides both signal 1 and signal 2 required for the production of bioactive IL-1b. EHEC-induced secretion of mature IL-1b was abrogated in ASC-and caspase-1 deficient macrophages (Fig. 1a, b) . To identify the specific inflammasome pathways that sense EHEC, we infected BMDMs deficient in various inflammasome sensors, such as NLRP3 and AIM2. NLRP3 senses intracellular perturbations caused by microbial and endogenous components [5] . Release of IL-1b and cleavage of caspase-1 was completely abolished in EHEC-infected NLRP3-/-macrophages. AIM2 was recently shown to recognize DNA in the cytosolic compartment and activate inflammasome [4, 6] . In our experiments, EHEC-induced caspase-1 cleavage and IL-1b secretion were dramatically reduced in AIM2-/-cells relative to infected WT cells. These data collectively indicate that NLRP3 and AIM2 each have an essential role in inflammasome activation by EHEC (Fig. 1a, b) . We and others have previously shown that AIM2 inflammasome detects the cytosolic pathogens [4] . Here, we provide evidence for the first time that AIM2 also plays an essential role in innate sensing of infection with a non-cytosolic bacterium. The role of AIM2 in sensing EHEC was unexpected because EHEC is an extracellular bacterium that is thought to be excluded from the cytosol [7] . To investigate the cellular localization of EHEC, we visualized the fate of GFP-expressing bacteria inside macrophages by confocal microscopy. EHEC was found in lysosomes within 15 min of infection. By 1 h post-infection we observed bacterial DNA but not whole bacteria in the cytosol (Fig. 2) . These observations indicate that bacterial degradation products such as DNA can become localized in the cytosol, where they may trigger the activation of AIM2 and NLRP3 inflammasomes. Fig. 2 Confocal microscopy of wild type macrophages infected with live GFPexpressing EHEC. EHEC DNA was stained with DAPI (blue) and lysosomes with fluorescent dextran (red) Next, we tested whether bacterial degradation in lysosomes is essential for inflammasome activation by EHEC. Following phagocytosis, acidification of lysosomal compartments leads to activation of proteolytic enzymes in the lysosomes and subsequent bacterial degradation. Prior to infection with EHEC, we treated BMDMs with the vacuolar H + ATPase system blocker, bafilomycin A, which inhibits lysosomal acidification and thus bacterial degradation. Treatment with bafilomycin completely blocked the release of EHEC DNA into the cytosol of macrophages (Fig. 3a) . Accordingly, bafilomycin treatment also markedly reduced the IL-1b secretion elicited by EHEC in a dosedependent manner, similar to its effect on inflammasome activation by silica, which depends on lysosomal acidification (Fig. 3b) . Another factor that has been implicated to contribute to bacterial lysis in lysosomes is type I interferon (IFN) signaling [8] . To test whether type I IFNs have a role in EHEC lysis in the lysosomes and thereby in inflammasome activation by EHEC, we infected WT and type I IFN receptor deficient (IFNR-/-) macrophages with EHEC and analyzed the inflammasome activation. EHEC-induced caspase-1 cleavage and IL-1b secretion were markedly decreased in IFNR-/-cells (Fig. 4a, b) . Interestingly, regulation of inflammasome activation by type I IFNs is considered to be a phenomenon restricted to cytosolic pathogens [8] . Our data demonstrates that inflammasome activation by extracellular pathogens such as EHEC is also regulated by type I IFNs. Fig. 3 Confocal microscopy of untreated cells and cells pretreated with 150 mM bafilomycin infected with GFP expressing EHEC in which DNA was tagged with DAPI (a). ELISA for IL-1b in supernatants of wild type BMDMs treated with 50-150 nM bafilomycin A or left untreated for 30 min before infecting with EHEC at MOI = 100 for 8 h (b) Fig. 4 ELISA for IL-1b (a) and immunoblot analysis for caspase-1 p10 (b) by wild type and IFNR-/-macrophages infected with 100 MOI of EHEC for 8 h or treated with the control nigericin for 1 h Discussion: Collectively, our findings reveal that the host innate system employs multiple inflammasome pathways that cooperatively sense EHEC, likely upon release of bacterial