key: cord-015147-h0o0yqv8
authors: nan
title: Oral Communications and Posters
date: 2014-09-12
journal: Inflamm Res
DOI: 10.1007/bf03353884
sha: 
doc_id: 15147
cord_uid: h0o0yqv8

nan

The Drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. Most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : (1) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? (2),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; (3) how does Drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. Over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the Drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. There is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. Several myelin-derived autoantigenic targets have been described and include myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein. There has been a particular focus on MBP for at least two reasons: MBP-specific CD4+ Tcell receptors (TCRs) have been found in multiple sclerosis brains, and cells presenting an immunodominant MBP(85-99) peptide in complex with HLA-DR2b have been shown to be present in multiple sclerosis lesions. Also, humanized mice expressing the HLA-DR2b gene and a human T-cell receptor (TCR) that recognises the MBP85-99 peptide in the context of HLA-DR2b either spontaneously or after immunization with MBP85-99 develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. This talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. To resolve the pathogenic mechanisms of rheumatoid arthritis (RA) we need to identify the causative genetic and environmental factors. This has however proven to be complex, with many factors and genes interacting. Inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as RA. Animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. There are several arthritis models, which each may reflect various variants of the heterogeneity of RA in humans. Examples are collagen induced arthritis (CIA) and pristane induced arthritis (PIA), which both fulfills the clinical diagnostic criteria for RA. Both diseases are genetically complex and the susceptibility is, as RA, dependent on many polymorphic genes operating in concert. So far 2 genes in this concert have been identified; the MHC class II Ab gene in the mouse (1) and the Ncf1 gene in the rat (2) and in the mouse (3) . The Ncf1 protein is a part of the NADPH oxidase complex mediating oxidative burst. The discovery of the Ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive T cells. Mice with the deficient Ncf1 allele are more susceptible to CIA, and also developed a chronic form of arthritis. Interestingly, the immune response to CII was enhanced by the Ncf1 deficiency linking the Ncf1 pathway to the adaptive immune response. Oxidation of T cell membranes seem to be a key event in the pathogenic mechanism as reduction of T cell membranes induces arthritis in rats (4). On the basis of these findings a new type of therapy Myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (AChR) at the neuromuscular junction. The antibodies reduce the number of AChR leading to failure of neuromuscular transmission and muscle weakness. The AChR antibodies as measured in conventional immunoprecipitation assays are IgG, high affinity, polyclonal and specific for human AChR. They reduce the numbers of AChR by antigenic modulation and by complement-mediated damage to the neuromuscular junction.

Myasthenia gravis has a very intriguing relationship with the thymus gland. In many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. Around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express AChRs. There are many B cells and plasma cells and thymic lymphocyte preparations synthesise AChR antibodies. The possibility that the thymic AChR induces the germinal centre formation and AChR antibody synthesis is supported by much evidence. Some patients, however, have thymic tumours and in these the role of the thymus is less clear. Moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age.

There are up to 20% of myasthenia patients that do not have the typical AChR antibodies. Some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. The pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed.

Finally, among the patients who have neither AChR nor MuSK antibodies by conventional testing, we have evidence for lower affinity antibodies to AChR which can now be detected using molecular approaches which will be described.

ARRY-886 (AZD6244) is an inhibitor of MEK1/2 currently in development for cancer. Phase 1 determined the MSD (100 mg) and the safety of the compound given continuously. In decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. Paired pre-and postdose tumor biopsies showed a reduction in pERK (-83%) and proliferative index (-46%). The trough plasma concentration (400 ng/ml) corresponded to~40% inhibition of pERK. About 45% of pts had stable disease after 2 months. These results demonstrate that ARRY-886 (AZD6244) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. There are several on-going Phase 2 studies, in melanoma, colorectal, lung and pancreatic cancer.

ARRY-162 is another potent, selective MEK 1/2 inhibitor, currently in development for inflammatory diseases. When human whole blood was stimulated with TPA, ARRY-162 inhibited TNFa, IL-1b and IL-6 (IC50s of 23, 21 and 21 nM, respectively). 50% inhibition of pERK required 280 nM. ARRY-162 was highly efficacious in CIA and AIA rat models, with ED50s of 3 and~10 mg/ kg, respectively. In normal volunteers ARRY-162 was well-tolerated and there was a dose-proportional increase in drug exposure. In ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of TPA-induced TNFa and IL1b. An on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of ARRY-162 monotherapy. In addition, we have initiated a clinical trial designed to evaluate ARRY-162 in combination with methotrexate in patients with rheumatoid arthritis. Rho kinases (ROCK) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.ROCKs prominent role in cytoskeletal architecture suggests that ROCK inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.We have synthesized small molecule inhibitors of ROCK which are specific for the ROCK-2 isoform.These ROCK-2 inhibitors, typified by SLx-2119 and SLx-3060, are potent (IC50 < 100 nM), selective for ROCK-2 (>100 fold selectivity for ROCK-2 over ROCK-1) and exhibit good oral bioavailability.This talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.SLX-2119 inhibits cell proliferation and migration in several tumor cell lines including HT-1080, PANC-1 and MDA-MB-231. Moreover in xenograft studies using nude mice, SLx-2119 significantly inhibited tumor growth with these same cell lines. In liver fibrosis, SLx-2119 prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.SLx-3060 is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.These data suggest that ROCK-2 selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as SLX-2119 and SLx-3060 will provide effective treatment for oncology and fibrotic disease. Cyclooxygenases (COX) catalyze the first step in the synthesis of prostaglandins (PG) from arachidonic acid.COX-1 is constitutively expressed.The COX-2 gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.Levels of COX-2 are increased in both inflamed and malignant tissues.In inflamed tissues, there is both pharmacological and genetic evidence that targeting COX-2 can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).Multiple lines of evidence suggest that COX-2 plays a significant role in carcinogenesis.The most specific data that support a cause-and effect relationship between COX-2 and tumorigenesis come from genetic studies.Overexpression of COX-2 has been observed to drive tumor formation whereas COX-2 deficiency protects against several tumor types.Selective COX-2 inhibitors protect against the formation and growth of experimental tumors.Moreover, selective COX-2 inhibitors are active in preventing colorectal adenomas in humans.Increased amounts of COX-2-derived PGE2 are found in both inflamed and neoplastic tissues.The fact that PGE2 can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of COX-2 contributes to both wound healing and tumor growth.Taken together, it seems likely that COX-2 induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. (1), K Hagihara(2), T Nishikawa(1), J Song(1), A Matsumura (2) (1) Health Care Center, Osaka University, Japan (2) Osaka University Graduate School of Medicine, Japan

It is still less known about the actual pathogenic role of IL-6 in the inflammatory status. To know the pathogenic role of IL-6 and the efficacy of IL-6 blockade in inflammation, a humanized anti-IL-6 receptor antibody, Tocilizumab, was used for the treatment in chronic inflammatory diseases, such as Castlemans disease, rheumatoid arthritis and Crohns disease. Since IL-6 blocking therapy improved the clinical symptoms and the laboratory findings, the IL-6 function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid A (SAA). SAAmRNA induction, SAA promoter activity and assembling of transcriptional factors on SAA promoter were analyzed by the real time RT-PCR, gel shift assay and DNA affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, IL-6, IL-1 and TNF-alpha. In result, IL-6 was an essential cytokine in induction of SAAmRNA through the activation of STAT3 which constructed the complex with NF-kappaB p65 and a cofactor P300. Although there was no STAT3 consensus region on SAA promoter, STAT3 bound at the 3 site of NF-kappaB RE. The above research proved that IL-6 signal is essential on the synergistic induction of SAA via newly discovered STAT3 transcriptional mechanism, suggesting the presence of this STAT3 mechanism in inflammation, and confirming the normalization of serum SAA level by IL-6 blocking therapy in inflammatory diseases. This research method develops a subsequent therapy for serious AA amyloidosis by inhibition of SAA production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases.

Takashi Wada(1), K Matsushima(2), S Kaneko (1) (1) Kanazawa University, Japan (2) University of Tokyo, Japan Accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. Pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.Locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.The possible positive amplification loop from CXC chemokines to CC chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.It is of note that monocyte chemoattractant protein (MCP)-1/ monocyte chemotactic and activating factor (MCAF)/ CCL2, a prototype of CC chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.Interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. New insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. In addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.The selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. We focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this Symposium. (1), P LaCamera (1), B Shea (1), G Campanella (1), B Karimi-Shah (1) , N Kim (1), Z Zhao(2), V Polosukhin (3), Y Xu(2), T Blackwell (3) Aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.Here we demonstrate that lysophosphatidic acid (LPA) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, LPA1, are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. The absence of LPA1 markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.The increase in vascular permeability caused by lung injury was also markedly reduced in LPA1-deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.These results demonstrate that LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.LPA1 therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. We have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. Our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. We have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. We have started to explore the role of TNF-alpha for adult neurogenesis. Infusion of an antibody to TNF-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the TNF-R2 receptor. We have shown that TNF-R1 is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. We have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. Rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. We found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. In contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. The role of inflammation for these differences is currently being explored. Proteinase-activated receptor-2 (PAR-2) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with SLIGKV that binds intramolecularly and activates the receptor. PAR-2 is implicated in innate defense responses associated with lung inflammation. We showed that PAR-2 is expressed by human alveolar (A549) and bronchial (16HBE) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide SLIGKV-NH2. In cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin G, two serine proteinases, and by Pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.We demonstrated that these three proteinases do not activate PAR-2, but rather disarm this receptor, preventing its further activation by trypsin but not by SLIGKV-NH2. Preincubation of a PAR-2 transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. Proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of PAR-2, generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. Our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of PAR-2 in the respiratory tract, thereby altering the host innate defense mechanisms.

caspase-3 -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. Ongoing research is investigating whether PAR agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways.

The intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.Given that protease-activated receptor 2 (PAR2) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.SCBN, Caco-II and T84 epithelial cells were grown to confluence on filter supports and mounted in Ussing chambers to study short circuit current (Isc) and transepithelial resistance (RTE).Cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to RTE.Apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in RTE and decreased the transepithelial flux of a 3000MW dextran.Interestingly, the effect of trypsin could not be replicated by activators of PARs 1, 2 and 4, suggesting that the effect on RTE was not due to activation of PARs.Subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase C.A trypsin-induced increase in intracellular calcium was not involved.Inhibition of PKC-zeta, but not of typical PKCs, also blocked the response.Our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. Physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. Further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation.

Clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. The high concordance in monozygotic twins (>55%), which is not seen in dizygotic twins (<5%) points to strong contribution of genetic susceptibility to the overall risk for disease. IBD represents a "complex disease" and may involve a large number of interacting disease genes. Crohns disease has become an example for the successful molecular exploration of a polygenic etiology. Crohns disease was not known before 1920. Incidence has increased since now leading to a lifetime prevalence of up to 0.5% in Western industrialized countries. The current hypotheses propose unknown trigger factors in the life style of Western industrialized nations that interact with a polygenic susceptibility. It appears that increased expression and production of TNF and an enhanced state of activation of the NFkB system are main drivers of the mucosal inflammatory reaction. The exploration of inflammatory pathophysiology of Crohns disease using full genome, cDNA and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. While this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. Genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome 16 being most consistent between different populations. In 2001 three coding variations in the CARD 15 gene were identified that are highly associated with development of the disease. All variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of NFkB in macrophages and epithelial cells. Interestingly, the three disease associated SNPs are never found on the same haplotype. In compound heterozygotes or homozygotes they result in a RR of > 35 to develop Crohns disease as an adult. A particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the CARD 15 gene. Variations in the CARD 15 gene do not fully explain the linkage finding in the pericentromeric region of chromosome 16. After stratification for CARD 15 variants, the broad linkage peak is reduced to two more defined peaks on 16p and 16q, respectively. While the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes 10 and 5, respectively). DLG-5 is the example of a low-risk susceptibility gene with a modest associated odds ratio (1.2-1.5) . Interestingly, the associa-tion signal appears to be confined to young males. SLC22A4/5 which encode the kation-transporters OCTN1 and 2 have been suggested to represent the disease gene in the 200+ kB haplotype block on chromosome 5q31. MDR1 has also been implicated as a disease gene in IBD. Although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes MDR1 likely as a low risk susceptibility gene. With the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. Recently a 330k Illumina scan has been published identifying IL-23R as a further disease gene. We used a genome wide candidate gene approach (with appr. 20.000 cSNPs) to identify ATG16L1 as a further disease genes. Both genes were confirmed and a further regulatory SNP involving PTGER4 was annotated by a Belgian genome wide scan. By the time of presentation three further genome wide SNP scans in Crohn disease will most likely have entered the public domain. The further exploration of Crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. The creation of a medical systems biology of disease will lead to new models and eventually new therapies. The chemokine receptor CCR9 plays a pivotal role in mediating the migration of T cells to the gastrointestinal mucosa. The ligand for CCR9, TECK, s highly expressed in the GI tract. The pathogenicity of intestinal CCR9+/ CD4+ T cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of Crohns and Celiac Disease patients. CCX282-B is a highly selective and potent, orally bioavailable, small molecule antagonist of CCR9.The compound proved to be highly efficacious in the TNF-aARE and MDR1a-/-murine models of inflammatory bowel disease (IBD). In Phase I trials, CCX282-B was well-tolerated, and no drug-related SAEs were reported.A 28-day placebo-controlled Phase II study was recently completed in patients with moderate to severe Crohns Disease. CCX282-B was shown to be both safe and to have encouraging clinical results: 56% of patients on CCX282-B (CDAI !250, CRP >7.5mg/L) exhibited a 70-point drop in CDAI compared to 29% on placebo. Furthermore, a CRP decrease of 11 mg/L was seen in the CCX282-B group compared to placebo. Colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. A mean decrease from baseline in the concentrations of TNF-alpha, IL-12 p70, IFN-gamma, and the chemokine RANTES was shown in the CCX282-B group while levels remained stable in the placebo group. CCX282-B is the first chemokine-based inhibitor of leukocyte trafficking to be tested in IBD. The compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of Crohns Disease. The activating receptor NKG2D seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for Type 1 Diabetes and Multiple Sclerosis. The aim of this study was to asses the role of NKG2D in a model of Inflammatory Bowel Disease, where CD4+CD25-T cells from BALB/c mice are adoptively transferred to SCID mice, and to evaluate the therapeutic effect of an Anti-NKG2D antibody therapy. The expression of NKG2D was evaluated by flow cytometry, immunohistochemistry and by PCR. We found a marked up-regulation of NKG2D on the cell surface as well as increased levels of NKG2D mRNA in CD4+ T cells from colitic SCID mice as compared to normal BALB/c mice. We next studied the effect of anti-NKG2D antibody (CX5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. CX5 treatment decreased the cellsurface expression of NKG2D and prophylactic administration of CX5 attenuated the development of colitis significantly. A moderate reduction in the severity of disease was observed after CX5 administration to mildly colitic animals, whereas CX5 did not attenuate severe colitis. Thus, NKG2D may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of NKG2D in CD4+ T cells increased markedly during the development of disease and since administration of CX5 early but not late in the course attenuated the disease severity. Proteins are used already for more than a century in the treatment of disease. The first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. The second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. The development of the recombinant DNA and cell fusion technology in the seventies of the 20th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. In 1982 human insulin was introduced as the first recombinant DNA derived biopharmaceutical and since than more than 160 have gained approval. The pipeline contains many more potential biopharmaceuticals and at present 1 in 4 new drug applications concerns a biotechnology derived product.

A major problem of therapeutic proteins is the induction of antibodies. For foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. However the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. And also the proteins which are homologues of endogenousfactors such as GM-CSF, interferons etc. induce antibodies, sometimes even in the majority of patients.

By definition we are immune tolerant to products which are copies of endogenous proteins. The products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. When human therapeutic proteins induce antibodies, they are breaking B cell tolerance, which starts with the activation of autoreactive B-cells. Presenting the self-epitopes in an array form is very potent activator of these B-cells. This explains why aggregates of human proteins are the most important factor in induction of antibodies.

These aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. There are only a few studies in experimental model systems on the properties of the aggregates which break B-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved.

We study the capacity of a protein product to break B-cell tolerance in mice made transgenic for the specific protein.

These mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. Although these models have helped to identify the factors important for breaking B-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. They also allow to study the involvement of Tcells in breaking B cell tolerance. All data obtained untilnow indicate this process to be T-cell independent.

Contact information: Dr Huub Schellekens, Central Laboratory Animal Institute, Utrecht University, Utrecht, The Netherlands E-mail: h.schellekens@gdl.uu.nl

BioMonitor ApS, and Institute for Inflammation Research (IIR), Rigshospitalet, Copenhagen, Denmark

Using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (Abs). Many have assumed that these drugs may be administered with little or no risk of triggering specific T-and/or B-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. Unfortunately, this is not the case, and even socalled 100% human biologicals are potentially immunogenic (1) (2) . I shall discuss two examples: 1) recombinant human cytokines (IFN-beta-1a and -1b), and 2) anticytokine Ab constructs (anti-tumor necrosis factor (TNF)-alpha).

IFN-beta has been used for treatment of patients with multiple sclerosis since the early nineties. Though initially neglected as a clinical problem, IFN-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. The reported frequencies and titers of anti-IFN-beta Ab vary considerably depending upon IFN-beta preparations and administration, and the types of assays being used (2-4). It took more than 10 years of clinical use before Abmediated decrease in bioactivity of IFN-beta, a condition in which the clinical effect of continued injection of rec. IFN-beta is minimized or abrogated, was universally recognized (5, 6).

2) Anti-TNF-alpha human Ab constructs TNF-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of TNF-alpha have long been a goal in the treatment of these conditions. Currently, there are three approved and two other anti-TNF-alpha biopharmaceuticals in clinical use. Unfortunately, response failure is frequently encountered. Thus, 30-40% of patients are primary non-or lowresponders to the anti-TNF constructs, and secondary response failure is commonplace, mostly due to induction of anti-Abs. Several different methods have been used to assess circulating levels of anti-TNF drugs as well as anti-Abs. Most of these have been based on ELISA technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc.

Interferon beta (IFNbeta) has been an important step forward in the treatment of multiple sclerosis(MS), an inflammatory disease of the human central nervous system. However, one of the problems of IFNbeta is its immunogenicity; a substantial percentage of MS patients treated this recombinant protein develop anti-IFNß antibodies, primarily of the IgG class. The level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. Most studies of these antibodies have measured their ability to neutralize IFNbetas effect in vitro, using assays in which sera from MS patients inhibit the protective effect of IFNbeta on viral killing of target cells. This antibody population is called neutralizing antibodies (NAbs).

Tests measuring binding of antibodies to IFNß in vitro are called binding antibody (BAb) assay. Anti-IFNbeta antibodies detected by BAb assays are present in a high percentage of MS patients, and can occur at low levels without any apparent adverse effect on IFNbeta bioactivity. The distinction between BAbs and NAbs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of BAbs and NAbs is a continuum with the assay systems simply measuring the strength of the antibody response. In many treated patients, the anti-IFNbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. High levels of anti-IFNbeta antibodies with high affinity results in loss of IFNbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or ADB. ADB can be considered the in vivo correlate of the neutralizing effect of the anti-IFNß antibody population, while the NAb assay measures the in vitro neutralization of this population of immunoglobulins in the serum. The three IFNbeta preparations have different incidences of NAbs and different patterns of appearance and disappearance over time of NAbs.

Because there is no direct correlation between NAb levels and bioactivity at moderate levels of NAb, in vivo bioactivity assays for IFNbeta have become increasingly utilized. In a large multicenter study in the US, called the INSIGHT study, bioactivity as measured by IFNbeta induced upregulation of the IFN-response genes MxA, viperin, and IFIT1, was shown to be highly correlated with NAb levels, confirming a single center study (Pachner, A.R., Pak,E., Narayan, K., Multiplex analysis of expression of three IFNbeta-inducible genes in antibody-positive MS patients, Neurology, 66:444-446, 2006) . Multiple studies, including a large multicenter study in Denmark and a recent study from our center using high resolution MRI of the brain once a month, have demonstrated that NAbs abolish the salutary effects of IFNbeta on clinical aspects of MS, especially inflammation. Recent guidelines for European neurologists recommend stopping IFNbeta in NAb-positive patients.

In order to maintain bioactivity of this important medication for MS, some neurologists have attempted to use immunosuppressives either to prevent the development of NAbs, or to treat them once they have developed. However, at this point in time, there is no clearly optimal way to treat NAbs. Major efforts have been underway to decrease the immunogenicity of IFNbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested.

proteinase-activated receptors (PARs). Endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin G, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. Thus, the expression of PARs on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of PARs as a part of the communication system in the skin during inflammation and the immune response. For example, PAR2 activates NF-kB in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. On sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. PAR1 and PAR2 also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. They also stimulate signal transduction pathways involved in cutaneous inflammation. In sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. Novel compounds regulating protease and PAR function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. Serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. In addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the Gprotein coupled receptor family called the proteinase activated receptors (PARs). These receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. Our study examined the effect of PAR4 activation in joint inflammation and pain. Male C57BL/6 mice received an intra-articular injection of either the PAR4 activating peptide AYPGKF-NH2 or the inactive peptide YAPGKF-NH2 (100 mg) into the right knee. Knee joint blood flow was then measured in these mice by laser Doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. Mechanical allodynia was also assessed in these animals by application of von Frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. Intra-articular injection of the PAR4 activating peptide caused knee joint blood flow to gradually increase by up to 25% over the succeeding 2hrs. Knee joint swelling was also observed as well as the development of a mechanical allodynia. All responses could be blocked by pre-treatment with the selective PAR4 antagonist pepducin P4pal10 (100 mg i.p.). The control peptide YAPGKF-NH2 had no discernible effect on joint inflammation or pain. These experiments show that peripheral activation of PAR4 receptors in mice knees causes joint inflammation and pain.

Vincent Lagente (1), E Boichot (2) (1) Air Liquide, Centre de Recherche Claude-Delorme, Jouy en Josas, France (2) Inserm U620, UniversitØ de Rennes 1

Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. These led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. Among metalloproteinases (MMPs) family, macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (COPD). Pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. Indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. In addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.Recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. Serine proteases cleaving specifically at an arginin site are able to activate Protease-Activated Receptors (PARs), which then send specific messages to cells. We have demonstrated that 3 members of the PAR family (PAR1, PAR2 and PAR4) are present on peripheral sensory neurons, where they can be activated by different proteases.The activation of PAR1 and PAR2 in isolated sensory neurons provokes calcium mobilization and the release of substance P and CGRP, while the activation of PAR4 inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.Thrombin and pancreatic trypsin caused inflammation respectively through a PAR1 and PAR2-dependent mechanism involving the release of neuropeptide.The extrapancreatic form of trypsin (mesotrypsin or trypsin IV) also caused neurogenic inflammation through a PAR2 and PAR1dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a PAR2-dependent mechanism.In contrast, activation of PAR4 on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. Taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of PARs on peripheral sensory neurons.Determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs.

Introduction: A scoring system for disseminated intravascular coagulation (DIC) in humans has been proposed by the International Society on Thrombosis and Haemostasis (ISTH). It was the objective of this study to develop and validate a similar scoring system for DIC in dogs in order to establish the dog as a spontaneous animal model.

Methods: For the developmental study, 100 consecutive dogs admitted to the intensive care unit (ICU) were enrolled prospectively (Group A). Blood samples were collected daily and a broad panel of coagulation assays performed. Diagnosis of DIC was based on the expert opinion of one human physician and two veterinarians. A multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of DIC. Integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the STARD criteria. The validation study prospectively enrolled 50 consecutive dogs (Group B).

Results: 37 dogs were excluded from group A where 23/63 dogs (37%) had DIC. Final multiple logistic regression model was based on aPTT, PT, D-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. Diagnostic accuracy of the model was sustained by prospective evaluation in Group B.

Conclusion: Based on generally available assays, it was possible to design an objective diagnostic model for canine DIC, which has both a high sensitivity and specificity. Such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients.

In 1997, a coagulation independent change in light transmittance (biphasic waveform [BPW] ) was reported in automated activated partial thromboplastin time assays (aPTT) in patients with disseminated intravascular coagulation (DIC). A Calcium-dependant precipitate of Creactive protein and Very-low-density-lipoprotein was causing the BPW. Our group recently identified this phenomenon in dogs also. Initially, BPW was introduced as a complementary tool to assist diagnosing DIC. However, recent studies reported that BPW may have a stronger potential as a prognostic marker for survival.

The aims of the study were to prospectively investigate (A) the diagnostic significance of BPW regarding DIC and (B) the significance of BPW to outcome, in dogs with diseases known to predispose for DIC. The study was performed as a prospective, observational study including 50 consecutive dogs with a final diagnosis known to predispose for DIC (20% were finally diagnosed with DIC). Outcome was 28-day survival.

BPW was assessed by means of a hirudin-modified aPTT assay (Kjelgaard-Hansen et al., JVIM 2006:20; 765-766) . Relative risk according to BPW (RR [95% confidence interval]) for (A) a DIC diagnosis and (B) 28-day mortality, were assessed. 28-day mortality in the study population was 44%. 28% were BPW positive. BPW was not a significant diagnostic factor for DIC (RR=0.64 [0.16; 2.67] ), but strongly so for outcome (RR=2.57 [1.46;4.52] ) with a 79% (11/14) mortality amongst BPW positive dogs.

In conclusion, BPW was observed in dogs predisposed to DIC, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. (1), B Hideo (2) (1) Department of Molecular Pathology, Kumamoto University, Japan (2) Department of Gastroenterological Surgery, Kumamoto University, Japan

Aeromonas species are facultative anaerobic Gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. Aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (DIC) is a life-threatening complication of sepsis patients, causing multiple organ failure.However, a mechanism leading to coagulation induction in the bacterial infection has not been known. To study the DIC induction by Aeromonas species infection, we investigated coagulation activity of a serine protease (ASP) from Aeromonas sobria, predominantly isolated in patients blood. Proteolytically active ASP shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of 30 nM. ASP activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate Boc-Val-Pro-Arg-MCA, but no enzymatic activity was produced from coagulation factors IX and X. Analysis by SDS-PAGE revealed that ASP released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. Western blotting using biotinylated Phe-Pro-Arg-chloromethylketone, a thrombin inhibitor, showed that ASP produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. Prothrombin incubated with ASP but not the protease itself caused platelet aggregation. These results indicate that ASP activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that ASP coagulation-inducing activity contributes to DIC development in sepsis caused by Aeromonas sobria infection. The present study shows a link between inflammation and coagulation mediated by a bacterial protease. Hemolytic episodes are often associated to high amounts of free heme in circulation (up to 20 uM) and the development of an inflammatory response that may develop to a chronic inflammation. Our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and NADPH oxidasederived reactive oxygen species (ROS) generation, as well as PKC activation and interlukin-8 expression (GraÅa-Souza et al., 2002) . Moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. Heme protective effect requires NADPH oxidase-derived ROS and involves the activation of MAPK, PI3K and NF-kB signaling cascades as well as heme oxygenase (HO) activity (Arruda et al., 2004) . More recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of Bax insertion into mitochondria and a dramatic increase on Bcl-XL/Bad protein ratio in a ROS-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. These findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. The recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.Financial support: FAPERJ, CNPq, CAPES. (1) (1) University of Melbourne, Victoria, Australia (2) Monash University, Victoria, Australia

We have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (Gpx1), show significantly larger infarcts after stroke. Recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. Nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. To explore this, Gpx1-/-mice were subjected to transient mid-cerebral artery occlusion (MCAo) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. After 1hr MCAo, leukocyte-endothelium interaction was significantly reduced in Gpx1-/-mice compared to WT counterparts during the second hour of reperfusion. Laser Doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in Gpx1-/-mice following transient MCAo. This suggests that the reduction in nutritive blood flow following stroke in Gpx1-/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. Furthermore, matrix metalloproteinase-9 (MMP9) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in Gpx1-/-mice to a greater extent than in WT mice after MCAo, suggesting a role for oxidative stress in cerebral microvascular injury. The data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of MMP9.

Chris Bolton(1), C Paul(2), S Barker (3), R Mongru (3) (1) William Harvey Research Institute, London, UK (2) University West of England, Bristol (3) Queen Mary University of London Adrenomedullin (AM) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.In vitro work has shown that AM beneficially regulates blood-brain barrier (BBB) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.Our preliminary studies in acute experimental autoimmune encephalomyelitis (EAE), a model of the human disease multiple sclerosis (MS), have demonstrated significant elevations in AM peptide levels corresponding with AM mRNA changes during late, neurological disease where AM production may be linked to the restoration of BBB function. However, AM is not exclusively produced as result of AM gene upregulation. Furthermore, AM peptide levels do not always match AM mRNA changes during other disease phases of EAE.The current study has investigated, more closely, the relationship between AM gene expression and subsequent levels of associated peptides. AM mRNA levels were determined, by RT-PCR, in the cerebellum, medulla-pons and spinal cord of normal and EAE-inoculated Lewis rats at the height of disease. AM and proadrenomedullin peptide (PAMP) levels were measured in the tissues by radioimmunoassay.All tissues examined showed an increase in AM gene mRNA compared to control levels.AM and PAMP changes were observed in the samples and differences between the peptide profiles were recorded.An understanding of alterations in the generation of AM and related peptides during neuroinflammation may provide insight into mechanisms affecting BBB permeability and be of relevance to the changes in neurovascular function seen during MS. Platelet-activating factor (PAF) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. Although, PAF is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that PAF may be a mediator of noxious signaling in spinal cord in case of neuronal injury. PAF-induced tactile allodynia may be mediated by ATP, glutamate and the generation of nitric oxide (NO). The present study elucidated down-stream signaling pathway for PAF-induced tactile allodynia. PAF-and glutamate-induced tactile allodynia was blocked by the pretreatment with NO scavengers and inhibitors of NO synthase, soluble guanylate cyclase or cGMP-dependent protein kinase (PKG). Recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. To explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a3 subunit (GlyR a3) by siRNA spinally transfected with HVJ-E vector was examined. In mice spinally transferred with siRNA for GlyR a3, the reduction of GlyR a3 was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pCPT-cGMP, PAF, glutamate failed to induce tactile allodynia in mice spinally transferred with siRNA of GlyR a3, while these compounds produced tactile allodynia in mice transferred with mutant siRNA of GlyR a3 as a control. Glycine tranporter inhibitors ameriolated PAF-and pCPT-cGMP-induced allodynia. These results suggest that glutamate-NO-cGMP-PKG pathway plays a key role for PAF-induced tactile allodynia in spinal cord and GlyR a3 may be a target molecule for PKG to induce allodynia. (1), R Leite(2), YS Bakhle (3) (1) Federal University of Minas Gerais, Belo Horizonte, Brazil (2) Medical College of Georgia, Augusta, USA (3) Imperial College, London, UK Selective cyclooxygenase 2 inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.We have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (CX). Male Holtzman rats (150-200g; 3-5 animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin B, cytochalasin B) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and 30 min later given CX (12mg/kg, s.c.). After a further 30 min, rats were injected (ipl) with the inflammatory stimulus, carrageenan (250 mg/paw). Mechanical pain threshold was hourly measured over the next 4 h, using the Randall-Sellitto method. The CXinduced hypoalgesia was reversed by low doses of latrunculin B or cytochalasin (latrunculin 100% reversal = 1.26 nanomoles), higher doses of microtubule inhibitors (taxol 100% reversal = 23.4 nanomoles) with no effect of acrylamide (7 up to 141 nanomoles).We conclude that 1) local changes in (paw) cytoskeleton occurred during CXinduced hypoalgesia and 2) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.Financial Support: CNPq, Fapemig and CAPES There are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. COX-2 in brain during neurodegenerative or neuropsychiatric disorders.In the present study, we examined the effect of nimesulide (a preferential COX-2 inhibitor) in subchronic immobilization stress. Mice were subjected to immobilization stress for 6 hrs daily for a period of seven days. Nimesulide (2.5 mg/kg, i.p.) was administered daily for 7 days before challenging them to immobilization stress. Behavioral analysis revealed the hyperlocomotor activity and increased anxious response. Subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. Biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. Chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. These results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. There is accumulated evidence for NGF role as a peripheral pain mediator. NGF is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. NGF increase was also observed in temporomandibular join (TMJ) after CFA injection, indicating its possible involvement in local hyperalgesic states. Therefore, the objective here was to evaluate if NGF participate in the TMJ nociception. To test this hypothesis, the NGF was injected into the TMJ alone or after carrageenan (Cg) and the spontaneous nociceptive behavior of head flinches was counted for up 30min. Further evidence for the NGF nociceptive activity was obtained quantifying the local production of NGF after Cg injection, by ELISA, and the FOS-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after NGF injection. Injections were performed in 0.25ul. NGF (0.2, 1 and 5ug) injected in the TMJ challenged 1h prior by Cg (100ug) induces a dose-dependent increase in the number of head flinches. This increase was reduced by k252A (1 and 2ug), indicating a trkA receptor-mediated effect. We detected a significant increase in the NGF production 1 and 3h after the TMJ Cg (300ug) injection. The TMJ injection of NGF (5ug) alone did not induce detectable spontaneous nociceptive behavior. However, the NGF (5ug) injection induces a significant increase in the FOS like immunolabeling (FLI) in the sensory trigeminal nucleus compared to the saline injection. These results indicate that the NGF participates in the nociceptive activity in the TMJ, specially in inflammatory conditions. MIF was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (RA) several years ago, but it now clear that MIF is also involved in the pathogenesis of systemic lupus erythematosus (SLE) and atherosclerosis. MIF-deficient lupus-prone MRL/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to MIF-expressing mice. Similarly, MIF-deficient atheroma-pone LDLR-deficient or ApoE-deficient mice are significantly protected from disease and antiMIF mAb therapy is beneficial. RA and SLE are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of MIF. Given the effects of MIF on atherosclerosis it can be hypothesised that MIF overexpression participates in the risk of atherosclerotic vascular disease in RA and SLE.

Recent data have provided insights into mechanisms of action for MIF relevant to all these concepts. Firstly, the newly described role of MIF in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to RA, SLE, and atherosclerosis, with evidence that MIF mediates macrophage recruitment in SLE and atherosclerosis. Secondly, glucocorticoid (GC) therapy is possible risk factor for atherosclerosis in patients with RA and SLE, and it is now clear that GC increase the expression and release of MIF, potentially implicating MIF in GC-related increases in atherosclerosis in RA and SLE.

Specific therapeutic targeting of MIF in RA and SLE may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases.

To enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.Cytokines such as tumour necrosis factor (TNF) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.However, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (MIF) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.Therefore we explored the ability of MIF to regulate leukocyte recruitment.This was achieved using intravital microscopy to examine the intact microvasculature in mice following local MIF treatment. These experiments showed that MIF induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly CD68+/F4/80-ve/CD11c-ve monocyte/ macrophage lineage cells.MIF did not induce upregulation of adhesion molecules P-selectin and VCAM-1, although their constitutive expression contributed to recruitment.In contrast, MIF-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, CCL2/MCP-1, and its receptor CCR2, and in response to anti-CXCR2.This was supported by in vitro experiments showing that MIF induced CCL2/MCP-1 release from cultured murine endothelial cells.Finally, mice lacking CD74, the putative MIF binding molecule, did not respond to MIF.These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from CD74.This function may be critical to the ability of MIF to promote diseases in which macrophages are key participants.

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kB and AP-1 activities. GM-BMM preferentially produced TNFa, IL-6, IL-12 and IL-23 while, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkBa degradation, RelA nuclear translocation and NF-kB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pre-treated with the other CSF prior to LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kB activity. Thus GM-CSF and M-CSF demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for M-CSF. Granulocyte macrophage-colony stimulating factor (GM-CSF), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. GM-CSF is able to prime macrophages for increased pro-inflammatory responses, including the increased release of TNFa and IL-12 following stimulation with, for example, LPS. In addition, GM-CSF has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. GM-CSF-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of GM-CSF with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. T cells appear to be the major cell type responsible for GM-CSF production required for arthritis, and GM-CSF appears important in the effector phase of disease, subsequent to T cell activation. Blockade of GM-CSF results in fewer inflammatory cells, particularly macrophages, and cytokines such as TNFa, at the site of inflammation. These findings suggest that blockade of GM-CSF may be an effective treatment in a range of inflammatory diseases. The autoimmune disease Type 1 diabetes mellitus (T1DM) is thought to be mediated by autoreactive T cells recognizing islet autoantigens, including GAD65, IA-2 and proinsulin. This disease arises on a distinctive genetic background, mapping most notably to the MHC, and is also open to strong environmental influence. To investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive T cells are important, we have developed an approach to epitope identification that is MHC allele and autoantigen specific, and operates for both CD4 and CD8 T cells. Utilizing this, we have uncovered populations of islet antigen-specific T cells that have the immunological credentials to be both pathogenic (eg Th1, Tc1) and protective (Treg) in the disease. We have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions.

TCR transgenic targeting B:9-23 cause diabetes 2. Knockouts of the insulin 2 gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin 1 gene (islet expression) prevents 90% of diabetes 3. Dual insulin knockout with transgenic insulin with altered peptide (B16:A) prevents all diabetes 4. Islets with native B:9-23 sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by T cells 5.Anti-B9-23 T cells have conserved Valpha and Jalpha chain usage but no conservation N region or beta chain 6. Alpha chain as transgene sufficient to engender anti-insulin autoantibodies 7. Kay and coworkers demonstrate insulin reactivity "upstream" of IGRP and IGRP reactivity nonessential.Future studies in NOD directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.In man we can now identify at birth genetic risk as high as 80% of activating anti-islet autoimmunity with MHC analysis and restricted heterogeneity suggesting dominant target.Insulin autoantibodies in prospective studies such as DAISY usually appear initially and levels are related to progression to diabetes.Analysis of cadaveric donors is underway to elucidate primary targets. (T1D) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. The Holy Grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. This has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of T-cell co-stimulation molecules) or form (as an altered peptide ligand). Compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (NOD) mouse model of T1D, and possibly in humans. Proinsulin/ insulin DNA, protein or T-cell epitope peptides administered in a tolerogenic manner to the NOD mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector T cells, induction of regulatory T cells). Administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory T cells in the rodent, is the most immediately translatable approach to humans. Initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. Recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for T1D. combination Autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established T1D. Leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). The initial paradigm of a 4-step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. Additionally, organ-specific differences regarding the role of distinct molecules were established. Finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. In-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. Numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-Lewisx, or ICAM-1 and LFA-1, have been proven sufficiently relevant to make them candidates for potential therapies. With the anti LFA-1 antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. Moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. Ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. Finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. Consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa.

Department of Dermatology, Heinrich-Heine-University, Düsseldorf, Germany Atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. The recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. During recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. With regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. Here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory T and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. The skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. A network of pro-inflammatory cytokines including IL-1 and TNF-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. Recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. Kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. Increased activity of MAPKs, in particular p38 MAPK, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. Progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. A careful examination of different signalling pathways in various inflammatory conditions is therefore needed. This presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin-1, TNF-µ, p38 MAPK, MSK1/2, MK2, NF-kappaB and AP-1. Histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. In contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. The discovery of the histamine H4 receptor (H4R) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. H4R deficient mice and mice treated with H4R antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in TH2 responses. Ex vivo restimulation of primed T cells showed decreases in Th2 cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the H4R was responsible for the the T cell effects. The influence of H4R on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and H4R deficient mice. The H4R was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the H1R was minor. Surprisingly the H4R effect was independent of mast cells or other hematopoetic cells. This work suggests that the H4R can modulate both allergic responses via its influence on T cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. The studies show that the H4R mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of H4R antagonists. (1), BSP Reddy (2) (1) Nizams Institute of Medical Sciences, Hyderabad; India (2) Genix Pharma, India Rupatidine, carries the majority of the histamine H1receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria.