components such as DNA into the cytosol. Notably, we demonstrate a role for a novel inflammasome pathway mediated by AIM2 in the detection of a noncytosolic bacterium. We also show that bacterial lysis in the lysosomes is essential for the activation of inflammasomes by EHEC. These insights raise the prospect of potential therapeutic intervention of EHEC-induced disease by modulation of IL-1b production. Mast cells, basophils and eosinophils represent the first line of host defence in allergic inflammation reaction. We investigated the mechanism by which the anti-allergic drugs di-sodium cromoglycate and sodium nedocromil inhibit histamine and eicosanoid release from human mast cells (HMCs) as well as peripheral blood basophils (PBBs) using biochemical techniques. Cord derived HMCs were cultured in vitro and PBBs obtained from human donors or cardiac puncture in mice. Subsequent antigen challenged led to a prompt release of histamine and prostaglandin D 2 (PGD 2 ), which was inhibited by cromoglycate, nedocromil, dexamethasone and human recombinant Annexin-A1 (Anx-A1). Treatment of HMCs with cromoglycate or nedocromil induced a rapid phosphorylation on the Ser 27 residue of Anx-A1, hence its secretion, secondary to inhibition of phosphatase activity and activation of PKC. Treatment of the HMCs or PBBs with a specific neutralising anti-Anx-A1 (but not irrelevant) monoclonal antibodies reversed the inhibitory effect of these drugs on histamine and PGD 2 -released. To highlight the crucial importance of Anx-A1 in this rapid effect of these drugs we tested the effect of nedocromil in Anx-A1 null mice. Cromones did not inhibit the release of histamine nor prostaglandin D2 from basophils from Anx-A1 null mice.To translate these new findings clinically, we have analyzed Anx-A1 pattern of expression in tears from patients with Vernal KeratoConjuntivitis (VKC). While this seasonal disease is associated with an increased amount of the clipped-or inactive-form of Anx-A1, VKC patients treated with the cromoglycate-like Alomide (or Lodoxamide) show an augmentation of the total-or active-form of the protein. Al-together, the use of in vitro, in vivo investigations and the translational outcome of VKC allergic disease, suggest strongly the anti-inflammatory Anx-A1 protein to be a component of rapid cromone actions on mast cells, basophils and eosinophils. Introduction: While the cromoglycate-like drugs can inhibit both the early and the late phase of the asthmatic reaction, their anti-asthmatic activity is attributed to their anti-inflammatory properties by most authorities [1] . Although cromoglycate, the prototype drug was developed in the 1960s the exact mechanism of action of this group of drugs has proved elusive. Early experiments [2] led to the concept that these drugs acted mainly on mast cells to suppress mediator release, but they also exert other therapeutically significant actions and mast cells are not their sole target. They are also effective in other models of inflammation and influence many facets of the inflammatory process unrelated to mast cell activation in vivo [3] or in vitro, e.g. eicosanoid generation [4] . We have recently reported that the ability of these drugs to inhibit leukocyte activation [5] and eicosanoid release by U937 cells [6] depends upon their ability to release the anti-inflammatory protein Anx-A1 secondary to an inhibitory action on the phosphatase PP2A. We demonstrate that this process is also required for the acute effect of the cromones to inhibit histamine and eicosanoid secretion by human mast cells and basophils thus supplying an important mechanistic explanation for the pharmacological action of these 50-year old drugs. Methods: Cord derived human mast cell culture Commercial purified CD34 + stem cells were cultured for the first 2 weeks in StemSpan (StemCell technologies) serum-free medium supplemented with 100 ng/ml human SCF, 50 ng/ml IL-6 and 1 ng/ml IL-3, and 100 lg/ml penicillin/streptomycin. After the 8 week, cells were cultured in StemSpan with 10% FCS. Assessment of Ser 27 -Anx-A1-P and PKC activation The total cellular protein was determined and supernatants analysed by conventional western blotting techniques. Immunodetection was accomplished using different antibodies recognizing either the full-length Anx-A1 protein, (polyclonal anti-Anx-A1 antibody), Anx-A1 phosphorylated on Ser27 (polyclonal anti-Ser27-Anx-A1), PKC phosphorylation (polyclonal anti-phospho-PKC antibody) and a-tubulin (monoclonal anti-a-tubulin). Measurement of Anx-A1, PGD 2 and histamine release An enzyme immunoassay was established to detect and quantify Anx-A1, PGD 2 or histamine-released in the supernatant. The method was conducted following the manufacturer's protocols. Results: Phosphorylation and secretion of Anx-A1 by HMCs. In Fig. 1a , all treatments on HMCs resulted in a significant increase (*2.5-to 3.5-fold) in the amounts of Anx-A1 and PKC phosphorylated hence an augmentation of Anx-A1 secretion in the cell culture fluid (Fig. 1b) . Okadaic acid, a known inhibitor of PP2A was included here as a positive control. Effect of human recombinant Anx-A1 protein on histamine and PGD 2 released from HMCs We next wanted to relate the increment of Anx-A1 protein secreted after treatment with nedocromil or dexamethasone (shown in Fig. 1 ) with the ability of these drugs to inhibit histamine and PGD 2 release. Figure 2a shows that human recombinant Anx-A1 protein at 1 and 10 nM is able to inhibit to the same extent as nedocromil (10 nM) and dexamethasone (2 nM) the level of histamine and PGD 2 release from IgE-activated HMCs. To ascertain the role of Anx-A1 in the mechanism of action of nedocromil sodium, the effect of a specific neutralizing anti-Anx-A1 antibody was tested. Figure 2b shows the effect of the co-incubation of HMCs with neutralising anti-Anx-A1 (and irrelevant mabs) on the effect of inhibitory action of nedocromil. Under control conditions the drug inhibited histamine and PGD 2 release by approximately 50%. This effect was completely abolished in the presence of the neutralising anti Anx-A1 mab (\6% inhibition), but not the irrelevant antibody. Effect of cromones on mediators released from human peripheral blood basophils and in Anx-A1 null mice Nedocromil suppressed Anx-A1 released by IgE challenged PBBs by approximately 45% and this effect is evident at 10 min. There was a concomitant increase in the amount of Anx-A1 released which rose by four-to fivefold with a maximum seen at 20 min by which time approximately 90% of the intracellular store was released. To extend this concept we turned to the use of transgenic mice lacking the Anx-A1 gene. Figure 3b shows that subsequent challenge on blood with anti-IgE for 30 min releases histamine (approximately 1.5-to 3.0-fold increase above basal levels). This was strongly (*50%) inhibited in the presence of nedocromil in blood taken from wild type animals but not that taken from Anx-A1 null mice. Anx-A1 content in tears from vernal keratoconjuntivitis patients The seasonal allergic disease is associated with 50% increase of Anx-A1 biologically inactive clipped form measured by ELISA in tears. This is reversed following treatment with the cromoglycate-like drug Alomide, which increases both the total amount and the relative proportion of intact Anx-A1 (Fig. 4) . Cromones promote externalisation of Anx-A1 from HMC cells. HMC cells were oultured as described and incubated with vehicle alone, nedocromil (10 nM), cromoglyoate (10 nM), ketotifen (10 nM), dexamethasone (2 nM) or okadaic acid (10 nM). Anx-A1 and PKC phosphorylation were assessed by Western Blotting (a). Anx-A1 released into the supernatant after 5 min incubation with the drugs was assessed using an ELISA assay and expressed as ng/ml culture fluid (b). * P \ 0.05 relative to vehicle treated cells; ANOWVbonferroni test. (Data redrawn from Yazid et al., submitted 2011) Fig. 2 Measurement of histamine and PGD, release from HMCs. a Human recombinant Anx-A1 protein inhibits histamine and PGD, release from HMCs. HMCs were cultured, sensitised with IgE and challenged with antigen. Vehicle or nedocromil (10 nM) or dexamethasone (2 nM) was added at the same time to the wells and their inhibitory effect compared with incubation of HMCs with human recombinant Anx-A1 protein at 1 and 10 nM. The cell culture supernatant was