Objectives: The aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response.

Methodology: 12 male volunteers were enrolled after written informed consent before to ethic committee approved protocol. In this randomised, double-blind, Single oral dose, cross overstudy, they were randomized to receive either 10 mg Rupatidineformulation after overnight fast. Washout was 10 days. Wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. Ten minutes later, wheal and flare were visualized under a bright lamp. Histamine induced wheal and flare skin test was performed before and regularly to 24hours after drug administration.

Results: Administration of reference and test formulations of Rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. The least square mean ratio (%) T vs R for peak activity Imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (AUC0-24 mmsq/hr and AUC 0-24 %/hr) both for untransformed and log transformed data were found to be within 80-125% of90% CI limits Both formulations well tolerated.

Conclusions: It can thus be concluded that be concluded that test formulation of Rupatidine tablet is bioequivalent to reference Rupatidine tablet (1), H Yoshimura(1), K Ohara (1), Y Mastui (1), H Hara (1), H Inoue (1), H Kitasato (1), C Yokoyama(2), S Narumiya (3), M Majima (1) (1) Kitasato University School of Medicine, Kanagawa, Japan (2) Tokyo Dental Medical Colledge, Tokyo, Japan (3) Kyoto University School of Medicine, Kyoto, Japan Thromboxane (TX) A2 is a potent stimulator of platelet activation and aggregation and vascular constriction. We have reported the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitorsconstitute revascularization. We hypothesized that TXA2 induces angiogenic response by stimulating SDF-1 and VEGF which derived from platelet aggrega-

Inflamm. Res., Supplement 3 (2007) tion.To evaluate this hypothesis, we dissected the role of the TXA2 in angiogenesis response using mouse hind limb model. Recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (C57Bl/6 WT) , Prostaglandin I2 receptor (IP) knock out(IPKO) and Thromboxane (TX) A2 receptor (TP) knock out(TPKO). Blood recovery in TP-/-significantly delayed compared to WT and IPKO. Immunohistochemical studiesrevealed that TP-/-mice were less stained against PECAM positive cells compared to WT and IPKOPlasma SDF-1 and VEGF concentration were significantly reduced in TP-/-mice. We observed during in vivo fluorescence microscopic study that compared to TPKO, IPKO and WT significantly increased platelet attachment to the microvessels around ligated area. TPKO translpanted WT bone marrow cells increased blood recovery compared to TPKO transplanted TPKO bone marrow cells. In addition, mice injected with TXA2 synthase c-DNA expressing fibroblast increased blood flow recovery compared to control mice. These results suggested that TP signaling rescues ischemic condition by inducing angiogenesis by secreting SDF-1 and VEGF from platelet aggregation. Purpose: The S100 calcium-binding proteins, A8, A9 and A12 are constitutively expressed in neutrophils and induced in activated macrophages. High levels are found in sera from patients with infection and several chronic inflammatory diseases. The calgranulin complex, A8/A9 is anti-microbial; A8 has oxidant-scavenging functions. A12 is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (MC). Effects of these mediators on MC and monocyte function were compared.

Methods: Human PBMC or murine MC were activated in vitro with S100 and mediator release and cytokine induction (assessed by quantitative RT-PCR/ELISA), determined. A Cys41 to Ala41 A8 mutant was used to determine whether effects on MC are mediated by redox. Immunohistochemistry was used to demonstrate S100s in asthmatic lung.

The S100s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. Strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of A8. A8, A9 or the A8/A9 complex had relatively low ability to induce IL1, TNF, IL6, and chemokines mRNA from PBMC compared to A12. Only A12 induced significant levels of IL13; none induced IL4 or GM-CSF mRNA compared to LPS. In contrast to A12 which is activating, A8 significantly inhibited MC degranulation provoked by IgE cross-linking; suppression was dependent on Cys41.

Conclusions: The cytokine profile generated by A12 in MC and monocytes strongly supports a role the pathogenesis of asthma. In contrast, results strongly support a role from A8 in oxidant defence, particularly to hypobromite generated by activated eosinophils. (1), D Mankuta(2), G Gleich (3), F Levi-Schaffer (1) (1) Hebrew University of Jerusalem, Israel (2) Hadassah University Hospital, Israel (3) University of Utah, USA

The onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. Eosinophil major basic protein (MBP), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. Characterized more than two decades ago, the exact nature of this activation has not been clarified as yet.

Here we demonstrate that MBP exerts its activating effect on human mast cells and basophils through CD29 and hematopoietic cell kinase (Hck). A genome-wide analysis showed that Hck displays shifts in mRNA levels specifically upon MBP-induced mast cell activation. Hck also shows a unique priming pattern prior to this activation. CD29 is phosphorylated specifically upon activation with MBP and deploys a signaling complex that critically depends on Hck. Extracellular neutralization of CD29 interferes with MBP entry into the cell, and this as well as RNA silencing of Hck results in defective MBP-induced activation. Finally, CD29 neutralization abrogates MBP-induced anaphylaxis in-vivo. These findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. Alexander Robinson (1), D Kashanin (2), F ODowd(2), V Williams(2), G Walsh (1) (1) Cellix Ltd, Institute of Molecular Medicine, Dublin, Ireland

(2) University of Aberdeen, Scotland, UK

Leukocyte adhesion to endothelial cell bound proteins, such as ICAM-1 and VCAM-1, is an initial step of the inflammatory response. We have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. Using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0-5 dynes/cm2). The adhesion profiles of resting and PMAstimulated peripheral blood lymphocytes (PBLs) were recorded, with respect to VCAM-1, ICAM-1, and BSA. Images at each shear stress level were captured using a digital camera, and analysed using our in-house Ducocell software package. Distinct morphological changes in PMA-stimulated PBLs, compared to non-stimulated cells, can be observed. These include a less rounded appearance of the PMA-stimulated PBLs, and evidence of "uropod" formation, which anchor the T cell to the endothelium as part of the migration process. Levels of adhesion to VCAM-1 are high (80-90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and PMA-stimulated PBLs.However, there is a distinct difference between the adhesion levels of non-stimulated and PMA-stimulated PBLs to ICAM-1, with PMA-stimulated cells showing a higher affinity for ICAM-1 than nonstimulated cells (approx. 70% and 40%, respectively).PBL adhesion to BSA is negligible. We present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. This platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. Introduction: Pulmonary aspiration of gastric contents is a common complication observed in ICU patients and a potential trigger of ARDS. In this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (GJ).

Methods: GJ was obtained from donor rats (pH 1.6). Male C57Bl/6 were instilled with 2 ml/kg of GJ. After 2 or 24 h, the animals were sacrificed and lung and BALF were collected. Control group consisted of non-manipulated mice. 287.3AE18.6, SG24h: 277.8AE63.8; pg/ml). Discussion: GJ aspiration induced an initial adherence of PMN to lung tissue that is correlated with increased TNF-a/IL-10 ratio in BALF at the 2nd h. The reduction of MPO activity is correlated with the decrease in TNF-a/IL-10 ratio. The late increase of PMN in BALF might be a consequence of the early production of TNF-a. The results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of TNF-a. Objectives: Intestinal I/R is implicated as a prime initiating event in the development of Acute Respiratory Distress Syndrome (ARDS) after trauma and hemorrhagic shock. We investigated the effects of LPS challenge to mice previously submitted to i-I/R, a two-hit model of acute lung injury.

Methods: Male C57Bl/6 mice were subjected to 45 min of intestinal ischemia and challenged with 0.1 mg/kg of intranasal LPS at the 4th hour of reperfusion (two-hit). BALF and culture of lung explants were performed 20 h after LPS challenge. Mice subjected to i-I/R or LPS alone were used as controls.

Results: Two-hit mice showed marked increase in lung Evans blue dye leakage compared to i-I/R (375.5AE53.9 vs 48.8AE3.5, mg/mg). Lung MPO was increased (0.30AE0.03 vs 0.15AE0.03; OD 450nm) whereas the neutrophil recruitment to BALF was inhibited in the two-hit group compared to LPS group (11.5AE2.6 vs 28.5AE3.4; x 10e3cells/mouse). The levels of NOx-in the two-hit group were significantly increased when compared to i-I/ R controls in BALF (10.6AE0.5 vs 6.5AE0.8; mM) and in lung explants (3.1AE0.3 vs 1.3AE0.1; mM/mg of tissue).

Conclusions: Intestinal I/R predisposes the animal to an exacerbated response to a low dose LPS insult. The exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. The results are suggestive that patients exposed to systemic inflammatory response might develop ARDS when in contact with secondary inflammatory stimuli. Nitric oxide may play an important role in this process. (1) (1) Novartis Institutes for BioMedical Research, Horsham, UK (2) University of Michigan, USA Obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. Thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like COPD. To test this hypothesis, we exposed high capacity runner (HCR) and low capacity runner (LCR) rats to 3 months of whole-body smoke exposures.The animals, bred over successive generations on the same background strain for high or low running capacity, differ by over 500% (p<1x10-37) for exercise capacity, measured by running on a treadmill.After 3 months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the HCR-and LCR-smokeexposed(SE) animals compared to air-exposed controls (p<0.001); however there was a 2-3-fold increase in the number of neutrophils and lymphocytes in the LCR-over the HCR-SE group (p<0.001).Histopathology revealed there was greater inflammation and lung damage present in the LCR-versus HCR-SE group (p<0.05). Metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both SE groups, oxidative damage to the lung surfactant layer was significantly more extensive for the LCR-SE. Systemic oxidative damage was also more apparent in the LCR-SE group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. The metabolic data suggest that repair processes maybe more effective in the HCRs. In summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. (1), AP Ligeiro-Oliveira(1), JM Ferreira-Jr(4), SR Almeida(1), W Tavares de Lima(1), SHP Farsky (1) (1) University of S¼o Paulo, Brazil (2) Regional Integrated University of Alto Uruguai and Missðes, Brazil Methods: Male Wistar rats were exposed to vehicle or HQ (50 mg/kg; ip.;daily, 22 days, two-day interval every five days). On day 10, animals were ip sensitized with ovalbumin (OA). Assays were performed on day 23.

Results: HQ-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro OA-induced tracheal contraction. The latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of OA. The OA-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound 48/80. In fact, lower levels of circulating OA-anaphylactic IgE antibodies were found in HQ-exposed rats. This latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules CD45 and CD6 on OA-activated lymphocytes indicated the interference of HQ exposure on signaling of the humoral response during an allergic inflammation.

Contact information: Ms Sandra Manoela Dias Macedo, Regional Integrated University of Alto Uruguai and Missðes / University of S¼o P, Department of Clinical and Toxicological Analyses, S¼o Paulo, Brazil E-mail: smdmacedo@yahoo.com.br (1) (1) Radboud University Nijmegen, Medical Centre, Nijmegen, The Netherlands (2) University Hospital, Zürich, Switzerland

Toll-like receptors (TLR) are essential in the recognition of invading microorganisms. However, increasing evidence shows involvement of TLR in autoimmunity, such as Rheumatoid Arthritis (RA), as well. Here we investigated whether synovial expression of TLR3 and TLR7 was associated with the expression of IFNa, TNFa, IL-1b, IL-12, IL-17, and IL-18 and studied in what way these receptors and cytokines were associated in vitro. Using immunohistochemistry, we found that TLR3 / TLR7 expression in synovial tissue was associated with the presence of IFNa, IL-1b and IL-18, but not TNFa, IL-12 and IL-17. To investigate whether IFNa, IL-1b and IL-18 could induce TLR3 / TLR7 upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined TLR3/7 mRNA expression. IFNa incubation resulted in significant TLR3 /TLR7 upregulation, whereas IL-1b and IL-18 did not. Pre-incubation with IFNa and subsequent stimulation of TLR3 /TLR7 significantly enhanced IL-6, TNFa and IFNa/b production, indicating that the IFN-induced TLR upregulation was functional. Low amounts of biologically active IL-1b were produced upon stimulation with ATP, but not upon TLR3 / TLR7 stimulation, although mRNA levels were high. Interestingly, IFNa-priming significantly increased the ATP-induced IL-1b production. Here, we demonstrated a dual role for IFNa in vitro, which could explain the association between TLR and IL-1b / IL-18 in synovial tissue. First, involvement in TLR3 /TLR7 regulation and second, involvement in ATP-induced production of biologically active IL-1b. These results suggest involvement of anti-viral immune responses in RA and IFNa as a key player in chronic inflammation. The pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (PPR) has been suggested recently. Here, we described the role of two members of the NACHT-LRR (NLR) family, NOD1 (nucleotide/ binding oligomerization domain) and NOD2 in model of acute joint inflammation induced by intraarticular injection of TLR2 (Toll-Like Receptor) agonist Streptococcus Pyogenes cell wall fragments. We found that NOD2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, NOD1 gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. To explore whether the different function of NOD1 and NOD2 occurs also in humans, we exposed PBMCs carrying either NOD1frameshift or NOD2frameshift mutation with SCW fragments in vitro. Both TNFa and IL-1b production was clearly impaired in PBMCs carrying the NOD2fs compared to PBMCs isolated from healthy controls. In line with the NOD1 gene deficient mice, human PBMCs bearing the NOD1 mutation produced enhanced levels of proinflammatory cytokines after 24h stimulation with SCW fragments. These data indicated that the NLR family members NOD1 and NOD2 have a different function in controlling TLR2-mediated pathways. We hypothesize that intracellular NOD1-NOD2 interactions determine the cellular response to TLR2 triggers. Whether lack of controlling TLR2-driven pathways by NOD1 signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (RA), remains to be elucidated. Leukocyte Immunoglobulin-like Receptors (LILRs) are a family of receptors with potential immune-regulatory function. Activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (RA). RA is a chronic inflammatory disease of joints caused by mediators (i.e. TNF-a) produced by activated leukocytes. We recently demonstrated expression of activating LILRA2 in synovial tissue macrophages from RA patients. The aim of this study was to determine expression and function of LILRA2 in monocytes and macrophages. Peripheral blood mononuclear cells (PBMC) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating THP-1 cells with vitamin-D3. LILRA2 expression was measured by flow cytometry before and after modulation with cytokines. Differentiation to macrophages significantly up-regulated LILRA2 expression (p=0.0239). Treatment of macrophages with LPS, TNF-a, IL-1b and IFN-g but not IL-10 caused significant down-regulation of LILRA2 (p<0.01). Function of LILRA2 was assessed by cross-linking with plate-immobilised LILRA2-specific mAb. Soluble TNF-a was measured by ELISA. Activation of cells elicited TNF-a production in a dose-dependent manner while time-course analysis shows maximal production at 18h. Correlation between LILRA2 expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. Decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. In RA, abnormal regulation of LILRA2 could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. Pharmacological blocking of LILRA2 could potentially provide therapeutic benefit. (1) (1) University of Valencia, Spain (2) Northwick Park Institute for Medical Research, UK CO-releasing molecules (CO-RMs) mimic the biological actions of CO derived from heme oxygenase activity. In the present work we studied the effects of a water-soluble CO-releasing molecule (CORM-3) on an animal model of human rheumatoid arthritis. DBA-1/J mice were treated with CORM-3 (5, 10 or 20 mg/kg/day, i.p.) from day 22 to 31 after collagen-induced arthritis (CIA) and sacrificed on day 32. Administration of CORM-3 resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. Histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. All these parameters were significantly reduced by CORM-3 treatment with the most pronounced protective effect observed at 10 mg/kg. The levels of pro-inflammatory mediators (PGE2, IL-1beta, TNFalpha, IL-2 and IL-6) in the hind paw homogenates were significantly inhibited by CORM-3. In addition, COMP levels in serum, a marker of cartilage degradation, was reduced by the CO-releasing agent. Our studies show that therapeutic administration of CORM-3 alleviates the clinical features of murine CIA at the late phase of this response. The beneficial action of CO liberated from CORM-3 appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. Aim: To setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis.

Methods: Full thickness bovine articular cartilage punches were cultured with or without 10 ng/ml IL-1a, TNF-a and oncostatin M. After three weeks the cartilage and culture medium were analyzed for weight changes, water content, DNA content, glycosaminoglycans (GAG), hydroxyproline (Hyp), damaged collagen molecules, MMP activity, CTX-II and COMP. Diclofenac, Dexamethasone, Pioglitazone, Remicade, Risedronate, Galardin and A77-1726 were tested for their effect on cartilage degradation.

Results: Exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active MMPs (all p < 0.01). COMP release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. Most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment.

Discussion: Stimulation of bovine articular cartilage explants with a cocktail of IL-1a, TNF-a and OSM results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. Further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. Toll-like receptors (TLRs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. Involvement of TLR2 and TLR4 activation in the expression of arthritis was studied using interleukin-1 receptor antagonist deficient (IL-1Ra-/-) mice, which spontaneously develop an autoimmune T-cell mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. After crossing with TLR knockouts, IL-1Ra-/-TLR2-/-mice developed more severe arthritis compared to IL-Ra-/-TLR2+/+ littermates; whereas, TLR4-/-IL-1Ra-/-mice were protected against arthritis. To clarify the mechanism by which TLR2 and TLR4 differentially regulated the disease expression, we studied the role of these TLRs in IL-23 production and Th17 development, both important in IL-1Ra-/-arthritis. Wild type bone-marrow-derived dendritic cells (BMDCs) produced similar levels of IL-23 upon stimulation with TLR2 and TLR4 ligands; however, IL-1Ra-/-BMDCs produced less IL-23 than wild type DCs upon TLR2 stimulation and more IL-23 than wild type DCs upon TLR4 stimulation. Furthermore, IL-1Ra-/-T cells produced lower amounts of IL-17 when cultured with TLR2-activated APCs and higher amounts of IL-17 when cultured with TLR4-stimulated APCs, both in combination with CD3 stimulation. FACS analysis of Th17 (CD4+/IL-17+) cells from both spleen and draining lymph nodes revealed 70% reduction in IL-1Ra-/-TLR4-/-mice compared to IL-1Ra-/-TLR4+/+ littermates. Specific CD3/CD28 stimulation of non-adherent splenocytes confirmed lower IL-17 production in IL-1Ra-/-TLR4-/-. These findings suggest important roles for TLR2 and TLR4 in regulation of Th17 development and expression of arthritis.

ProstaglandinE2 (PGE2) stimulates the transactivational activity of p53 through p38MAP kinase-dependent Ser15 phosphorylation (JBC 2006) .p53 controls cell-cycle progression, in part, by differential regulation of AP-1 proto-oncogenes (Jun/Fos).Currently we studied PGE2 control of cyclin D1 promoter activity with particular attention to the role of AP-1 oncogenes.PGE2 induced a 7.6 fold increase in JunB mRNA expression (northern blot), a 2.1-fold increase in JunB promoter activity (luciferase assay), and increased Ser259JunB phosphorylation in human synovial fibroblasts (HSF) (western blot).c-Jun was strongly inhibited while JunD, c-Fos, Fra1/ 2, and FosB expression were upregulated by PGE2.In cell-cycle experiments, transformation with a constitutively active Ha-Ras construct (Ras G12V) resulted in a 3.9 fold increase in cyclin-D1 promoter activity, cyclin-D1 synthesis, Thr202/Tyr204 phosphorylation of ERK1/2 (6.2 fold) and AP-1 (c-Jun)-dependent transactivity (3.1 fold); cyclin D1/CDK4-6 inhibitor p16INK4a synthesis was suppressed. Addition of excess RasS17N dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.Ectopic expression of c-Jun, c-Fos and especially JunD expression constructs stimulated cyclin D1 promoter activity/protein synthesis, blocked p16INK4a synthesis; the latter effects were reversed by the addition of excess JunB.PGE2 exerted temporal and bi-phasic dose-dependent control of the cyclin D1 promoter activity, largely through differential AP-1 activation and promoted cell cycle arrest and apoptosis in HSF at high physiological concentrations.The results provide further insight into the biology of the cPLA2/ COX/PGES biosynthetic axis and highlight the complexity of PGE2 action in terms of cell-cycle progression. Di-glucopyranosylamine (diGa) is an antikeratitic (Roberts et al., 1990 , ACVO Conference, Scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (Bolton et al., 2005 , Inflammation Res. 54(S2) S121) and unknown mechanism of action.Interestingly, anti-TNF therapy is anti-keratitic.-diGA hydrolyses to monoglucosylamine (MGA) and glucose, which is prevented by N-acetylation (NAcdi-GA).Lider (1995, PNAS. 92: 5037-41) showed that sulphated disaccharides are orally active, inhibit TNF synthesis and the DTH reaction.We have investigated the anti-TNF and anti-rheumatic activity of the sulphated and free diGAs.Human whole blood (HWB) was stimulated with PHA (5mg/mL) to synthesise TNF.Antigen induced arthritis (AIA) was induced in methylated bovine serum albumin (mBSA) sensitised C57bl/6 mice challenged i.a. into the stifle joint.Collagen arthritis (CIA) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day 21. HWB TNF synthesis was inhibited by diGA, MGA and NAcdiGA(IC50 <0.1mM). diGA (10mL, 1mM) i.a. prevented 24 hour AIA (-0.32+/-0.06mm).diGA at 100mg/kg reduced AIA when administered i.v. (-0.40+/-0.13mm, p<0.05) and i.p. (-0.34+/-0.13mm, p<0.05), but is hydrolysed p.o. (0.24+/-0.08mm NS). Polysulphated diGA (diGA8S) was unstable, but stabilised by N-acetylation (NAcdi-GA8S).TNF synthesis was potently inhibited by both NAcdiGA and NAcdiGA8S (IC50 <0.1mM).NAcdiGA8S (100mg/kg p.o.) inhibited AIA (-0.43+/-0.13mm), and NAcdiGA8S with lower degrees of sulphation (Mw 800 and 1200 kDa) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. Sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of TNF synthesis and possess oral anti-rheumatic activity. Excessive NO appears to play a key role in the pathogenesis of chronic inflammatory diseases. In this study we aimed to evaluate NO synthesis in rheumatoid arthritis (RA) before and after therapy. It was performed on 92 persons, divided into 6 groups: a negative control group of healthy volunteers, a positive control group with RA, a group with RA and physiotherapy (PHYS), a group with RA and low doses of cimetidine (CIM) + doxycycline (DOX), a group with RA and combined treatment PHYS + CIM + DOX, and a group treated with usual doses of ibuprofen (IBU). Serum nitrite/nitrate (Griess) was measured in order to evaluate NO synthesis.

Results: Compared to the positive control group, in all the treated groups NO synthesis decreased significantly. There was no significant difference between PHYS and CIM+DOX effect alone. The combined treatment, PHYS + CIM + DOX had a much better inhibitory effect on NO synthesis. Between the PHYS, CIM + DOX and PHYS + CIM + DOX groups and that treated with ibuprofen, there was no significant difference in reducing NO synthesis.

Conclusions: 1) In RA PHYS + CIM + DOX treatment was as efficient as ibuprofen in reducing NO synthesis. 2) The low doses of CIM and DOX may allow a longer treatment due to the lower side effects risk Enhanced SOCS1 expression following exposure of murine macrophages to LPS implicated SOCS1 in the control of LPS-mediated signaling. SOCS1 regulates NFkB signaling in murine macrophages, blocking at the level of Mal or IkBa phosphorylation. We investigated the role of SOCS1 in regulating the production TNF by LPS and Pam3CSK4-activated primary human monocytes. Blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing SOCS1 (AdV-SOCS1), control vector (AdV-GFP) or left untreated. AdV-SOCS1 monocytes were exposed to TLR4 and TLR2 ligands, LPS (500 ng/ml) or Pam3CSK4 (300 ng/ml). FACS analysis demonstrated infection efficiencies of 50AE4% and 67AE4% (n=16, mean AE SEM) of monocytes expressing AdV-GFP or AdV-SOCS1 at MOI 50. AdV-SOCS1 blocked LPS and Pam3CSK4 induced TNF mRNA and protein production in a dose-dependent manner. In contrast, IL-6 and IL-10 production by AdV-SOCS1-infected monocytes was not blocked. AdV-SOCS1 also blocked LPS and Pam3CSK4induced TNF production by macrophages isolated from synovial fluid. Infection efficiencies of 67AE1% or 72AE1% were obtained. Quantitative Western blot analysis revealed that the classically defined NFkB pathway was not altered at the level of IkBa or p65 activation. Furthermore, the kinetics of LPS and Pam3CSK4induced IkBa phosphorylation and degradation in AdV-SOCS1 monocytes remained unaffected (n=5 and 2 donors, respectively). Further, analysis of parallel MAPK pathways demonstrated no block in p38 or ERK MAPK pathways. These data suggest that SOCS1 regulation of LPS and Pam3CSK4-induced TNF production by human monocytes occurs downstream of TLRs, possibly at the level of transcription. Recently, beta-NAD+ has emerged as a novel extracellular player in the human urinary bladder. beta-NAD+ is the natural substrate of CD38 which catalyzes the conversion of beta-NAD+ to cADPR. Under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-NAD+ compared to intracellular levels (200-1000 mM). During inflammation cell lysis may cause bursts of high local beta-NAD+ levels. However, the effect of beta-NAD+ on the human detrusor smooth muscle cells (HDSMC) was unknown. The effect of beta-NAD+ on cultured (explant technique) HDSMC was determined by: 1) Measuring cytosolic free calcium ([Ca2+] i) in fura-2 loaded HDSMC using spectrofluorometry and 2) force measurements in 10-20 mg detrusor strips. HDSMC responded to beta-NAD+ (40-100 mM) with an immediate and transient increase in [Ca2+] i. The Ca2+ transient was followed by one or two much slower and transient increases in [Ca2+] i, indicative of beta-NAD+ enzymatic conversion into cADPR. The Ca2+ responses persisted in the absence of extracellular calcium. The Ca2+ responses to beta-NAD+ were not affected by exposure of HDSMC to ATP supporting the notion that the effects of beta-NAD+ were not mediated via P2X7 purinoceptors. Furthermore, beta-NAD+ caused a concentration-dependent detrusor muscle relaxation. This is the first study to report that extracellular beta-NAD+ affect intracellular calcium homeostasis and force in HDSMC. These powerful actions of beta-NAD+ suggest a role for beta-NAD+/cADPR system as a novel extracellular player in the human detrusor during inflammation. AIDS remains a worldwide threat more than two decades after identification of HIV as the etiological agent. Its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. HIV trans-activator (Tat) is one of the regulatory proteins that mediates HIV replication and dysregulates cellular functions such as apoptosis and cytokine expression. For example, Tat induces tumor necrosis factor (TNF) and enhances gp120-induced neurotoxicity. We recently showed that Tat induces the overexpression of IL-10 via cellular kinase PKR and activation of transcription factor Ets-1. In this study, we examined whether Tat plays a role in perturbing interferon-& (IFN&) signal transduction. We showed that Tat impaired IFN&-induced STAT1 tyrosine phosphorylation, but had no effects on the serine residue of STAT1 and Jak kinases in primary human blood monocytes. Furthermore, we found that the nuclear translocation of phospho-STAT1 was abrogated by Tat. The inhibition of phospho-STAT1 led to the deformation of STAT1 homodimers and subsequent STAT-DNA complex. To investigate the cellular consequences, we measured the expression of IFN&-stimulated genes including human leukocyte antigen (HLA) and 2,5oligoadenylate synthetase (2,5OAS), a key enzyme in the activation of latent ribonuclease L. The results showed that Tat inhibited transcriptional activation of 2,5OAS and HLA. Taken together, we identified a new role for Tat in which it impairs IFN& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to HIV persistence, opportunistic infections and disease progression.

Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin-18 (IL-18), a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. In vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p38 MAPK dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK dependent but caspase-1 independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1 dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK dependent but capsase-1 independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1 dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. Prostaglandin E2 (PGE2) regulates the stability of cyclooxygenase-2 (COX-2) mRNA through adenylate/uridylate-rich elements (AREs) in the 3untranslated region (3UTR) by a positive autocrine/paracrine feed-forward loop. The principal objective of this study was to elucidate the molecular mechanisms involved in the PGE2dependent stabilization of COX-2 in human synovial fibroblasts (HSFs). Transfection of well-known ARE binding proteins (AUBPs) demonstrated that tristetraprolin (TTP) potently destabilized a [luciferase-COX-2 3UTR] reporter fusion mRNA (71AE5.7% decrease in luciferase activity vs. control). TTP protein levels in HSFs remained constant despite IL-1b-induced changes in TTP mRNA levels, thus suggesting translational regulation of its expression. PGE2 did not affect the transcription or translation of this gene in HSFs. Western blot analysis of HSF TTP demonstrated the existence of a specific, covalent~55kDa heterocomplex containing TTP (TTPhcx). Although TTPhcxs exact composition and stoichiometry is yet to be defined, PGE2 selectively regulated the amount of this heterocomplex in a time-dependent manner. Furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that PGE2 can induce export of a small nuclear pool of TTP-GFP. Finally, transfection of TTP into HSFs also influenced COX-2 gene transcription, thus enabling TTP to regulate COX-2 gene expression at both the transcriptional and post-transcriptional level. In conclusion, we have demonstrated that TTP is an RNA binding protein capable of influencing COX-2 mRNA stability and transcription and whose localization and interaction with other factors is regulated by PGE2. These data can provide important insight into deciphering the role of PGE2 in fine-tuning physiological and pathophysiological gene regulation. (1) (1) Chinese Academy of Sciences, Shanghai, PR China (2) Ohio State University, USA Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. MAP phosphatases (MKP)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates MAP kinases. Induction of MKP-1 has been implicated in attenuating the lipopolysaccharide (LPS) and Peptidoglycan (PGN) responses, but how the expression of the MKP-1 is regulated is still not fully understood. Here, we show that inhibition of p38 MAP kinase by specific inhibitor SB 203580 or RNA interference (RNAi) markedly reduced the expression of MKP-1 in LPS or PGN-treated macrophages, which is correlated with prolonged activation of p38 and JNK. Depletion of MAPKAP kinase 2 (MK2), a downstream substrate of p38, by RNAi also inhibited the expression of MKP-1. The mRNA level of MKP-1 is not affected by inhibition of p38, but the expression of MKP-1 is inhibited by treatment of cycloheximide. Thus, p38 MAPK plays a critical role in mediating expression of MKP-1 at a posttranscriptional level. Furthermore, inhibition of p38 by SB 203580 prevented the expression of MKP-1 in LPStolerized macrophages, restored the activation of MAP kinases after LPS restimulation. These results indicate a critical role of p38-MK2-dependent induction of MKP-1 in innate immune responses. The I-kB kinase (IKK) complex regulates the activation of NF-kB a key transcription factor in inflammation and immunity. Whilst IKKa activity is necessary for proinflammatory and anti-apoptotic gene expression, IKKa has distinct roles in lymphorganogenesis and B cell maturation. Here we describe a role for IKKa in cell mediated immunity (CMI). Paw inflammation in methylated BSA-induced CMI was significantly reduced in transgenic mice expressing a mutant IKKa protein that cannot be activated (Ikka AA/AA ) compared to wild-type (WT). Antigen-induced IL-2 and IFNg production by Ikka AA/AA splenocytes and Ikka AA/AA T cell:DC cocultures were also significantly reduced ex vivo. This could be normalised by using WT T cell: Ikka AA/AA dendritic cell (DC) but not Ikka AA/AA T cell:WT DC combinations. This suggests that reduced CMI in Ikka AA/ AA mice is due to a defect in Ikka AA/AA T cells not DCs. This is not due to a requirement of IKKa in TCRmediated activation of T cells, since anti-CD3/CD28 mediated activation of Ikka AA/AA T cells was unaltered. However, LPS-induced production of the important Th1 cytokine IL-12 is impaired in Ikka AA/AA DCs. We are currently addressing the hypothesis that IKKa activity may be required for the generation and maintenance of antigen-specific T cells in vivo.

Recently we described a role for IKKµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. IKKa may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. As a latent transcription factor, NF-kB translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. Although extensive studies have been done regarding how NF-kB is triggered into nucleus, little is known about how it is regulated inside nucleus. By twohybrid approach, we identify a prefolding-like protein SNIP that is expressed predominantly and interacts specifically with NF-kB inside nucleus. We show that RNAi knockdown of SNIP leads to impaired nucleus activity of NF-kB and dramatically attenuates expression of NF-kB dependent genes. This interference also sensitizes cells to apoptosis by TNF-a. Furthermore, SNIP forms a dynamic complex with NF-kB and is recruited to the NF-kB enhancesome upon stimulation. Interestingly, SNIP protein level correlates with constitutive NF-kB activity in human prostate cancer cell lines. The presence of NF-kB within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous SNIP. Our results reveal that SNIP is an integral component of NF-kB enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of NF-kB regulation. Bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. An imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. Osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. Osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. In vitro, osteoclast formation from bone marrow macrophages is induced by RANKL (receptor activator of NF-kappa B ligand) in the presence of M-CSF (macrophage colony stimulating factor). Osteoclastogenesis is markedly enhanced in bone marrow macrophages from IFNAR1-/-and IFNAR2-/-mice and results in increased number of multinucleated cells positive for osteoclast marker, TRAP (tartrate-resistant acid phosphatase). Consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density.

These findings suggest that the IFN alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that RANKL signaling is essential for the induction of osteoclast differentiation. ATP acting on P2X7 receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin-1beta (IL-1b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. Here we identify pannexin-1, a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1b induced by P2X7 receptor activation. Furthermore, maitotoxin and nigericin, two agents considered to evoke IL-1b release via the same mechanism were studied. Maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin-1-independent phase induced by P2X7 receptor activation, and this was unaltered by pannexin-1 inhibition.Nigericin did not induce dye uptake.Inhibition of pannexin-1 blocked caspase-1 and IL-1b processing and release in response to this two stimuli.Thus, while pannexin-1 is required for IL-1b release in response to maitotoxin, nigericin and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes. Introduction: SAA is a classic acute-phase protein upregulated during inflammatory response. SAA is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine IL-8. Here we verify the effect of SAA on the mRNA expression and release of MIP-3alpha, a chemokyne involved in the recruitment of dendritic cells.

Methods: Peripheral blood mononuclear cells (PBMC) isolated from peripheral blood by density gradient were cultured in RPMI medium in the presence of SAA. MIP-3alphaconcentration was determinated in the supernatant of cell cultures by ELISA. mRNAwas analyzed bythe Ribonuclease Protection Assay (RPA).

Results: PBMC stimulated with SAA (20 ug/mL) induced the expression of MIP-3alpha mRNA at 2, 4 and 12 hours. MIP-3alpha protein was found in the suppernatant of 24 and 42 hours cultures (p<0,05) and the addition of SB 203580 (p38 inhibitor) and PD 98059 (ERK 1/2 inhibitor) completely abolished the release of MIP-3alpha.

Conclusions: SAA is an inducer of MIP-3alpha expression in PBMC and p38 and ERK1/2 are important pathway signaling to this effect. SAA is one of the factors responsible by the recruitment of dendritic cells. The p38 pathway is activated in numerous inflammatory conditions, including RA, IBD asthma, acute coronary syndrome, and COPD, and its activation helps drive the production of inflammatory mediators. Inhibitors of p38 decrease mediator production and therefore can produce profound anti-inflammatory effects. ARRY-797 is a potent inhibitor of p38 enzyme (IC50 < 5nM) with a novel pharmacophore and physiochemical properties distinct from those of other p38 inhibitors, being very water soluble. It is extremely potent in human whole blood, blocking LPS-stimulated TNF production with an IC50 < 2nM.In animal models of rheumatoid arthritis (CIA and AIA) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ED50~3 mg/kg). A Phase I single ascending dose clinical study was run in healthy volunteers. After an oral dose of 50, 100, 200, 300 or 400 mg), blood was drawn at various times, stimulated ex vivo with LPS, and analyzed for cytokines and inflammatory mediatorsfj ARRY-797 was well-tolerated and drug exposure was proportional to dose. In ex vivo samples, there was both a time-and concentrationdependent inhibition of IL1, PGE2 and TNFfz with >80% inhibition observed at the 50 mg dose level. The plasma concentrations of drug peaked at~20 ng/mL at the 50 mg dose and cytokine inhibition was sustained for >4 hours, showing that low doses of ARRY-797 produced profound effects on clinical biomarkers. Further evaluation of ARRY-797 in patients with inflammatory diseases is planned. Introduction: We demonstrated that in vivo chronicle blockage of NOS (L-NAME, 20mg/Kg; oral route; 14 days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. These effects may be depending, at least in part, on decreased expression of L-selectin on leukocytes and PECAM-1 on endothelium. Aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of L-NAME treatment on secretion of TNF and IL-1b; by circulating leukocytes and migrated peritoneal neutrophils.

Methods: Male Wistar rats were treated with L-NAME (20mg/Kg; oral route; 14 days) or sterile saline (control). Circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained 4 hours after i.p. injection of oyster glycogen (1%;10mL). NO (Griess reaction) and cytokines (ELISA) were quantified in supernatants of 1 x 106 cultured cells before and 18 hours after LPS stimulation (5m;g/mL).

Results: Levels of NO, TNF and IL-1b; were reduced in circulating leukocytes from L-NAME-treated rats in both basal and LPS stimulated conditions. On the other hand, only secretion of IL-1b; was impaired by migrated neutrophils.

Conclusions: Results show that in vivo L-NAME treatment, which partially reduces NO production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. However, the same pattern of inhibition is not detected if neutrophils are in vivo primed. Objectives: To investigate the ability and mechanism of IFN-g to suppress interleukin-1 (IL-1)-induced MMP-13 expression in articular chondrocytes.

Methods: Human chondrocytes were treated with IFN-g or IL-1beta alone or in combination. MMP-13 mRNA was analyzed by RT-PCR. MMP-13 protein, phospho-STAT1 and p44/42 MAPK levels were measured by Western blotting. MMP-13 promoter-luciferase, CMV-CBP/p300 plasmids and STAT1 siRNA were transfected by Calcium phosphate method. AP-1 activity was monitored by ELISA. STAT1-CBP/p300 interaction was studied by immunoprecipitation.

Results: IFN-gpotently suppressed IL-1-induced expression of MMP-13 and promoter activity. Blockade with neutralizing IFN-gR1 antibody revealed that MMP-13 inhibition by IFN-¼ was mediated by the IFN-¼ receptor. IFN-beta-stimulated activation of STAT1 was directly correlated with MMP-13 suppression. Knockdown of STAT1 gene by specific siRNA or its inhibition with Fludarabine partially restored the IL-1 induction of MMP-13 expression and promoter activity. IFN-g did not alter activator protein (AP-1) binding ability but promoted physical interaction of STAT1 and CBP/p300 co-activator. P300 overexpression reversed IFN-g inhibition of endogenous MMP-13 mRNA expression and exogenous MMP-13 promoter activity.

Conclusions: IFN-g through its receptor activates STAT1, which binds with CBP/p300 co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of MMP-13 gene in chondrocytes. IFN-g and its signaling pathways could be targeted therapeutically for (1), P Asmawidjaja (1), R Hendriks(2), Erik Lubberts (1) (1) Erasmus Medical Center, Department of Rheumatology, Rotterdam, The Netherlands (2) Erasmus Medical Center, Department of Immunology, Rotterdam, The Netherlands

The objective of this study was to identify the role of IL-23 in Th17 polarization in the prone autoimmune DBA-1 mice with and without collagen-induced arthritis and to evaluate Th17 specific cytokine and transcription factor expression. IL-23 induced Th17 cells in vitro from spleen cells of naïve and collagen-type II (CII) immunized DBA-1 mice. The percentage of Th17 cells is markedly higher in CII-immunized versus naïve DBA-1 mice. Adding IL-23 to TGF-beta/IL-6 stimulated CD4 + T cells did not significantly increase the percentage of Th17 cells. TGFbeta/IL-6 in contrast to IL-23 induced a relatively high percentage of IL-17+/IFN-gamma-cells and low IL-17-IFN-gamma+ cells. TGF-beta/IL-6 did not increase IL-23 receptor expression, which may explain why adding IL-23 directly or two days after TGF-beta/IL-6 did not result in an increase in the percentage of Th17 cells. Elevated expression of IL-17A and IL-17F as well as the Th17 specific transcription factor RORgammat was found under IL-23 as well as TGFbeta/IL-6 conditions. Interestingly, IL-23 but not TGF-beta/IL-6 is critical in the Th17 cytokine IL-22 expression in T cells from CIIimmunized DBA-1 mice. These data show that IL-23 was more pronounced in inducing IL-17+/IFN-gamma-(Th17) cells under CII-immunized conditions. Furthermore, IL-23 did not markedly increase the percentage of Th17 cells induced by TGF-beta/IL-6. However, IL-23 is critical for the induction of IL-22 expression, suggesting a unique role for IL-23 in the induction of specific Th17 cytokines EBI3 was initially discovered as a transcriptionally activated gene in Epstein-Barr virus-infected human B lymphocytes, and similar to p40 of IL-12. EBI3 protein has been shown to form heterodimers with p28. p28/EBI3 termed IL-27, can influence the function of multiple T cell subsets, including naive, effector, regulatory and memory T cells. However, previous studies showed that the overlapped expression of EBI3 and p28 is very limited. These data lead to the hypothesis that EBI3 may play a role independently from its association with p28. Thus, to define the function of EBI3, we generated EBI3 transgenic (Tg) mice expressing in multiple tissues. EBI3 Tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory CD4+, CD8+ T cells, B cells, NK cells and NKT cells. CD4+T cells isolated from spleens of EBI3 Tg mice, however, produced less IFN-g than cells from WT (wild type) control mice after in vitro stimulation with anti-CD3 and anti-CD28 antibodies. In vivo studies, delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) responses were significantly reduced in EBI Tg compared with WT mice. Moreover, the CHS responses in EBI3 Tg mice were recovered with anti-EBI3 polyclonal antibody. Notably, CHS reaction in WT mice was increased by anti-EBI3 antibody. In contrast, anti-p28 antibody suppressed CHS responses in WT mice. These data suggest that EBI3 acts in different from IL-27, and reduces Th1 responses. (1), O Thaunat(2), X Houard (1), O Meilhac (1), G Caligiuri(2), A Nicoletti (2) (1) INSERM U698 and University Denis Diderot-Paris 7, CHU Xavier Bichat, Paris, France (2) INSERM UMR S 681, UniversitØ Pierre et Marie Curie-Paris 6, Centre de recherche des Cordeliers, Paris, France

Arteries are composed of three concentric tissue layers which exhibit different structures and properties. Because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). In contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. In the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. Several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. These mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. We propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. Hence, an adventitial adaptive immune response predominates in chronic rejection. Inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. Centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. Adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. These adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. Atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial COX-derived prostaglandins such as PGE2 and prostacyclin acting on EP and IP receptors, respectively.Activation of vascular IP receptors is especially important in limiting the atherogenic properties of thromboxane A2 acting on TP receptors.More recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.As well as the complex crosstalk between G-protein-coupled receptors (GPCRs), recent evidence indicates that many GPCRs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.We have found that EP3-I, TP and ghrelin receptors readily form homodimers, but that co-transfection of HEK293 cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.Since inflammatory conditions are thought to change the relative expression levels of GPCRs in the vasculature, and since varying the expression levels of GPCRs will affect their ability to form heterodimers, then one might predict that GPCR heterodimerization would indeed influence the reactivity of vascular tissue during inflammation.

[This work was fully supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (CUHK4267/02M and 2041151 Vascular inflammation leads to formation of leukotrienes through the 5-lipoxygenase pathway of arachidonic acid metabolism. Leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the BLT and CysLT receptor subtypes. Recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. Leukotriene B4 (LTB4) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. Initially identified on neutrophils, BLT receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in LTB4-induced atherogenesis. Targeting LTB4-induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. Furthermore, BLT receptor expression has been demonstrated on T-cells, suggesting LTB4 as a potential link between innate and adaptive immunological reactions. Taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. Further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. In normal physiological conditions, the prostanoid (prostaglandin (PG) and thromboxane (Tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (COX-1). This synthesis and release happen few minutes after cell or tissue stimulation. In vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (COX-2) and other prostanoid synthases can be observed. As an illustration of the previous experimental results, there is an increased presence of COX-2 and the inducible enzyme responsible for PGE2 synthesis (mPGES-1) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. In vascular cells in culture, PGI2 is the major biological active prostanoid produced in the normal physiological conditions. However, when COX-2 is induced, PGI2 and PGE2 are equally produced. The role of COX-1, COX-2 and mPGES-1 activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. There is increasing evidence for the presence and a role of the EP receptor subtypes (EP1, EP2, EP3 or EP4) preferentially stimulated by PGE2 in the vascular wall. For these reasons, we have characterized the receptors activated by PGE2 in human mammary arteries. In these vessels incubated with a pro-inflammatory cytokine (interleukin-1â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. This effect is abolished by treatment of the vascular preparations with a selective COX-2 inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. Rheumatoid Arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. Two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for RA).We show that the different HLA class II alleles contribute to the development of anti-CCP-positive and anti-CCP negative RA.The SE alleles do not independently contribute to the progression to RA, but rather contributed to the development of anti-CCP antibodies. Next we determined the effect of smoking and observed that smoking only conferred risk to contract RA in the CCP-positive group and not in the anti-CCP negative group. For the risk factor PTPN22 (a gene that regulates treshold of lumphocyte activation) the allele C1858T only contributed to CCP-positive RA. In contrast to HLA two other risk factors were found to be associated with both CCP-positive and CCP-negative RA. The risk factor in the FCRL-gene as has been identified in the Japanese population was also tested in 931 Dutch RA cases and 570 unrelated Dutch controls. Carrier analysis of the SNP (rs7528684) revealed association of CC genotype with higher risk of developing RA as compared to TT & TC carriers (P =0.039 and OR =1.31). In a meta-analysis of all studies comparing 9467 individuals, the OR for the CC genotype to develop RA was 1.2 and the P-value < 0.001. In conclusion, different steps in pathogenesis of the syndrome RA can be delineated This talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (Crohns disease and ulcerative colitis). Proven genes containing genetic variants predisposing to Crohns disease include IBD5/5q31, CARD15/NOD2 and IL23R. Data is suggestive but not yet as convincing for many other genes. A common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. Only for CARD15/NOD2 is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. Some mouse models (gene targeted) of CARD15 appear to show opposite effects to other models and human systems. In humans, CARD15 mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. It is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. Pathogen-recognition receptors (PRRs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. Since their discovery in vertebrates, Toll-like receptors (TLRs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. Recently a new family of intracellular PRRs, the NOD-like receptors (NLRs), which include both NODs and NALPs have been described. Mutations within the NALP3/cryopyrin/ CIAS1 gene are responsible for three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA/NOMID. The NALP3 protein associates with ASC and caspase-1 (thereby forming a molecular machine termed inflammasome that displays high proIL-1beta-processing activity. Macrophages from Muckle-Wells patients spontaneously secrete active IL-1beta. Increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with NALP3-dependent autoinflammatory disorders. Here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. Allergic inflammation (AI) is a complex phenomenon initiated by allergen binding to IgE sensitized mast cells and consequent mast cell activation. This causes the symptoms of the early phase of AI and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. Their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the AI becomes a chronic process. We defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. In the synapse these two old cellular players of AI have a cross talk via soluble mediators and receptor-ligand interactions. This results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. In addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. We propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. (1) (1) Erasmus MC, Rotterdam, The Netherlands (2) Department of Immunology Weizmann Institute of Science, Rehovot, Israel

Allergic asthma is one of the most common chronic diseases in Western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with Th2 cells, eosinophils, and mast cells. If we are to devise new therapies for this disease, it is important to elucidate how Th2 cells are activated and respond to intrinsically harmless allergens. Dendritic cells (DCs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. We have shown that intratracheal injection of ovalbumin (OVA) pulsed DCs induces sensitization leading to eosinophilic airway inflammation upon OVA aerosol challenge. We investigated the role of DCs in the secondary immune response in a murine asthma model. OVA aerosol challenge in OVA-DC sensitized mice, induced an almost 100 fold increase in the number of airway DCs as well as an increase in eosinophils and T cells. To investigate the functional importance of DCs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous DCs in sensitised CD11c-Diphtheria toxin (DT) receptor (CD11cDTR) transgenic mice by airway administration of DT 24 h before OVA aerosol (4x) challenge or during an ongoing inflammation (depletion after 3x OVA aerosols continued with 3 additional OVA aerosols). Numbers of BALF eosinophils, Th2 cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway DCs during secondary challenge. 

Karolinska Institute, Stockholm, Sweden NK cells are innate lymphocytes with potent immunoregulatory functions. They are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. Even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. For example, they are involved in, and affect, acute as well as chronic inflammatory responses. In the present talk, a general overview on our current knowledge of NK cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. Basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity IgE receptors (Fc<epsilon>RI) with allergens. Although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate Th2-type cytokines (e.g. IL-4 and IL-13), subsequently supporting IgE synthesis and underlying atopy. Importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. S. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing Th2-type adaptive immune responses. While we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. Recent advances, however, have shed light upon the major intracellular signal transduction processes involved in Fc<epsilon>RI activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. An important discovery in this regard is the phosphatase SHIP, which downregulates PI 3-kinase signalling in both basophils and mast cells. Recent data shows that SHIP expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with SHIP recruitment (CD200R, CD300R). Identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. Mitogenesis and proliferation of VSMC play an important role in atherogenesis. Pro-inflammatory secretory phospholipases A2 (sPLA 2 ) hydrolyse glycerophospholipids of HDL and LDL and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.sPLA 2 s lipolysis products localize in vascular wall in vicinity of VSMC.We have tested the impact of sPLA 2 , HDL and LDL and of their hydrolysis products on mitogenesis and PGE2 and LTB4 release from VSMC.Mitogenesis was significantly enhanced by native HDL, and LDL, and by group V sPLA 2 . sPLA 2 hydrolysis of HDL and LDL enhanced mitogenic activity in order V>X>IIA.The release of PGE2 from VSMC was enhanced by group X sPLA 2 s but not IIA or V.The greatest effect was seen for HDL hydrolysed by group V and X sPLA 2 .Native LDL and its sPLA2 hydrolysis products enhanced the release of PGE2 in order X>V>IIA.The release of LTB4 from VSMC was markedly increased by native LDL and HDL, and hydrolysis products of group V and X, but not IIA sPLA2.Migration of VSMC was significantly enhanced by sPLA 2 IIA and inhibited by HDL.This study demonstrates a complex interaction of HDL and LDL with pro-inflammatory sPLA2s, which affects mitogenesis, eicosanoid release and migration of VSMC.Study of biocompatible sPLA2 blockers in the therapy of atherosclerosis is indicated.

Contact information: Professor Waldemar Pruzanski, University of Toronto, Department of medicine, Toronto, Ontario, Canada E-mail: drwpruzanski@bellnet.ca (1) (1) IPMC-CNRS UMR6097, Valbonne, France (2) University of Washington, Seattle, USA (3) INSERM UMRS 525, Paris, France (4) University of Naples, Italy

The superfamily of phospholipase A2 comprises at least 9 intracellular enzymes and up to 12 secreted PLA2s (sPLA 2 s). Elucidating the biological roles of each PLA2 member is currently the most challenging issue in the PLA2 field. The different sPLA 2 s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). Increasing evidence suggests that sPLA 2 s IIA, III, V, and X are involved in inflammatory diseases including atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (LDL). On human alveolar macrophages, hGX sPLA 2 can trigger secretion of TNF alpha, IL1 and IL6 in a non-enzymatic manner. On colorectal cancer cells, hGX sPLA 2 stimulates cell proliferation, produces potent eicosanoids including PGE2, and activates the transcription of key genes involved in inflammation and cancer. The enzyme can also hydrolyze PC and Platelet-Activating Factor (PAF) of LDL particles very efficiently. Finally, hGX sPLA 2 is present in human atherosclerotic lesions and converts LDL into a proinflammatory particle that induces macrophage foam cell formation, as well as MAP kinase activation, arachidonic acid release, and expression of adhesion molecules in HUVEC cells. Some other key molecular features of sPLA 2 s including hGX will be presented. We have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by sPLA 2 s, but the mechanism of this selectivity is not known. Since both sphingomyelin (SM) and lysoPtdCho inhibit the activity while increasing fatty acid specificity of other PLA2s, we have examined fatty acid release by sPLA2sIIA, V and X in relation to relative increases in proportion of endogenous SM and lysoPtdCho during lipoprotein digestion. The analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (LC/ESI-MS) and LC/collision induced dissociation (CID)/ESI-MS using conventional preparations ofLDL and HDL. The highest preference for arachidonate release from LDL by group X sPLA 2 was observed when the residual SM/ PdCho molar ratio had reached 1.2 compared to a starting ratio of 0.4.Group V sPLA 2 showed preferential release of linoleate at residual SM/PtdCho molar ratio 1.5, while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. The relative increases in lysoPtdCho and SM during the digestion with sPLA 2 IIA were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. These results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of PtdCho. The residual SM/PtdCho ratios reached during group V and X sPLA 2 digestion are similar to those observed for lesional LDL, which promote release of ceramides by SMase leading to LDL aggregation. The above findings support a potential role of sphingomyelins in atherogenesis. Although sphingomyelin (SM) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. Because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (PC),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (PLA). We showed that SM inhibits several lipolytic enzymes including secretory PLA2 IIa, V, and X, and hepatic and endothelial lipases, all of which hydrolyze PC. Treatment of SM in the substrate with SMase C not only relieved the inhibition but also activated the PLA2 reaction further, suggesting that ceramide, the product of SMase C, independently stimulates PLA2, possibly by disrupting the bilayer structure. SMase D treatment, which produces ceramide phosphate, did not stimulate the sPLA2. The fatty acid specificity of PLA2 is significantly affected by SM. Thus sPLA2X exhibited enhanced specificity for the release of arachidonic acid (20:4) in presence of SM, due to a preferential inhibition of hydrolysis of other PC species. In contrast, SM inhibited the release of 20:4 by sPLA2V. Ceramide selectively stimulated the release of 20:4 by both enzymes. Only the long chain ceramides (> 10 carbons) were effective, while ceramide phosphate did not stimulate sPLA2 activity. SM-deficient cells released more 20:4 in response to sPLA2-treatment than normal cells, and pretreatment of normal cells with SMase C increased their susceptibility to sPLA2 attack. These studies show that SM and ceramide regulate the activity and specificity of PLA, and consequently the inflammatory response. Secretory phospholipase A2 (sPLA2) types IIA, V, or X, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.Given the link between sPLA2 and atherogenesis, a mouse model of atherosclerosis (apoE-/-) was used to study the effects of A-002, an inhibitor of sPLA2 enzymes, on atherosclerosis and cholesterol levels over 16 weeks of treatment. Mice were fed with a high-fat, high cholesterol diet alone during the study (21% fat; 0.15% cholesterol, 19.5% casein) and were treated with vehicle or A-002 bid at 30 mg/kg or 90 mg/kg by oral gavage. Total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.Treatment with A-002 significantly reduced aortic atherosclerotic plaque formation in apoE-/-mice fed a high fat diet when compared to the untreated control by approximately 40 %. In a different model that used angiotensin II in conjunction with a high fat diet in a background of apoE-/-deficient mice for 4 weeks, oral dosing of A-002 (30 mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. These data suggest that A-002 is a potential novel therapeutic agent for the treatment of atherosclerosis. (1), S Doty(2), C Antonescu (3), C Staniloae (1) (1) Saint Vincents Hospital Manhattan, New York, USA (2) Hospital for Special Surgery, New York, USA (3) Sloan-Kettering Institute for Cancer Research, New York, USA TNF-stimulated gene 6 (TSG-6) is induced by TNF-a during inflammation and its secreted product TSG-6 glycoprotein is involved in immune-mediated inflammatory diseases and fertility. It regulates COX-2 and prostaglandin synthesis, and participates in extracellular matrix remodeling. Considering the chronic inflammatory nature of atherosclerosis we hypothesized that TSG-6 is expressed in atherosclerotic plaques and investigated TSG-6 protein expression and cellular distribution on 12 superficial femoral artery endarterectomy specimens from 6 diabetic and 6 non-diabetic patients with peripheral vascular disease. Six histologically normal radial artery specimens were analyzed as control. Paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-TSG-6 antibody. TSG-6 expression was consistently present in all atherectomy specimens but not in control specimens. A distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. Cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. Patchy TSG-6 expression was noted in the extracellular matrix. Within the inflammatory plaques from diabetic patients, TSG-6 stained the foamy macrophages. TSG-6 expression was also confirmed and quantified by qRT-PCR that showed a significant up-regulation of TSG-6 gene (more that 100 fold induction compared to housekeeping genes). Our study identifies for the first time the preferential expression of TSG-6 in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. TSG-6 is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. (1), R Krohn (1), H Lue (1), JL Gregory(2), A Zernecke (1), RR Koenen (1), T Kooistra (3), P Ghezzi(4), R Kleemann (3), R Bucala(5), MJ Hickey (2), C Weber (1) (1) University Hospital of the RWTH Aachen, Germany (2) Centre for Inflammatory Diseases, Monash University, Melbourne, Australia Mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (CLF)-chemokines. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. The underlying molecular mechanisms are poorly understood, but, interestingly, MIF displays structural features resembling chemokines. We have identified the chemokine receptor CXCR2 as a functional receptor for MIF. MIF triggered Galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through CXCR2, inducing rapid integrin activation. MIF directly bound to CXCR2 with high affinity (Kd of 1.4 nM). Monocyte arrest mediated by MIF in inflamed or atherosclerotic arteries involved CXCR2 as well as CD74, a recently identified membrane receptor moiety for MIF. Accordingly, CXCR2 and CD74 were found to occur in a receptor complex. In vivo, MIF deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. Thus, MIF displays chemokine-like functions by acting as a non-cognate ligand of CXCR2, serving as a regulator of inflammatory and atherogenic recruitment. These data harbor an intriguing novel therapeutic prospect by targeting MIF in atherosclerosis and add a new dimension to MIF and chemokine receptor biology. (1), R Toes (3), H van Bockel(2), Paul Quax (1,2) (1) TNO Bioscienses, Leiden, The Netherlands (2) Department of Vascular Surgery, Leiden University Medical Center, The Netherlands (3) Department of Rheumatoly, Leiden University Medical Center, The Netherlands

The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in Jµ281knockout mice that lack NK1.1+ Natural Killer T (NKT)cells, indicating that NK-cells, rather than NKT-cells are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ Tlymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1treated MHC-class-II-deficient mice that lack both NKand CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, two strains with different bias in immune responsiveness. Correspondingly, fewer CD3-positive lymphocytes accumulated around collaterals in BALB/c mice. These data show that both NK-cells and CD4+ T-cells play an important role in arteriogenesis. Moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. Post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (IH) and accelerated atherosclerosis, often causing graft failure. Inflammation is an important trigger for these processe. Complement is an important part of the immune system and participates in regulating inflammation. Although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. The involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic ApoE3Leiden mice. In these veins IH and accelerated atherosclerotic lesions develop within 28 days, consisting mainly of foamcells and SMC. To study the functional role of complement in vein graft remodeling, Cobra Venom Factor (CVF:20U daily) was used to deplete complement starting one day prior to vein graft surgery. CVF-treatment reduced vein graft thickening by 64% (p=0.02), when compared to saline treated controls (n=6).To confirm that the reduction by CVF was due to hampered complement function and not a direct effect of CVF, complement activation was blocked using Crry-Ig (inhibiting C3 convertases). Crry-Ig (3mg every other day) led to 50% decrease in vein graft thickening (p=0.03) compared to controls receiving non-relevant control IgG. These data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. (1), M-C Koutsing Tine(1), P Borgeat(2), H Ong (1), Sylvie Marleau (1) (1) Universite de Montreal, Quebec, Canada (2) Centre de Recherche en Rhumatologie et Immunologie, Canada

We have previously shown that EP 80317, a growth hormone-releasing peptide (GHRP) analogue binding selectively to the scavenger receptor CD36, elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoE-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. We investigated the effect of GHRP analogues on I/R-elicited remote lung injury in 18week-old apoE-/-mice fed a high fat high cholesterol (HFHC) diet from 4 weeks of age. At 18 weeks old, mice were treated daily with a s.c. injection of EP 80317 (300 mg/kg) for 14 days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for 30 minutes, followed by 180 minutes reperfusion. Our results show that EP 80317 significantly reduced leukocyte accumulation by 56% in the lungs, from 3.6 (AE 0.7) in vehicle-treated mice to 1.6 (AE 0.6) x 107 leukocytes/g lung in EP 80317-treated mice (n = 6-7 per group), as assessed by myeloperoxidase assay. This was associated with a 56% reduction of opsonized zymosan-elicited blood chemiluminescence. In contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoE-/-/CD36-/-deficient mice, from 1.9 (AE 0.5) in vehicle-treated mice to 1.9 (AE 0.8) x 107 leukocytes/g lung in EP 80317-treated mice. We conclude that EP 80317 protects I/R-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a CD36-mediated pathway. Glycogen synthase kinase 3beta (GSK-3beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. Dysregulation of GSK-3beta has been implicated in the pathogenesis of several diseases including sepsis. Here we investigate the effects of two chemically distinct inhibitors of GSK-3beta, TDZD-8 and SB216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. Male Wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmHg for 90 min) and subsequently resuscitated with shed blood for 4 h. Hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. Treatment of rats with either TDZD-8 (1 mg/kg, i.v.) or SB216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. The protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. In addition, TDZD-8, but not SB216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6. Neither of the GSK-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. Thus, we propose that inhibition of GSK-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. (1), Y Ito (1), H Yoshimura (1), H Inoue (1), N Kurouzu (1), H Hara (1), Y Mastui (1), H Kitasato (1), S Narumiya(2), C Yokoyama (3), M Majima (1) (1) Kitasato University School of Medicine, Japan (2) Kyoto University School of Medicine, Japan (3) Tokyo Medical and Dental University, Japan Thromboxane (TX) A2 is a potent stimulator of platelet activation and aggregation and vascular constriction. We have reported cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors constitute the major determinant of revascularization. We hypothesized TXA2 induces angiogenic response by stimulating SDF-1 and VEGF which derived from platelet aggregation. To evaluate this hypothesis, we dissected the role of the TXA2 in angiogenesis response using mouse hind limb ischemia. Recovery from acute hind limb ischemia, as assessed in wild type mice (C57Bl/6 WT) , Prostaglandin I2 receptor (IP) knock out mice (IPKO) and Thromboxane (TX) A2 receptor (TP) knock out mice (TPKO) by using lase doppler. Blood recovery in TPKO significantly delayed compared to WT and IPKO. Immunohistochemical studies revealed that the number of CD31positive cells in the ischemic quadriceps were less stained in TPKO compared to WT and IPKO.Plasma SDF-1 and VEGF concentration were significantly reduced in TPKO mice. We observed during in vivo fluorescence microscopic study that compared to TPKO, IPKO and WT significantly increased platelet attachment to the microvessels around ligated area. TPKO translpanted WT bone marrow cells increased blood recovery compared to TPKO transplanted TPKO bone marrow cells. In addition, mice injected with TXA2 synthase c-DNA expressing fibroblast increased blood flow recovery compared to control mice. These results suggested that TP signaling rescues ischemic condition by inducing angiogenesis by secreting SDF-1 and VEGF from platelet aggregation. Administration of selective TP agonist may open new therapeutic strategy in regenerative cardiovascular medicine. During renal ischemia/reperfusion (I/R) injury, apoptosis has been reported as a very important contributor to the final kidney damage. The determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. The aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. Based in a rat kidney I/R model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the I/R derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. Thus, the control in the peroxynitrite generation during the I/R could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal I/R injury. Metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. Using NMR spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. Serum from 101 patients with synovitis of "T 3 months duration whose outcome was determined at clinical follow-up was used. Vitreous samples were from 31 patients undergoing vitrectomy for vitreoretinal disorders. One dimensional 1H NMR spectra were acquired. Principal Components Analysis (PCA) of the processed data was conducted along with a supervised classification. With the arthritis serum there was a clear relationship between each samples score in the PCA analysis and the level of CRP. Supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. A similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. In this case differences were not due to a straightforward relationship with inflammatory markers (IL-6, CCL2), which did not correlate with PCA in these samples. Similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. These results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. 1H-NMR-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. (1), AM Artoli(2), A Sequeira(2), C Saldanha (1) (1) Instituto de Medicina Molecular,Faculdade de Medicina de Lisboa, Portugal (2) CEMAT, Instituto Superior TØcnico, Universidade de TØcnica de Lisboa, Portugal

The recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. The precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. In this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. Leukocyte-endothelial cell interactions in post-capillary venules of Wistar rats cremaster muscle were monitorized by intravital microscopy. From these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice Boltzmann solver for shear thinning fluids. The dynamics of leukocyte clusters under non-Newtonian blood flow with shear thinning viscosity was computed and discussed. In this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. We have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. We verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. It was found that the lattice Boltzmann solver used here is fully adaptive to the measured experimental parameters. This study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. Macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. One family of transcripts that are highly expressed in activated macrophages are members of the Schlafen (Slfn) gene family; a recently identified family whose function is still unknown. This study examined the mRNA expression of Slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (CIA) in vivo. Real-time PCR expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) over a time course, revealed differential expression of individual Slfn family genes. In particular, Slfn-1, Slfn-2, and Slfn-4 were maximally induced after 24 hours. The maximal induction of Slfn-8 and Slfn-9 was observed after 4 hours of LPS treatment. Individual members of the Slfn family were also differentially expressed in CIA, a model of rheumatoid arthritis. mRNA levels of Slfn-1, Slfn-2, Slfn-4 and Slfn-5 were elevated in joints affected by CIA. To investigate the role of Slfn-4, we have generated a transgenic mouse line, which over expresses Slfn-4 specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and Gal4. Further characterisation of the Slfn-4 over expressing mouse line will be used to assess the function of Slfn-4 in macrophage biology and inflammation, and its potential as a therapeutic target. Macrophages play an important role in resolving inflammation. It is known that the resolution of inflammation requires alternative activation of macrophages. But the precise events of phenotype switching in macrophages remain poorly understood. We show that Lipocalin 2, Lcn-2, is able to provoke a switch in macrophage activation. In an in vitro co-culture model for renal epithelial cells and macrophages, we detected by siRNA technique that the presence or absence of Lcn-2 determines proliferation processes in damaged renal epithelial cells. The proliferative response was dependent on proinflammatory or anti-inflammatory environment. As Lcn-2 is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. We anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by Lcn-2. (1), Y Cao (1), S Adhikari (1), M Wallig (2) (1) National University of Singapore, Department of Pharmacology, Singapore (2) University of Illinois at Urbana Champaign, USA It has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. Our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. The current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. Acute pancreatitis was induced in the mouse by administering hourly injections of caerulein (50 mg/kg) for 3, 6 and 10 hours respectively. Neutralizing monoclonal anti-IL-10 antibody (2.5 mg/kg) was administered either with or without crambene (70 mg/kg) 12 hours before the first caerulein injection. RT-PCR, western blotting and immunostaining were performed to detect CD36 expression. Apoptosis in pancreatic sections was visualized by TUNEL. Severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (MPO), water content, and morphological examination. Pancreatic levels of inflammatory mediators were examined by ELISA. The protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as MCP-1, TNF-a and IL-1â and up-regulating anti-inflammatory mediators like IL-10. Phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor CD36.The anti-inflammatory mediator IL-10 plays an important role in crambene-induced protective action against acute pancreatitis. The release of anti-inflammatory mediator IL-10 is downstream of phagocytosis. These results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. (1,2) (1) Northern Ontario School of Medicine, Thunder Bay, Ontario, Canada (2) Lakehead University, Canada

Integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including Pseudomonas aeruginosa. We have recently established that beta1 integrins in lung epithelial cells (LEC) provide co-stimulatory signals regulating inflammatory responses (Ulanova et al, Am J Physiol, 2005, 288: L497-L507). We hypothesized that LEC integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to P. aeruginosa. To determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of P. aeruginosa infection of A549 cells. To investigate interactions of bacteria with LEC, P. aeruginosa strain PAK was chromosomally labeled with a green fluorescent protein gene using a mini-Tn7 delivery system.Using several fluorescence-based detection systems, we established that the natural beta1 integrin ligand, fibronectin, mediates bacterial adhesion to LEC.P. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta4 integrin expression followed by the increased cell surface protein expression. The surface expression of integrin beta1 increased shortly following bacterial exposure without alterations of mRNA expression, suggesting rapid protein redistribution within the cells. The data indicate that P. aeruginosa are capable to modulate integrin gene/protein expression in LEC, potentially using fibronectin to alleviate bacterial binding to beta1 integrins. Upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. Integrin receptors in LEC may represent significant therapeutic targets in pulmonary infection caused by P. aeruginosa. The purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. Neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. Through activation of the A2A receptor (A2AR) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. Also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene B4 formation while potentiating that of prostaglandin E2 through the up-regulation of the cyclooxygenase (COX)-2 pathway. Moreover, our laboratory determined that A2AR engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including TNF-and MIPs. In mice lacking the A2AR, migrated neutrophils expressed less COX-2 than their wild type counterpart while displaying higher mRNA levels of TNF-and MIP-1. Mononuclear cells from A2AR knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. Signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following A2AR engagement, implicate pivotal metabolic pathways such as intracellular cyclic AMP, p38 and PI-3K. Together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. The newest developments regarding adenosines effects on neutrophil functions will be presented.This work is supported by the Canadian Institutes of Health Research (CIHR). Human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (AMPs). To identify human skin AMPs, we analysed extracts of healthy persons stratum corneum by reversed phase-HPLC and purified a novel 9 kDa AMP that showed sequence similarity to mouse hornerin. Suggesting that it originates from the human ortholog, we cloned it. Human hornerin encodes a 2850 amino acid protein that contains a S100 domain, an EF-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused S100 protein family. Strongest constitutive hornerin mRNA expression was seen in differentiated keratinocyte cultures. To follow the hypothesis, that hornerin fragments represent AMPs, we recombinantly expressed three hornerin peptides, rHRNR2 (tandem repeat unit B), rHRNR3 (tandem repeat unit A) and rHRNR4 (C-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. The rHRNR3 peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The other peptides were found to be not or nearly not antimicrobially active. Our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed AMPs, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. (1), N Lu(1), R Jonsson(2), D Gullberg (1) (1) Department of Biomedicine, University of Bergen, Norway (2) The Gade Institute, University of Bergen, Norway a11ß1 is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. Previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. In this study we investigated the factors that regulate mouse integrin a11ß1 expression. Specifically, we have analyzed the influence of cell passage, growth factors and the 3-D microenvironment. Using SV40 immortalized as well as primary fibroblasts, we show that a11ß1 integrin is up-regulated when these cells are cultured within stressed collagen type I lattices. However, a11ß1is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. We also show here that a11 is upregulated by TGF-a on planar substrates. These findings suggest that mechanical tension and TGF-a are important factors in the regulation of a11ß1 that need to be to taken into consideration when evaluating the role of a11ß1 in wound healing and fibrotic disorders. (1), N Vergnolle (1), P Andrade-Gordon (2) (1) Inflammation Research Network, University of Calgary, Canada (2) RW Johnson Pharmaceutical Research Institute, Canada

The objective of this study was to investigate the effects of PAR2 deficiency in various models of colonic inflammation in order to elucidate the role of endogenous PAR2 in the process of inflammation in the gut.Colonic inflammation in C57BL6 wildtype and PAR2 -/-mice was induced by treatment with 2.5% DSS (in drinking water) or TNBS (1mg or 2mg in 100ul of 40% ethanol, single intracolonic injection) or pre-sensitizing mice with 3% oxazolone (in olive oil) applied to the skin of the abdomen, and 7 days later, a single intracolonic injection of 1% oxazolone (dissolved in 50% ethanol).Intravital microscopy was performed, 7 days (TNBS/DSS) or 4 days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.Lastly, various parameters of inflammation were assessed following the intravital microscopy.PAR2 -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in DSS, and TNBS challenge. In all three challenges, MPO activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to PAR2 -/-.Our evidences indicate that deficiency in PAR2 attenuates inflammatory responses in the experimental models of colitis associated with either Th1 (TNBS/DSS) or Th2 (oxazolone) cytokine profile.Therefore, PAR2 deficiency in the gut exerts antiinflammatory properties that are independent of Th1 or Th2 cytokine profile.The present study further highlights PAR2 as a potential target for inflammatory bowel diseases. (1), N Vergnolle (1), P Andrade-Gordon (2) (1) Inflammation Research Network, University of Calgary, Canada (2) RW Johnson Pharmaceutical Research Institute, Canada

In a previous study, inflammatory responses induced by three different models of colitis (TNBS/DSS/oxazolone) were significantly attenuated in mice deficient for PAR2 (PAR2-/-). Among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by PAR2 deficiency. Aim of this study was to assess the effects of PAR2 deficiency (via PAR2-/-mice) on the recruitment of leukocyte in colonic venules. In anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fMLP (100 mM) or PAF (100 nM) or by intraperitoneal injection of TNF-a; (0.5 mg -given 3 hours before the intravital microscopy). Using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of PAR2 -/-versus PAR2+/+ mice. fMLP and PAF as well as TNF-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. PAR2 -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fMLP topical administration. Leukocyte adherence induced by fMLP and TNF-a; was significantly lower in PAR2 -/-mice compared to wild types as well. No difference was observed between PAR2 -/-and wildtype for leukocyte rolling/adherence-induced by PAF. The lack of functional PAR2 attenuated leukocyte trafficking in response to fMLP and TNF-a; but not to PAF. The involvement of PAR2 activation in mouse colon leukocyte trafficking highlights PAR2 as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. (1), KK Hansen(2), K Chapman(2), N Vergnolle (2), EP Diamandis (1), MD Hollenberg (2) (1) Advanced Center for Detection of Cancer, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada (2) Proteinases and Inflammation Network, University of Calgary, Calgary, AB, Canada Kallikreins (KLKs) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. We have recently shown that KLKs can activate proteinaseactivated receptors (PARs), a family of G-protein coupled receptors associated with inflammation. We hypothesized that like trypsin, kallikreins can trigger inflammation via the PARs. We studied the ability of KLKs 6 and 14 to activate PARs 1, 2 and 4 in vitro and to cause oedema in a mouse model of paw inflammation in vivo. We found that KLK14 is able to activate both of PARs 2 and 4 and to prevent thrombin from activating PAR1. On the other hand, KLK6 was a specific activator of PAR2. Kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. The oedema was accompanied by a decreased threshold of mechanical and thermal nociception. Our data demonstrate that by activating PARs 2 and 4 and by inactivating PAR1, kallikreins, like KLKs 6 and 14, may play a role in regulating the inflammatory response and perception of pain. (1), D Park (1), B Short(2), N Brouard(2), P Simmons(2), S Graves (1), J Hamilton (1) (1) Melbourne University, Melbourne, Victoria, (2) Peter MacCallum Cancer Institute. Melbourne, Australia

Mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the Sca-1 antigen. Such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. Using this model we investigated the influence of the proinflammatory cyto-kines, TNFa or IL-1b, on early osteogenesis in vitro. Under osteogenic conditions, IL-1b was found to inhibit cell proliferation in a dose dependent manner, whereas TNFa exhibited no effect. Histochemical examination revealed the presence of either TNFa or IL-1b to dramatically decreased mineralization in a dose dependent manner. Q-PCR analysis indicated that in the presence of IL-1b, despite increased expression of bone-specific alkaline phosphatase (Akp2) mRNA, levels of other osteogenesis markers (Runx2, Col1a and Sp7) were decreased. In the presence of TNFa, levels of Akp2, Runx2 and Sp7 were all decreased. Our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. Using ARE-driven and NF-kB-targeted reporter genes, transfection of the NF-kB p65 subunit and Nrf2 into HepG2 or other cells, as well as siRNA technique to knockdown endogenous p65 in cells, we found that NF-kB p65 subunit repressed the anti-inflammatory and anticarcinogenetic Nrf2-ARE pathway at transcriptional level. In p65-overexpressed cells, the ARE-dependent expression of heme oxygenase-1 was strongly repressed. In the cells where NF-kB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized Nrf2 transcriptional activity. The p65-mediated ARE inhibition was independent of the transcriptional and DNA-binding activities of p65. Co-transfection and RNA interference experiments revealed two mechanisms which coordinate the p65-mediated repression of ARE: (1) p65 selectively deprives CREB binding protein (CBP) from Nrf2, but not MafK, by competitive interaction with the CH1-KIX domain of CBP, resulting in inactivation of Nrf2 transactivation domain and concomitant abrogation of the Nrf2-stimulated coactivator activity of CBP; (2) p65 promotes recruitment of histone deacetylases 3 (HDAC3) to ARE by enhancing the interaction of HDAC3 with either CBP or MafK, leading to inactivation of CBP and deacetylation of MafK. This study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the ARE-dependent gene expression by p65 subunit. Since various inflammatory and tumor tissues constitutively overexpresses p65 in their nuclei, the finding in this study implies a strong repression of ARE-dependent gene expression must take place in those tissues. In this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. Dendritic cells (DC) play a pivotal role in the induction of immune response and tolerance. It is less known that DC accumulate in atherosclerotic arteries, where they might activate T-cells and contribute to the progression of disease. The serine protease thrombin is the main effector protease of the coagulation cascade. Thrombin is also generated at sites of vascular injury and during inflammation. Hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. Thrombin activates various cells via protease-activated receptors (PARs). Immature DC do not express PARs. Upon maturation with LPS, TNFalpha, or CD40L, only LPS-matured DC expressed PAR1 and PAR3 on their surface. Stimulation of DC with thrombin, PAR1-or PAR3-activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alphamatured DC. The thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. Stimulation of PARs with thrombin or respective receptoractivating peptides led to activation of ERK1/2 and Rho kinase I (ROCK-I) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (MLC2). The ERK1/2-and ROCK-I-mediated phosphorylation of MLC2 was indispensable for the PAR-mediated chemotaxis as shown by use of pharmacological inhibitors of ROCK, ERK and MLC kinases. In addition, thrombin significantly increased the ability of mature DC to activate proliferation of naive T-lymphocytes in mixed leukocyte reactions. In conclusion, our work demonstrates expression of functionally active thrombin receptors on LPS-matured DC. We identified thrombin as a potent chemoattractant for mature DC, acting via Rho/ ERK-signaling pathways. Data concerning the role of circulating modified low density lipoproteins (modLDL) in atherogenesis and other pathologies are scarce. One reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modLDL in vivo. We report a novel approach for specific labeling of human native LDL (nLDL) and modLDL (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated LDL) with the positron emitter fluorine-18 (18F) by either NH2-reactive N-succinimidyl-4-[18F]fluorobenzoate or SH-reactive N-[6-(4-[18F]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, 15-40%; specific radioactivity, 150-400 GBq/ mmol). Radiolabeling itself caused neither additional oxidative structural modifications of LDL lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. The approach was evaluated with respect to binding and uptake of 18F-nLDL and 18F-modLDL in cells overexpressing various lipoproteinrecognizing receptors. The metabolic fate of 18F-nLDL and 18F-modLDL in vivo was delineated by dynamic small animal PET studies in rats and mice. The in vivo distribution and kinetics of nLDL and modLDL correlated well with the anatomical localization and functional expression of LDL receptors, scavenger receptors, and receptors for advanced glycation end products. The study shows that LDL modification, depending on type and extent of modification, in part or fully blocks binding to the LDL receptor, and reroutes the modLDL to tissuespecific disease associated pathways. In this line, 18Flabeling of modLDL and the use of small animal PET provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. The p38 MAPK signaling pathway, which regulates the activity of different transcriptions factors including Nuclear Factor-ÞB (NF-ÞB), is activated in lesional psoriatic skin. The purpose of the present study was to investigate the effect of fumaric acid esters on the p38 MAPK and the down stream kinases MSK1 and 2 in cultured human keratinocytes. Cell cultures were incubated with dimethylfumarate (DMF), methylhydrogenfumarate (MHF) or fumaric acid (FA) and then stimulated with IL-1b before kinase activation was determined by Western blotting. A significant inhibition of both MSK1 and 2 activations was seen after pre-incubation with DMF and stimulation with IL-1b whereas MHF and FA had no effect. Also, DMF decreased phosphorylation of NF-kB / p65 (Ser276), which is known to be transactivated by MSK1. Furthermore, incubation with DMF before stimulation with IL-1b resulted in a significant decrease in NF-kB binding to the IL-8 kB and the IL-20 kB binding sites as well as a subsequent decrease in IL-8 and IL-20 mRNA expression. Our results suggest that DMF specifically inhibits MSK1 and 2 activations and subsequently inhibits NF-kB induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. These effects of DMF explain the anti-psoriatic effect of fumaric acid esters. A humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking B, T and NK cells. In this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. Inflammation is associated with the expression of activation markers and inflammatory medi-ators such as TNF-alpha, HLA-DR and CD1a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of Ki-67 and CK-16. Epidermal hyperplasia is a typical readout in this model. In a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of TNF-alpha, antibodies directed against growth factors, MMP-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine A.In addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (Caspary et al. Brit J Dermatol 2005; 153, 937-944) . Employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.Due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. 