sampled and the net % release of histamine and PGD 2 was assessed by ELISA. *Signifies P \ 0.05 and **P \ 0.01 relative to vehicle treated cells. ANOVA/Bonferroni test. (Data redrawn from Yazid et al., submitted 2011) . b The inhibitory effect of nedocromil on antigen-induced histamine and PGD, release is Anx-A1 dependent. Vehicle, nedocromil (10 nM) alone or in the presence of 10 lg/ml neutralising anti-Anx-A1 mab (or an irrelevant control) was added to wells containing HMCs and 5 min before challenge with anti-IgE. Histamine and PGD2 release into the cell culture fluid was assessed using ELISA and expressed as a percentage of net histamine released from HMCs *P \ 0.05 relative to the appropriate controls. ANOVA/ Bonferroni test. (Data redrawn from Yazid et al., submitted 2011) Fig. 3 Inhibition of histamine released from peripheral blood basophils is Anx-A1 mediated. a Vehicle or nedocromil (10 nM) was added to the wells for 5 min and the whole blood cells were challenged for 30 min with anti-IgE. At various time points (10, 20 30 min), the cell culture supernatant was sampled and the histamine concentration (expressed as nM) and the Anx-A1 concentration (expressed as ng/ml), was assessed by ELISA. The total cell lysate histamine and intracellular Anx-A1 concentrations are included for comparison purposes. *P \ 0.05; **P \ 0.01 relative to the challenged but untreated control cells. ## P \ 0.01 relative to the WBCs histamine content of untreated control WBCs. ANOVA Bonferroni test. (Data from Yazid et al., submitted 2011) . b PBBs were obtained by cardiac puncture from wild type or Anx-A1 null mice (n = 6 mice/group). PBBs were incubated with 10 nM nedocromil for 5 min prior to challenge with anti-lgE. Histamine release from cell suspensions was assessed by ELISA and expressed as nM. PBBs from both wild type and Anx-A1 cells released histamine in response to IgE challenge. Nedocromil totally inhibited the release from the wild type but not the Anx-A1 deficient cells (data from Yazid et al., submitted 2011) Fig. 4 Cromoglycate-like Alomide (Lodoxamide) increases Anx-A1 in tears from patients with VKC. Tears from VKC patients contain increased amount of the cleavage (inactive) form of Anx-A1 This is reversed (as a measure of intact form) when patients treated with the cromone-like Alomide. **p \ 0.01, ***p \ 0.001 ANOVABonferroni test. (n = 17 control group, n = 23 VKC group and n = 11 Alomide treated group). Yazid et al., unpublished data, 2011 Discussion: Our data here clearly indicates that cromone drugs promote the phosphorylation, externalisation and release of Anx-A1 and that this process is mandatory for these drugs to bring about their inhibition of histamine and eicosanoid release in HMCs as well as blood basophils. That this mechanism is common to several cell types is shown by our previous observations using U937 cells and PMN, where a similar Anx-A1 dependent inhibition of cell function was observed. I propose that this mechanism underlies the acute inhibitory effect of these drugs on mast cell mediator release and accounts for the aspect of their pharmacology. Disclosure of interest: None declared The presents of CTD made necessary assessment of status of cardiovascular system in all patients. Results: All patients had CAP caused by atypical pathogens with indistinct clinical manifestations and symptoms. Recurrent course of CAP was in 54 (84.4%) patients. CAP caused by Chlamydophila pneumonia (Cp) was more frequent in group A-in 15 (93.7% of the group) patients. CAP caused by Mycoplasma pneumonia (Mp) was more frequent in group B-in 44 patients (91.6% of the group), in 6.8%-together with Cp, in 27.3%-with Cytomegalovirus. Asthma was diagnosed in 29 (45.3%) patients mainly of group B, with recurrent CAP in 10 (34.5% of patients with asthma); 62.1% of these children had pulmonary hypertension (PH). 