Kristian Otkjaer (1), E Hasselager(2), J Clausen(2), L Iversen (1), K Kragballe (1) (1) Aarhus University Hospital, Denmark (2) Novo Nordisk A/S, Denmark

Interleukin-20 (IL-20) is assumed to be a key cytokine in the pathogenesis of psoriasis. Increased levels of IL-20 are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. Whether IL-20 is derived from antigen-presenting cells or keratinocytes remains unsolved. The aim of the present study was, therefore, to characterize IL-20 expression in non-lesional psoriatic skin ex vivo. 3 mm punch biopsies from nonlesional psoriatic skin were collected. Biopsies were transferred to CaCl enriched keratinocyte basal media and cultured with vehicle or IL-1beta (10ng/ml) for 0, 1, 2, 4, 6, 12 and 24 hours, respectively. The samples were analyzed by in situ hybridisation, qRT-PCR, immunofluorescent staining and ELISA. Incubation with IL-1beta rapidly induced IL-20 mRNA expression in the biopsies. The highest level of IL-20 mRNA was detected after 4 hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of IL-20 mRNA. Increased levels of IL-20 protein were detected in the supernatant of the IL-1beta stimulated biopsies. Immunofluorescent staining of the biopsies showed no IL-20 protein in the keratinocytes, whereas the IL-20 protein was present in epidermal CD1a positive dendritic cells. Our data emphasize the keratinocyte as the cellular source of IL-20 expression in human skin. Interestingly, immunofluorescent staining of our cultured biopsies showed IL-20 protein in epidermal dendritic cells whereas no IL-20 was detected in the keratinocytes. This indicates that epidermal dendritic cells are the target for keratinocytederived IL-20. One response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (MMPs) that participate in tissue remodeling. Excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. Calcitriol, the hormonally active form of vitamin D, is known to have beneficial effects during cutaneous inflammation. We hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive MMP proteolytic activity. Our experimental model consists of HaCaT keratinocytes cultured with TNF to simulate an inflammatory state. pro-MMP-9 was quantified by gelatin zymography and mRNA by real-time PCR. The levels and activation of signaling proteins were determined by immunoblotting. The increase in pro-MMP-9 activity and mRNA levels induced by TNF was inhibited by~50% following 48h treatment with calcitriol. Using specific inhibitors we established that the induction of MMP-9 was dependent upon the ERK pathway, while p38-MAPK and PKC inhibited, and Jun-kinase, PI-3-kinase and Src did not affect it. Levels of c-Fos, a component of AP-1 transcription complex known to mediate MMP-9 induction, were elevated by TNF and further increased by calcitriol. The induction of MMP-9 by TNF was abolished by inhibition of the EGFR tyrosine kinase attesting to the requirement for EGFR trans-activation. Calcitriol also inhibited the induction of MMP-9 by EGF. We conclude that calcitriol inhibits the induction of MMP-9 gene expression by TNF in keratinocytes by affecting an event downstream to the convergence of the EGFR and the TNF signaling pathways. (1), P Verzaal (1), T Lagerweij (1), C Persoon-Deen (1), L Havekes (1), A Oranje (2) (1) TNO Pharma, Department of Inflammatory and Degenerative Diseases, Leiden, The Netherlands (2) Erasmus Medical Center, Department Dermatology and Venereology, Rotterdam, The Netherlands

Mice with transgenic overexpression of human apolipoprotein C1 in liver and skin display a strongly disturbed lipid metabolism. Moreover, these mice show a loss of skin-barrier function evident from increased Trans Epidermal Water Loss. These mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. Both hyperplasia of epidermis and dermis are observed. Histological analysis shows increased numbers of CD4+ T cells, eosinophils, mast cells and IgE-positive cells in the dermis. Serum levels of IgE are increased as well. Cytokine profiling of draining lymph nodes is in favor of a Th2-mediated disease. Development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. Moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. Impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.This model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. Topical immunosupppressants such as Elidel and Protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-When delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. We speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.Accordingly, we have designed and discovered a series of "soft" cyclosporin A (CsA) derivatives as potentially safer alternatives.In general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.In this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.The results or our drug discovery efforts around soft CsA derivatives will be presented. (1), Y Sawanobori (2), U Bang-Olsen (3), C Vestergaard(4), C Grønhøj-Larsen (1) Background: A strain of Japanese fancy-mice, NC/Nga, serves as a model for atopic dermatitis. Under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. Scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of IL-31 mRNA has been shown. Also, transgenic mice over-expressing IL-31 exhibit increased scratching behaviour and develop severe dermatitis. Consequently we decided to explore the therapeutic effect of an anti IL-31 antibody on scratching behaviour and dermatitis in NC/Nga mice.

Methods: Prior to clinical manifestation of dermatitis, we commenced treatment of NC/Nga mice with IL-31 ratanti-mouse 10 mg/kg intraperitoneally every fifth day for seven weeks. Clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. Serum analysis for IgE and IL-13 as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point.

Results: Taken over the entire intervention period, treatment with anti IL-31 antibody in NC/Nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. However, post hoc analysis revealed a significant reduc-tion of scratch by the anti IL-31 antibody treatment in the time interval day 22-43.

Our results suggest an anti pruritic role for IL-31 antibody in an atopic dermatitis-like animal model. Anti IL-31 antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. (1), P Ferro (1), HM Asnagli (1), V Ardissone (1), T Ruckle (2), F Altruda (3), CH Ladel (1) (1) RBM Merck Serono/University of Torino, Italy (2) Merck Serono Pharmaceutical Research Institute, Geneva, Switzerland (3) University of Torino, Dipartimento di Genetica, Biologia, Biochimica, Italy Class-I phosphoinositide 3-kinases (PI3Ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. Since PI3K-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of PI3K-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. In our study the inhibitory properties of a selective PI3K-g inhibitor AS605858 on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. Two mouse models were used: the first, Irritant Contact Dermatitis (ICD), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. The second, Contact Hypersensitivity (CHS) is a T-cell dependent model, modeling in part T-cell-mediated skin diseases such as psoriasis. We demonstrated the therapeutic effect of PI3Kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective PI3K-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (ICD) -effective dose 0,3mg/kg p.o. once -as well as in T-cell mediated skin pathology (CHS)effective dose 1mg/kg p.o. bid. We conclude that the mechanism of action related to inhibition of PI3K-g are demonstrable after oral administration of selective inhibitors like AS605858 in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. Introduction: High Mobility Group Box 1(HMGB1) has recently been identified as a late mediator of endotoxin lethality. We newly developed an extra corporeal HMGB1 absorber. The purpose of this study was to test the hypothesis that HMGB1 removal could prevent or reduce endotoxin induced lethality or tissue injury of rats.

Methods: All experiments were conducted in accordance with the Institutional Care and Use Committee.Male Wistar rats were randomly allocated into three groups; HMGB1 absorber group (Group I),HMGB1 nonabsorber group (Group II), and Vacant column group (Group III).We applied these columns for each groups at 6 hours after LPS injection. The rats were sacrificed 48 hours after LPS injection for pulmonary histology. We statistically analyzed survival rate with Kaplan-Meier and the levels of HMGB1 with ANOVA.

Results: Survival rate was 67% in the Group I at 48 hours after LPS injection, as compared with 0% in the Group II and 11% in the Group III. The pulmonary histology in both Group II and Group III showed acute inflammatory injuries, whereas Group I showed less inflammatory changes.The level of HMGB1 in the Group I was significantly lower than those of Group II and III. Discussions:These results demonstratethat specific absorption of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines.

Contact information: Dr Hideo Iwasaka, Oita University, Anesthesiology and Intensive Care Unit, Yufu City, Japan E-mail: hiwasaka@med.oita-u.ac.jp (1) (1) Department of Pharmacology, National University of Singapore, Singapore (2) DSO National Laboratories, Singapore

Hydrogen sulfide (H2S) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. In this study, we have investigated the role of H2S in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. Male Swiss mice were subjected to cecal ligation and puncture (CLP) induced sepsis and treated with saline, DL-propargylglycine (PAG, 50 mg/kg i.p.), an inhibitor of H2S formation or sodium hydrogen sulfide (NaHS) (10mg/kg, i.p.), a H2S donor. PAG was administered either 1 hour before or 1 hour after induction of sepsis while NaHS was given at the time of CLP. Using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of PAG significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mRNA and protein levels of adhesion molecules (ICAM-1, P-selectin and E-selectin) in lung and liver. In contrast, injection of NaHS significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. On the other hand, normal mice were given NaHS (10mg/kg i.p.) to induce lung inflammation with or without pretreatment of NF-x×B inhibitor, BAY 11-7082. H2S treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. These alterations were reversed by pretreatment with BAY11-7082. Moreover, expression of CXCR2 in neutrophils obtained from H2S treated mice was significantly upregulated leading to an obvious elevation in MIP-2 directed migration of neutrophils. Therefore, H2S act as an important endogenous regulator of leukocyte trafficking during inflammatory response. Transient receptor potential vanilloid 1 (TRPV1) is primarily found on sensory nerves. We have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. Selective TRPV1 antagonists are not available for use in the mouse in vivo, thus established TRPV1 knockout (-/-) mice were used. C57BL6 WT and TRPV1-/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (LPS). The response was monitored for 4h. Blood pressure, measured before and at intervals after LPS in conscious mice via a tail cuff was reduced in both WT and TRPV1-/-mice, with TRPV1-/-mice showing an enhanced drop at 4h. In a separate group temperature, a proposed pre-mortality marker was also reduced by 4h, again with a significantly increased drop in TRPV1KO. Furthermore higher levels of two inflammatory markers TNFa and nitrite (as an indicator of NO) were measured in peritoneal lavage and higher levels measured inTRPV1-/-as compared with WT samples. Finally aspartate aminotransferase (AST) levels also enhanced in TRPV1-/-versus WT mice, although markers for kidney and pancreatic damage were similar in both genotypes. We conclude that TRPV1 plays a protective role in sepsis. TRPV1 is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. (1), DA Souza-Junior(2), L De Paula (1), MC Jamur (2), C Oliver (2), SG Ramos (2), CL Silva (2), Lucia Helena Faccioli (1) ( H37Rv) . Infected Balb/c mice developed an acute pulmonary inflammation and higher levels of TNF-a, IL-1, KC, MCP-1 and MIP-2 were detected in the lungs by day 15. In vivo degranulation of mast cells by C48/80 led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. The magnitude of cellular immune response was also partially impaired in infected mice and treated with C48/80. Histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with C48/80. Of interest, the number of mycobacterial bacilli recovered from the lungs was 1 log higher after treatment of infected mice with C48/80. These findings suggest that MCs participate in host defense against M. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. (1), MS Chadfield (2), DB Sørensen (1), H Offenberg (1), M Bisgaard (1), HE Jensen (1) (1) Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Denmark (2) Novo Nordisk A/S, Cell Biology, Gentofte, Denmark

Introduction: Pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. The aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of P. multocida of different origin in an aerogenous murine model. Materials and Methods: 30 female BALB/ c-J mice (app. 20 g, Taconic, Denmark) were infected intranasally with two clinical isolates of P. multocida of avian (VP161) and porcine (P934) origin, at three different levels of inocula concentration. After euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. Furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase MMP-9 and metalloproteinase inhibitor TIMP-1.

Results: All mice infected with the avian strain were euthanized after 24 hours. Viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. In the liver, disseminated necrosis with formation of microabscess was also seen. On the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after 24, 48 and 72 h. Furthermore, differences were seen in the nature of the lesions caused by the two strains. There was a difference in expression of MMP-9 and TIMP-1 between infected and noninfected mice.

The model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of MMP-9 and TIMP-1. (1), R Molinaro (1), A FranÅa (1), M Bozza (1), F Cunha(2), S Kunkel (3) (1) Universidade do Rio de Janeiro, Brazil (2) Universidade de S¼o Paulo, Brazil (3) University of Michigan, USA Introduction and Objectives: Studies reveal that regulatory T (Treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. CCR4 knockout mice (CCR4 -/-) are more resistant to LPS shock. So, our aim is to study the mechanisms involved in the resistance of CCR4-/-subjected to severe sepsis by cecal ligation and puncture (CLP) and how Tregs modulate this effect.

Results: C57/BL6 mice were subjected to CLP model, whereby the cecum was partially ligated and puncture nine times with a 21G needle. Sham-operated mice were used as control. Mice subjected to CLP and SHAM surgery were treated with antibiotic since 6h after and until 3 days. CCR4 -/-mice subjected to CLP presented an increase in survival rate (78%) compared with wildtype mice (17%), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. Besides, Tregs from CCR4-/-CLP mice did not inhibit proliferation of T effector cells as observed for Treg from wild CLP mice, at a proportional ratio of Teffector:Treg. Interesting, Treg from CCR4-/-CLP mice did not inhibit neutrophil migration to BAL when co-injected with fungal challenge as secondary infection, while the Treg from wild CLP mice did, as expected.

Conclusions: These results suggest that Treg cells from CCR4-/-mice did not present suppressive response and it could be an important factor in their survival.

Inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. Inflammation has been associated with diseases like cancer, diabetes and many other. Proinflammatory cytokine (TNF -µ and IL-1â) and NO are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. Thus inhibition of pro-inflammatory cytokines and NO production are important targets for treatment of inflammatory disorders. Nowadays due to the emerging side effects of COX inhibitors, these targets have been paid more attention for the treatment of these conditions. Some medicinal plants such as Curcuma longa (1), Commiphora mukul (2) In humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. Antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated B lymphocytes, eliminated after a few days or weeks by apoptosis. However, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without DNA-synthesis and refractory to signals from antigen or antigen-antibody complexes. The lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. The niche provides survival signals like IL-6, CXCL12 and TNF. Within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. The number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. Thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. This competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like CXCL12, which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. Plasmablasts and plasma cells express CXCR4, the receptor for CXCL12. While plasmablasts migrate in response to CXCL12, plasma cells depend on it for survival in the niche, but are no longer migratory. Thus once disloged from their niche, they will die. Plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express CXCR3, the receptor for the interferon-gamma-induced chemokines CXCL9, 10 and 11, which may lure the plasmablasts into inflamed tissue. The switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. Inflamed tissue contains survival niches for plasma cells.

In the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. Upon resolution of the inflammation the plasma cells will be dislodged and die. Longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory B cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. In chronic inflammation, this mechanism can contribute to pathogenesis. Thus in the NZB/W model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. Later, in established disease, autoreactive plasma cells are shortlived and continuously generated. They do not compete with the longlived plasma cells, and both populations coexist as prominent populations. Interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. This may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, (1), H Lee (2) C5a is a potent inflammatory mediator produced during complement activation. Unregulated C5a signalling through its receptor (C5aR) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. Considerable effort has gone into development of C5aR antagonists for human therapy. We took neutrophils from genetically modified human C5aR knock-in mice in which the mouse C5aR coding region was replaced with human C5aR sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs inhibit C5a-induced neutrophil migration and calcium-flux, and bind to a region of the 2nd extracellular loop of C5aR loop that seems to be critical for receptor activity. This study investigated the effectiveness of these mAbs in the K/BxN serum-transfer model of inflammatory arthritis. Human C5aR knock-in mice were given 1-10 mg/kg mAb intraperitoneally, before or after inflammatory arthritis developed. Mice treated with anti-C5aR mAb one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. Histopathology of the joints in anti-C5aR mAb-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. Furthermore, and most significantly, a single 1 mg/kg dose of anti-C5aR mAb given 5 days after initiation of disease completely reversed inflammation. In the collagen-induced arthritis (CIA) model, injection of anti-C5aR mAb after development of inflammation also reversed inflammation to baseline. These potent new antibodies to human C5aR are in preclinical development. The cytokine macrophage migration inhibitory factor (MIF) participates in fundamental events in innate and adaptive immunity. The profile of activities of MIF in vivo and in vitro is strongly suggestive of a role for MIF in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (RA), asthma, and sepsis. MIF also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of MIF is in fact induced by glucocorticoids. Thus, MIF functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. Therapeutic MIF antagonist may therefore provide a specific means of steroid sparing. Since MIF are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.We developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against MIF.The antibody can neutralize MIF activity in cell based assays, and is very effective in a LPS induced mouse sepsis model. Using this antibody as a tool, we are studying the function of MIF in comparison with the function of LPS.We found that LPS induced iNOS expression and NO secretion are dependent on the secretion of MIF.We also found that although both LPS and MIF induce G1 arrest in macrophage cell line Raw264.7, their functions are independent to each other. structure-based small molecule drug design. An effective agent would be the first orally active cytokine antagonist.

Methods: Collagen-induced arthritis (CIA) was induced in DBA-1 mice by immunisation with bovine type II collagen/adjuvant on day 0 and 21. COR100140, synthesized on the basis of computer modeling of MIF protein X-ray crystallographic data, was administered by daily oral gavage from day 21. Etanercept (8mg/kg IP q2d) was used as a positive control. The MEK-ERK pathway is activated in numerous inflammatory conditions, including RA, IBD and COPD. ARRY-162 is a potent (IC50 = 12 nM), selective, ATP-uncompetitive MEK1/2 inhibitor.ARRY-162 is highly efficacious in CIA and AIA rat models, with ED50s of 3 and 10 mg/kg, respectively, equal to or better than standard agents. Addition of ARRY-162 to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. In TPA-stimulated human whole blood, this compound inhibited TNF, IL-1 and IL-6 production (IC50s of 23, 21 and 21 nM, respectively). In contrast, inhibition of pERK required 280 nM to achieve a 50% reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to pERK inhibition.In clinical studies, healthy volunteers were administered a single oral dose of 10, 20, 30, 40 or 80 mg); blood was drawn at various times after dosing and stimulated ex vivo with TPA. ARRY-162 was well-tolerated and drug exposure was dose-proportional. In ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of TPAinduced IL1 and TNFfz with >80% inhibition observed at plasma concentrations of 50 and 150 ng/ml, respectively. Similar inhibition of pERK required 300 ng/ml. A multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of ARRY-162 and helped define tolerability. Clinical evaluation of ARRY-162 in combination with methotrexate in patients with rheumatoid arthritis is on-going. p38 (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as TNFand IL-1a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. Inhibition of p38 activity is expected to regulate the levels of TNF-a and IL-1b thereby alleviating the effects of inflammation in RA. A new class of p38 inhibitors based on the naphthyridininone scaffold have been discovered. X-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template.

This presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. (1), H Aaes (1), W-H Boehncke(2), J Pfeffer(2), T Skak-Nielsen(1), I Teige (1), K Abell (1), PH Kvist (1), E Ottosen (1), TK Petersen (1), Lars Svensson (1) (1) Discovery, LEO Pharma, 55 Industriparken, Ballerup, Denmark (2) Department of Dermatology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany P38 MAP kinase plays an important role in mediating an inflammatory response in mammalian cells. As a consequence of activation, several inflammatory mediators are released including IL-1B] and TNFa. Both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. Approximately 30% of psoriasis patients develop psoriatic arthritis. LEO15520 is a member of newly developed class of selective p38 MAP kinase inhibitors. The compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. The in vivo models selected include the CIA arthritis model, the human psoriasis xenograft SCID mouse model, the UVB-induced dermatitis model, the LPS induced TNFa model and a local GvH model. Treatment with LEO15520 led to an amelioration of the ongoing inflammation in all investigated in vivo models. In the CIA model, a clear dose response effect was observed on the developing arthritis in both rats and mice (65 % reduction in mice and 77 % in rats at 10 mg/kg p.o.). In the humanised psoriasis model, LEO15520 at a dose of 20 mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by 41%) and on the infiltrating inflammatory cells. The anti-inflammatory effect of LEO15520 was even close to the effect of systemically delivered steroids in both models. We believe that the new highly selective class of p38MAP kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. SLx-2119 is a potent, selective, orally bioavailable inhibitor of the Rho-kinase ROCK-2. Its IC50 for ROCK-2 and ROCK-1 inhibition is 100 nM and >10 mM respectively. The ability of SLx-2119 to inhibit septic liver injury was investigated in C57BL/6J mice challenged with lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Mice were given LPS (10 mg/mouse) and D-Gal (18 mg/ mouse) i.p and 5.5 hours later were sacrificed for analysis of liver injury. Mice challenged with LPS/D-Gal had a >10 fold increase in serum ALT and AST levels. This increase was reduced by >90% in mice pretreated with SLx-2119 either orally (100 mg/kg, 2 and 20 hrs prechallenge) or i.p. (10-100 mg/kg, 15 min pre-challenge). SLx-2119 inhibited the increase in hepatic levels of TNFfalpha produced by LPS/D-Gal by >50%. To assess the kinetics of SLx-2119s benefit, SLx-2119 (100 mg/kg i.p.) was given 15 min prior to, or 15 or 60 min after the LPS/ D-Gal. SLx-2119 was effective at inhibiting the rise in ALT and AST levels at all 3 time points suggesting that inhibiting ROCK-2 even after the initiation of the LPS/D-Gal driven cascade protects against septic liver injury. In a survival study, 9 out of 10 mice given LPS/D-Gal were dead by 8 hrs whereas in mice given SLx-2119 (100 mg/kg orally) 1 animal died at 10 hours and the remaining 9 mice were alive 48 hrs later. These results show that specific inhibitors of ROCK-2 may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.They potently inhibit protein kinase C activity in vitro as demonstrated by inhibition of IL-2 secretion in human purified T-cells stimulated with anti-CD3 and anti-CD28 or whole blood SEB challenge.The PKC-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. (1), J Zhang (1), K Henley (1), M White (1), D Hilton (1), B Kile (1) ( To investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human Fcgam-maRIIa in inducible and passively transferred models of arthritis. Transgenic mice developed severe RA-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. Disease could be reduced by the administration of either specific monoclonal antibodies to FcgammaRIIa or small chemical entities (SCE) designed to bind to the Fcgam-maRIIa dimer. To investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to C57BL/6 control mice was examined using phagocytosis of fluorescent beads coated with OVA or OVA/anti-OVA immune complex or opsonised sheep red blood cells. In both assays macrophages from FcgammaRIIa transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at 4 hours.This difference diminished over time andwas only seen where particles were opsonised. Treatment of macrophages with specific FcgammaRIIa blocking monoclonal antibody fragments 8.7 F(Ab)2 or IV.3 F(Ab) reduced phagocytosis to background levels . Macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines TNF-alpha and IL-1beta when stimulated in vitro with heat aggregated immunoglobulin (HAGG). Moreover, this response was also blocked by specific FcgammaRIIa monoclonal antibody fragments or SCE. Thus, expression of the FcgammaRIIa transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased TH1 cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. Harald Burkhardt (1), U Hüffmeier (2), I Kçnig (3), J Lacorz (2), A Reis (2), K Reich (4) (1) Johann Wolfgang Goethe University, Frankfurt, Germany

(2) Friedrich-Alexander-Unisversity of Erlangen-Nuremberg, Germany Results: Whereas the earlier described strong association of allele TNF*-238A with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the PSORS1 risk allele. For PsA, but not psoriasis vulgaris without joint involvement strong association with the allele TNF*-857T was detected (OR=1.956, 95% CI 1.33-2.88; pcorr=0.0025) also in patients negative for the PSORS1 risk allele.

Our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. While the previously reported association between TNF*-238A and psoriasis seems to primarily reflect LD with PSORS1, TNF*-857T may represent a risk factor for PsA independent of PSORS1. (1), N Modi (1), M Stanford (1), E Kondeatis (1), R Vaughan (1), F Fortune (2), W Madanat (3), C Kanawati(4), P Murray (5) Methods: DNA was obtained from 167 patients with BD, 61 from the UK and 106 from the Middle East (ME), and 159 controls individuals, 129 from the UK and 30 from ME. DNA was prepared by proteinase K digestion, and salt extraction and -318 and 49 SNP were detected by a PCR-SSP.

Results: There was no significant difference in expression of -318C/T or 49A/G when all BD patients were compared to all control individuals, (p=0.3 and 0.9, respectively). As we have previously shown differences in SNP expression in different patient groups we tested the UK and ME patients separately. However, there was no significant difference in either SNP in UK BD patients, (p=0.15, p=0.39) or ME BD patients (p=0.7, p=0.6) when compared to the appropriate controls. (1), DA Brown (1), H Johnen (1), MR Qiu (1), T Kuffner (1), PGM Curmi (1), L Brown (1), M Mazzanti (2), SN Breit (1) ( Introduction: Cerebral palsy (CP) is a nonprogressive motor disorder caused by white matter damage in the developing brain. It is often accompanied with neurocognitive and sensory disabilities. The cause and pathogenesis of CP is multifactorial and continues to be poorly understood. Chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for CP both in term and preterm infants. IL-16 gene is single copy gene located on chromosome 15q26.1-3 in humans. Interleukin-16 is synthesized by a variety of immune (T cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. It is also detected in organ-specific secretions in a number of inflammatory processes. Amniotic fluid interleukin-16 concentrations decreased with advancing gestational age. But, women with preterm labor and women with chorioamnionitis have higher interleukin 16 amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. The aim of our study was to estimate allelic frequency for regulatory IL16 -295 SNP in the children with the CP.

Methods: DNAs obtained from peripheral blood of 46 CP patients and 182 unrelated healthy volunteers were genotyped for the IL16 -295 SNP PCR-RFLP method.

Results and Conclusions: IL16 -259 genotype fnCC was more common in the population with cerebral palsy in the comparison to healthy volunteers. The significance of the association between IL16 fngene polymorphisms and cerebral palsy has to be investigated in the future studies. (1), D Delbro (1), E Hansson (2) (1) Kalmar University, Sweden (2) Gçteborg University, Sweden

Background: Acetylcholine (ACh) is a major signalling molecule, binding partly at nicotinic receptors (nAChRs; a family of ions channels with nicotine as a selective ligand). One subtype, the alpha7nAChRs, has antiinflammatory effects by way of down-regulation of TNFalpha release from macrophages. The alpha7nAChRs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. Aim. In rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: 1. The expression of alpha3nAChRs and alpha7nAChRs by immunofluorescence and Western blot. 2. Intracellular Ca2+-transients spread within the astroglial networks after stimulation with nicotine. 3. Pro-inflammatory cytokines, Il-1b, Il-6, and TNFalpha, released from microglial cells after stimulation with nicotine.

Results: alpha7nAChRs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. The Ca2+ transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. These Ca2+ responses were blocked by the alpha7nAChR antagonist alpha-bungarotoxin. The release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine.

Conclusion: alpha7nAChRs appear to be involved in some of the effects of nicotine administration to rat astrocytes. An anti-inflammatory action of cholinergic nerves on the astrocytes via nAChRs seems probable, which may have therapeutic implications.

University, Department of Natural Sciences, Kalmar, Sweden E-mail: ann.pettersson@hik.se

Gedeon Richter Plc., Budapest, Hungary

Tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of NSAIDs. Review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (Cohran Database 2006). Efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. Our aim was to assess oral efficacy of tramadol at side effect free doses in rats. Anti-hyperalgesic effect was determined in Complete Freunds Adjuvant (CFA) induced thermal hyperalgesia test (TH) and in model of CFA-induced knee joint arthritis. Effect on inflammation was characterized in carrageenan induced paw edema test (ET). For the characterization of side effect accelerating rotarod assay (ARR) was used. Significant impairment in ARR was noted at 55 mg/kg dose, and above. In the TH test weak 35% effect was seen at side effect free dose (40 mg/kg). In the arthritis model b.i.d. 40 mg/kg dose of tramadol given on days 3-7 after CFA caused a maximum 86% effect on weight bearing incapacitance (Day 4). However, its effect seemed to diminish (34% on Day 7) upon repeated treatment. In ET tramadol had significant anti-inflammatory effect at 100 but not at 30 mg/kg dose. These results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. Chronic relapsing experimental allergic encephalomyelitis (CR EAE) is a disease that bears striking similarities to the human condition multiple sclerosis (MS). In particular, CR EAE and MS have major inflammatory events in the central nervous system (CNS) that culminate in demyelination and disruption of axonal function.One group of mediators involved in the progressive CNS inflammation are the prostaglandins (PGs).PG generation is regulated in experimental non-immune conditions of the CNS by N-methyl-D-aspartate (NMDA) receptor activation.NMDA receptor-mediated events are also evident in EAE and may therefore have the potential to influence PG production.The study was designed to profile PGE2 and PGD2 in CNS tissues during the development of CR EAE and to examine the role of the NMDA receptor in PG production through the use of the specific antagonist MK-801.Biozzi mice were inoculated for CR EAE and CNS tissues were sampled during the course of disease.Enzyme immunoassay of processed samples revealed PGE2 and PGD2 levels within normal limits during the acute and subsequent remission phases of CR EAE.In contrast, dramatic changes in PG concentrations were observed with a relapse of symptoms and a remission of disease.MK-801 was therapeutically administered to CR EAE-diseased mice at the onset of relapse and changes in CNS PG levels were recorded.The relapse phase of CR EAE, but not the acute stage of disease, is characterised by an increase in CNS PG production that may be influenced by NMDA receptor activation.

Livia L Camargo(1), LM Yshii (1) (1), SK Costa (1) (1) University of S¼o Paulo, Brazil (2) King's College, London, UK (3) Butantan Institute, S¼o Paulo, Brazil

Objectives: The neuropeptide substance P (SP) released by capsaicin-sensitive nerves (CSN) plays a pivotal role in neurogenic inflammation. Despite the prevalence of arthritis, the contribution of SP to the progression of arthritis has not been established. This study investigated the effect of CSN ablation and SR140333, a SP antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin (10 %, 5 h time course) in female Wistar rats.

The kaolin-injected knee (ipsilateral -IPSI) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. In addition, increased pain score and high levels of myeloperoxidase (MPO, marker of neutrophil accumulation) and both pro-inflammatory (IL-1b and IL-6, but not TNFa) and anti-inflammatory (IL-10) cytokines was detected in the IPSI synovial fluid of these animals. Both destruction of knee joint CSN fibres by neonatal capsaicin treatment and i.a. injection of rats with SR140333 (1 nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. In contrast, the same treatment caused increased MPO activity and cytokine concentrations measured 5 h post kaolin injection.

Conclusions: Peripheral release of SP after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. However, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. (1) (1) University of S¼o Paulo, S¼o Paulo, Brazil (2) Butantan Institute, S¼o Paulo, Brazil

Objectives: Previously, intra-tracheal (i.tr.) injection of DEP or 1,2-naphthoquinone (1,2-NQ) evoked plasma extravasation and cell influx in rat airways. We now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. We also determined the ability of DEP and 1,2-NQ-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (RTA) and corpus cavernosum (RCC) reactivity using organ bath assays.

Results: Capsaicin-or vehicle-treated male Wistar rats received i.tr. injection of DEP (1 mg/kg) and 1,2-NQ (35 nmol/kg). After 15 min, DEP and 1,2-NQ produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. In capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for C-fibres, primarily tachykinins. Increased MPO and cytokine levels were detected in bronchi of capsaicin-treated rats following 3 h treatment with pollutants. This treatment contributes to augment the ACh (10-9-10-4 M)-induced relaxation in the RTA but not in the RCC. In capsaicin-treated rats, the RTA response to the pollutants was not affected but it was capable of evoking a marked relaxation in RCC in animals challenged with pollutants.

Conclusions: 1,2-NQ exacerbates DEP-induced plasma extravasation and MPO activity in the airways, indicating a neurogenic mechanism through tachykinins. The exposure to DEP and 1,2-NQ affects the endotheliumdependent response in RTA without interfering with RCC. Neuropeptides are unlikely to affect the pollutantsinduced changes in the RTA. Acknowledgements: CAPES, CNPq, Fapesp.

We thank MA Alves for technical assistance. Trans-resveratrol (RV) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. The purpose of this study was to determine the effect of RV on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (LPS) in glial cells. RT-PCR showed that RV (5, 25, 50 mM) dose-dependently inhibited 500 ng/ml LPS induced TNFa, IL-1b, IL-6, MCP-1 and inducible nitric oxide synthase mRNA expression. RV also inhibited LPS-induced production of these cytokines (ELISA), nitric oxide and reactive oxygen species in a dose-dependent manner. Western blot analyses showed that resveratrol could inhibit LPS-stimulated phosphorylation of ERK1/2 and JNK but not p38. NF-kB reporter assay showed RV could inhibit NF-kB activation by LPS in microglia and astrocytes. These results suggest that RV may inhibit LPS-induced microglial and astrocyte activation through ERK1/2, JNK and NF-kB signaling pathways. Therefore, RV is a natural product with therapeutical potential against disease conditions in CNS that involve an overproduction of proinflammatory cytokines and reactive oxygen species. (1), CS Patil(2), SV Padi (1), VP Singh (1) (1) University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India (2) Pharmacology R & D, Panacea Biotec Ltd. Lalru, Punjab, India Persistent stimulation of nociceptors and C-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. The important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (NO). The aim of the present study was to test the sensitivity of PDE5 inhibitor sildenafil in chronic constriction injury (CCI) model, a rat model of neuropathic pain. Sciatic nerve injury is associated with development of hyperalgesia 14 days after the nerve ligation. Sildenafil (100 and 200fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. The hyperalgesic effect of sildenafil was blocked by L-NAME and methylene blue (MB), which on per se treatment showed antinociceptive effect in nerve ligated rats. The results from the present study indicated that the major activation of NO cGMP pathway in the chronic constriction injury model of neuropathic pain. The aggravation of hyperalgesic response might be due to the increased cGMP levels resulting in PKG-I activation and its upregulation. Glycine transporter (GlyT) 1 and GlyT2 are expressed in glia and neurons, respectively. GlyT1 make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to NMDA receptors, while GlyT2 supplies glycine into synaptic vesicles in glycinergic neurons. Therefore, GlyT inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. The present study examined the effects of GlyT inhibitors on pain in animal models. Inhibitors of GlyT1 (sarcosine and ORG25935) and GlyT2 (ALX1393 and ORG25543) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. GlyT1 inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in Complete Freund Adjuvant (CFA)-induced inflammation mice but the antiallodynia effects appeared after latent period. On the other hand, GlyT2 inhibitors produced antiallodynia effects immediately after the i.t. injection in CFA-treated mice. These inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.Pretreatment of specific antagonists of glycine site of the NMDA receptors disappeared the latent periods of GlyT1 inhibitors and potentiated the antiallodynia effect. Glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected GlyT inhibitors. These results suggest that both GlyT1 and GlyT2 inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. The tumour microenvironment in particular tumour associated macrophages (TAMs) play a role in determining tumour outcome. Despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of TAMs in this disease. We have utilized our mouse model of gastric tumourigenesis, the gp130757FF mouse to assess the effect of the gp130757FF mutation on macrophage function and ascertain the role of macrophages in tumor formation. This mouse has a knockin mutation in the IL-6 family cytokine receptor gp130 preventing SHP2/ras/ERK signaling and leading to constitutive STAT3 transcriptional activation. Tumour development is inflammation dependent, there is a requirement for IL-11, and development is inhibited by NSAID treatment or microbial eradication, however an adaptive immune response is S410 Inflamm. Res., Supplement 3 (2007) Posters dispensable for tumorigenesis. In gastric antrum the gp130757FF mutation results, decreased ERK1/2 activation and constitutive phosphorylation of STAT3, abnormal signaling is replicated in macrophages. Antral STAT3 activation is unaffected by depletion of IL-6. However in macrophages an absence of IL-6 results in higher STAT3 activation demonstrating the anti-stimulatory role of IL-6 on macrophages. The gp130757FF mutation results in macrophages with decreased IL-6 and increased iNOS mRNA expression reflecting a more basally activated phenotype. Manipulations of the gp130757FF mouse that reduce tumor size (eg. antibiotic or NSAID treatment or STAT3 hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. Macrophages of gp130757FF mice display aberrant gp130 signaling potentially resulting in exaggerated response to stimuli. The key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. (1), Y Riffo-Vasquez(2), S Brain(2), S Costa (1) (1) University of S¼o Paulo, Brazil (2) King's College London, UK Objectives: We have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. This study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (WT) and TRPV1 knockout (KO) mice exposed to DEP and 1,2-NQ. DEP (1 mg/kg) and 1,2-NQ (35 nmol/kg)-induced airway inflammation was assessed via 125I-labelled albumin 1 h post pollutants injection. MPO assay and histopathology were performed 3 h after treatment. Staining of lung and trachea specimens with H&E provided a profile of cell infiltration in both WT and KO animals.

Results: Injection of DEP and 1,2-NQ evoked a potent plasma extravasation into the trachea and lung of WT, but not TRPV1 KO, suggesting these pollutants act via a TRPV1-mediated mechanism. In contrast, MPO activity in the airways of TRPV1 KO mice was exacerbated compared to WT mice data. Likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of TRPV1 KO mice compared to WT.

Conclusions: Inhibition of increased microvascular permeability in the airways of TRPV1 KO mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. However, neutrophils/macrophages accumulate more in TRPV1 KO, indicating that the lack of TRPV1 receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for TRPV1 receptors.

Acknowledgements: CAPES, CNPq, Fapesp. (1), N Sato(1,2), Y Endo (1), S Sugawara (1) (1) Division of Oral Immunology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, Sendai, Japan (2) Division of Fixed Prosthodontics, Department of Restorative Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan

Biotin is a water-soluble vitamin of the B complex and functions as a cofactor of carboxylases.Biotin deficiency causes alopecia and scaly erythematous dermatitis.Moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.However, immunological effects of biotin on allergic inflammation remain unclear.In this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (Ni)-allergy model mouse and a murine macrophage cell line J774.1. Female BALB/c mice (4 weeks old) received a basal diet or a biotin-deficient diet for 8 weeks.Ten days after sensitization with intraperitoneal injection of LPS and NiCl2, the mice were challenged with intradermal injection of NiCl2 into the pinnas.Allergic inflammation was measured by ear swelling.The Ethical Board for nonhuman species of the Tohoku University Graduate School of Medicine approved the experimental procedure followed in this study.J774.1 cells were cultured in biotin-sufficient or deficient medium for 4 weeks. Ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.IL-1 beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.Moreover, biotin-deficient J774.1 cells produced IL-1 beta significantly higher than biotinsufficient J774.1 cells.To investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for 2 weeks.Ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.These results indicated that biotindeficiency deteriorates allergic inflammation.The

augmentation of IL-1 beta production is probably involved in those deteriorations. One of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. Applying the ELISA technique, levels of IL-18, ICAM-1 and E-selectin were studied in 77 patients with acute pancreatitis. Mediators levels were studied in arterial, venous and pancreatic ascites samples. According the Atlanta criterion the mild pancreatitis was established in 33 patients and severe -in 44 patients. The highest levels of IL-18 were noted in ascites and lowest in arterial samples. The highest concentration of adhesion molecules was in venous samples and lowest in ascites. It was a clear correlation between levels of IL-18 and adhesion molecules and severity of pancreatitis. During first week the levels of IL-18 gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. ICAM-1 levels gradually increased during first three day with the following decrease after this term. The highest levels of E-selectin were noted at the time of admission. The clear correlation between IL-18 and adhesion molecules was noted in both groups of patients. Besides that, the clear strong correlation was observed between IL-18 and quantity of circulating granulocytes and between E-selectin and hematocrite in patients with necrotizing pancreatitis. Our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. Subsequently Eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. This is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. MC/Eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. We have previously described an MC/Eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. Still, pathways that mediate MC/Eos cross-talk in allergy are not fully characterized.