44.4% of patients with PH had evidence of pulmonary fibrosis (PF) Cp CAP was more frequent in children up to age of 3 years, Mp CAP-in children aged More than one-third of patients with asthma had recurrent CAP Asthma in children with CAP and CTD was more severe with development of PH, PF, PS, SP. Disclosure of interest: None declared Brazil Introduction: Upon the lack of more efficient therapies, great interest has been shown in the therapeutic use of mesenchymal stem cells (MSC) towards Acute Kidney Injury (AKI) treatment. In this work, we aimed to study the effect of mesenchymal stem cell (MSC) Mesencult cell mediaÒ (Stem Cell Technologies). Before administration of these cells, MSC were characterized by immunophenotyping assays for CD34, CD45, CD73 and CD105, and also by differentiation assays for , morphologic study and PCR analysis (TNF-a, IL-1b and IL-6) Monteiro 1 1 Department of Physiology and Pharmacology, 2 Department of Pharmacy G8-P90 (3M ESPE) applied in the same volume and unpolymerized into transwell membranes for resin composite or added to the culture medium for adhesive systems. G9-control group (medium alone) was also evaluated. Cell metabolic activity was evaluated by succinic dehydrogenase (SDH) activity, which is a measure of the mitochondrial respiration of the cells. For such purpose, the methyltetrazolium (MTT) assay was used at times of 6 and 24 h. The data were compared by Two-way ANOVA (p \ 0.05) and Bonferroni post-test (p \ 0.05). Results: The results were given in optical density Conclusion: At 24 h, the decrease in cell metabolism was statistically significant for G1, G3, G6, G7 and G8 when compared with the same groups in 6 h Inflammatory mediators and the failing heart: past, present, and the foreseeable future Inflammation as a therapeutic target in heart failure? A scientific statement from the Translational Research Committee of the Heart Failure Association of the European Society of Cardiology MicroRNAs: new regulators of immune cell development and function Requirement of bic/ microRNA-155 for normal immune function Regulation of the germinal center response by microRNA-155 MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development Cathepsin G: the significance in rheumatoid arthritis as a monocyte chemoattractant Cathepsin G activates protease-activated receptor-4 in human platelets Dipeptidyl peptidase I activates neutrophilderived serine proteases and regulates the development of acute experimental arthritis Proteinase-activated receptors Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice Proteinaseactivated receptor-4 (PAR4) activation leads to sensitization of rat joint primary afferents via a bradykinin B2 receptor-dependent mechanism The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation Thrombin receptor: An endogenous inhibitor of inflammatory pain, activating opioid pathways Acute mesenteric ischemia: a clinical review Protease-activated receptors: contribution to physiology and disease Proteinase-activated receptors (PARs) in infection and inflammation in the gut Differential expression of matrix metalloproteinases and their tissue inhibitors in colon mucosa of patients with inflammatory bowel disease Selective ablation of matrix metalloproteinase-2 exacerbates experimental colitis: contrasting role of gelatinases in the pathogenesis of colitis Matrix metalloproteinase-9-mediated tissue injury overrides the protective effect of matrix metalloproteinase-2 during colitis Pancreatic trypsin increases matrix metalloproteinase-9 accumulation and activation during acute intestinal ischemia-reperfusion in the rat Molecular mechanisms of Escherichia coli pathogenicity Enterohemolysin operon of Shiga toxin-producing Escherichia coli: a virulence function of inflammatory cytokine production from human monocytes The inflammasomes The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses Inflammasomes as microbial sensors AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157 Type I interferon signaling is required for activation of the inflammasome during Francisella infection Anti-inflammatory therapy for asthma Disodium cromoglycate (FPL 670) ('Intal'): a specific inhibitor of reaginic antibody-antigen mechanisms An antiallergic activity of disodium cromoglycate unrelated to mast cell activation Nedocromil sodium prevents the release of 15-hydroxyeicosatetraenoic acid from human bronchial epithelial cells exposed to toluene diisocyanate in vitro Antiallergic cromones inhibit