Methods: Human cord-blood derived MC were cocultured with peripheral blood Eos, and activated with anti-IgE. MC/Eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. Expression of surface molecules was analyzed by FACS. MC activation was measured by chromogenic assays for â-hexosaminidase. Relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation.

Results: MC and Eos physically interact, forming welldefined couples in-vitro and in-vivo. In the presence of Eos, MC are more releasable under baseline or IgEactivating conditions. This effect is partially mediated by CD48 and DNAM-1 on MC, and 2B4 and Nectin-2 on Eos.

We describe a novel physical interaction between MC and Eos that we name "the allergic synapse". This synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. Methods: MC were obtained from cord blood mononuclear cells (6-8 weeks with SCF, interleukin-6 and prostaglandin E2, CBMC). CBMC were activated, after their culture for 5 days with myeloma IgE (2mg/ml), by rabbit anti-human IgE antibodies (5mg/ml), for 1, 3 and 6h at 378C. Activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. The expression of FLIP, MCL-1, Bcl-2, Bcl-xL, BAK and BAX was assessed by immunoblot analysis.

Results: Two anti-apoptotic proteins were found to be upregulated: FLIP, which is involved in the extrinsic apoptotic pathway and MCL-1 that is mainly implicated in the intrinsic apoptotic pathway. In contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. Bcl-2 and Bcl-xL) was not altered. Likewise, the expression of pro-apoptotic proteins from the bcl-2 family (i.e. BAK and BAX) was either undetectable or unchanged.

Conclusions: Our findings reveal that IgE-dependent activation of human MC mainly induces the selective increase in the expression of two pro-survival molecules. This may be one of the mechanisms that underlay MC hyperplasia in the chronic allergic inflammation. (1), C Armishaw(2), Z Yang (1), H Cai (1), P Alewood(2), C Geczy (1) (1) University of New South Wales, Faculty of Medicne, Sydney, Australia (2) University of Queensland, Institute for Molecular Biosciences, St Lucia, Australia

Purpose: Human S100A8, S100A9 and S100A12 are closely related proteins associated with inflammation. S100A12 is expressed in the human genome, but not in the mouse. S100A12 is a potent monocyte chemoattractant and mast cell (MC) activator. Mouse (m) S100A8 and human (h) S100A8 share 58% structural identity, but the hinge domains are more divergent. The mS100A8 hinge (mS100A842-55) is a potent chemoattractant for leukocytes whereas this sequence in hS100A8 (hS100A843-56) is inactive.

Methods: S100A12 hinge domain and its alamine scan mutants were synthesized and their activities were tested by using THP1 cells or murine MC in vitro and mouse footpad injection.

Results: S100A12 hinge domain (S100A1238-53) was chemotactic for monocytes and MC and provoked MC degranulation in vitro and oedema, and leukocyte recruitment in vivo. In contrast to S100A12, the hinge domain only weakly induced cytokine production (IL6, IL8) by macrophages. Residues essential for oedema were hydrophobic in nature (Leu40, Isoleu44, Isoleu47 and Isoleu53). N42 and I44 were essential for responses provoked by 10-12M S100A1238-53 whereas mutation of K38, L40, N46, I47, D49, K50 and I53 significantly reduced migration with 10-9M S100A1238-53.

Conclusions: S100A12 and mS100A8 may be functional chemattractant equivalents; S100A12 may have arisen by duplication of human S100A8. Isoleucine residues in S100A12 hinge domain are essential for its proinflammatory properties. (1), G Kiriakopoulou (1), E Tsimara(2), A Voultsou(2), K Zarkadis (1) (1) General Hospital of Zakynthos, Greece (2) Medical Centre of Katastari, Zakynthos, Greece Schistosome is a disease which pests in 74 tropical countries. The endemic areas are South America, Far and Middle East, and Africa. Our aim is to present an incident which concerning a parasitic infection, not endemic in Greece. Patient: Man 30 years old who immigrated from Pakistan (at the side of the AparKenar river) in Greece, a year ago. He turned out with anlage, headache and weight loss. He is a swimmer. He mentioned also a fever before migrating to Greece. Clinically: Largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. Laboratory examination: leucocytes 9.030 Eo 24.8%, Hb 8.2g/dL, Ht 27.2%, MCV 65,6fL, MCH 19.1pg, Thrombocytes 419.000, blood sedimentation 10mm, Fe 10mg/mL, Ferritin 2.98ng/mL. Normal biochemical examinations. U/s: Livers size lightly increased 16.5mm, hepatoportal vein with normal amplitude. Parasitology of feces: In the immediate confection with Lugol of the second sample, there were observed: big scattering oval ovules (130 -60mm) with a thin wall and quills on the side, as well as three imago worms (>10 mm): Schistosoma mansoni. Treatment: Praziquantel was given. Recheck in a months time: Blood examinationleucosites 11.230, Eo 9.8%, Hb 10,2g/dL, Ht 33.6%. Parasitology of feces is negative.

In the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis Posters Inflamm. Res., Supplement 3 (2007) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. Bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as T lymphocytes and eosinophils. Recently, high-sensitivity CRP (hs-CRP) has become available for detecting small changes in CRP levels within the normal range, allowing for assessment of subclinical inflammation. This study was undertaken to investigate the relationship between hs-CRP and bronchial asthma. We collected blood samples from 109 patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ECP) and pulmonary function as well as serum hs-CRP. Serum CRP levels in patients without attacks (average 0.473 mg/ L; p < 0.001) and with attacks (average 0.908 mg/L; p < 0.001) were significantly higher than those of normal controls (average 0.262 mg/L). Serum hs-CRP levels were inversely correlated with FEV1.0% in asthmatic patients (r = -0.4915, p < 0.01). In conclusion, these results indicate that serum hs-CRP as well as ECP may be related to the state of asthma exacerbation and allergic inflammation. Objectives: The PSHR assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type I hypersensitivity reaction. In this study we tested methods for removing IgE-antibodies (stripping) from donor basophils. Moreover a method was developed for improving the antigen specificity profile in PSHR. Proof-of-concept was provided using absorption with complete allergen extracts.

Methods: Buffy coats were screened to exclude reactivity against relevant allergens. Subsequently PBMCs were purified and basophils were stripped with either lactate or a phosphate buffer. Cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. Released histamine was measured spectrofluorometrically (HR-Test, RefLab). Cutoff was 10% HR. For absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and BSA as control). After centrifugation the supernatant was used to passively sensitize stripped basophils. HR was measured as described above.

Results: Stripping experiments using the two buffers only partially removed surface IgE but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. Absorption experiments showed that it was possible to specifically remove peanut specific IgE from patient serum. Removal of specific IgE reactivity to peanut extract was verified by Western blotting.

Conclusions: Using peanut extract as a model it was demonstrated that in PSHR antigen specificity can be modified. (1), SK BK(1), V Sharma(2), RP Bhandari (3) (1) Nepal Medical College Teaching Hospital, Nepal (2) All India Institute of Medical Sciences, New Delhi,

Background: Nepal has one of the highest maternalmortality rates in the world. This study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women.

Methods: Women with thromboembolic diseases were identified and their case records retrieved and reviewed S414

Inflamm. Res., Supplement 3 (2007) Posters from January 1998 to December 2006. Demographiccharacteristics were compared between women with and without thromboembolism.

The total number of deliveries over the study period was 16,993, giving an incidence of 1.88 per 1000 deliveries. There were two cases of pulmonary-embolism and one resulted in a maternal-death. The others had deep-vein-thrombosis of which over 80% were limited to calf veins only. The ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were 1.62 and 10.7 per 1000 deliveries (P <.001);the corresponding cases of deepvenous-thrombosis diagnosed were 0.29 and 2.94 per 1000 deliveries, respectively (P <.001). The majority (75%) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. Women with venous-thromboembolism were older, had higher BMI, and a higherincidence of preeclampsia. There were approximately twice as many postpartum as antepartum events. Bloodgroup A, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of <36 weeks, a BMI of25, or more and maternal-age of 35 or over were all found to increase incidence of venous-thromboembolism.

The long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis Most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like Nepal. (1), CH ladel (1), T Ruckle(2), C Rommel(2), R Cirillo (1) (1) RBM-Merck Serono, Colleretto Giacosa, Italy (2) Serono Pharmacological Research Institute, Geneva, Switzerland Rheumatoid arthritis (RA) is a severe articular disease. Massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. Blockade of PI3K signalling pathway has been demonstrated to be curative in a murine model of RA, collagen-induced arthritis (CIA). In this study we explored the molecular mechanisms by which PI3K signalling inhibition resulted in clinical amelioration of disease symptoms. AS605858, a novel isoform non-selective, yet specific class-I-PI3K inhibitor, administered at 15 mg/kg twice-a-day for 8 days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. At the end of treatment, post-arthritic paws were removed and phosphrylation levels of AKT (p-AKT), downstream target in PI3K-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. AKT phosporylation resulted to be significantly enhanced by disease induction and AS605858 was able to decrease its levels down to values comparable with naïve animals. Controls and AS605858treated mice were bled before treatment, after two days of treatment and at treatments end. No changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. T cell number was not affected, however a significant decrease of natural-killer, memory and regulatory T cells was observed after AS605858 administration. Finally, a non-significant, moderate reduction of Bcell number was also observed. These data demonstrate that efficacy of AS605858 in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via PI3Kg) and immunosuppressive (via PI3Kd/a/b) activity. (1) (1) Institute of Biomedical science, University of Sao Paulo, Brazil (2) IBILCE -Sao Paulo State University, Brazil Epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. We investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. Seven days after being ovariectomized (OVx), groups of rats were sensitized with ovalbumin (OA). Fourteen days after sensitization, the animals were OAchallenged and used 1 day thereafter. Allergic,shamoperated animals were used as controls. Some OVx animals were treated with estradiol (280 ìg/Kg) or progesterone (200 ìg/Kg) 24 h before being challenged. Mast cell degranulation was quantified in samples of isolated, OA-challenged bronchi. The airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. OVx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. Estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. Cultured BAL and bone marrow cells from allergic rats increased Posters Inflamm. Res., Supplement 3 (2007) S415 the release of IL-10 and reduced that of IL-1 and TNF. The release of IL-4 by bone marrow cells was significantly reduced. These effects were reverted by estradiol, and progesterone reduced IL-4 and increased IL-10 production in BAL and increased that of IL-1 and TNF in bone marrow cells. Bronchial mast cell degranulation upon direct contact with OA in OVx rats was less than in controls. It is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. 

Objectives: To study the participation of FSH on modulation of E-and L-selectin, ICAM-1 and Mac-1 expression in ovariectomized (OVx) rats made allergic.

Methods: Female rats were sensitized (OA/alumen) after 1 (OVx-1) or 7 days (OVx-7) of OVx or sham-operated (Sh). Fourteen days thereafter animals were challenged (OA, 1%; aerosol) and sacrificed 24 h after. Bronchoalveolar lavage (BAL) was collected and flow citometry analyses ofICAM-1, Mac-1 and L-selectin expression was studied. In parallel, lungs were frozen and sections were analysedfor E-selectin expression by immunohistochemistry.

Results: At day 1, E-selectin expression increased(OVx-1=1,8AE0,08) and at day 7, decreased (OVx-7=1,3AE0,1) as compared to respective controls (Sh-1=1,45AE0,08; Sh-7=1,9AE0,08). Estrogen treatment reverted this profile in both groups (OVx-1+E=1,37AE0,09; OVx-7+E=1,8AE0,1). Mean fluorescence intensity of BAL cells showed increase of Mac-1 expression (OVx-1=19AE0,5 vs Sh-1=15,5AE1,0), ICAM-1 (OVx-1=18,1AE1,1 vs Sh-1=13,3AE0,2) and L-selectin (OVx-1=11,2AE0,8 vs Sh-1=9,5AE0,12) at day 1 i.e., OVx-1 group. On the other hand, we observed a decrease in ICAM-1 (OVx-7=14,2AE0,75 vs Sh-7=19AE1,4) and Mac-1 expression (OVx-7=20,7AE1,4 vs Sh-7=27,5AE1,5) was seen in OVx-7 group.

Conclusions: Oscillation of hormone levels during immunization with OA increased (OVx-1) and decreased (OVx-7) expression of adhesion molecules. Estradiol treatment reverted this effect. This results suggest that FSH modulates the ALI in rats by acting on cell (1), S Lim (1), Y Lin (1), BP Leung (1), C Thiemermann (2), WSF Wong (1) (1) National University of Singapore (2) The William Harvey Research Institute, London, UK Glycogen synthase kinase 3b (GSK-3b) is known to regulate various cellular functions including inflammatory responses. We hypothesized that inhibition of GSK-3b may have anti-inflammatory effects in a mouse asthma model. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. TDZD-8, a non-ATP competitive GSK-3b inhibitor, was administrated by i.v. injection one hour before OVA challenge. TDZD-8 significantly reduced the OVA-induced eosinophilia in a dose-dependent manner and inhibited the levels of IL-5, IL-13 and eotaxin in bronchoalveolar lavage (BAL) fluid. TDZD-8 also suppressed the mRNA levels of ICAM-1, VCAM-1 and chitinase proteins in the lung. Histological studies revealed that TDZD-8 substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. TDZD-8 also decreased OVA-specific IgE level in the serum. In addition, OVA-induced increase in airway resistance and reduction in dynamic compliance were attenuated by TDZD-8. Our findings suggest that inhibition of GSK-3b may have therapeutic potential for the treatment of allergic airway inflammation. (1), A Yildirim (1), F Ercan (2), N Gedik (3), M Yuksel(4), Inci Alican (1) ( Materials and Methods: Sprague-Dawley rats (200-250 g) were exposed to 90 oC (Burn Group) or 25 oC water bath (Control Group) for 10 s under ether anesthesia. ADM (100 ng/kg; s.c) was administered 10 min before burn and all rats were decapitated 24 h after the burn insult. Trunk blood was collected for the measurement of TNF-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (LP), myeloperoxidase (MPO) activity and formation of reactive oxygen metabolites (ROMs) using chemiluminescence assay.

Results: Burn resulted in severe morphologic damage in tested tissues. LP increased in lung and kidney (p<0.01-0.001) and MPO activity showed a marked increase in all tested tissues (p<0.001) of the Burn Group. ADM reversed these parameters effectively (p<0.05-0.001).

Luminol chemiluminescence levels showed increases in both ileum and lung (p<0.01-0.001) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p<0.01) of the Burn Group. ADM treatment was also beneficial in reducing chemiluminescence levels near to controls (p<0.01). ADM reduced plasma TNF-alpha level (p<0.01) which showed a significant increase in burned animals compared to controls (p<0.001).

Conclusions: ADM is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, ROM generation, LP, and the release of proinflammatory cytokine TNF-alpha. Kalpana Panday (1), SD Joshi (2), KR Reddy (3) ( We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, with anti-inflammatory activities in vivo, at 1.45 resolution. HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH in the quaternary complex. HQL-79 inhibited H-PGDS competitively against the substrate PGH2 as well as noncompetitively with respect to GSH. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 10-fold higher in the presence of GSH and Mg2+ than in their absence. HQL-79 inhibited selectively PGD2 production by human H-PGDS-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 inhibited antigen-induced production of PGD2, and airway inflammation in mice without affecting the production of PGE2 and PGF2f¿. Knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically. Introduction: It has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. This study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung.

Methods: Male Wistar rats (250-275g) were anesthetized and paralyzed, and the two lungs were independently intubated. Differential ventilation was applied for 3h (70 breath/min). One lung was subjected to hyper-ventilation (20mL/Kg/lung) and the other was ventilated with a normal volume (5mL/Kg/lung). In a control group, both lungs were ventilated with a normal volume. After sacrifice, samples of lung, plasma and liver were collected. The expression of the pro-inflammatory chemokine MIP-2 was evaluated by RT-PCR and the edema was assessed by the ratio between the wet and dry weight of the lung. Systemic inflammation was estimated in liver by measuring the expression of TNFa by RT-PCR as well as its levels in plasma.

The hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in MIP-2 expression compared to the normal ventilated lung. No differences were found in edema, neither expression of MIP-2 between the normally ventilated lung and control lungs. No significant changes were observed in liver expression and plasma levels of TNFa as a consequence of unilateral lung hyper-ventilation.

The over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. The normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. (1), J-Y Gillon(2), V Lagente (1), E Boichot (1) (1) University of Rennes 1, Rennes, France (2) Serono International S.A., Geneva, Switzerland

Macrophage elastase (MMP-12) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with COPD (Chronic Obstructive Pulmonary Disease). The mechanism of action of MMP-12 in the development of pulmonary inflammatory process is still unknown. In the present study, we investigated the effect of recombinant human MMP-12 (rhMMP12) on IL-8/CXCL8 release from alveolar epithelial cell line A549 and we explored the underlying mechanisms. A549 cells were stimulated with rhMMP-12 (1.10-3-2.10-1 U/ml) during 6 hours and IL-8/CXCL8 level in supernatant was determined by ELISA. Involvement of MAP (Mitogen activated protein)-kinases was studied by Western-blotting and also using chemical inhibitors. NFkB activation was examined with the TransAM NFkB p65 Kit. We observed that MMP-12 elicited IL-8/CXCL8 release in a dose-dependent manner. This production could be prevented by a pretreatment for 1hour with a selective MMP-12 inhibitor (AS111793 1-30 mM) or with the nonselective inhibitor Batimastat (1-30mM) . The IL-8/CXCL8 production was also inhibited by actinomycin D (5mg/ml), ERK1/2 inhibitors (U0126 5mM and PD98059 10mM) and NFkB inhibitors including BAY11-7082 (10mM) and NFkB activation inhibitor (10nM), whereas P38 kinase inhibitor (SB203580) had no effect. Stimulation with MMP-12 was rapidly followed by a phosphorylation of ERK1/2 (5min) and an NFkB nuclear translocation and activation (1h). The NFkB activation was not inhibited by a treatment with U0126. These data suggest that alveolar epithelium is a target of MMP-12 since it upregulates gene expres-S418 Inflamm. Res., Supplement 3 (2007) Posters sion and release of IL-8/CXCL8, via ERK1/2 and NFkB activation, but these two pathways appears to be distinct. Agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (LPS), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (IL)-8, in particular, is often used a measure of relative toxicity.In this study we have compared mRNA expression and mediator release in NCI-H292 human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (TPM), LPS, bleomycin, diesel exhaust particles, residual oil fly ash (ROFA), carbon black and vanadyl sulphate.Polystyrene, poly(methyl-methacrylate) and the TPM vehicle, dimethyl-sulphoxide were used as negative controls.Confluent monolayers of H292 cells were exposed to serial dilutions of test agents in serum-free medium for 24 hours.The conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by Luminex technology.The levels of gene expression of IL-8, matrix-metalloprotease-1 (MMP-1), the gel-forming mucin MUC5AC, heparinbinding epidermal growth factor-like growth factor (HB-EGF) and the cytochrome P450s CYP1A1 and CYP1B1 were determined by quantitative-polymerase chain reaction.All of the toxicants induced similar responses whereas the negative controls were largely ineffective.-Such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-Moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of IL-8 alone, particularly in the case of agents such as TPM, where the conventional vehicle is found to have some biological activity. Respiratory tract infections are a major public health issue. Prevention in high risk populations relies mainly on vaccination. Vaccination is highly recommended in decrease absenteeism. Immunomodulating drugs are important tools in the treatment of infectious diseases. Immunomodulatory agents are probably contributive in decreasing exacerbation rates. The authors present different classes of immunomodulators that are currently in use. The vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. By preventing infections, vaccines prevent the development of a strong T helper (Th1) response. The challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. Deoxypodophyllotoxin (DPT) is a medicinal herbal product that is isolated from Anthriscus sylvestri. that inhibits cyclooxygenase-2 (COX-2) and COX-1dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC50 values of 1.89mM and 65.3mM, respectively. This compound inhibited COX-1 and 2-dependent conversion of the exogenous arachidonic acid to PGD2 in a dose-dependent manner with an IC50 values of 0.01mM and 12.1mM, respectively. However, DPT did not inhibit COX-2 protein expression up to a concentration of 30mM in the BMMC, indicating that DPT directly inhibits COX-2 activity. Furthermore, this compound consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 0.37mM. These Posters Inflamm. Res., Supplement 3 (2007) S419 results clearly demonstrate that DPT has a dual COX-2/5-LOX inhibitory activity in vitro. Therefore, this compound might provide the basis for novel anti-inflammatory drugs. In order to determine anti-allergic and antiasthmatic activity of DPT in vivo, we used rat PCA model which was activated by anti-dinirophenyl IgE and ovalbumin/alum induced mouse asthmatic animal model, respectively. As a result, DPT strongly inhibited PCA reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. Furthermore, DPT decreased the mRNA levels of the Th2 cytokines in a murine asthmatic model. In addition, northern blot analysis showed that DPT also reduced both the eotaxin and arginase I mRNA levels in a dose dependent manner. These results suggest that DPT may be beneficial in regulating various inflammatory diseases. An imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), a smokingrelated disorder associated with accelerated lung function decline.In particular, the activity of matrix metalloproteases (MMPs) have been implicated in driving both the inflammation and parenchymal destruction observed in COPD patients.Here, we tested whether a broad spectrum MMP inhibitor, PKF-242484, could block the inflammation induced by an acute exposure to cigarette smoke in 2 strains of mice, balb/c and c57/bl6.Animals were administered the compound (1-10 mg/kg) either per os (p.o.) or intranasally (i.n.) 1 hour before and 5 hours after exposure to smoke on three consecutive days.Bronchoalveolar lavage (BAL) was performed and lungs were flash frozen for inflammatory marker analysis.PKF-242484 dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~45% at 10 mg/kg; p < 0.01)or i.n. (~60% at 10 mg/kg; p < 0.01).However, the compound had no clear effect on BAL neutrophil infiltration in c57/bl6 mice by either route of administration.Interestingly, in both strains BAL macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < 0.05). Examination of lung tissue cytokine levels revealed that while smoke-exposure increases IL-1beta, KC, and MIP-2, PKF-242484 had little effect on these cytokines.These data suggests the ability of broad spectrum MMP inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. To investigate the role of sEH in the regulation of the pulmonary inflammatory response, we have used sEH deficient mice in a locally administered LPS model.

Male sEH deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled LPS.Four hours later they were sacrificed and BAL was performed. Differential counts and cytokine levels in BAL were evaluated.

Results: LPS induced a significant increase in total cell number and neutrophil number in BAL in the sEH deficient mice and in wt mice;no significant differences between the groups were seen (Table 1 ) Cytokine analysis showed significant increases in TNFalpha, IL-6, KC, GM-CSF, MCP-1, IL-1beta and Rantes in the LPS-exposed wt mice. No significant differences were seen between LPS exposed wt and ko mice except for a significant increase in TNFa in ko mice.

Our results show that sEH has no pivotal role for the regulation of the acute inflammatory response to lung administered LPS. fam.Liliaceae. Based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. The antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by Freunds adjuvant in rats. In the first test, the antiinflammatory effect of steroidic saponins 600 mg/kg is weak, statistically insignificant. In the arthritis test, steroidic saponins 600 mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after 16 days). In both tests, the suprarenal glands weight was modified. Objectives: Dendritic cells (DCs) are professional antigen presenting cells. Many types of DC with subtle difference in phenotype have been reported in several organs. Existence of DC was reported in synovial tissue of rheumatoid arthritis (RA), however, the details in RA DC still remains unclear. In this study, we generated a new lineage of DC with GMCSF (+TNFa) and investigated their functions. Furthermore the ability of osteoclastgenesis was examined.

Methods: Monocyte-derived DCs or macrophage were generated in (1) TNFa + GM-CSF (2) GM-CSF (3) IL-4 + GM-CSF (4) MCSF. The phenotypes of these cells were analyzed by morphological examination and flow cytometry (FACS Calibur). Cell proliferation was examined by WST-8 assay. DC functions were assessed in antigen presenting ability (MLR assay), cytokine production (ELISA), and endocytosis (FITC-dextran uptake).

Concerning osteoclastgenesis, monocyte-derived DCs were incubated with RANKL and MCSF. TRAP stain was performed and the resorption ability was assessed on Osteologic Cell Culture System and dentine slices.

Results: These cells were dendritic-like and their surface markers were CD1a low CD11b + CD11c + CD14 + CD86 + CD83 low HLA-DR + DC-SIGN low and different from conventional DC or macrophage. They had an antigen presenting ability to induce na*ve CD4+ T cell proliferation, IL-12 production and endocytosis. In the presence of RANKL and MCSF, they differentiated multinuclear TRAP-positive cells with bone resorption ability, which was strengthened by TNFa.

We generated a new lineage of DC with GM-CSF (+ TNFa). The DC seemed to play a pivotal role in inflammatory arthritis under TNF immunity. The clinical effectiveness of rituximab and other B cellattenuating rheumatoid arthritis (RA) therapies has increased interest in understanding the role of B cells in RA pathogenesis.The possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the Entelos RA PhysioLab platform, a mathematical model of the joint of an RA patient.The platform dynamically integrates the contributions of immune cells (T cells, B cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in RA.The B cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.The dynamics of these B cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from RA patients (e.g., ACR score and radiographic progression rates).An assessment of the contribution of individual B cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in RA patients. In contrast, proinflammatory cytokine production by B cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.Immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to T cells.Biosimulation research in the RA PhysioLab platform is advancing our understanding of the mechanisms underlying effective B cell-targeting RA therapies, and may guide the development of improved second-generation therapeutic approaches. Methods: The hMSCs were obtained at the operation.The mononuclear cells were extracted and the colony forming assay were performed after 2weeks. The hMSCs were cultured and in passage1,the cell surface antigens of both groups were analyzed by flow cytometory.In passage2, in control group, the cells were cultured with beta glycerophosphate(bGp) and in osteogenic group, the cells were cultured with bGp and ascorbic acid(AA) and dexamethasone(Dex).After 2weeks, ALP staining and activity were measured in each group.After 3weeks, alizarin red S assays were performed.RNA was extracted from the cells cultured with bGp and AA and Dex for 2weeks and 3weeks and the gene expressions of bone formation markers were examined by real time PCR. The Mann-Whitney test was used for the statistical analyses.

The colony forming assays showed no significant differences in OA and RA. In flow cytometory, the cell surface antigens in OA and RA were almost same.In ALP activity,there were no significant differences in OA and RA.In alizarin red S, there were significant differences.In real time PCR,the gene expressions showed no significant differences in OA and RA.

Conclusions: The hMSCs of RA will be able to use for regenerative therapy. Silje Vermedal Høgh (1), HM Lindegaard (2), GL Sorensen (1), A Høj (3), C Bendixen (3), P Junker (2), U Holmskov (1) ( Cytosolic phospholipase A2 (cPLA2) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. In addition, cPLA2 regulates the phagocyte NADPH oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. Collageninduced arthritis (CIA) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling Rheumatoid Arthritis in humans. Previous studies show that cPLA2-deficient mice are resistant to CIA. Thus we aimed to study whether cPLA2 is up-regulated during the development of CIA and to detect its exact location in the inflamed joints. Immunoblot analysis revealed an increase in the level of joint cPLA2 protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. Immunohistochemistry with specific anti-cPLA2 antibodies revealed low positive cPLA2 protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. In the joints of the CIA mice, large amounts of inflammatory infiltrate containing cPLA2 were detected. In addition, robust cPLA2 protein expression in the skeletal muscles surrounding the joints and strong cPLA2 positive staining in sebaceous glands were detected. The high correlation between the severity of inflammation and the elevated cPLA2 protein, due to an inflammatory infiltrate and increased cPLA2 expression, suggests an important role of cPLA2 in the development of arthritis. Rheumatoid arthritis (RA) is the complex disease depending on environmental as well as genetic factors.

In spite of the large research efforts we know in fact only small number of genes involved in this disease. Animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. The aim of the current project is to identify the genes and their functional role importance for arthritis.

Two loci associated with arthritis were identified using a cross between the B10.Q (intermediate susceptible) and NOD.Q strains (resistant to arthritis). One on chromosome 2 a disease protective locus (Cia2) and another on chromosome 1 a disease-promoting locus (Cia9). NOD.Q allele at Cia9 promotes arthritis whereas Cia2, has a protective effect in contrast to the B10.Q allele on chromosome 2. A promising candidate gene in Cia2 locus is complement factor 5 (C5) as the NOD.Q allele produced defective C5 protein. Cia9 locus contains several genes of potential importance for disease such as Fc RIIb, Fc RIII, Fc RIV Ncf2, fH. The results of CIA (collagen induced arthritis) and CAIA (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains Fc R region showed a significant difference in incidence and severity of arthritis. The disease is controlled in a recessive pattern. The fragment devoid of Fc R region seems to be protective. The subcongenic for the Cia2 locus has been generated recently which will be tested for CIA and CAIA susceptibility. The results from these experiments will be discussed in detail.

Angela Pizzolla, KA Gelderman, R Holmdahl

Ncf1 is a component of the NADPH oxidase complex, which produces reactive oxygen species (ROS) upon activation into phagosomes and extracellularly. Polymorphisms in the Ncf1 gene that impair the capacity to produce ROS, enhance susceptibility of both mice and rats to arthritis. Activation of autoreactive T cells drives arthritis development but neither Ncf1 expression nor oxidative burst have been detected in T cells. We hypothesize that antigen presenting cells influence T cell activity by producing ROS during antigen presentation. We aimed to clarify the role of ROS produced by dendritic cells (DC) on T cell activation. DC were grown from bone marrow from Ncf1 mutated and wildtype mice. We could show that Ncf1 mutated DC proliferated and differentiated better, had higher expression levels of costimulatory molecules and MHC II upon stimulation as compared to Ncf1 wildtype DC. In addition, Ncf1 mutated DC induced higher levels of IL-2 production by hybridoma T cells. To analyze the role of Ncf1 in DC on arthritis, mice were developed expressing functional Ncf1 restricted to DC (B10.QDCN). These mice are characterized for burst, Ncf1 expression and ability to present antigen. We published that immunization with myelin oligodendrocyte glycoprotein (MOG) protein resulted in higher disease severity than immunization with MOG peptide in Ncf1 deficient mice. This suggests that Ncf1 plays a role in the uptake and processing of Posters Inflamm. Res., Supplement 3 (2007) S423

antigens, probably by DC. This will be further investigated with the B10.QDCN mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. Purpose: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. It is expressed in human RA synovial tissue and its suppression inhibits T or B cell dependent animal models of RA. We investigated the role of MIF in K/BxN serum transfer arthritis.

Methods: Arthritis was induced by injection of K/BxN serum on days 0 and 2 in littermate WT and MIF-/-mice. Arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. Sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage.

Results: WT mice exhibited arthritis as early as day 1 and 100% incidence was observed on day 7. MIF -/-mice exhibited delayed arthritis, with onset on day 3 and 100% incidence on day 9. MIF -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( Methods: Osteoarthritis was induced by bilateral transection of the medial meniscus in Dunkin-Hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding.

Results: The first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. Twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. Cartilage destruction due to meniscal transection was also histologically detected. However, biomarkers for cartilage destruction (CTX-II, HP/LP ratio, COMP) were not increased. Treatments aiming at different processes in osteoarthritis, such as bone destruction (Risedronate), inflammation (Pioglitazone and Anakinra), and cartilage destruction (Galardin) were not effective in this model.

The early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. Further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. The ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. The meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments.

Livia L Camargo (1), A Denadai-Souza (1), LM Yshii (1), A Schenka (2), MA Barreto (1), D Boletini-Santos (3), C Lima (3), V Rioli (3), MN Muscar (1), E Ferro (1), SK Costa (1) ( Methods: AIA was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. Knee oedema and pain score were assessed daily in controls and animals treated with hemopressin (10 or 20 ìg/day; i.a.). Histopathological changes, cell number and cytokines in the synovial fluid of AIA rats were determined at day 4.

Results: AIA rats developed a severe mono-arthritis characterized by a joint oedema and pain. At day 4, there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by ELISA. Both doses of hemopressin significantly reduced the knee oedema, but only 20 mg of hemopressin attenuated the pain score. Acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.).

Conclusions: Hemopressin has potential for treating acute signs of AIA by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis.

Calcitriol, the hormonal metabolite of vitamin D and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. The MAP kinase ERK plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.We hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on ERK activation in the presence or absence of inflammatory mediators. Our experimental model was immortalized non-tumorigenic human HaCaT keratinocytes cultured in the absence of exogenous growth factors or active mediators. Inflammation was mimicked by exposure to TNF. Level and activation of signaling molecules were determined by immunoblotting. By using the specific EGF receptor (EGFR) tyrosine kinase inhibitor, AG1487, we established that TNF activates ERK in an EGFR-dependent and EGFRindependent modes. The EGFR-dependent activation resulted in the induction of the transcription factor, c-Fos, while the EGFR-independent activation was of a shorter duration and did not affect c-Fos expression. Treatment with calcitriol alone increased ERK activation and c-Fos induction. Pretreatment with calcitriol enhanced EGFR-dependent ERK activation and tyrosine phosphorylation of the EGFR, but completely abolished EGFR-independent TNF-induced ERK activation. Pretreatment with calcitriol increased the rate of de-phosphorylation of activated ERK, accounting for the inhibition of EGFR-independent ERK activation by TNF. It is possible that effects on the ERK cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation.

Christina Barja-Fidalgo (1), R Saldanha-Gama (1), JA Moraes (1), R Zingali (2), C Marcienkewicz (3) (1) Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil (2) Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (3) Temple University, Philadelphia, USA Neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. Integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.Acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. Disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an RGD sequence, a cell attachment site of ECM and cell surface proteins recognized by integrins. They are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ECM interactions. We reported that RGD-disintegrins, selectively interact with integrin aMb2, a5b1 and/or avb3) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways.

Recently showed that, a selective ligand of a9/a4b1 integrins, VLO5, induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. These effects are mediated VLO5 interaction with a9b1 integrin, activating the focal adhesion cascade. VLO5 effects on the delay of neutrophil is modulated by PI3K, ERK-2 MAPK and NF-kB pathways that seems to interfere with the balance between anti-and pro-apoptotic Bcl-2 family members and with mitochondrial membrane potential. Data emphasize mechanistic details of the role of a9b1 integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (FAPERJ, CNPq, CAPES, IFS-Sweeden) (1), H Serezani (2), M Peters-Golden(2), Sonia Jancar (1) '(1) University of S¼o Paulo, Brazil (2) University of Michigan, USA

It is been shown that leukotriene (LT) B4 and cysteinyl LT (LTC4, LTD4 and LTE4) enhances FcR-mediated phagocytosis in alveolar macrophages (AM), dependent on protein kinase C (PKC). In contrast, LTB4 but not cysLT effects are exclusive on syk activation. In the present study we investigated the role of specific PKC isoforms and its upstream and downstream targets involved in LT-enhanced FcR-mediated phagocytosis. To this purpose, AMs were pretreated or not with inhibitors of PKC-d (Rottlerin -6 uM), PKC-a (Ro-32-0432-9 nM), PI3K (Ly290042-10 uM and Wortmannin-10nM), ERK 1/ 2 (PD98059 -2 uM), cPLA2 (AACOCF3-10 uM), p38MAPK (SB20190-10 uM) and Ca++ (BAPTA/AM-10 uM), before stimulation with LTB4 or LTD4 and addition of IgG-opsonized red blood cells for 90 min. Activation (phosphorylation) of signaling molecules by LTs were analyzed by Western blot. Our results demonstrate that LTB4 -enhanced phagocytosis is dependent on PKC-a, while LTD4 effects are mediated by PKC-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. While galectin-1 mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin-3 is known as a strong pro-inflammatory and anti-apoptotic signal. We have recently recognized galectin-3 as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. In this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin-1 at gene and protein level in monocytic THP-1 cells. We have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from E. coli (LPS). The targeted mRNA level was evaluated using quantitative RT-PCR technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. The results showed that immunomodulatory drugs affected the expression of galectin-1 on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. The modulatory effects of the applied drugs on galectin-1 and -3 expressions were also observed in the cells activated by LPS. These findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin-1 and-3 expressions, as well as the role of these lectins in the physiology of monocytes. Introduction: Pancreatitis-Associated Protein (PAP) has been recently described as an endogenous mechanism involved in the regulation of inflammation. In the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by PAP.

The pancreatic human cell line PANC1 was incubated with PAP (500ng/ml) and/or TNFa (100ng/ml). Total RNA was obtained and the expression of TNFa was examined by RT-PCR. In addition, the effect of PAP on NFkB activation was measured by inmunofluorescence in cells. Western Blot analysis was used to determine the expression of NFkB mediators: phosphorylated IKK, IkBa and p65.

Results: We observed that PAP administration to cells prevented NFkB translocation to the nucleus as well as the TNFa-induced TNFa gene expression. When TNFastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of TNFa gene expression was completely restored. On the other hand, PAP had no effect on IKK phosphorylation or IkBa degradation.

Conclusions:In this study we have provided evidence that PAP modulates the inflammatory response by blocking NFkB translocation to the nucleus. This PAP-induced NFkB inhibition requires a JaK/STAT-dependent de novo protein synthesis. Objectives: This study investigated the inhibitory mechanism of hyaluronan (HA) on lipopolysaccharide (LPS)stimulated production of proinflammatory cytokines in U937 macrophages.

Methods: HA was added to U937 macrophage cultures in the presence of LPS, with or without pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody. Secreted levels of tumor necrosis factor a (TNFa), interleukin (IL)-1b, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-kB, IkBa, and mitogenactivated protein kinases (MAPKs) was analyzed by immunoblotting.

Results: LPS stimulated production of TNFa, IL-1b, and IL-6. In contrast to 800 kDa HA, 2700 kDa HA at 1 mg/ ml inhibited LPS-induced cytokine production. Anti-ICAM-1 antibody blocked the effects of HA on the LPS actions on U937 cells. LPS activated NF-kB and MAPK pathways, whereas HA down-regulated p65 NF-kB and IkBa phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-kB for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-kB and IkBa.

Conclusions: HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-kB and IkB. Exogenous HA into arthritic joints could act as an anti-NF-kB agent by the mechanism demonstrated in the present study. The principal eicosanoid product of endothelial COX-2 is prostacyclin (PGI2), which is a potent vascular patency factor. Induction of endothelial COX-2 under hypoxic conditions is well documented. This response, along with associated PGI2 release, is likely an important protective homeostatic response. In order to explore the role of candidate signalling agents in COX-2 expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the COX-2 promoter region in HUVEC. COX-2 induction under hypoxic conditions was confirmed with the wild type construct. Strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (HIFs) were more important for COX-2 expression than those for NFkB. Furthermore, expression of COX-2 was increased under normoxic conditions by transfection of HUVEC with normoxia-stable HIF mutants. EMSA showed HIF binding in nuclear extracts from untransfected hypoxic HUVEC. Under these hypoxic conditions, increased release of PGI2 but not vaso-occlusive thromboxane A2 was seen. Thus the putative protective induction of COX-2 in endothelial cells in response to hypoxia involves signalling by HIFs. Crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of Th1 immune response plays a crucial role. However, the therapeutic efficacy of the regulation of Th1-predominant immune responses remains to be investigated. Therefore, the effects of a Th1 selective inhibitor TAK-603 were investigated in a model of crescentic glomerulonephritis in WKY rats.

Methods: TAK-603 was administered orally, starting at the time of induction of glomerulonephritis. In group 1, the drug was administered daily for the initial 6 days. TAK-603 was administered on day 0 only in group 2, and from day 3 to 5 in group 3. In each group, nephritic rats were killed on days 6 and 56.

Results: In group 1, glomerular damage, including crescent formation, was improved on day 6, with reductions in the numbers of CD4, CD8 and ED-1 positive cells, as well as in urinary protein excretion. Protein and transcript levels of Th1 cytokines in the diseased kidneys were markedly decreased by TAK-603 treatment. Renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. Elevated levels of serum creatinine showed concomitant improvement. In group 3, in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. Treatment only on the first day in group 2, partially rescued renal dysfunction.

Conclusions: These results suggest that the initial inhibition of Th1 immune response has an appealing therapeutic potential for crescentic glomerulonephritis.

Parasitic nematodes and their hosts are now known to produce a wide range of galectins. Whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. Studies at Moredun have demonstrated that both the endoparasitic helminth, Haemonchus contortus, and the ectoparasitic mite, Psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. Excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. Separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. In the case of H. contortus, there was homolgy with known EST sequences, which allowed subsequent cloning and expression studies to be undertaken. A functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. This may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear S428 Inflamm. Res., Supplement 3 (2007) Posters to provide the primary nutrient source for their survival. Moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. The aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (LPS)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mRNA levels in these tissues. Male CD-1 mice were injected intraperitoneally with 50 mg/kg LPS alone or concomitantly with an intravenous dose of pentoxifylline (100 mg/kg), lisofylline (100 mg/kg) or prednisolone (10 mg/kg). The tissues were harvested 0, 1, 4, 6, and 24 h following LPS administration and stored at -808C. Relative quantification of tumor necrosis factoralpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma) mRNA levels was performed by real-time RT-PCR. The highest levels of cytokine mRNA were observed at 4 or 6 h after LPS administration, whereas the expression of TNF-alpha and IL-1beta in lungs and IL-6 in liver reached the peak values at 1h and then decreased gradually. In addition, the LPS effects on cytokine mRNA were more pronounced in liver when compared to lungs. All administered compounds inhibited the LPSinduced TNF-alpha mRNA expression (by up to approximately 50%), whereas lisofylline significantly increased IFN-gamma mRNA levels in both tissues at most investigated time points. For other cytokines, the observed differences did not reached statistical significance. In conclusion, with the exception of IFN-gamma, the time course of cytokine mRNAs differed considerably depending on the type of tissue. In the murine model of LPS-induced septic shock, only TNF-alpha gene expression was suppressed by all compounds under investigation.

Maria Sanz(1), M Losada (1), C Company (1), C Lope-Gines(1), L Piqueras(2), J Cortijo (3) The migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. Fractalkine/ CX3CL1 (FR) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. Clinical studies and animal disease models have shown that FR is also involved in the pathogenesis atherosclerosis. We have demonstrated that angiotensin-II (AII) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. In the present study we have investigated whether AII can cause the synthesis and expression of FR on human umbilical arterial endothelial cells (HUAECs). HUAECs were stimulated with Ang-II 1 microM or with TNFalpha (20 ng/ml) for 1, 4 and 24 h. FR was determined in the culture supernatants by conventional sandwich ELISA. FR was only detected after 4 h and 24 h stimulation with TNFalphafnwhereas AII was unable to provoke the cleavage of the chemokine. Semiquantitative RT-PCR analysis of HUAECs showed increased FR mRNA expression in AII-stimulated cells for 1 and 4 h. These effects were caused by the interaction of AII with its AT1 receptor since they were abolished by losartan (AT1 receptor antagonist). TNFalpha also increased FR mRNA. Immunohistochemical analysis of the cultured endothelial cells showed a clear expression of FR in HUAECs stimulated with AII or TNFalpha for 4 and 24 h. These results suggest that FR could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by AII. The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD).Among organisms of the Malassezia species, M. globosa and M. restricta are particularly dominant on the skin of AD patients. Our previous study has demonstrated that human keratinocytes responded to the two Malassezia species with different Th2-type cytokine profiles, i.e. M. globosa induced IL-5, IL-10, and IL-13 secretion from the keratinocytes, whereas M. restricta induced IL-4 secretion.These findings suggest that M. globosa and M. restricta play a synergistic role in triggering or exacerbating AD by stimulating the Th2 immune response. Pattern recognition receptors (PRRs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. In this study, we assessed the role of PRRs for cytokine production by human keratinocytes in response to Malassezia species. Human keratinocytes were pretreated with various anti-PRR monoclonal antibodies (mAbs) and stimulated with M. globosa or M. restricta. Cytokine secretion from keratinocytes was measured by using Fast Quant ELISA kit. Exposure of human keratinocytes to M. globosa and M. restricta resulted in enhanced secretion of IL-5 and IL-4, respectively. The M. globosainduced increase in IL-5 secretion was inhibited by mAbs against CD14 and CD91. In case of M. restricta, mAbs against Toll-like receptor 2 (TLR2) and CD14 suppressed significantly IL-4 secretion from keratinocytes. These findings suggest that the distinct PRRs interactions with fungal pathogen-associated molecular patterns (PAMPS) are key factors in differential cytokine secretion from keratinocytes stimulated with Malassezia species. Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease associated with allergy. MDC (macrophage-derived chemokine/CCL22) and TARC (thymus and activation-regulated chemokine/CCL17) are Th2type cytokines, and it has been reported that serum MDC and TARC levels are associated with AD disease. In present study, we investigated the effect of Prunus yedoensis Matsum barks on the inflammatory chemokines (MDC and TARC) and Jak-STAT pathway in HaCaT keratinocytes. As a result, EtOAc fraction and E5 sub-fraction inhibited the mRNA expression and protein level of MDC and TARC in a dose-dependent manner. Also, E5 sub-fraction showed inhibitory activity on the STAT1 protein level. These results suggest that P. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (MDC & TARC).

The IL-1 family now consists of 11 members, most of which have assigned functions.There are 10 members of the IL-1 receptor family (including the decoy receptor type II IL-1R).Many of the IL-1 family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.The IL-1 family members IL-1F6, F8 and F9 signal through a complex of the IL-1R family member RP2 in association with IL-1R AcP to activate common inflammatory pathways.The specific activity is is low, on the order of 1-10ug/ml EC50.We have found that removal of a few N-terminal amino acids from IL-1F5, F6, F8 and F9 can increase their bioactivity approximately 1000-fold. The location of the N-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.In addition, N-terminally truncated IL-1F5 is capable of antagonizing signaling via IL-1Rrp2, but full-length F5 is inactive. (1) University of Ulsan, Japan Kyoto University, Japan

Inflammation plays a pathogenic role in the development of obesity-related complications such as type II diabetes and atherosclerosis. Tumor necrosis factor alpha (TNFa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. TR2 (HVEM/TNFRSF14), which is a member of the TNF receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT/TNFSF14), is a potent mediator of inflammatory responses. The purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting TR2 pathway. C57BL/6 TR2 knockout mice and their wild-type control were fed a high-fat diet for 12 weeks and the obesity phenotypes were determined in the obese TR2 knockout mice and the control. The obese TR2 mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. Expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese TR2 knockout mice compared with those of the control. Our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese TR2 knockout mice fed a high-fat diet. Objectives: The present study was undertaken to investigate the role of insulin on allergic airway inflammation.

Methods: Diabetic male Wistar rats (alloxan, 42 mg/Kg, i.v.) and controls were sensitized with OVA (100 ìg) and Al(OH)3 (8 mg, s.c.) 10 days after alloxan or saline injection. The animals were challenged 14 days later by the intratracheal instillation of OVA (1 mg/0.4 mL). The following analyses were performed 6 h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid; (b) quantification of TNF-alpha and IL-1 beta in the BAL by ELISA; and (c) immunohistochemistry for P-and E-selectins on lung vessels.

Results: Compared to the control animals, diabetic rats exhibited reduced number of neutrophils (90%) and mononuclear cells (50%); reduced levels of TNF-alpha (45%) and IL-1 beta (30%), and reduced P-selectin expression (30%) in response to OVA challenge. These abnormalities were corrected after treatment of diabetic rats with a single dose of NPH insulin (4 IU, s.c.), 2 h before OVA challenge. Although we did not find differences in E-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin.

Conclusions: Data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of TNF-alpha and IL-1 beta production and selectin expression. Supported by FAPESP and PRONEX. Hormonally active vitamin D derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. Their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.We examined whether vitamin D also interferes with the pro-inflammatory action of the keratinocytes themselves. Human HaCaT keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to TNF to simulate an inflammatory challenge and their response was monitored by assessing mRNA levels of the cytokine TNF, the chemokine IL-8 and the adhesion molecule ICAM-1 by real-time PCR. ICAM1 and IL-8 were induced rapidly peaking after 2h, their mRNA levels increased again from 8h to reach a plateau between 16h to 24h after exposure to TNF, whereas TNF mRNA levels increased steadily between 2h and 24h. 24h pretreatment with Calcitriol, the hormonal form of vitamin D, inhibited induction of IL-8 but did not affect that of ICAM-1 or TNF 2h following exposure while calcitriol markedly inhibited the induction of all 3 pro-inflammatory genes 16h after the TNF challenge.Calcitriol inhibits the activation of Jun kinase (JNK) and p38 by TNF. This action was mimicked by the Posters Inflamm. Res., Supplement 3 (2007) S431

JNK inhibitor SP600125 and the p38 inhibitor SB203580.The combination of the two Inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. We conclude that vitamin D attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the JNK and p38 cascades. (1), CM Lotufo (2), P Borelli (1), ZS Ferreira (2), RP Markus (2), SHP Farsky (1) (1) Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of S¼o Paulo, Brazil (2) Department of Physiology, Bioscience Institute,University of S¼o Paulo, Braziil

Introduction: We showed that endogenous glucocorticoids (EG) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of L-selectin on segmented cells. Aims: We evaluated the role of EG on endothelial cells (EC) and the molecular mechanisms responsible for hormonal actions in neutrophils and EC.

Methods: Neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and EC were cultured from testis vessels. Cells were obtained from adrenalectomized (ADX), RU 38486-treated, shamoperated, vehicle-treated and non-manipulated (NM) Wistar rats.

Results: Circulating neutrophils and segmented cells from RU 38486-treated rats presented elevated and decreased expressions of L-selectin vs cells from control animals, respectively. The effects were not dependent on alterations of L-selectin mRNA levels. EC from ADX animals presented more ability to adhere neutrophils from NM rats and enhanced mRNA levels and membrane expressions of ICAM-1, VCAM-1 and PECAM-1. Participation of the glucocorticoid cytosolic receptor(GCR) on these effects was shown by similar results in cells from RU 38486 treated rats. NFkappaB translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in EC from ADX or RU 38486-treated rats.

Conclusions: Data show the participation of the GCR on events involved in neutrophil mobilizations, but NFkappaB transcription is only involved on EC. (1), Y Naito(2), T Okuda (3), K Mizushima (3), T Okayama (3), I Hirata (3), H Tsuboi (3), T Suzuki (3), O Handa (1) Background: Despite the inhalation of CO at high concentrations had been considered as a toxic gas, the inhalation of CO at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. However, it is unclear whether the direct exposure of CO to the intestinal inflamed mucosa is effective or not. In this study, we investigated the therapeutic efficacy of the rectal CO administration for rat colitis model.

Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. CO(200ppm-10ml) was intrarectally administrated twice a day after the induction of colitis. Rats were sacrificed at 3 days after the administration of TNBS. The distal colon was removed, and the ulcer lesions were measured. Thiobarbituric acid (TBA)-reactive substances and tissueassociated myeloperoxidase (MPO) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. Moreover, we evaluated the expressions of CINC-1 mRNA/protein and p-p38 MAPK protein.

The intracolonic administration of CO ameliorated TNBS-induced colonic ulceration. The increases in TBA-reactive substances and MPO activity after TNBS administration were both significantly inhibited by treatment with CO. Moreover, the rectal administration of CO significantly inhibited the increased expression of CINC-1 mRNA/protein and p-p38 MAPK protein after the induction of TNBS-induced colitis.

The rectal administration of CO protected from the intestinal inflammation in rats. Based on these data, the beneficial effects of CO on the intestinal mucosal injury may be attributed to its anti-inflammatory properties.

Alessandra Gambero (1), M Maróstica (1), M Saad(2), J Pedrazzoli Jr (1) (1) S¼o Francisco University Medical School, Brazil (2) State University of Campinas, Brazil

Recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines.

The adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. In this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic TNBS instillations were evaluated. The adipocyte size was measured after collagenase digestion. The basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. The colitis animals showed higher mesenteric fat mass (1.02+-0.06 and 0.78+-0.09 % of body weight for colitis and control, respectively; p<0.05) in despite of the lower body weight. The mesenteric adipocytes from colitis animals presented reduced diameter (31.67+-0.28 and 35.69+-0.37 um for colitis and control, respectively; p<0.01), higher basal lipolysis (41.67+-6.89 and 18.77+-3.66 ug.mg-1 for colitis and control, respectively; p<0.05) and TNF-alpha production (8.64+-0.77 and 5.51+-0.86 ng.mg-1 for colitis and control, respectively; p<0.05). Perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis (72.48+-15.38 and 25.96.77+-3.05 ug.mg-1 for colitis and control, respectively; p<0.05), increased TNFalpha (46.34+-5.82 and 15.71+-2.15 ng.ml-1 for colitis and control, respectively; p<0.01), leptin (400.41+-77.10 and 201.01+-52.01 pg.ml-1 for colitis and control, respectively; p<0.05) and adiponectin production (15029+-2340 and 5918+-498 ng.ml-1 for colitis and control, respectively; p<0.01). The mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. Perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one.

This work was financially supported by FAPESP. Inflammatory Bowel Disease (IBD) is a group of chronic inflammatory disorders of the intestine. The role of the pro-inflammatory p38 MAPK signalling cascade in the pathogenesis of IBD is highly controversial. We therefore aimed to investigate the role of p38 MAPK in Chronic Dextran Sodium Sulfate (DSS) induced colitis, an experimental model of IBD. Chronic intestinal inflammation was induced by oral cyclic administration of 3 % DSS in SJL mice. Clinically, the DSS treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. At the molecular level, this was accompanied by an up-regulation of TNFa, IL-1â, IL-6, IL-17, KC, Cox-2, IgG heavy chain, and phospho-Stat3 in the DSS treated mice.The clinical and molecular features described above recapitulate findings reported in human IBD. In order to assess the role of p38 MAPK, the activation of the p38 MAPK signalling cascade was analysed by Western blot analysis. The expression and phosphorylation levels of both p38 MAPK and of MK2 and HSP27, two down-stream targets, were not increased in DSS treated animals compared to controls. LEO 15520, a potent inhibitor of p38 activity in vivo, was dosed as pretreatment and after completion of DSS treatment. Pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. Collectively, these results strongly suggest that the p38 kinase pathway only plays a minor role, if any, in the DSS model. (Sp) were GMCSF differentiated, DCs purified through Gr1+ cell depletion, and spleen Tcells isolated by pan Tcell negative selection. SpDCs or BMDCs were stimulated +/-100ng/ml LPS. For MLR, Balb/c Tcells were added for 5 days. Cells were incubated with SB203580 (4-(4-Fluorophenyl)-2-(4-ethylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, SB) or ML3403 ((RS)-{4-[5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]pyridine-2-yl}-(1 -phenylethyl)amine, ML) and washed prior to LPS stimulation (BMDCs) or cell mixing (T Cells). 3Hthymidine incorporation was measured, cell viability by MTT assay, TNFa and IL-12 production by ELISA. MLR Tcell proliferation inSpDCs or BMDCs was inhibited by SB (IC50 SpDC 0.06mM, BMDC 1.0mM) and ML (IC50 SpDC 0.03mM, BMDC 0.03mM). Preincubation with DCs had no effect, despite reduced LPS stimulated IL-12 and TNFa synthesis by SB (IC50 IL-12 1.2mM, TNF 0.16mM) and ML (IC50 IL-12 0.9mM, TNF 0.11mM). Preliminary data shows that preincubation of T cells with SB and ML modifies the MLR response. p38 plays a role in the interaction of DCs and T cells in antigen recognition. However, pre-incubation of drugs with DCs was ineffective. The role of T cell p38 MAPK in the MLR is under investigation. p38 inhibitors may possess disease modifying properties because of reduced Tcell antigen reactivity to DC antigen presentation. (1), S Luik(2), S Laufer(2), M Seed (3), V Holan(4), S Fiorucci (5) (1) Synovo GmbH, Tübingen, Germany (2) University of Tübingen, Germany In vivo anti-inflammatory activity of certain p38 kinase inhibitors is limited by bioavailability. However, it is possible that they may be useful in the therapy of IBD should it be possible to mediate there uptake in and around bowel lesions. We reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (WBC) associated with lesions. We prepared prodrugs of p38 inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. Pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically (2 d post boost) or in a DSS or TNBS model of IBD in the mouse. In both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at 15mmolkg-1d-1. We propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. Despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. The plasma levels of interleukins (IL-8, IL-18) and adhesion molecules (E-selectin and ICAM-1) were measured in 36 patients with necrotizing pancreatitis. The measurement was performed immediately after admission, at the 3, 7, and 14 day. All patients were divided on two groups: first group compiled 20 patients, in which dexamethasone (16 mg/day during 7-8 days) was applied in the complex management of acute pancreatitis, and control group -16 patients that did not receive corticosteroids. All patients received the initial therapy. The increased levels of IL-6, IL-8, IL-18, ICAM-1, and Eselectin were noted in both groups of patients at the time of admission. The gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. Its levels clear correlated with the severity of MODS and spreading of necrotic processes confirmed by CT. Starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. The incidence of contamination of necrotic foci had no difference in both groups of patients. The ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of NF-kappa B pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. The objective of this study was to examine whether T cell specific overexpression of the Th2 transcription factor GATA-3 can inhibit Th1/Th17 cell mediated experimental mBSA arthritis. mBSA-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day 7. At day 1, T cell specific GATA-3 tg mice did not show any difference in arthritis score compared to wild type mice. However, at day 7, wild type mice had developed severe joint inflammation having the maximum arthritis score. In contrast, GATA-3 tg mice showed only mild joint inflammation, suggesting that T cell specific overexpression of GATA-3 protects against development of severe joint inflammation. FACS analysis revealed low levels of IL-17 +/IFN-gammacells in wild type as well as in GATA-3 tg mice at day 1. As expected, IL-4 positive cells were higher in GATA-3 tg mice compared with wild type mice. Interestingly, at day 7, the percentage of IL-17 +/ IFN-gamma-cells were markedly increased in wild type mice but not in GATA-3 tg mice, suggesting prevention of Th17 expansion under GATA-3 overexpression in vivo. These data revealed that T cell specific overexpression of the Th2 transcription factor GATA-3 protects against progression of severe joint inflammation during mBSAinduced arthritis. Furthermore, IL-17 +/IFN-gammacells play a critical role in the progression of joint inflammation in this model and GATA-3 overexpression in T cells prevents expansion of the IL-17 +/IFN-gamma-T cell subset. Pingping Jiang (1), PT Sangild(2), T Thymann (2), HH-Y Ngai (1), W-H Sit (1), K-L Chan (3), JM-F Wan (1) ( Necrotizing enterocolitis (NEC) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. Preterm delivery and enteral milk formula feeding are factors predisposing to NEC. To understand the pathophysiology of NEC, two-dimensional gel electrophoresis (2D PAGE) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous NEC occurring in response to 2 days of parenteral feeding followed by 1 day of enteral formula feeding. The intestinal proteomes of pigs with clinical symptoms of NEC (n = 3) were compared with corresponding pigs remained healthy (n = 4). SyproRuby staining was used and differently expressed proteins were identified by MALDI-TOF MS or MALDI-TOF/TOF MS. The proteins with significantly different expression between NEC and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase 2 and Chain A, Medium-Chain Acyl-Coa Dehydrogenase with 3-Thiaoctanoyl-Coa etc.), inflammation (peptide-binding protein 74 (PBP74) and snail homolog 3), signal transduction proteins (thyroid hormone binding protein precursor, PARK7 protein and chain B, structure of the rho family GTP-binding protein cdc42 in complex with the multifunctional regulator RhoGDI etc.) and anti-oxidation (manganese-containing superoxide dismutase(SOD)). These data underscore the significant impact of intestinal proteomics in unraveling NEC pathophysiology and biomarker discovery.

blood are used to monitor the progression of inflammation. The aim of our study were to investigate systemic markers of disease in a rat model of LPS induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. Animals were exposed to bacterial lipopolysacharide (E.coli 026:B6) by inhalation. The lungs were lavaged 6 hours post provocation and the level of cell influx was determined. Relevant mediators of acute pulmonary inflammation were analysed with standard ELISA technology in bronchoalveolar lavage fluid and in blood. In addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. Results showing the effects of LPS challenge on local and systemic parameters will be presented and the possible link to LPS responses in man discussed. Pulmonary inflammation models are widely used in pharmacological research. However, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. Also, results derived from bronchioalvelar (BAL) fluid are subject to variability if the retrieval techniques are non standardized. Here we describe a non-invasive standardized method for retrieval of BAL fluid to be used in mice. We present the characterisation of these techniques using the inflammatory response to LPS and propose that this non invasive method can be used to refine LPS and other challenge models. The objectives were to evaluate the dynamic response after a single intra-tracheal administration of of 5mg (50ml/animal) of LPS (P.aeruginosa) to C57BL/6J mice. Control animals received a single dose of sterile 50ml 0.9% saline/animal. The mice were terminated 4, 24, 48, 72 and 96h after instillation using a non invasive and operator independent lavage technique.

Results showing the effects of single LPS challenge on BAL parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. These techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from BAL fluid retrieval.

The human psoriasis xenograft SCID mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. The model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. In order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient SCID mice. Transplanted mice were treated with Daivonex /Dovobet (Calcipotriol) and BMS (Betamethasone). The results show a strong antipsoriatic efficacy after treatment with BMS (epidermal thickness reduced by 68%). Treatment with Daivonex / Dovobet also showed an anti psoriatic effect with a 31% reduction in epidermal thickness. Serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. The observed effects are in concordance with clinical results of treatment of psoriasis. It is concluded that the model is useful for testing topical treatments. We have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (Barja-Fidalgo; Inflamm Res 52 (11): 2003) . Here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (UN-Group) or 22% protein diet (C-Group) during the first 10 days of lactation. UN rats showed lower number of blood PMN, though no difference in bone-marrow neutrophils number was observed. Blood neutrophils from UN-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed iNOS and spontaneously produced O2-, NO and TNF-alpha. In vivo treatment with LPS, at non-lethal doses, significantly increases TNF-alpha and superoxide production by neutrophils, compared with controls. LPS increased NO production by neutrophils from both groups, inducing iNOS expression in control cells, but no further increase in iNOS expression in UN rats. Nucleare NF-kB is constitutively augmented in UN rats. UN animals presented a higher survival rate in a model of CLP-induced severe sepsis. These results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. Transgenic mice over-expressing vascular endothelial growth factor (VEGF) in the epidermal basal layer under the human keratin 14 (K14) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. Important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of T-cells in the dermis and epidermis and also increased dermal angiogenesis. The aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with T-cells in transgenic K14/VEGF mice. We induced a cutaneous inflammation in the ear skin by repeated topical treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), in order to investigate the inflammatory response. The in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone-17-valerate, was also investigated. The ear thickness was significantly increased in transgenic animals compared to wild type animals following TPA-induction. The epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. Furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. A marked infiltration with CD3-positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. Finally, topical treatment with betamethasone-17-valerate significantly reduced the ear swelling and epidermal thickness. We conclude that over-expression of VEGF in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. The model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. Background and aims: The diabetes-prone BB (BBDP) rat spontaneously develops insulin-dependent diabetes resembling type 1 diabetes (T1D) in man. The BBDP rat is T-cell lymphopenic with a profound lack of regulatory T cells. The recent thymic emigrants in BBDP rapidly undergo apoptosis unless rescued from apoptosis by TCR stimulation. The increase in apoptosis is due to a frameshift mutation in Gimap5 which causes a severe truncation of the protein. The mutation is the strongest genetic factor for rat T1D. We aim to detect how Gimap5 affects the lifespan of T cells. Results: Overexpression in C58 cells of both wt Gimap5 and Gimap5 with the BBDP mutation causes an increase in apoptosis -the latter with a very rapid onset. Reduction of human Gimap5 by RNA-interference in Jurkat cells did not affect the number of apoptotic cells. Overexpression of human Gimap5 causes apoptosis in Jurkat cells and primary naïve T cells but not in activated T cells. Finally, Gimap5-mRNA is upregulated in in vitro activated human primary T-cells (detected by RT-PCR).

Conclusions: Based on the phenotype of the BBDP, rat Gimap5 was expected to be anti-apoptotic. However, we report here that overexpression of both mutated and wt Gimap5 causes rapid death of the cells. This suggests that Gimap5 is pro-apoptotic. The results with human wt Gimap5 support this Conclusion: Recently, much focus has been on the cellular CD38/ cADPR signaling system during inflammatory processes. The CD38/cADPR system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine IL-8. To our knowledge, the expression and function of the CD38/cADPR signaling system in the human detrusor muscle have not been described. CD38 protein expression in cultured (explant technique) human detrusor smooth muscle cells (HDSMC) was demonstrated by western blot (WB) and confocal microscopy (CM). Cytosolic free Ca2+ concentration ([Ca2+] i) in HDSMC and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. WB and CM showed that HDSMC expressed cell surface CD38 which could be upregulated by IL-8 (20 ng/ml). In HDSMC briefly activated with IL-8 (20 ng/ml) cADPR induced a rapid, transient dose-dependent increase in [Ca2+]i. Cyclic ADPR-mediated Ca2+ increase was greatly reduced in Ca2+ free medium suggesting Ca2+ entry as well as Ca2+ release. Cyclic ADPR -elicited Ca2+ increase was mimicked by 3-deaza-cADPR, and blocked by 8-bromo-cADPR, a cADPR antagonist, but not by nifedipine or verapamil. In the presence of IL-8, cADPR caused concentration-dependent relaxations of detrusor muscle. We report for the first time that 1) HDSMC express cell surface CD38, 2) the expression and function of CD38 are augmented by IL-8, 3) externally added cADPR elicited a rapid, IL-8 -dependent, and 8-bromo-cADPR-inhibitable Ca2+ mobilization, 4) cADPR induces relaxation of human detrusor muscle. The study indicates a role of CD38/cADPR in human urinary bladder inflammation. Miao Lin is a formulation of Sen Miao San and Lingzhi that consists of Cortex Phellodendri, Atractylodisa Rhizoma, Radix Achyranthis Bidentatae, and Ganoderma lucidum. These ingredients are reported to have anti-inflammatory and analgesic effects. In this study, we have investigated the anti-arthritic property of Miao Lin in an animal model of arthritis induced by unilateral injection of Freunds complete adjuvant (FCA) into rat knees. Contents of the Miao Lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for 7 days before induction of arthritis and 7 days after. Extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. Assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. Single unilateral injection of FCA into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. Intra-peritoneal injection of Miao Lin (50 mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration (500 mg/kg/day) suppressed oedema and hyperaemia. Histological examination showed that both routes of administrations of Miao Lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered Miao Lin also attenuated synovial tissue proliferation. These findings suggest treatment with intra-peritoneal or oral Miao Lin could provide significant anti-arthritic effects. An extract of the anti-arthritic Thermalife Cream contains 13 trace elements. Diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. Non-penetrating trace elements were discarded from the test formula (T2), and compared with the original formula (T1) for in vitro anti-inflammatory efficacy (TNF-a secretion in LPS-challenged human monocytes).

Methods: Human epidermis was mounted in vertical Franz type diffusion cells (stratum corneum facing up). T1 cream (n=4) or no cream (n=4) was applied to the donor compartment of diffusion cells, with PBS in the receptor compartment (3.0ml ; stirred continuously at 37 C). 240 Min after administration the receptor fluid was analysed for presence of metal ions by ICP-MS. A replication study used a different skin donor. Subsequently, human monocyte cultures (10% FCS, 5% CO2) were either stimulated with 500ng/ml LPS (E.coli 0111:B4,) or not in the presence of 10% T1, 10% T2, or no treatment.24 Hours after incubation, culture media were collected, centrifuged, and assayed (cytokine ELISA). Statistical analyses used a Treat by LPS ANOVA (p < 0.05).

Results: Zinc was the only trace element to penetrate the human epidermis significantly. Both formulations strongly suppressed LPS-induced TNF-a secretion. T2 with zinc only was more effective than T1 (Treat:F2,12 = 57.13, p < 0.0001; LPS:F1,12 = 245.47, p < 0.0001; Treat by LPS:F2,12 = 70.01, p < 0.0001).

Conclusions: Anti-TNF efficacy from Thermalife extracts was retained with zinc chloride as the only trace element. (1) (1) Osprey Pharmaceuticals Limited, Canada (2) Probetex, Inc., Texas, USA

The CCL2/CCR2 chemokine/receptor axis, infiltrating monocytes/macrophages (M/M), Th1 cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. The eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. A recombinant fusion protein comprised of the human CCL2 chemokine fused to a truncated form of the enzymatically active A1 domain of Shiga toxin has been developed. The CCL2 portion binds specifically to CCR2-bearing leukocytes and enters the cells, where the SA1 portion inhibits protein synthesis. The compound was tested in a model of Anti-Thymocyte Serum (ATS)-induced mesangioproliferative glomerulonephritis. Male rats were injected with ATS on day 0 and treated intravenously with vehicle, 50 or 100mg/kg of the recombinant protein Q2D from day 2 until day 8. Urine and blood collections were made prior to ATS injection and on days 5 and 9. Animals were sacrificed on day 9. No treatment related effects on body weight or signs of clinical toxicity were observed. Urine protein levels were decreased in treated animals. Histopathological analyses of kidney sections revealed maximum reductions of 40, 36, 38, and 28% for glomerular lesions, M/M count, fibronectin and µ-smooth muscle actin, respectively. The latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. These results indicate a significant renal-protective effect in this model of nephritis. Further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. Inflamm. Res., Supplement 3 (2007) Posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the Sca-1 antigen. Such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. Using this model we investigated the influence of the proinflammatory cytokines, TNFa or IL-1b, on early osteogenesis in vitro. Under osteogenic conditions, IL-1b was found to inhibit cell proliferation in a dose dependent manner, whereas TNFa exhibited no effect. Histochemical examination revealed the presence of either TNFa or IL-1b to dramatically decreased mineralization in a dose dependent manner. Q-PCR analysis indicated that in the presence of IL-1b, despite increased expression of bone-specific alkaline phosphatase (Akp2) mRNA, levels of other osteogenesis markers (Runx2, Col1a and Sp7) were decreased. In the presence of TNFa, levels of Akp2, Runx2 and Sp7 were all decreased. Our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. Coronary artery disease (CAD) is characterized by enrichment of inflammatory cells in the vessel wall. We hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. In vivo transmigration was studied by use of a skin blister model. Blisters are raised by suction and cells are analysed the following morning (0h blister) and after additional ten hours of incubation with PBS or autologous serum, corresponding to intermediate and intense blister. Monocytes were analysed by flow cytometry for the expression of CD11b, before and after in vitro fMLP stimulation. Chemokines in serum and blister fluid was analysed in parallel. CD11b expression on resting monocytes harvested from 0h blister was lower in patients as compared to controls (p=0.001). Lower expression of CD11b in patients was also observed in the intermediate and intense blisters after stimulation with fMLP (p=0.04 and p= 0.005, respectively). The number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. Serum concentration of MIP-1µ was higher among patients (p=0.01) and similar levels were seen in blister fluids. Concentration of MCP-1 was similar in both serum and blister fluid. We demonstrate that monocytes from patients with CAD have a reduced expression and ability to up-regulate the adhesion molecule CD11b at sites of inflammation. These differences may modulate events related to the transmigration process and indicate a changed activation pattern. To which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. In inflammation, nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) induced by bacterial products and cytokines, and NO acts as a regulatory and proinflammatory mediator. One of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of NO production. The aim of the present study was to investigate the mechanisms how glucocorticoids inhibit iNOS expression and NO production. Dexamethasone and a dissociated glucocorticoid RU24858 inhibited NO production, and iNOS protein and mRNA expression in murine J774 macrophages exposed to bacterial lipopolysaccharide (LPS). In the presence of a glucocorticoid receptor (GR) antagonist mifepristone, the effects of dexamethasone and RU24858 on NO production were reduced. The role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (HDACs); non-selective trichostatin A and apicidin, and HDAC1 selective MC1293. HDAC inhibitors reversed the effects of dexamethasone and RU24858 on iNOS expression or NO production. Stably transfected A549/8 cells containing human iNOS promoter were used in promoter-activity studies. Cytokine-induced iNOS promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin A. These results suggest that glucocorticoids inhibit iNOS expression and NO production by a GR-mediated and GRE-independent manner possibly through histone deacetylation and transcriptional silencing. We are investigating mechanisms involved in TNFainduced hyperalgesia in the mouse paw. Previously, we have seen that TNFa causes a TRPV1-dependent bilateral hyperalgesia. Here we investigate the role of COX in this process. Female CD1 mice (25-30g) were given intraplantar injections (i.pl.) of TNFa (10pmol/50microl) and Tyrode (as vehicle, contralateral paw; 50microl). Thermal hyperalgesic thresholds were measured using the Hargreaves technique before and 1h after injection. Indomethacin (20mg/kg) was co-injected with TNFa whilst contralateral paw was injected with Tyrode and corresponding amounts of indomethacin vehicle (5% NaHCO3). Another group of mice were injected with TNFa i.pl. plus 5% NaHCO3 with the contralateral paw injected with Tyrode plus indomethacin (20mg/kg). Results are expressed as mean AE s.e.m and statistical analysis performed using Students t-test. TNFa (10pmol) leads to significantly reduced (p<0.05 compared to baseline values) paw withdrawal latency in both paws 1h after injection i.e bilateral hyperalgesia. However, local injection of indomethacin (20mg/kg) with TNFa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. Interestingly, indomethacin co-injected with Tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. The same results were seen using the selective COX-2 inhibitor, nimesulide. In conclusion, COX-2 derived prostaglandins are important in the development of hyperalgesia. Local COX-2 inhibition at the site of TNFa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. Hydrogen sulfide (H2S) is synthesized naturally in the body from cysteine by cystathionine g lyase (CSE). H2S has been variously reported to exhibit both pro-and antiinflammatory activity. In an attempt to obtain further information about the role of H2S in inflammation we examined the effect of dexamethasone on lipopolysaccharide (LPS)-mediated endotoxic shock. Male Sprague Dawley rats (240-280g) were administered dexamathasone (1 mg/kg, i.p.) either 1 h before or 1 h after LPS (4 mg/kg, i.p.) injection. Animals were killed 3 h after LPS administration and plasma and tissues harvested. As expected, LPS injection significantly increased plasma TNFa and IL-1b as well as liver and lung myeloperoxidase (MPO) activity. LPS also increased plasma nitrate/ nitrite (NOx), H2S concentration and liver and kidney H2S synthesis from exogenous cysteine indicative of upregulation of CSE in these tissues. Either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma H2S and tissue H2S synthesizing activity. In separate in vitro experiments, exposure of rat peripheral leucocytes to LPS (100 ng/ml, 3 h, 37oC) resulted in upregulation of both CSE and iNOS (measured by Western blot). Dexamethasone (100 nM) significantly (P<0.05) reduced expression of both CSE and iNOS. These data provide further evidence that H2S is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of H2S production. (1), U Jalonen (1), H Kankaanranta (1), R Tuominen(2), E Moilanen (1) (1) The Immunopharmacology Research Group, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland (2) The Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Tristetraprolin (TTP), also known as Nup475, TIS11, G0S24 and Zfp36, is a factor that binds to 3UTR of mRNA of some transiently expressed inflammatory genes and regulates mRNA stability. TTP has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. However, the regulation of the expression of TTP itself is largely unknown. In the present study, we investigated the role of classical protein kinase C (cPKC) isoenzymes in the regulation of TTP expression.

In J774 macrophages TTP expression is induced by lipopolysaccharide (LPS) and this can be further enhanced by addition of 100 nM phorbol myristate acetate (PMA). This additive effect of PMA on TTP was abolished by a prolonged preincubation with a higher S442

Inflamm. Res., Supplement 3 (2007) Posters concentration of PMA for 24 h, which also downregulated the expression of PKCa, PKCbI and PKCbII isoenzymes. PKC inhibitors RO318220 (inhibits PKCb, & and e), GÖ6976 (inhibits PKCa, b and &) and CGP53353 (inhibits PKCbII) reduced LPS + PMA -induced TTP protein and TTP mRNA expression. PKCbII inhibitor CGP53353 did not affect TTP mRNA half-life and therefore we measured the effects of CGP53353 on the activation of transcription factors involved in TTP expression. CGP53353 had no effect on the activation of NF-kB, EGR1 or Sp1. In contrast, CGP53353 reduced the activation of transcription factor AP-2, which may explain its inhibitory action on TTP expression.

The results suggest that PKCbII is involved in the regulation of TTP expression in activated macrophages, possibly through the activation of transcription factor AP-2. The most widespread Gracilaria verrucosa in the sea of Korea is the attached form of red algae growing on a rockly substrate. In this study, we isolated fourteen compounds from G. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (TNF-a IL-6, IL-1 and NO) in RAW264.7 cells. Among them, 10-oxooctadec-8-enoic acid and 11-oxooctadec-9-enoic acid inhibited the production of TNF-a, IL-6, IL-1 and NO at the concentration of 20 mg. Also, these two compounds showed inhibitory activity on the mRNA expression and protein level of inflammatory markers (TNF-a IL-6, IL-1 and iNOS) in a dose-dependent manner. These results suggest that G. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and iNOS.

Lene Jensen(1), P Hjarnaa (1), J Fensholdt (1), P-P Elena(2), K Abell (1), TK Petersen (1) (1) Discovery, LEO Pharma, Ballerup, Denmark (2) IRIS Pharma, La Gaude, France

Angiogenesis is known to play an important role in many inflammatory diseases including arthritis. Additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (AMD). We have synthesized a potent angiogenesis inhibitor, LEO-A, targeting kinases related to angiogenesis, e.g. VEGFR-2. Additionally, LEO-A has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. The compound was tested systemically in inflammatory in vivo models in mice and rats. The in vivo models selected include the CIA arthritis model (mice and rats), the local GvH rat model, the LPS induced TNFa model (mice and rats), the anti-CD3 induced IL-2 response mouse model and the rat argon laser-induced choroidal neovascularisation (ChNV) model, a model for AMD. The following results were obtained after systemic treatment with doses of up to 30 mg/kg i.p. or 50 mg/kg p.o. once daily: In the local GvH model, LEO-A significantly inhibited the growth of the local lymph node by 50 %. In the CIA model, LEO-A had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). In the LPS induced TNFa model in mice, high doses of LEO-A were found to inhibit the TNFa release. In the ChNV model, a significant effect was obtained following systemic treatment. In conclusion, LEO-A has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved.