neutrophil recruitment onto vascular endothelium via annexin-A1 mobilization Cromoglycate drugs suppress eicosanoid generation in U937 cells by promoting the release of Anx-A1 Animal work was performed according to Home Office regulations and was approved by the Queen Mary University of London Ethics Committee. Human cells and clinical sample studies were prepared according to a protocol approved by the East London & the City Local Research Ethics Committee SP-25, P-129 SP-25 P-436 SP-05, OC-117, OC-072, P-471, P-476 OC-063, P-302 OC-083, P-498, P-500, P-504, P-158 OC-126, OC-084, P-361 OC-114, P-170, P-182, P-506, P-498 OC-075, P-288, P-405 OC-069, P-018 SP-23, SY-31, P-145, P-434 OC-063, P-300, P-301, P-417 OC-083, P-498, P-504 OC-100, P-089 OC-063, OC-129, P-300, P-301 SP-41, P-414 OC-075, P-288, P-405 P-405 OC-075, P-288, P-405 OC-059, P-143 OC-127, P-473, P-134 P-192, P-193, P-036 OC-024 OC-129, P-298, OC-063, P-300, P-301, P-417 SP-25, P-251, P-129, P-175, P-176 P-241, P-156, OC-106 OC-083, P-158, P-577, P-498 SP-22, OC-121, P-261 SP-23, SY-31, P-434, OC-105, P-238, P-261 P-077 SP-25, OC-114, P-129, P-503, P-452 OC-059 OC-024, P-122, P-127, P-172 M. SP-01, P-205 SP-38, P-409 SP-25, P-251, P-436 KL-02, OC-102 SP-37, OC-019, OC-111, P-128, P-445, P-259 OC-063, P-300 OC-009, P-298, OC-063, P-300, P-301, P-417 INVITED SPEAKERS Discussion: We show for the first time that leukocytes mediate cardiac hypertrophy and failure, and propose a novel pathway involving miR-155/SOCS1/IL6/STAT3 to mediate this effect. Restoration of cardiac hypertrophy in the absence of miR-155 by inhibition of SOCS1 will yield final proof of this mechanism. MiR-155 provides an ideal therapeutic candidate, offering a pure leukocyte target to inhibit detrimental cardiac inflammation and failure. Many cells and tissues in the body including those at barrier sites express the vitamin D receptor (VDR) and 1-alpha-hydroxylase (CYP27B1), the enzyme required to convert inactive 25(OH)D 3 into active 1,25(OH) 2 D 3 . These can activate and locally produce 1,25(OH) 2 D 3, which acts as an immunomodulatory hormone. The eye is an immune privileged site maintained by blood-aqueous (ciliary body and iris) and blood-retinal barriers. Corneal endothelium contributes to barrier function between corneal stroma and aqueous humor. This research aims to study the role of 1,25(OH) 2 D 3 in ocular barrier function. To investigate this, human adult retinal pigment epithelial , non-pigmented ciliary body epithelium (ODM2), and human corneal endothelial (HCEC-12) cell lines were cultured, and gene expression was examined by conventional and real-time polymerase chain reaction (RT-PCR) both in the absence and presence of TLR ligands and vitamin D substrate (25(OH)D 3 ). Protein expression was analysed by immunohistochemistry. Production of cytokine and 1,25(OH) 2 D 3 was measured by enzyme immunoassay. ARPE-19, ODM2, HCEC-12 cells expressed mRNA for VDR, CYP27B1, and the catabolizing enzyme, 24-hydoxylase (CYP24A1). This was confirmed by immunohistochemistry in non-pigmented ciliary body epithelium and corneal endothelium tissue sections and by immunofluorescence in ARPE-19 cells. ARPE-19, ODM2, and HCEC-12 converted 25(OH)D 3 into active 1,25(OH) 2 D 3 with HCEC-12 showing higher conversion rates. ARPE-19 cells responded to TLR ligand stimulation but this did not influence elements of vitamin D gene expression. Conversely, the addition of 25(OH)D 3 did not affect cytokine production by these cells. This novel research has identified that ocular barrier cells can convert inactive 25(OH)D 3 into active 1,25(OH) 2 D 3 and this is not influenced by TLR ligand stimulation. Vitamin D 3 produced by ocular barrier cells may possibly play a role in ocular immune privilege. Disclosure of interest: None declared. Chronic inflammation is an important factor in the development of insulin resistance, type 2 diabetes mellitus and fatty liver disease. Recent progress has led to the identification of several adipose tissue inflammatory components such as immune cells (e.g. macrophages, T cells) and cytokines/chemokines that account for obesity-induced inflammatory responses and insulin resistance. However, the intrinsic factors that trigger adipose tissue inflammation are not yet completely understood. 