Mice lacking PI3Kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. We show here that mice harbor PI3Kg gene depletion and PI3Kd kinase-inactive mutation, pik3cgd KOI, exhibited thymus atrophy, similar to previously reported PI3Kg and d double knockout (p110g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory T cell activity. In particular, serum IgG1/ IgG2a ratio and IgE level were elevated in pik3cgd KOI mice corresponding to a skewed Th2 profile in vitro. Histological analysis revealed eosionophil-and T celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik3cgd KOI mice, but organ-specific auto-antibody was not detected in circulation. On the contrary, when mature WT T cells were treated with PI3K d or together with PI3K g selective inhibitors, while Th1 cytokines were suppressed Th2 cytokines were not augmented in vitro. Thus, T cell development, but not peripheral T cell proliferation or cytokine production, requires cooperativity of PI3Kg and d. Genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type 2 Ig and T cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote Th2 reaction but attenuate Th1 medicated disorders. Platelet activating factor (PAF) is an important mediator in several pathophysiological processes. PAF receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. PAF can up-regulate kinin B1 receptor expression by various mechanisms. Our aim was to investigate the role for kinases in PAF-induced kinin B1 receptor up-regulation. Wistar rats were treated with PAF, or left untreated as controls, 6h before i.d. injection of 0.1ml PBS containing des-Arg9-bradykinin (DAPK, 100nmol right hind paw) and 0.1ml PBS (for control, left paw). Various kinase inhibitors were administered to the rats after PAF treatment and oedema was measured by the use of a plethysmometer (Ugo Basile) 10-120 minutes after DAPK-injection. Oedema was expressed in ml as difference between right and left paws.Additionally paw samples were taken for Western blot analysis for total and phosphorylated forms of JNK and ERK1/2. DABK-induced paw oedema after PAFinjection is significantly inhibited by the selective JNK SP600125 and ERK1/2 PD98059 inhibitors. Western blot analysis shows that phosphorylation of JNK and ERK1/2 is important in the up-regulation of B1 receptors. Our results clearly show that the phosphorylation of both ERK1/2 and JNK MAPKinases is an important step for the in vivo up-regulation of B1 receptors by PAF. However, the exact mechanisms (transcriptional and post-transcriptional) by which PAF can trigger kinase phosphorylation and then up-regulate the B1 receptor require further investigation. Continued interest in development of small molecule inhibitors of p38 mitogen-activated protein (MAP) kinase is based on the central role this enzyme plays in inflammatory cell signaling. Activation of p38 leads to increase production of pro-inflammatory cytokines such as TNF-a and IL-1b making it an prominent target for antiinflammatory drug discovery. A virtuell screening approach identified 3-(2-chlorophenyl)-6-((4-methoxyphenoxy)methyl)- [1, 2, 4] triazolo [3,4-b] [1, 3, 4] thi adiazole as a potential hit. This was confirmed by synthesis and testing. To explore further SAR, a first set of derivates was prepared by cyclization of the 5-substituted-4-amino-3-mercapto-4H-1,2,4-triazoles with carboxylic acids in presence of phosphorus oxychloride. The synthetic strategy used allows both variation at position 3 and 6. Synthesis and SAR will be presented. Cytokines like IL-1b and TNFa play central roles in inflammatory diseases like rheumatoid arthritis. Production of cytokines in monocytes, macrophages and other cells is triggered by factors such as LPS, UV-light, osmotic and cellular stress or physical and chemical attraction. In particular IL-1b and TNFa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. Involved in this signal pathway, p38MAPK as a pivotal enzyme is considered to be a validated drug target and therefore, p38MAPKinhibitors are of therapeutical interest. In this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p38MAPK-inhibitors with promising in vitro activity. The assay procedure involves defined blood cell stimulation by LPS and isolation of TNFa or IL-1b, which are subsequently quantified by TMB-ELISA technique via photometric measurement. The validation of the assay conditions involved well characterized p38MAPK inhibitor SB203580 and a highly active compound developed in our lab. A data set was generated by determining 18 whole blood samples consisting of in each case three male and female individuals on three different days. Statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. P38 mitogen-activated protein (MAP) Kinase is required for the biosynthesis and release of pro-inflammatory cytokines IL-1 and TNF a. Inhibition of p38 MAP Kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. Trisubstituted pyridinylimidazoles are potent inhibitors of the p38 MAP Kinase. Scope of this work was to investigate 2-thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. We synthesised 2-alkylsulfanyl, 4-(4-fluorophenyl), 5-(2-aminopyridin-4-yl) substituted imidazoles as p38 MAP Kinase inhibitors. As substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. The synthesis and biological testing of effective the 2-aminopyridin-4-yl imidazoles with low inhibitory concentrations are described. Biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.Variation at the 2-thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone NH group of Met109, for inhibitory activity. Less clear is the importance of the hydrogen bond between N3 of the imidazole ring and Lys53 of p38 MAP kinase.To investigate the role of Lys53 in interacting with the scaffold we prepared two sets of 4,5diaryl-substituted isoxazoles. These data suggest a dynamic interaction of the core heterocycle with Lys53, contrary to the observation on the compound VK-19911 and p38 MAP kinase, that a nitrogen atom bearing a lone pair in position 3 of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of Lys53 rather than to form an attractive interaction with p38 MAP kinase. To complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of 3-substituted and unsubstituted 4,5-diarylisoxazoles investigating the interaction with the hydrophobic pocket II of p38. These data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. Despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. It is a consequence of acute inflammatory response to lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria. Natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. In this study, we investigated the effect of ferulaldehyde (FA), a natural compound of red wine, on LPS-induced endotoxic shock in mice and on LPS-stimulated murine macrophage-like RAW 264.7 cells. Treatment of C57BL/6 mice with FA significantly attenuated the LPS-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines TNF-a and IL-1b in the serum. The serum level of the anti-inflammatory cytokine IL-10 was higher in mice treated with FA and LPS compared to LPS treatment alone. LPS-induced phosphorylation and thereby activation of Akt, and JNK was also strongly inhibited by FA treatment whereas the phosphorylation level of Erk1/ 2 and p38 MAPKs remained unaltered. Activation of nuclear factor-kappaB (NF-kB) in liver of FA-treated mice were significantly suppressed. Although FA had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in RAW 264.7 cells either, it decreased the LPS-induced ROS and nitrite production in a dose-dependent manner. Our results suggest that FA has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress NF-kB activity via the down-regulation of Akt and JNK. Myeloperoxidase (MPO), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. Therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. On the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. The formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. Membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. We investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI TOF MS). Specific reaction products play an important role in the modulation of the immune response.

Comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. Objectives: To study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (RA).

Methods: Twenty patients with active RA received leflunomide 100 mg for 3 days followed by 20 mg daily for 32 weeks. At week 2 all patients started infliximab 3 mg/kg, and received a further four infusions at weeks 4, 8, 16 and 24.

Results: The commonest adverse event was pruritis associated with an eczematous rash. There was no relationship between the serum concentration of A77 1726, the active metabolite of leflunomide, and adverse events. The mean Disease Activity Score (DAS28) fell from 7.18 at week 0 to 5.18 (P<0.0001) at week 4 and remained between 3.85 and 4.85 up to week 32. In those patients remaining on treatment, more than 80% achieved an ACR20 response from week 8 to week 28, and up to 46% achieved an ACR70 response.

Conclusions: Infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult RA. However, widespread use may be limited by adverse events, which were common and in some cases severe. Objective: The transcription factor nuclear factor-kB (NFkB) regulates the expression of proinflammatory cytokines such as TNFa and IL-1 those play pivotal roles in pathogenesis in rheumatoid arthritis. Parthenolide, a sesquiterpene lactone, was reported to inhibit the DNA-binding of NFkB. The objective of this study is to investigate the potential of Parathenolid to inhibit the pathogenesis of collagen-induced arthritis.

Methods: Mice were injected i.p. with a cocktail of 4 anticollagentype II mAbs on day 0, followed by i.p. injection of LPS on day 3 to induce anti-collagen mAb-induced arthritis. The mice were orally administrated with Parathenolide (50 mg/kg/day) starting on the day of first immunization (Day 0) in prophylactic treatment group and after the onset of arthritis (Day 4) in the therapeutic treatment group. Clinical disease score, radiographic and histological scores were evaluated. mRNA expression of IL-1b and TNFa in the affected joints were measured by real-time PCR.

Results: Clinical disease scores were significantly reduced both in prophylactic treatment group (7.4AE3.7) and therapeutic treatment group (7.25AE3.1) compared to untreated group (13.6AE2.7, p = 0.0163 and p = 0.0084 respectively). Histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p<0.05). Steady state mRNA levels of IL-1b and TNFa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p<0.05).

The results in this study suggest that NFkB is an important therapeutic molecular target for treatment of inflammatory arthritis. Fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. The concentration of this glycoprotein increase in inflammatory conditions. A fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. The objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. Neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine 123 (1ìM) to detect oxygen free radical production. The cells (1,0x 106 cell/mL) were then incubated with a range of concentrations of fibrinogen (0-400mg/dL) for 15 minutes. Our results show that Posters Inflamm. Res., Supplement 3 (2007) S449

fibrinogen leads to an increase in neutrophil activation as measured by free radical production. This effect becomes evident at borderline-high concentrations (300-400mg/ dL), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. We hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. Cyclooxygenases (COX-1 and COX-2) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. The constitutively expressed isoform COX-1 is mainly involved in homeostatic processes, while the inducible isoform (COX-2) is associated with inflammatory reactions. Various in vitro assays have been developed in order to define the selectivity against COX-1 and COX-2 of nonsteroidal anti-inflammatory drugs (NSAIDs). However, these in vitro assays can give discordant results related to several parameters. The aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new NSAID candidates. First, in an enzymatic COX assay, the arachidonic acid concentration (AA; COX substrate), and the species of COX enzymes tested (ovine vs. human), two factors able to conceal the anti-COX activity of NSAIDs, have been evaluated and optimized. Next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. This cell-based assay allows concomitant measurement of anti-COX-1 and anti-COX-2 effects by prostaglandin E2 (PGE2) measurement after A23187 (calcimycin) and bacterial lipopolysaccharide (LPS) stimulations, respectively. Both assays have been calibrated and compared by testing 7 reference NSAIDs, selective or not for COX-1 or COX-2. Fifty % inhibitory concentration (IC50) values against COX-1 and COX-2 and COX-2:COX-1 ratios obtained were in accordance with the previously described NSAID specificity and coherent between both assays (r= 0.93). In conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new NSAID candidates against human COX-1 and COX-2. For increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. The human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. We aimed to study the translational aspects of these models.

Material and Methods: In humans, epidermal skin chambers were stimulated with autologous serum for 10 hours. In rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day 6. The inflammatory response was measured after 4, 8 and 24 hours. The cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated.

Results: At 8/10 hours the cellular distribution was similar in air pouch and skin chambers. The major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. Both in human and rats the concentrations of MPO and MCP-1 were increased. Furthermore, transmigrated cells displayed a different chemokine receptor pattern. In rats transmigrated cells expressed CD11b, were CD45lo, SSClo and RP-1+ (granulocyte marker). In humans, transmigrated granulocytes expressed CD16 and CD11b. These cells had a significantly higher CD11b expression compared to corresponding cells in peripheral circulation.

Our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. These models may be useful for bridgingpreclinical and clinical drug discovery. Furthermore, they may work as translatable proof of mechanism (PoM) models for drug candidates targeting different inflammatory components. Objectives: To analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke.

Material and Methods: We investigated 86 consecutive patients with mean age 67AE7.8 years who were admitted within 24 h after ischemic stroke. A control group of 37 patients with mean age 58AE4.9 years without ischemic stroke was also tested. Measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay.

Results: Patients with acute ischemic stroke had significantly higher serum levels (mean value+SD) of neopterin than those without acute ischemic stroke: 9.6AE1.2 and 7AE0.8 nmol/L. Correlation analysis revealed p<0.01.

Discussion: Immune mechanisms contribute to cerebral ischemic injury. The finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin.

Our results indicate increased macrophage activation after ischemic stroke. In patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. However, these preliminary results need be confirmed by controlled studies. produced a marked (p<0.01) reduction in the number and duration of ventricular tachycardia (VT) during both ischemic and reperfusion phases. The total number of ischemic ventricular ectopic beats (VEBs) reduced from 667ffAE116 in the control to 66ffAE22 at the concentration of 50 ffmg/ml (p<0.001). In the ischemic phase, Cynodon Dactylon (50 ffmg/ml) also decreased the incidence of VT from 100% (control) to 33%. In addition, incidences of reperfusion-induced VT, total VF and reversible VF duration were significantly lowered by the same concentration (p<0.01 for all). The results show that Cynodon Dactylon has a protective effect against I/R-induced cardiac arrhythmias in isolated rat hearts. Regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of Cynodon Dactylon probably is due to its anti-inflammatory properties.Key words: Cynodon Dactylon; arrhythmias; anti-inflammatory; isolated heart; rat Objectives: Intestinal ischemia-reperfusion (IRI) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. We thus decided to investigate the effects of IRI on NOS and COX isoforms, neutrophil infiltration (MPO), lipoperoxidation (TBARs) and protein tyrosine nitration (NT) in different brain areas and diaphragm muscle of Wistar rats.

Methods: IRI comprised the occlusion of superior mesenteric artery during 45 min followed by 2 h of reperfusion. Sham animals were submitted to the surgical procedure with no interference on the blood flow.

Results: IRI resulted in increased expression of mRNA for nNOS (cortex) and COX-2 (hypothalamus) associated to a marked reduction of Ca2+-dependent NOS activity in cortex, hypothalamus and hippocampus (but not in cerebellum). TBARs contents were also reduced in cortex and hypothalamus. Neither MPO activity nor NT was altered by IRI in the brain. Diaphragms from animals with IRI exhibited increased MPO and Ca2+-dependent NOS activities, as well as TBARs content and NT. In contrast, eNOS protein expression and both gene and protein nNOS expression were reduced. No effects were observed on COX isoforms or eNOS gene expression.

Conclusions: These findings suggest that, within the first 2 h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. In the CNS, distinctive susceptibility to the IRI seems to occur in the different areas, probably as a defensive strategy aimed to counteract the IRI-mediated systemic injury.

Anne-Sofie Johansson (1), H Qui (2), M Wang (2), I Vedin (1), JZ Haeggstrçm (2), J Palmblad (1) ( (1), R Carnuccio(2), P Romagnoli (3), F Rossi (1) (1) Second University of Naples, Italy (2) University of Naples, Italy (3) University of Florence, Italy

We previously found that several inflammatory markers, e.g., nuclear factor-kB (NF-kB), were increased and a neointima was formed in a model of carotid surgical injury (1) . The purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. To this aim we measured COX-2, NF-kB, platelet aggregation and neointima formation in rats administered rosiglitazone (10 mg/kg/ die, by gavage) for 7 days before carotid injury and 21 days after injury. Control rats received physiological solution. 14 days after injury COX-2 expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p<0.0001). Rosiglitazone also caused a significant decrease of NF-kB/DNA binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. Platelet aggregation was reduced by 30% in treated versus control carotids (p<0.0005). The influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting 7 days after rosiglitazone treatment. The results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. The aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (EAE). Rats of EAE-susceptible Dark Agouti and EAE-resistant Albino Oxford strain were immunized with guinea pig spinal cord homogenate (DAGPSC and AOGPSC), while non-immunized rats served as controls (DANIM, AONIM). On day 15 after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. Macrophages from AONIM rats exhibited lower adherence capacity and higher phagocytosis and H2O2 production then macrophages from DANIM rats. Immunization decreased adherence and phagocytosis and increased H2O2 production in macrophages from AO rats, but did not influence these activities in macrophages from DA rats. Our results suggest that inflammatory activities of macrophages from AO rats could be considered as regulatory mechanisms connected with the resistance to EAE induction ( (1), B Sehnert (2), H Lanig(2), S Päßler(2), R Holmdahl (3), H Burkhardt (1) (1) Johann Wolfgang Goethe University, Frankfurt, Germany

(2) Friedrich-Alexander University of Erlangen-Nuremberg, Germany (3) Lund University, Sweden

Objectives: The aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine IgG mAB CIIC1that is highly somatically mutated and its epitope on type II collagen (CII, aa359-369).

Methods: The establishment of a dynamic simulation modelling of a CIIC1 single-chain fragment (scFV) in complex with the triple helical CIIC1 epitope permitted structural insights into immune complex formation. The computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the C1scFvs and the respective CIIC1 epitope that were produced as recombinant constructs. The binding affinities of the mutated scFVs were determined by ELISA and surface plasmon resonance measurements.

The mutation experiments confirmed the predicted interaction sites of CII in the CDR2 and CDR3 regions of both heavy andlight chain. Surprinsingly also the model prediction, that the conversion of the C1scFv sequence into the respective germline does not affect CII binding affinity (KD 3x 10-8) could be confirmed experimentally by the mutagenesis of 13(!) positions.

Our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. The molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding CDRregions of the antibody considerably compared to globular antigens. These sterical constraints might provide an explanation why somatic mutations have no obvious impact on CII recognition by the arthritogenic autoantibody. Moreover, the structural insights into CII-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. We observed a significant association between the MBP-elicited CD4+ T-cell proliferation and active brain lesions, on the one hand, and IL-4, IL-5 and IFN-gamma, on the other. When grown in the presence of standard serum from a healthy donor, PBMC from healthy individuals responded to MBP with a higher IL-10 production than PBMC from MS patients. Thus, normal PBMC respond to MBP with production of TNF-alpha, IFN-gamma and IL-10, but MS is associated with enhanced TNF-alpha-, IFN-gammaand decreased IL-10 responses, and disease activity is associated with MBP-induced proliferation of CD4+ T cells. (1), K Goula (1), P Georgakopoulos (2) (1) Renal Unit, St. Anrdew Hospital, Patras, Greece (2) Intensive Care Unit, St. Anrdew Hospital, Patras, Greece

Background: Urethritis is an infection of the urethra. Most cases are sexually transmitted. Haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. Patients with underlying diabetes are also a specific population at risk. Urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (E. coli or klebsiella). Viral causes of urethritis include herpes simplex virus and cytomegalovirus. Neisseria gonorrhoeae and C trachomatis account for most cases of urethritis in men (70%). The aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. We also determined diabetes as a coexisting factor in the infected patients.

We retrospectively reviewed all cases of urethritis of 72 maintenance haemodialysis patients at our center over the past 5 years. The diagnosis was made according to patients symptoms and signs but also using urine specimens for culture.12 patients (16.6%) from the study group were diabetic.

Results: 4 cases of urethritis were determined. All infected patients were diabetic. Isolated microorganisms were E. coli (2 cases), Enterobacter aerogenes (1 case Objectives: To explore the ability to use paquinimod as a steroid sparing drug in an animal model for SLE.

Methods: Mice were initially treated with a high dose of prednisolone (2 mg/kg/day). Thereafter the amount of steroid was reduced to 0.5 mg/kg/day and a low dose of paquinimod (0.2 mg/kg/day) was added. The development of glomerulonephritis was measured as hematuria during the experimental period. Serum was collected for analysis of anti-dsDNA antibodies. Kidneys were collected and histopathological observations were performed. Organ weight and lymphocyte sub-populations were assessed in the spleen.

Results: When treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. A significant reduction in the level of hematuria, in spleen enlargement and in the total number of CD4+, CD8+ and on CD4-CD8-T cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. The development of glomerulonephritis was also significantly reduced in these mice. An almost complete inhibition of anti-dsDNA in serum was seen in all treated groups.

Conclusions: When high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, T-cell sub-populations and development of glomerulonephritis were examined. This setting could be of great importance in future treatment of human SLE in order to reduce the steroid dose needed in the treatment of this disease. and La(SSB). The purpose of this study was to screen for novel antibodies against cell surface antigens in primary SjS. Proteins (MP) were isolated from cell membranes (HeLa cells), and were tested with sera from SjS patients or healthy blood donors individually in western blot (WB) at 1:100. MP were separated on 2-D gels and tested in WB (1:100) to locate the appropriate spots for mass spectrometry (MS) analysis. Paraformaldehyde fixed HeLa cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. Antigens were isolated at around 35, 50, 75, 100 kDa (64 total positive/79 tested patients). The dominant antigen was at 50 kDa. Large quantities of endogenous proteins were obtained and the membrane fraction was enriched. One of the main obstacles to further study possible surface antigens as M3 muscarinic receptor was overcome. Proteins were separated on 2D-gels and tested in WB to locate the relative spots for MS. The correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. In conclusion, membrane or membrane-associated antigens were recognised by sera from SjS patients. One of them might correspond to M3 muscarinic receptor. This identification might help in developing a diagnostic assay for SjS. Osamu Handa, S Kokura, K Mizuahima, S Akagiri, T Takagi, Y Naito, N Yoshida, T Yoshikawa

Aim: Various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. However the precise mechanism of cytotoxicity of these additives are not known. In this study, we investigated the effects of ultraviolet-B (UVB) exposure on additives-treated human normal skin keratinocytes (HaCaT).

Most popularly used additives in cosmetics such as methylparaben (MP), octandiol (OD) and phenoxyethanol (PE) were used. HaCaT keratinocyte was cultured in MP-containing medium for 24 h, exposed to UVB and further cultured for another 24 h. Subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with Hoechst 33342 and propidium iodide or annexin-V. Same experiments were done using OD and PE respectively instead of MP under same condition. In addition, gene chip analysis was performed in each group.

Results: UVB exposure enhances cytotoxicity of these additives even at low concentration. Gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group.

These results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight.

these kinases in the pathogenesis of psoriasis. Recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase 1 (MSK1) was demonstrated in psoriatic epidermis. The purpose of this study was to investigate MSK2 and the transcription factor cAMP-response-element-binding protein (CREB) in psoriatic skin and in cultured normal human keratinocytes. Keratome and punch biopsies were taken from patients with plaque-type psoriasis. Normal human keratinocytes were cultured and stimulated by interleukin-1â (IL-1ß) or anisomycin. Some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. On human skin inflammation, matrix metalloproteinase-1 (MMP-1) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. In the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on MMP-1 activity and MMP-1 expression were examined. From the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant MMP-1 with the IC50s of 39.6 -43.7 ìM, while the flavones such as apigenin and wogonin showed weak inhibition. When the effects of flavonoids on MMP-1 induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin (12.5 -25.0 ìM) strongly inhibited MMP-1 induction from TPA-treated human dermal fibroblasts, but naringenin (flavanone) did not. By gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, AP-1, whereas naringenin did not. Among MAPKs, quercetin inhibited the extracellular signal-regulated protein kinase (ERK) and p38 MAPK activation, and kaempferol inhibited the p38 MAPK and c-Jun N-terminal kinase (JNK) activation. On the contrary, the flavones and naringenin did not inhibit the activation of these three MAPKs. All these results indicate that the capacity of MMP-1 inhibition and MMP-1 down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. (1) (1) University of Valencia, Spain (2) Istituto di Chimica Biomolecolare CNR, Napoli, Italy

Avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. Recently, its derivative avarol-3-thiosalicylate (TA) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.It is interesting to note that avarol and TA inhibited NF-ÞB activation in HaCaT keratinocytes. Now, the effect of avarol and TA was investigated in the TPA-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. Topical treatment with TA (20 mg/ml) produced a 97 % inhibition of oedema and a strong reduction of PGE2 (100 %), LTB4 (100 %) and MPO activity (65 %) in skin homogenates. The inhibitory effect of avarol at the same dose was 79 % for oedema, 90 % for PGE2, and 50 % for LTB4 and MPO activity. Histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to TPA treatment. Besides, the reduction of cutaneous TNF-a by avarol and TA was also detected by immunohistochemical analysis. These compounds were also capable of suppressing NF-ÞB nuclear translocation in mouse skin. In summary, our results suggest that inhibition of proinflammatory metabolites by TA and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. Its mechanism of action is related to the inhibition of NF-ÞB activation and can be mediated by the downregulation of intracellular signal-transduction (1), AMS Silva(2), CMM Santos(2), DCGA Pinto(2), JAS Cavaleiro(2), JLFC Lima (1) (1) REQUIMTE, Departamento de Química-Física, Faculdade de Farmµcia da Universidade do Porto, Porto, Portugal (2) Departamento de Química, Universidade de Aveiro, Aveiro, Portugal 2-Styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. Several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. However, the anti-inflammatory potential of 2-styrylchromones has not been explored so far. Thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic 2-styrylchromones by studding their influence on different systems that are related to the inflammatory process. The putative inhibitory effects of several 2-styrylchromones on the proinflammatory enzymes cyclooxygenase 1 (COX-1), cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) was evaluated in vitro and compared with structurally related flavonoids. The capacity of the studied 2-styrylchromones to scavenge reactive oxygen (ROS) and nitrogen species (RNS) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. From the tested 2-styrylchromones, those having a catecholic Bring where shown to be the most effective scavengers of ROS and RNS, being, in some cases, more active than flavonoids. No considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. The results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. The usefulness of 2-styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. The importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. Primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. We therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (PET) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. Uptake of radiotracers 18F-fluorodeoxyglucose (FDG), 3-O-methyl-6-18F-fluoro-L-DOPA (OMFD), and 18F-labeled native/oxidized low density lipoproteins (nLDL, oxLDL) in single-or cocultivated human myeloid (monocytic) leukemia cell line THP-1 was compared with that by squamous cell carcinoma (FaDu), mamma (MCF-7) and colorectal adenocarcinoma (HT29) cell lines (without or in the presence of specific inhibitiors). Several THP-1 phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. Differentiated THP-1 cells show, when compared with tumor cells, a comparable FDG accumulation, a considerably lower OMFD uptake, and a significantly higher oxLDL uptake. On the other hand, during differentiation and retrodifferentiation THP-1 cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. The observed differences in uptake of several radiotracers in vitro in-between THP-1 phenotypes and between THP-1 phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. Genomic and full-length cDNA sequences provide opportunities for understanding human gene expression. Determination of the mRNA start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. Although the mRNA start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. Recently, we have developed a 5-end SAGE (5SAGE) that can be used to globally identify the transcriptional start sites and frequency of individual mRNAs. A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (HDAC) inhibitor is currently in clinical trials. However, how epigenetic drugs mediate its effects is poorly understood. To assess the effects of epigenetic drugs, the gene expression by 5SAGE in colon cancer cell lines treated with epigenetically affecting agents, 5-aza-2deoxycytidine, a potent inhibitor of genomic and promoter-specific DNA methylation and trichostatin A, a HDAC inhibitor was investigated. Epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. Colon cancer is one of the most frequently diagnosed cancers in Western Societies. Interleukin-6 (IL-6) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. IL-6 is produced by many different cell types. The main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. A variety of studies have demonstrated that over expression of IL-6 contributes to the pathogenesis of various inflammatory diseases as well as cancer. It has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce IL-6. Serum levels of IL-6 are elevated in patients with colorectal cancer, however serum levels of IL-6 were found to be independent of IL-6 mRNA expression in tumor tissue. In this study we analyzed IL-6 mRNA expression by real-time PCR in 50 sporadic colon cancer tissue as well as corresponding normal mucous tissue. IL-6 mRNA expression in tumor tissue was lower than in the corresponding normal mucous tissue (p=0,0074). There was no correlation betweenIL-6 mRNA expression and tumor grade or stage. Thus we can conclude that IL-6 produced at the tumor site is not involved in sporadic colon cancer progression. (1), T Aiamsa-ard (1), V Chinswangwatanakul (2), Ki Techatraisak (3), S Chotewuttakorn (1), A Thaworn (1) ( Material and Methods: HUVEC were cultured as standard techniques and grown to confluence until use. Serum was obtained from cholangiocarcinoma patients and normal healthy subjects. HUVEC were treated with 10% of serum and incubated for 24 hours. Cells were analyzed by using [3H] thymidine and immunoblotting assay for cell proliferation and COX-2/NOS-2 protein expression, respectively.

Results: Serum of CCA patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. On the protein expression, CCA serum significantly increased the expression of COX-2 but not NOS-2 in HEVEC. However, the proliferate effect on endothelial cells by CCA sera did not correlate with the expression of COX-2.

Conclusions: This result suggested that some factors in serum of cancer patients could induce COX-2 protein expression in HUVEC. The increasing of COX-2 might be one of various factors involve in the proliferation process. Aim: Superoxide is responsible for the neutrophil-mediated tumoricidal activity. The aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of OK-432@on neutrophil-derived superoxide production and tumor growth.

Methods: AH109a rat hepatocellular carcinoma cells were implanted into the hind leg of male Donryu rats. PMNs were harvested from rat peritoneal cavity 6 h after intraperitoneal injection of oyster glycogen. Superoxide production were measured by the method of CLAdependent chemiluminescence, which has high sensitivity and specificity to superoxide.

The counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after 18 days of tumor inoculation. Both PMA and OZ-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. The suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. The subcutaneous administration of OK-432, a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression.

Our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. Furthermore, the effect of OK-432 on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of OK-432. (1), M Jokic (2), V Zjacic-Rotkvic(1), S Kapitanovic (2) (1) University Hospital Sestre Milosrdnice, Bucharest, Romania

(2) Division of Molecular Medicine Rudjer Boskovic Institute, Bucharest, Romania

Introduction: IL-6 is a pleiotropic cytokine mapped to chromosome 7p21-24. Its promoter SNP -174 G/C is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. We tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (GEP-NETs).

Patients and Methods: DNAs from 80 patients diagnosed with GEP-NET and 162 age and sex-matched volunteers were analyzed for -174 G/C SNP of the IL-6 gene. To analyze IL6 -174 C/G polymorphism we used PCR -NlaIII RFLP method. For statistical analysis Ä2 test and Fishers Exact test were used. The level of significance was 0.05.

Results: There were no differences observed in the frequencies of the -174 high expression (GC and GG) genotypes between the patients and healthy volunteers (p=0.5518), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p=0.3762). -174 G/C genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (PETs) than in those with functional PETs (p=0.0246).

Conclusions: High expression genotypes of IL-6 -174 SNP are more frequent in non-functional PETs and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. Their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. How mast cells influence tumor growth is not well understood. The neuroendocrine peptide, neurotensin (NT) is a potent secretagogue of MC that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its GPCR NT-type 1 receptor (NTS1). Here we show that HMC-1 human MC express NT-precursor (proNT) mRNA and protein, and secrete immunoreactive NT when stimulated. RT-PCR on HMC-1 cell RNA yielded a band with 99% sequence identity to proNT and a band corresponding to the proNT processing enzyme, PC5a.Immunocytochemistry on HMC-1 cells showed specific staining for proNT. Stimulation of HMC-1 cells with A23187 + PMA, PGE2, C48/80 or mastoparan released immunoreactive NT.RT-PCR on HMC-1 cell RNA yielded a band with 99% sequence identity to human NTS1. Western blotting gave bands corresponding to unglycosylated (49 kDa) and glycosylated (55 kDa) NTS1.Immunocytotochemistry on HMC-1 cells showed specific staining for NTS1. These finding have significance for the role of mast cells in tumor growth. (1), J Buddenkotte (1), MP Schçn(2), M Steinhoff (1) (1) University Hospital Münster, Germany (2) University Hospital Würzburg, Germany

The proteinase-activated receptor PAR-2 has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. However, the role of PAR-2 in cutaneous cancerogenesis is still unknown. Here we could show a protective role of PAR-2 in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. To this end, PAR-2-deficient and wild-type mice were painted once with DMBA (7,12-dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester PMA (phorbol myristate acetate (12-O-tetradecanoylphorbol-13-acetate)) twice per week at the same sites. Tumors started to appear after eight weeks. After 13 weeks, PAR-2-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild-type (eight tumors per mouse). Analysis of possible signal transduction pathways activated upon PAR-2 stimulation in HaCaT keratinocytes showed an involvement of extracellular signal regulated kinase 1/2 (ERK1/2) and profound epidermal growth factor receptor (EGFR) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta 1 (TGF-â1). Thus, our results provide the first experimental evidence for a tumor-protective role of PAR-2. (1), MA Arbós (1), A Fraga (1), I de Torres(2), J Reventós (1), J Morote (3) ( Pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer (PCa) is still unresolved, although chronic inflammation may play a significant role in disease progression. Prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. To better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. We analysed 20 primary human fibroblast cultures from normal transition zone (TZ), normal peripheral zone (PZ), benign prostatic hyperplasia (BPH), and pathologically confirmed prostate cancer (CA). Cells of different origin displayed distinct mRNA expression profiles for the core proteins of proteoglycans and both SDF1/CXCR4 and MCP1/CCR2 chemokine axis. When incubated with monocyte-conditioned medium all four cell types significantly changed SDF1/CXCR4 and MCP1/CCR2 expression in a fibroblast population dependent manner. Monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. Monocytes adhered rapidly to fibroblasts and preferentially to BPH and PZ cells. In addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. Our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. Supported by the Spanish Urology Society (Madrid, Spain). We have recently shown that PAF-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with PAF-like molecules present on the surface of these cells. Removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. In the present study we investigated the impact of apoptotic cells or treatment with PAF-R antagonist on Ehrlich ascitic tumor (EAT, ip) and melanoma B16F10 (sc). PAF-R antagonist, WEB2170 (5mg/Kg, ip) was given daily for 6 days. We found that EAT growth was significantly reduced by pretreatment with WEB2170, and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by WEB2170 pretreatment. EAT growth was accompanied by increased production of prostaglandin E2, VEGF and NO which was reduced significantly by WEB2170 treatment. In B16F10 melanoma, WEB2170, alone or in association with an apoptosis inducer chemotherapeutic agent, Dacarbazin (DCB, 40 ug/Kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. In association with DCB, WEB-2170 reducedactive caspase-3 expression in the tumor andmarkedly increased the survival of tumor-bearing mice. The data obtained here show that during tumor growth, activation of PAF-R by molecules present in the surface of apoptotic/necrotic cells, or by PAF produced in the milieu, favors tumor growth and suggests that PAFantagonists could be useful in tumor treatment, particularly when in association with chemotherapy.

Financial suport by FAPESP and CNPq. (1), MT Quiles (1), A Figueras(2), R Mangues(2), F Vinals(2), JR Germa(2), G Capella (2) (1) Institut de Recerca Vall de Hebron, Barcelona, Spain (2) Translational Research Laboratory, IDIBELL -Institut CataladOncologia, Spain

The malignant potential of tumor cells may be influenced by the molecular nature of K-Ras mutations. We have previously shown that codon 12 mutations associate with an increased resistance to apoptosis. We hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the CXCL12(SDF-1)/ CXCR4 chemokine axis. To do so we have combined in vitro and in vivo studies using stable CYS12 and ASP13 NIH3T3 transfectants. CYS12 tumors showed a higher microvessel density associated with shorter latency period. Prominent vessels with µ-smooth muscle actin positives cells surrounded by F4/80 macrophages were only observed in ASP13 tumors associated with a shorter growth period. ASP13 tumors displayed increased VEGF expression both at the RNA and protein levels, mainly produced by tumor cells. TSP-1 protein levels were similarly diminished in both transfectants. Higher MMP9 and MMP7 activities and expression were observed in ASP13 tumors probably produced by macrophages or stromal cells. Total and active MMP2 levels were higher in CYS12 tumors. The expression of SDF-1 and CXCR4 remained unchanged while SDF-1g isoform was selectively induced in CYS12 tumors, suggesting SDF-1a or b are induced in ASP13 tumors. These results show distinct K-Ras mutations induce specific angiogenic phenotypes. The differential stimulation of VEGF expression, metalloprotease activities and SDF-1 expression observed is the result of the joint action of tumor cells and the local microenvironment.

Contact information: Dr Maria A Arbos Via, Institut de Recerca Vall de Hebron, Unitat de recerca Biomedica, Barcelona, Spain E-mail: maarbos@ir.vhebron.net Incisional hernias (IHs) represent a common complication of laparotomies, involving remarkable healthcare costs. Representative IH animal models are lacking and characterization of human tissue resources is scant. This limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of IHs. Here, we compared tissue specimens (carefully obtained > 5cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without IH hernia. The most prominent morphologic characteristics of IH tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ECM), and a paucity of fibroblasts. In IH muscles, inflammatory infiltrates were observed. Other significant changes were: decreased collagen type I/III ratio; differential proteoglycans mRNA expression; enhanced metalloproteinases/ endogenous inhibitors ratio (MMPs/TIMPs); and upregulation of apoptosis effectors (caspase-3 and substrates; TNF-alpha; IL-6). In vitro, hernia fibroblasts (IHFs) exhibited significantly higher (2-fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. Moreover, IHFs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. Based on these descriptive results in human tissues, a novel hypothesis emerges regarding IH formation. Specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. Overall, this may be causally involved in the mechanisms of ECM destruction yielding IH (Supported by FIS PI_ 030290 and GenCat_Agaur_ XT_0417). (1), M Spinola(2), C Pignatiello(2), W Cabrera (1), OG Ribeiro (1), N Starobinas(1), T Dragani (2) (1) Butantan Institute, Sao Paulo, Brazil (2) Istituto Nazionale Tumori, Milan, Italy

AIRmax and AIRmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. In a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, AIRmin mice developed a persistent subacute lung inflammation and a 40fold higher lung tumor multiplicity than AIRmax mice, which showed a transient lung inflammatory response. We have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant C57Bl/6 and lung cancer susceptible A/J mouse strains. Gene expression profile analysis of urethane-treated and untreated animals was performed using the Applied Biosystems Mouse Genome Survey Microarray containing 32,000 mouse transcripts. mRNA expression of candidate differentially expressed genes was validated by quantitative real-time PCR and the over-represented biological themes were analyzed with the EASE software. Urethane treatment modulated the gene expression profile in all four lines. Among the confirmed genes, vanin3 (Vnn3) and major histocompatibility antigen E alpha (H2-Ea) resulted common to both mouse models. The most represented gene categories in AIR model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. MHC/antigen process and presentation and immunoresponse were the major themes in the inbred model. Moreover, a gene cluster in chromosome 3 (45.0 cM) was observed. The study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. Inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.It is well established that the VEGF signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. However, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of VEGF signalling only. We have developed a nanomolar inhibitor (compound A) of the receptor tyrosine kinase VEGFR-R2 (KDR), which was subsequently shown to be a potent inhibitor of closely related kinases (VEGFR-1 and -3, PDGFR, KIT, CSF-1R) but also unrelated soluble tyrosine kinases (SRC-familily of kinases and Raf). Compound A potently inhibits VEGF stimulated endothelial cell proliferation but has no effect on non-EC proliferation, which is suggestive of a selective antiangiogenic potential. The unique kinase inhibitory profile of compound A combined with excellent oral bioavailability (87%) has translated into superior in vivo anti-

Inflamm. Res., Supplement 3 (2007) Posters tumour efficacy when compared to the relatively selective KDR inhibitor PTK787. Thus, treatment of nude mice implanted with either commercial ATCC derived tumour cells (A431 and DU-145) or low passage patient derived tumors (CXF 1103; colon cancer, RXF 631; renal cancer) with compound A resulted in inhibition of tumour growth which was significantly better than for PTK787 treated mice. Compound A is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. (1), P Bobrowski(2), M Shukla (3), T Haqqi (3) (1) Albany Medical College, USA (2) Rainforest Nutritionals, Inc, USA (3) Case Western Reserve University School of Medicine, USA Background: The Amazonian medicinal plant Sangre de grado (Croton palanostigma) has traditional applications for wound healing and inflammation. We sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants.

Methods: Acute oral safety and toxicity was tested in rats according under OECD protocol #420. Proanthocyanidin oligomers were quantified by HPLC and progrados antioxidant activity assessed by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were treated with IL-1b (10 ng/ ml) to induce matrix degradation and glycosaminoglycans (GAG) release. Progrado (2-10 mg/ml) was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes.

Results: Progrado displayed no evidence of toxicity (2000 mg/kg po) leading to GSH safety rating of 5/unclassifiable. Oligomeric proanthocyanidin content was high (158 mg/kg) with the majority of oligomers > 10mers.Progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and IL-1b induced glycosaminoglycan release from human cartilage explants. Progrado prevented IL-1b induced suppression of IGF-1 production from human cartilage explants as well as stimulating basal IGF-1 production (P<0.05). Comparable changes in IGF-1 gene expression were noted in cultured human chondrocytes.

Conclusions: Progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, IGF-1. This suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. The solvent extracts from Korean fermented soybean (Chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. The activities of Chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. In addition, the protective effects against H 2 O 2 -induced cytotoxicity and oxidative DNA damage in the NIH/3T3 fibroblasts line were examined. The extracts from Chungkukjang and soy isoflavones inhibited the generation of 1,1-diphenyl-2picryl hydrazine (DPPH) radicals, and had an inhibitory effect on LDL oxidation. The extracts from Chungkukjang and soy isoflavones strongly inhibited H 2 O 2 -induced DNA damage in the presence or absence of endonuclease III and FPG. Furthermore, they showed cytoprotective effects against H 2 O 2 , without cytotoxicity except for the hexane extract at high concentrations (> 450 mg/ml). The ethanol and n-butanol extracts appeared to have most potent antioxidant activities. These in vitro results show that the extracts of Chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative DNA damage without having cytotoxic effect. Moreover, the extracts of Chungkukjang inhibited MDA formation in the liver, DNA damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in KBrO 3 -treated mice. These in vivo results were similar to those of in vitro experiments. Therefore, Chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress.

(Supported by BK21 project from Korea Research Foundation). SIRT1 is a histone deacetylase, involved in oxidative stress and aging. Because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the SIRT1 activity, comparing heart (H) and adipose (A) tissue of sedentary young (n10), sedentary old (n10) and trained old (n10) rats. The trained old rats performed a 8-weeks moderate training on treadmill. On H and A tissue of all rats SIRT1 activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (MDA) and protein-aldehyde adducts 4-hydroxynonenal (4-HNE), MnSOD, catalase and FOXO3a by western blot, and GADD45a, Cyclin D2 and FOXO3a mRNA by RT-Pcr. Aging reduced SIRT1 activity in H (p<0.0001) without effects in A, producing an increase of MDA (H, p<0.0005; A, p<0.0001) and 4-HNE (H, p<0.005; A, p<0.0005), and a decrease of Mn-SOD (p<0.02) and catalase (p<0.0001) expression in both H and A. Aging did not affect FOXO3a protein expression in H, and FOXO3a mRNA in A. Exercise produced an increase in H FOXO3a protein expression (p<0.02) and in A FOXO3a mRNA, associated to higher Mn-SOD (H, p<0.01; A, p<0.005) and catalase (H, p<0.0001; A, p=0.01) levels in both H and A of aged rats. In heart exercise-induced higher SIRT1 activity bring on decrease in Cyclin D2 and increase in GADD45a mRNA expression. In A we found a similar decrease in Cyclin D2, without changes in GADD45a mRNA expression. These findings suggest that exercise is able to increase SIRT1 activity in aged rats. (1), T Horiguchi(1), K Abe(2), H Inoue(2), T Noma (1) (1) Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan (2) Minophagen Pharmaceutical Co. LTD, Japan

Objectives: Glycyrrhizin (GL) is a major component of Glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. GL has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. However, the molecular mechanisms of the effects remain unclear. To analyze the molecular basis of GL signaling, we performed the microarray analysis using CCl4-induced mouse hepatitis models.

Methods: Eight-week-old ICR male mice were treated intraperitoneally with 100 f×l/ kg BW of CCl4 w/wo 250 mg/ kg BW of GL. After 24 hours and 72 hours, livers and serum were collected and analyzed. For microarray analysis, the expression patterns of genes between 72 hour-treated-livers (CCl4 or CCl4 and GL) and no treated-livers were compared.

Results: GL-treatment dramatically decreased the GPT activity in plasma at 24 hours compared to that in CCl4treated plasma. However, the levels of mRNA expression of inflammatory genes such as phospholipase A2, HSP47, and procollagen were still very high in GL-treated liver. After 72 hours, the mRNA levels of them were significantly reduced in GL-treated mice compared to those of CCl4-treated liver. Then, we screened 41,000 genes by microarray and found that 71 genes were up-regulated and 63 genes were down regulated in CCl4+GL compared to CCl4 treatment. Interestingly, ROS scavenger-related genes were significantly up-regulated in CCL4 + GL. Detail analysis is currently ongoing.

We found the unique relationship between GL activity and ROS regulation. This finding suggests a novel way to treat inflammatory diseases including hepatitis. Objectives: Experimental autoimmune encephalomyelitis (EAE) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the CNS. It is widely employed as an animal model of human multiple sclerosis. Interestingly, the number of studies relating these diseases with peripheral organs is limited. We thus investigated the consequences of EAE on the degree of lipoperoxidation (TBARs) and MPO activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder).

University of Waikato, Hamilton, New Zealand

Mitochondria play a fundamental role in the life and death of all eukaryotic cells. Cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn60). This protein is increasingly being implicated to play a role in modulating cellular inflammation. We have developed an in vitro model cell system using THP-1 monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn60 expression and modulation of proinflammatory cytokine responses. We have found that the ability of cpn60 to modulate TNF-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our THP-1 cells. We also demonstrate that such modulation involves both ERK1/2 and p38mapk pathways. The significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. (1), B Arnold(2), G Opdenakker (3) (1) Jagiellonian University, Department of Evolutionary Immunobiology, Krakow, Poland (2) German Cancer Research Center, Department of Molecular Immunology, Heidelberg, Germany (3) Rega Institute for Medical Research, University of Leuven, Laboratory of Immunobiology, Leuven, Belgium

We showed that in mice genetically deprived of metalloproteinase 9 (MMP-9-/-) at least one compensatory mechanism operates as there are elevated levels of PGE2 of COX-1 origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. Also infiltration of peritoneal cavity by inflammatory neutrophils is changed in MMP-9-/-mice as at 6 hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. In contrary, at 24 hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. Thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in MMP-9-/-mice. For this numbers of apoptotic (Annexin V) and necrotic (7-AAD) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. The results revealed that both, numbers of apoptotic cells and levels of active caspase 3 were significantly lowered in MMP-9-/-mice while levels of caspase 7, 8 and 9 were significantly elevated in comparison to control animals. We conclude that an impairment of apoptosis is observed in MMP-9-/mice during zymosan peritonitis and it is due to the decreased levels of active caspase 3. The increased activity of other examined caspases is most probably independent of apoptosis. (1), H James (1) The selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(NOS) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. The aim of this study was to evaluate the activity of NOS inhibitors in experimentally induced inflammation, pain and hyperalgesia. The effect on acute inflammation was studied in carrageenan-induced paw edema in rats. The effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. NOS inhibitor NG-nitro-L-arginine methylester (L-NAME), 10 and 20 mg/kg produced a dose-dependent inhibition of paw edema (42% and 57% at 2h; 45% and 57% at 4h). A marked reduction in paw edema was observed with NG-monomethyl-L-arginine acetate (L-NMMA), 10 mg/kg(79% at 2h; 91% at 4h). Selective inducible NOS(iNOS) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of 20 mg/ kg(59% at 2h; 64% at 4h) but not with a dose of 10 mg/kg . The effects were comparable to nonselective COX inhibitor indomethacin 5 mg and 10 mg/kg (40% and 77% at 2h; 29% and 72% at 4h respectively) and selective COX-2 inhibitor rofecoxib, 5 mg/kg (49% and 78% respectively). NOS and iNOS inhibitors significantly increased the pain threshold latency in the tail-flick test. These inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. However, carrageenan-induced paw hyperalgesia was not inhibited. The results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both NOSand iNOS inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. (1), P Hart(2), J Edwards (1), C Quirk (1) (1) Molecular Pharmacology Limited (USA), Australian Division, Perth, Western Australia (2) Telethon Institute for Child Health Research, Perth, Western Australia Thermalife Cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. A novel product, derived from Thermalife, was assessed on its therapeutic potential in oxsoralen-UVB burns. As a possible mechanism for the sunburn efficacy, suppression of TNF-a and IL-1â production by human monocytes was assessed in vitro.

Methods: Sunburn: Four sites were marked on the arm of the subject. Three sites were exposed to oxsoralen (1%) plus UVA/UVB light, one site was exposed to oxsoralen only. Cream was applied at 5min, or at 4hrs after injury. A third injury site was not treated. Photographs were taken before, 24hrs, and 7weeks after injury. Cytokines: Human monocyte cultures (10% FCS, 5% CO2) were either stimulated with 500ng/ml LPS (E.coli 0111:B4) or not in the presence of 0% or 10% active ingredient.24Hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine ELISA).

Results: At 24hrs after oxsoralen-UV, the 5min treatment site showed slight erythema, the 4hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. 7 Weeks after injury, the 5min site was normal, the 4hr site was a dark colour, whereas the untreated site had a significant scar. Oxsoralen alone had no effect on the skin. The novel product suppressed LPS-induced TNF-a and IL1â secretion by 48.7% and 71.1%, respectively.

Conclusions: A novel Thermalife-derived product reduced total injury after oxsoralen enhanced UVA/ UVB burns, which is possibly related to cytokine suppression. (1), P Hart(2), J Snowden (1), Maud Eijkenboom (1) (1) Molecular Pharmacology Limited (USA), Australian Division,Perth, Western Australia (2) Telethon Institute for Child Health Research, Perth, Western Australia A mixture of bovine plasma protein fractions and zinc chloride (Bov-zn) was assessed for its ability to regulate cytokine production by LPS-stimulated monocytes. Dosereponse curves for TNF-a suppression were generated. Further, competition with FCS in the culture medium and the metabolism of monocytes under influence of Bov-zn were assessed.

In all experiments the culture medium environment was similar. Human monocyte cultures (10% FCS, 5% CO2) were either stimulated with 500ng/ml LPS (E.coli 0111:B4) or not in the presence of 0%, 2.5%, 5%, 7.5%, 10%, 20% or 40% Bov-zn (two pooled experiments). 24 Hours after incubation, culture media were collected, centrifuged, and assayed (cytokine ELISA). A competitive inhibition design for the standard TNF-a assay was set up for 0%, 1%, 5%, 10% FCS against 0%, 2.5%, 5%, 10% Bov-zn. The culture media were treated as above. Metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (Promega CellTiter 96) in treated and untreated cell cultures over 0-45 hrs at intervals.

The IC50 for TNF-a suppression was reached at 2.5% Bov-zn in each of two experiments. FCS did not compete with Bov-zn in suppressing TNF-a in LPSstimulated monocytes. At low FCS concentrations Bov-zn stimulated TNF-a production in the absence of LPS. This TNF-a increase was countered with increasing concentrations of FCS. Metabolism of cells was not affected by 10% Bov-zn.

Conclusions: Bov-zn could reliably and effectively reduce TNF-a secretion in vitro, without competing with the FCS in the culture medium, and without disturbing the metabolism of monocytes.

Inflammatory diseases such as rheumatoid arthritis (RA) result from overproduction of cytokines including TNF-£\ and IL-1fÒ. These cytokines are known to be regulated by the stress-activated p38fnfnMAP kinase pathway. Because of this, inhibition of p38 MAP kinase has been one of the most compelling targets for the treatment of inflammatory disease. Over the last 10 years, numerous groups have reported on the development of p38 MAP kinase inhibitors. X-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. Regions of the binding site are known to be unique to p38 vs other kinases, enabling the development of p38 selective molecules. Inflamm. Res., Supplement 3 (2007) Posters anti-inflammatory activities. A series of 11 labdane-type diterpenoids (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) with various patterns of substitution were tested for potential anti-inflammatory activity.Of these compounds, 4 and 11 were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. These derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of 50 and 90 % were noted in the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ear oedema in mice. In addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. The diterpenoids also reduced the production of nitric oxide, prostaglandin E2, and tumour necrosis factor-alpha in bacterial endotoxin-activated RAW 264.7 macrophage cells with IC50 in the range 1-10 mM. Inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase-2, as determined by western-blot analysis and RT-PCR. Since nuclear factor-kappaB (NF-kB) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. Our results indicate that both compounds interfere with the phosphorilation of IKBalpha and IKBß, resulting in inhibition of their degradation. In summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking NF-kB activation and provide potentially therapeutic perspectives in inflammatory conditions. The aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of 100 mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. To fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. A comparison with diclofenacum at a dosage of 8 mg/kg and substance ffW-755Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect (41%) at a dosage of 16 mg/kg) was made. The results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. Fepolen revealed the highest effect during first 30 min and 1 hour of inflammation that was higher then action of diclofenacum. These data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. During next hours therapeutic effects of fepolen and diclofenacum were at the same level. Fepolen showed the same dynamics of anti-inflammatory action as ffW-755Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. In summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. The aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. The gastrointestinal inflammatory condition Crohns disease involves a Th1-response with increased levels of proinflammatory cytokines like TNFa and IL12, and mouse models of Crohns disease show that the balance of IL10/12 is crucial for disease progression. We have used mouse bone marrow derived dendritic cells (BMDC) to model a proinflammatory Crohns disease like condition in vitro with cocktail-induced BMDC secretion of IL12, IL6 and TNFaf jThe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin D2, which were able to suppress the cocktail induced IL12 secretion. Further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress TNBS induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. Among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. In summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development.

P11-12 INFLAMMATION AND HUMAN INCISIONAL HERNIA PATHOPHYSIOLOGY

Maria Antonia Arbos Via(1)

EAE was induced by immunization of female Lewis rats with guinea-pig myelin basic protein (MBP) in complete Freunds adjuvant (CFA) and the animals were studied at the stage III of the disease (characterized by complete paralysis of the hind-limbs)

Compared to CFA rats, EAE resulted in increased MPO activity (U/mg tissue) in kidney (280 AE 75 vs. 121 AE 14

179 AE 35; p<0.001), and higher TBARs contents (nmol MDA/mg tissue) in liver

Acknowledgements: CAPES, CNPq, FAPESP. Contact information: Ms Simone Teixeira, University of S¼o Paulo, Department of Pharmacology, Campinas, Brazil E-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption

Supported by BK21 project from Korea Research Foundation) Contact information: Mr Young Hoon Kim

Here we report on the development of the PGE2 mimetic combination therapy (DP-837953) that inhibits basal and LPS/TLR4 induced TNF-?, IL-1ß, and MMP-1, 8, 9, 13 in human and murine synovial membranes and peripheral macrophages.In a murine model of chronic synovitis (dorsal skin air pouch), DP837953 dramatically inhibited IL-1ß, TNF-?, MIP2, MCP-2 and IL-8 expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.In a model of inflammatory arthritis (collagen induced arthritis, CIA), DP837953 markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.In addition, TNF-?, IL-1ß, MMP-8 and to a lesser extent MMP-9 expression/synthesis levels were strongly suppressed as judged by RT-PCR and ELISA measurements

We have developed two RIAs, one for functional blood levels of the above mentioned anti-TNF-alpha constructs, and one for anti-Abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/Remicade and etanercept/Enbrel ; I shall present some of these data (7, 8 

Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec, Canada Neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate LTB4 from the 5-lipoxygenase pathway, PGE2 through the inducible cyclooxygenase (COX-2) pathway and cytokines/chemokines as TNF-alpha, IL-1beta, IL-8, MIP-1alpha, MIP-1beta, MIP-2alpha and MIP-3alpha. Engagement of the adenosine A2A receptor (A2AR) blocks the in vitro synthesis of LTB4 while it potentiates the COX-2 pathway in fMLP-treated neutrophils. In addition, it selectively prevents the expression and release of TNFalpha, MIP-1alpha, MIP-1beta, MIP-2alpha and MIP-3alpha in toll-like receptor-4-stimulated human neutrophils. Little effect was observed on IL-1beta and IL-8. Using the murine air pouch model of inflammation with A2AR-knockout mice, we observed that the activation of A2AR positively impacts the expression of COX-2 in vivo, with particular magnitude in inflammatory leukocytes. In mice lacking the A2A receptor, neutrophils that migrated into the air pouch 4h following LPS injection expressed higher mRNA levels of TNF-alpha, MIP-1alpha and MIP-1beta than neutrophils from wild type mice. Together, these results indicate that neutrophils are important mediators of adenosines protective effects. Given the uncontrolled inflammatory phenotype observed in A2AR-knockout mice and in view of the potent inhibitory actions of PGE2 on inflammatory cells, an increased COX-2 expression and a prevented release of TNF-alpha, MIP-1alpha and MIP-1beta caused by A2AR activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. Sepsis induced by endotoxins including lipopolysaccharide (LPS) is a big problem in clinical medicine. For a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF to follow the changes of significant proteins in a murine macrophage cell line -RAW 264.7 after challenged with LPS (Escherichia coli 026:B6) for 12, 18 and 24 hours. We identified 21 proteins from approximately 500 detected protein spots with either increased or decreased in relative abundance as a result of LPS treatment. The proteins identified with increased expression are the retinoblastoma binding protein 7, Capg protein, Poly(rC) binding protein 1, isocitrate dehydrogenase 3 (NAD+) alpha, lactate dehydrogenase 1, A chain, guanine nucleotide binding protein (G protein), beta polypeptide 2 like 1, triosephosphate isomerase 1 and proteasome alpha 5 subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein P0, malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type 1 and Rho, GDP dissociation inhibitor (GDI) beta). Many of these altered proteins have interesting functions in inflammation. With the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. The ubiquitous mitogen-activated protein (MAP) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. They are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. Based on a virtual screening approach we identified 6-amino-1-benzyl-4-(4-bromophenyl)- 3-methyl-1,4-dihydropyrano[2,3c] pyrazole-5-c arbonitrile as a potential novel lead structure as p38 MAP kinase inhibitors. A set of compounds were prepared starting from different substituted pyrazol-5(4H)-ones via a base-catalyzed condensation with aldehydes and CH acids, such as malononitrile, to provide them for biological tests in a p38 enzyme assay. First structure-activity relationship confirm the value of this novel lead. This study was conducted to determine the physiological C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. Serum CRP levels were measured by ELISA and AAG levels were measured in healthy beagles of various ages by TIA, and then separately -in pregnant beagles -by SRID. Serum CRP levels ranged from 1.5 to 16.0 ìg/ml in male, and from 1.8 to 18.9 ìg/ml in female dogs. No significant sex-related differences were observed in the values. Further, there were no significant age-related differences either. Serum CRP levels increased during pregnancy and peaked at 70.2-90.4 ìg/ml 30 or 45 days after ovulation, demonstrating two characteristic features of CRP levels change in pregnant dogs. Serum AAG levels ranged from 40 to 960 ìg/ml in male, and from 47 to 833 ìg/ml in female dogs, without any significant sex-or age-related variation. Serum AAG levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 ìg/ml. Despite a high value of 1,210 -1,360 ìg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40 ìg/ml). No significant sex-/-age related differences were observed in serum both CRP and AAG levels and these levels increased during pregnancy. The results of AAG levels in umbilical cords were below the detection levels suggest AAG is not transported to the placenta. Polymorphonuclear neutrophils (PMNs) play a key role in the inflammatory response against infectious agents.However, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.In this study, we examined the effect of PBI-1393, a low molecular weight immunomodulatory molecule, on PMN activation by LPS both in vitro and in vivo. We measured by ELISA the production of TNF-a by human LPS-activated PMN in the presence or absence of PBI-1393.The ability of PBI 1393 to modulate PMN activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.Exudates from different groups of animals (controls and PBI-1393 treated animals, n=6) were used to assess leukocyte infiltration and to measure by ELISA TNF-µ, MCP-1 and PGE2 production.In vitro, PBI-1393 is able to significantly decrease by 28.8% AE 0.08% (p<0.05), TNF-a production by human LPSactivated PMN.In vivo, PBI-1393 significantly decreased the production of TNF-a (41.4% AE 7.2%; p<0.005), MCP-1 (16.3% AE 8.4%; p<0.05) and PGE2 (29.2% AE 13.5%; p<0.05) induced by LPS injection.However, PBI-1393 did not significantly inhibit leukocyte infiltration.These results show that PBI-1393 is able to modulate PMN activation and inflammatory response and suggest potential use as anti-inflammatory agent. (1), LJ Lowenstine,(2), AJ Norris(2), T Spangler(2), LM Woods (2) (1) Zoological Society of San Diego, USA (2) Department of Pathology, Microbiology, and Immunology, University of California, Davis, USA This study investigated the role of a novel reovirus in a 2004 outbreak of necrotizing typhlocolitis in American Crows in California. Included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. Complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. Feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. Two control groups (n=10 each) were selected for parasitology and EM (group 1), and gross and histopathology (group 2). All outbreak cases and group 2 controls tested negative for West Nile virus by PCR.All outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.Two cases had multifocal hepatic necrosis. Negative contrast EM revealed reovirus particles in 100 % (4/4) of outbreak cases and in 0% (0/ 10) of controls. Supplemental tests failed to suggest other concurrent or confounding etiologic agents.Overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.There was a noteable absence of similar typhlocolitis in 65 archived crows submitted to the VMTH from 1994-2004, suggesting an emerging corvid disease in California, which bears further investigation. Mitogen-activated protein kinase (MAPKs) pathways play an important role in the signalling system activated by proinflammatory cytokines. Among the most important cascades the activation of ERK1/2 by MEK1/2 is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. AS701820, a selective MEK1 inhibitor, demonstrated anti-inflammatory properties in reducing TNF-alpha production induced by LPS injection (IC50 2 mg/kg). Therefore, primary aim of the present study was to assess the therapeutic strength of the AS701820 in a mouse model of collagen-induced rheumatoid arthritis (CIA) assessing the effect of the compound on structural changes related to the cartilage. CIA is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. AS701820 treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for 7 days (twice daily), by oral route at the doses of 10, 30 and 100 mg/kg. AS701820 at 30 and 100 mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. At histology, vehicle-treated animals showed severe inflammation and joint surface erosion. Administration of AS701820 significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. In conclusion, the results obtained in this study clearly demonstrate that the selective blockade of MEK1 could be considered as an innovative therapeutic approach to treat rheumatoid arthritis.

Experimental evidences have shown that the toxicity of Ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ROS) and carcinogenicity. Neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. However, relatively little is known about the potential of nickel salts in activating human neutrophils.Thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. The measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ROS and reactive nitrogen species (RNS). Enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. The obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. In the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (O2-.), hydrogen peroxide (H2O2), hydroxyl radical (HO.) and perchloric acic (HOCl). The observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. (1), H Spalteholz (1), U Reibetanz (1), P Salavei (1), M Fischlechner (1), H-J Glander(2), J Arnhold (1) (1) University of Leipzig, Medical Faculty, Institute for Medical Physics and Biophysics, Germany (2) University of Leipzig, Derpartment of Dermatology, Andrology Training Centre of the European Academy of Andrology, Germany Unintentional childlessness often caused by common reasons as inflammation affects 15 -20 % of German couples. Inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (PMN), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (AR) and apoptosis as well as changes in the lipid structure and reduced mobility. Stimulated PMN release the strongly cationic heme protein myeloperoxidase (MPO), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (PS). A population of freshly prepared spermatozoa shows only a very small amount of cells with MPO binding ability as well as externalization of PS. The number of spermatozoa able to bind MPO raises considerably in samples containing predamaged cells or introducing the AR as could be observed with rhodamine B isothiocyanate (RITC)labelled MPO and antibody techniques by fluorescence microscopy as well as flowcytometry. The activation ofMPO with its substrate hydrogen peroxide (H2O2) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (HOCl) and enhanced markedly the number of annexin V positive and non-vital cells. Components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of MPO. The coincidence of PS externalization and MPO binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. Recent findings suggest a crucial role of Proteinaseactivated receptor-2 (PAR2) in inflammation and innate immunity. PAR2 is the second member of a novel G protein-coupled receptor subfamily with seven putative trans-membrane domains. This subfamily is characterized by a unique mechanism of receptor activation. Accessible serine proteases cleave the receptor to expose a new, previously cryptic, N-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. Tryptase, trypsin, and bacterial serine proteases are capable of directly activating PAR2. PAR2 is expressed by human neutrophils, however its functions on these cells remained unclear. The data of our present study indicate that PAR2 agonists enhance interferon gamma (IFNa)-induced up-regulation of cell surface Fca;RI, one of the key receptors involved in neutrophil phagocytic activity. Moreover, PAR2 agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. Additionally, there is a significant increase of PAR2 expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. Together, our results indicate that PAR2 may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and Fca-receptor expression. 

Aim: To ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process.

We determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal I/R could restore reparation and assessed the influence of inflammation in the process.Results: I/R provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and PCR mRNA of stathmin and PCNA. The cytokine profile revealed the influence of the inflammatory environment on kidney repair. Regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation (72 hours). Pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.Conclusions: Macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. (1), K Bendtzen (1), F Sellebjerg (2), CH Nielsen (3) ( Antibodies against myelin basic protein (MBP) are present in sera from patients with multiple sclerosis (MS), but the role of these antibodies is controversial. We collected sera from 22 MS patients and 17 healthy individuals and found that both groups contained IgM anti-MBP antibodies, while MS sera contained small amounts of IgG anti-MBP. However, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. Addition of MBP to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (PBMC) resulted in a significant deposition of IgM on CD14+ monocytes, indicating that formation of MBP/IgM complexes had occurred. This deposition was strongly inhibited by addition of 10 mM EDTA to the sera, indicating that it was complement dependent. The PBMC produced significant amounts of IL-10, TNF-alpha and IFN-gamma upon stimulation with MBP, and the extent of the cytokine production did not depend upon whether sera from MS patients or from healthy controls were present. However, disruption of the tertiary structure of MBP by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to MBP. We propose that natural IgM autoantibodies may form complexes with MBP, facilitating the uptake of MBP by antigenpresenting cells (APC). Since sera from MS patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. We currently investigate this possibility.

Loredana Postiglione(1), G Tarantino(2), A Spanò(2), P Ladogana (1), FL Perrone(1), S Padula(2), A Riccio (2) (1) Federico II University Medical School of Naples, Department of Molecular and Cellular Biology and Pathology L.Califano, Naples, Italy (2) Federico II University Medical School of Naples, Departmentof Clinical and Experimental Medicine, Naples, ItalyBackground: Hepatitis C Virus (HCV) infection can induce immunological disorders with different clinical expression such asarthritis, Sjogren Sindrome and various form of vasculitis.Aim: To study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-CCP) in a group of patients affected by HCV-related arthritis and the eventual correlations with rheumatoid factor (RF) and/orantinuclear antibodies (ANA), and articular involvement. Study Design: 30 patients with arthritis were selected in a population of 380 subjects affected by HCV infection. Each patients was evaluated by clinical examination (23 denoted poliarticular and 7 mono-oligoarticualr involvement), by X-graphic aspects of joint involvement (8 patients presented join erosions), by ANA, RF and anti-CCP positiveness.Results: 33,3% of patients presented positivenessfor anti-CCP, without significant correlation between suchparameter and ANA, RF and articular involvement. Anti CCP resulted positive in 4 out of the 8 patients with joint erosions, and only in 6 out of the 22 patients without joint erosions. Such frequency analyzed by chi square ended up in no significant differences.

Our patients presented an interesting prevalence of the positiveness for anti-CCP. These data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. 

Expression of NKG2D on CD4+ T cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, Crohns disease and an animal model of type 1 diabetes. The monoclonal antibody CX5 recognizes murine NKG2D and has been shown to block ligandbinding and mediate internalization of NKG2D. Furthermore, CX5 can inhibit and/or ameliorate disease in animal models of type 1 diabetes and inflammatory bowel disease. Thus, it is very likely that NKG2D plays an important role in the development of inflammatory and autoimmune diseases. Since little is known about the pharmacokinetics and pharmacodynamics of the CX5 antibody, we decided to study this in both regular BALB/ c mice and immunodeficient CB17.scid mice. Different doses of CX5 antibody was injected intraperitoneally and PK and PD was measured by ELISA (anti-CX5 ELISA in serum) and flow cytometry (down-regulation of NKG2D on CD49b+ NK cells) for up to two weeks after administration. We found that CX5 very efficiently down-regulate NKG2D on CD49b+ NK cells and that the effects of the antibody can be seen for more than two weeks after one single injection. Finally, we propose a model which may be helpful in predicting the effects of different doses of CX5 antibody in vivo. (1), K Mehta(2), N Deo(2), J Chaudhary(2), P Bobrowski (3) (1) Albany Medical College, USA (2) Vedic Lifesciences, USA (3) Rainforest Nutritionals, Inc, US Background: The efficacy and safety of Reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a Mumbai-based multi-center, randomized, double-blind study.Methods: Subjects (n=95) were screened and randomized to receive glucosamine sulfate (n= 41, 1500 mg/day) or reparagen (n=38, 1800 mg/day), a polyherbal consisting of vincaria (Uncaria guianensis) and RNI 249 (Lepidium meyenii) administered orally, twice daily. Primary efficacy variables were WOMAC scores, visual analog score (VAS) for pain, and response to treatment defined as a 20% improvement in WOMAC pain, with assessments at 1, 2, 4, 6 and 8 weeks. Secondary variables were Results: Subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (P<0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (42-49% reduction in total WOMAC or VAS scores). The response rate was substantial for both glucosamine (88%) and reparagen (92%), which exceeded placebo responses (55%, p <0.01) and supported by investigator and subject assessments. Tolerability was excellent and safety parameters were unchanged. Rescue medication use was significantly lower in the reparagen group (p <0.01), and serum IGF-1 levels were unaltered.Conclusions: Both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. Response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. We speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. Inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. Growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. We investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. Tested nutrient mixture (NM) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. Systemic inflammation in mice challenged with bacterial lipopolysaccharide (LPS) was monitored by blood plasma levels of fourteen key inflammatory cytokines. Two week supplementation with 250 mg NM/kg body weight prior to LPS challenge provided significantly greater protection than did supplementation with ibuprofen. Induction of interleukin-6 (IL-6) and monocyte chemoattracting protein-1, two cytokines especially responsive to LPS challenge, was reduced in NMsupplemented animals by 58% and 86%, respectively. Corresponding reduction in ibuprofen group was 34% and 43%. Protective mechanisms involved were assessed in human cultured U937 macrophages stimulated with LPS.The cytokines most responsive were tumor necrosis factor alpha (71% and 17% reduction by supplementation with NM and ibuprofen, respectively) and IL-12 (66% and 15% in corresponding reduction). NM supplementation dramatically reduced prostaglandin E2 secretion by stimulated macrophages along with cyclooxigenase-2 (COX2) cellular protein expression. mRNA levels forCOX2 and inflammatory cytokines were also dramatically reduced. Quercetin was the most effective nutrient when tested individually. However, NM appeared to surplus the combined effect of individual components. We conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. (1), HP Kim (1), KH Son (2) (1) College of Pharmacy, Kangwon National University, South Korea (2) Department of Food and Nutrition, Andong University, South Korea Chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. Recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (iNOS)-catalyzed NO production in cell cultures. In the present study, to find the optimal chemical structures and to elucidate their action mechanisms, 41 synthetic chalcones having the substituent(s) on A-and B-rings were prepared and their effects on iNOS-catalyzed NO production were evaluated using LPS-treated RAW 264.7 cells. Among the tested compounds, 2-methoxy-3,4-dichlorochalcone (Ch15), 2-hydroxy-6-methoxychalcone (Ch29), 2-hydroxy-3-bromo-6-methoxychalcone (Ch31) and 2-hydroxy-4,6-dimethoxychalcone (Ch35) potently inhibited NO production (IC 50 s, 7.0 -9.9 mM). The favorable chemical structures were found to be a methoxyl substitution in A-ring at adjacent position (2 or 6) to carbonyl moiety with/without 2-(or 6-)hydroxyl group and 3-halogen substitution in B-ring. When the cellular action mechanisms of Ch15, Ch31 and Ch35 were further examined, it was revealed that Ch15 and Ch31 clearly down-regulated iNOS expression while Ch35 did not. Moreover, Ch15 and Ch31 were proved to suppress the nuclear transcription factor-kB activation. From the results, it is suggested that certain chalcone derivatives potently inhibit iNOS-catalyzed NO production by the different cellular mechanisms, iNOS down-regulation or iNOS inhibition, depending on their chemical structures. These chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. An oligomeric stilbene alpha-viniferin (AVF) was isolated from root of Carex humilis (Cyperaceae) as an inhibitor of cyclooxygenase (COX)-2 activity by bioassayguided fractionation. The AVF was later found to downregulate lipopolysaccharide (LPS)-induced COX-2 expression as well as to inhibit nuclear factor (NF)-kB activation, in addition to its inhibitory effect on COX-2 activity. Furthermore, the compound exhibited antiarthritic effect in vivo. AVF is a trimer of resveratrol and contains benzofuran moieties in its central part. Starting from benzofuran and its related chemicals, 2cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1, 3] oxathiol-4-one (LYR-64) was discovered to inhibit LPSinduced NF-kB transcriptional activity in macrophages RAW 264.7. The LYR-64 reduced LPS-induced DNA binding activity and nuclear translocation of NF-kB as well as inhibited LPS-induced degradation and phosphorylation of inhibitory kB (IkB) protein. These results suggest that LYR-64 could suppress LPS signaling molecule, putatively IkB kinase (IKK) complex, upstream IkB degradation in NF-kB activating pathway. LYR-64 inhibited in vitro kinase activity, GST-IkB phosphorylation, of wild type IKKbeta or a constitutively active IKKbeta mutant (C/A, Cys-179 to Ala) but did not affect that of another constitutively active IKKbeta mutant (SS/EE, Ser-177 and 181 to Glu). Therefore, LYR-64 could inhibit LPS-induced NF-kB activating pathway by targeting Ser-177 and/or 181 residues on the activation domain of IKKbeta. As pharmacological actions, LYR-64 prevented NF-kB-dependent expression of inducible nitric oxide synthase, COX-2, or inflammatory cytokines at the transcription level in LPS-stimulated macrophages RAW 264.7. Furthermore, LYR-64 protected LPSinduced septic shock in vivo.

Faculty of Medicine, Institute of Pharmacology, Ljubljana, Slovenia A part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. In mammals histamine is mainly degraded by two enzymes: histamine-N-methyltransferase (HNMT) and diamine oxidase (DAO). The aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. Their effects on enzyme activity and mRNA expression were studied in guinea pig tissues. Plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. Specific enzymatic activities of DAO and HNMT were determined by radiometric assay. In addition, guinea pigs were treated with saline or amitriptyline (4 mg/kg, ip), afterwards DAO and HNMT mRNAs were detected by PCR in different tissues. Results showed thatamitriptyline, 100 nM, 50, 100 and 500 mM, increased guinea pig plasma DAO activity by 5, 7, 17 and 11 %, respectively, while sertraline increased it at 30 mM (by 15 %). At higher concentrations (100 and 500 mM) sertraline decreased DAO activity. In the guinea pig tissues HNMT activity changes were found only when incubated with amitriptyline; sertraline had no effect. At 10 and 50 nM amitriptyline the activity of HNMT increased by 20 and 9 %, respectively. In animals treated with amitriptyline an induction of DAO and HNMT mRNA expression was noticed in several tissues. Our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. The effect might be the opposite with higher amitriptyline concentrations.

Steven Hefeneider(1,2), C MacArthur (1), D Trune (1), S McCoy (2) (1) Oregon Health and Science University, Portland, Oregon, USA (2) Targeted Gene Delivery, Inc., Portland, Oregon, USA Engagement of Toll-like receptors (TLRs) by bacterial components such as LPS and DNA initiates inflammation.The current study examines a novel anti-inflammatory peptide, termed P13, for treatment of inflammation induced by either LPS or bacteria.Peptide P13 was derived from an immunoregulatory protein of vaccinia virus, and interferes with TLR signaling.In this study we examined the efficacy of P13 to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (AOM).We demonstrate in the sepsis model, that in vivo treatment of mice with P13 inhibited LPS-induced production of serum inflammatory mediators.Moreover, P13 treatment, administered after initiation of inflammation, significantly increased survival of mice injected with LPS.In the AOM model, peptide P13 significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by H. influenza.Assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide P13.Simultaneous injection of bacteria and peptide P13 resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.Fluid accumulation within the Eustachian tubes was also significantly reduced following P13 treatment.-Subcutaneous and oral administrations of P13, but not intravenous administration, were also efficacious in reducing inflammation. Administration of P13 after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.Taken together, these results demonstrate the therapeutic potential of peptide P13 to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. (1), C Zhou(2), Y Zhang(2), M Sun(2), X Wan (1), H Yu(2), X Yang(2), RD Ye (3), J-K Shen (1) Formyl peptide receptor-like 1 (FPRL1) is a structural homologue of FPR, which binds chemotactic peptides of as small as 3 amino acids (e.g., fMet-Leu-Phe, fMLF) and activates potent bactericidal functions in neutrophils. In comparison, FPRL1 ligands include peptides of 6-104 amino acids, such as Trp-Lys-Tyr-Met-Val-[D]Met (WKYMVm) and other synthetic peptides. To determine the core peptide sequence required for FPRL1 activation, we prepared various analogues based on WKYMVm and evaluated their bioactivities in an FPRL1-transfected cell line. Although substitution of D-Met6 resulted in loss of activity, removal of Val5 together with D-Met6 produced a peptide that retained most of the bioactivities of the parent peptide. The resulting peptide, WKYM, represents a core structure for an FPRL1 ligand. Further substitution of Lys2 with Nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both FPRL1 and FPR. Based on these structure-activity studies, we propose a model in which the modified tetrapeptide Trp-Nle-Tyr-Met (WNleYM) binds to FPRL1 through aromatic interactions involving the side chains of Trp1 and Tyr3, hydrophobic interaction of Nle2, and the thio-based hydrogen bonding of Met4, with the respective residues in FPRL1 which have not been identified. The identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for FPRL1-related disorders.There is a growing awareness of the interaction of food constituents with the immune system. The present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. Mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. Ear swelling, as a measure for inflammation, as well as IL-1, TNF-a and IL-6 levels in the ear and the number of TNF-a producing spleen cells were analyzed.-Glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of TNF-a producing spleen cells).Glycine effects (20, 50 and 100 mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses (0.1 and 1 mg/mouse/ day) inhibited the inflammatory response significantly. Surprisingly higher doses of lactoferrin (5 and 25 mg/ mouse/day) failed to influence the inflammatory reaction. A combination of both nutrients (lactoferrin 0.1mg/ mouse/day in combination with glycine 20 or 50 mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. Additionally an additive effect of both components was seen on the number of TNF-a producing spleen cells. The present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.Moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of TNF-a producing spleen cells. (1), P Sambrook(1), K Fukudome(2), M Xue (1) (1) University of Sydney, St Leonards, NSW, Australia (2) Saga Medical School, Saga, Japan Objectives: To investigate the i) expression of endothelial protein C receptor (EPCR) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and ii) role of EPCR and its ligand, activated protein C (APC), on the function of monocytes from RA patients.Methods: EPCR, CD68 and PC/APC in synovial tissues were detected by immunostaining and in situ PCR. Monocytes were isolated from peripheral blood of patients with RA and treated with APC, lipopolysaccharide (LPS), and/or EPCR blocking antibody, RCR252. Cells and supernatants were collected to analyze the expression/activation of EPCR, nuclear factor NF-kB and tumour necrosis factor TNF-a.Results: EPCR was expressed by both OA and RA synovial tissues but was markedly increased in RA synovium. EPCR was colocalized with PC/APC mostly on CD68 positive cells in synovium. In RA monocytes, APC upregulated EPCR expression reduced monocyte chemoattractant protein-1-induced chemotaxis of monocytes by approximately 50%. APC also completely suppressed LPS-stimulated NF-kB activation and attenuated TNF-a protein by more than 40% in RA monocytes. The inhibitory effects of APC were reversed by RCR252, indicating that EPCR modulates the inhibitory effects of APC.Conclusions: Our results demonstrate for the first time that EPCR is expressed by synovial tissues, particularly in RA, where it co-localizes with PC/APC on monocytes/ macrophages. In addition, APC inhibits the migration and activation of RA monocytes via EPCR. These inhibitory effects on RA monocytes suggest that PC pathway may have a beneficial therapeutic effect in RA.