4-1BB/CD137 (TNFRSF9), a member of the tumor necrosis factor receptor superfamily expressed on immune cells, provides a bidirectional inflammatory signal through binding to its ligand 4-1BBL. 4-1BB and 4-1BBL have been shown to play an important role in the pathogenesis of various inflammatory diseases. However, the role of 4-1BB/4-1BBL in obesity-induced inflammation has not been established. Here we demonstrate that 4-1BB deficiency protects against high fat diet (HFD)-induced obesity, glucose intolerance and fatty liver disease. 4-1BB-deficient mice fed an HFD for 9 weeks showed less body weight gain, adiposity, adipose infiltration of macrophages/T cells, and tissue levels of inflammatory cytokines (e.g., TNFa, IL-6, MCP-1) compared to HFD-fed control Neutrophil accumulation contributes to the pathogenesis of rheumatoid arthritis. The aim of this study was to examine the ability of hydrogen peroxide (H 2 O 2 ) to influence neutrophilic inflammation in a model of antigen-induced arthritis (AIA) in mice. AIA was induced by administration of antigen into the knee joint of previously immunized mice. Neutrophil accumulation was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity in tissues surrounding the knee joint. Apoptosis was determined by morphological and molecular techniques. The role of H 2 O 2 was studied by using mice that do not produce reactive oxygen species (gp91 phox-/mice) and drugs which enhance H 2 O 2 generation (superoxide dismutase) or its degradation (catalase). Antigen challenge of immunized mice induced neutrophil accumulation that peaked at 12-24 h after challenge. H 2 O 2 production peaked at 24 h, concomitant with the resolution of the joint inflammatory process. Neutrophil recruitment was similar in WT and gp91 phox-/mice; however we observed a delayed resolution in gp91 phox-/mice or after administration of catalase. In contrast, administration of H 2 O 2 or SOD accelerated the resolution of neutrophilic inflammation. Resolution of inflammation induced by SOD or H 2 O 2 was accompanied by increase in number of apoptotic neutrophils. Apoptosis was caspase-3-and bax-activation-dependent and secondary to PI3K/AKT activation. Levels of H 2 O 2 increase during neutrophil influx and are necessary for natural resolution of neutrophilic inflammation. Mechanistically, enhanced levels of H 2 O 2 (endogenous or exogenous) decreased p-AKT and induced apoptosis of migrated neutrophils. Modulation of H 2 O 2 production may represent a novel strategy to control neutrophilic inflammation in the joints. Disclosure of interest: None declared. It has been reported that coniferaldehyde isolated from Fraxinus rhynchophylla exhibits inducible nitric oxide synthesis inhibitory activity. But, it is not clear how coniferaldehyde down-regulate inducible nitric oxide synthesis. In the present study, we investigated the anti-inflammatory effect and the underlying molecular mechanisms of coniferaldehyde in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Coniferaldehyde dose-dependently reduced the productions of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) induced by LPS. Consistent with these findings, coniferaldehyde significantly suppressed the LPS induced expressions of inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E 2 synthase (mPGES-1). In addition, coniferaldehyde attenuated LPS-induced DNA binding and the transcriptional activities of nuclear factor-kappa B (NF-jB), which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa B-a (IjB-a) and consequently by decreased nuclear translocation of p65 subunit of NF-jB. Taken together, these results suggest that the anti-inflammatory effect of coniferaldehyde in LPS treated RAW 264.7 macrophages is associated with the suppression of NF-jB transcriptional activity. Disclosure of interest: None declared. It has been already reported that fucosterol, found in several brown algae, have anti-diabetic and anti-oxidant effects. However, the anti-inflammatory effects of fucosterol are not widely known. In this study, we investigated the anti-inflammatory effects of fucosterol in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. Fucosterol was found to inhibit LPS-induced nitric oxide (NO), prostaglandin E 2 , interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) production in a dose-dependent manner. In addition, fucosterol suppressed the LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) at the mRNA and protein levels. Moreover, fucosterol attenuated LPS-stimulated DNA binding activity and the transcription activity of nuclear factor-kappa B (NF-kappa B) and decreased phosphorylation of inhibitor kappa B (Ikappa B)-alpha which is regulating NF-kappa B. Furthermore, fucosterol attenuated the phosphorylations of p38 mitogen-activated protein kinase (p38) in LPS-stimulated RAW 264.7 cells. Taken together, these results suggest that the anti-inflammatory effect of fucosterol in LPStreated RAW 264.7 cells is associated with the suppression of NFkappa B transcriptional activity via the inhibitory regulation of Ikappa B and p38 phosphorylation. Disclosure of interest: None declared. Data are means ± SEM G + PepducinType III10 ± 29 ± 14 ± 112 ± 219 ± 34Type IV17 ± 510 ± 11 ± 0.220 ± 625 ± 52Nd13 ± 1--17 ± 224 ± 23Data are means ± SEM Introduction: Acute mesenteric ischemia is a potentially fatal abdominal emergency. Although mesenteric ischemia accounts only for about 1-2% of gastrointestinal illnesses, it still causes a high in-hospital mortality rate (60-80%). Interruption of blood supply to the gut results in an ischemic injury, characterized by an acute and massive inflammatory reaction [1] . Proteases represent 2% of the human genome and a number of in vitro evidences have indicated that their release is rapidly mobilized in damaged or hypo-oxygenized cells. Indeed, proteases participate to several inflammatory states [2] . In particular, activation of members of the proteinase-activated receptors (PARs) family, which are G protein-coupled receptors activated by the cleavage at an Arginin-site of their N-terminus extracellular domain, has been implicated in leukocyte recruitment at inflammatory sites [3] . Other proteases belonging to the matrix metalloprotease (MMPs) family, such as MMP-2 and MMP-9 are up-regulated in the inflamed gut [4] . Despite the strong rationale for a potential role of proteases in inflammatory response to ischemia, the profile and functions of proteases in intestinal ischemic tissues has never been investigated, nor their relationship with inflammation. The aim of our study was to investigate the proteolytic profile associated with intestinal ischemia/reperfusion injury, measuring Arginine-cleavage and MMP proteolytic activities, and to study their possible correlation with inflammatory parameters. Methods: C57Bl6 male mice (8-week-old) were subjected to 90 min of intestinal ischemia by occlusion of the superior mesenteric artery followed by 0 or 2 h reperfusion (I/R). Sham-operated (SO) animals served as controls. Myeloperoxidase (MPO) activity was measured in intestinal tissues as an index of neutrophil tissue infiltration. Hydrolysis of tosyl-Gly-Pro-Arg-p-nitroanilide substrate was measured as an index of Arginin-cleavage proteolytic activity in plasma or intestinal tissues. MMP-2 and MMP-9 protein expression was evaluated by western-blot, while their activity was measured by gelatine zymography (0.1% gelatine). Intestinal ischemia-reperfusion injury induces inflammation in intestinal tissues: Intestinal ischemia-reperfusion caused inflammation to ischemic tissues, as observed by macroscopic features of inflammation: redness, and swelling, but also increased MPO activity, a marker of granulocyte infiltration. This increased granulocyte infiltration was not observed after the 90-min ischemic period, but only after ischemia + reperfusion (2 h) (Fig. 1 ). Sham MPO mU/mg protein 0 50 100 0hR 2hR *** § 25 75 Fig. 1 Intestinal MPO activity after 90-min of ischemia only (0 h), or after 90-min of ischemia followed by 2-h reperfusion (2 h). Data are mean ± SEM of 8-10 animals per group. *Significant difference compared to the corresponding Sham-operated group (***P B 0.001). § Significant difference between I/R groups ( § P B 0.05)