No description available.

No description available.

No description available.

No description available.

Sjogren's syndrome (SjS) is a systemic autoimmune disorder specifically targeting the exocrine glands, primarily the salivary and lacrimal glands. The autoimmune attack upon these organs is characterised by lymphocytic infiltration of the salivary and lacrimal glands, leading to an impairment of secretory function. Little is known about the mechanisms underlying glandular hypofunction, because the condition is very difficult to detect at an early stage, rendering treatment difficult. Recent studies in salivary glands have identified a mechanism whereby the inflammatory mediator NO, acting through cGMP and cADPr, can modulate the fluid secretory process. Thus NO production may contribute to the glandular hypofunction associated with SjS. The likelihood that NO plays a key role in the aetiologvof SjS would be greatly increased if it may be demonstrated that NO perturbed fluid secretion in lacrimal, as well as salivary gland acinar cells. Using microfluorimetric and patch clamp techniques I have shown that the acute response of lacrimal acinar cells exposed to NO is an amplification of the muscarinic agonist evoked Ca2+ signal. Also, that this amplification is probably mediated through increased ryanodine receptor sensitivity. Chronic exposure to NO in salivary gland acinar cells has been shown to inhibit the response to muscarinic agonist. Observation of the same effect in lacrimal gland cells would suggest that increased levels of NO may be a unifying factor between two separate secretory organs for the glandular hypofunction associated with SjS. Inhibition of secretion by anti-muscarinic receptor autoantibodies represents a complimentary mechanism of glandular hypofunction and one which also has the potential to aid in early detection of the condition. These autoantibodies are very difficult to detect using conventional immunological techniques, but represent an attractive target for being the basis of a diagnostic test. My data demonstrate a similar pattern of response in lacrimal to salivary gland acinar cells, but also demonstrate that lacrimal acinar cells are capable of endogenous NO production. The mechanism by which NO modulates secretory mechanisms appears to be a universal feature of the exocrine glands affected by SjS. However, my data suggest at least quantitative differences in the role of NO in glandular function. These data represent further elucidation of the role of NO in glandular hypofunction in SjS, highlighting fundamental differences between two similar gland systems affected by SjS, potentially affecting avenues of treatment. I have also demonstrated that anti-muscarinic receptor autoantibodies may be detected using conventional microfluorimetric techniques and I have developed a semi-automated fluorimetric tool that allows rapid screening and detection of autoantibodies within the isolated IgG fraction of patient serum. Preliminary data indicate that the sensitivity and specificity of detection of antimuscarinic autoantibodies was not sufficiently high enough to offer significant improvement over current methods for detecting SjS.

Sjogren's syndrome is a systemic autoimmune disorder which specifically targets the glands, primarily the salivary and lacrimal glands and leads to an impairment of secretory function and is characterised by lymphocytic infiltration of the salivary and lacrimal glands. Until relatively recently it was believed that the glandular function was a direct consequence of immune mediated destruction of the :inar tissue but most recent data suggests that the glandular hypofunction is more 5ly to be the result of immune mediated inhibition of acinar function, which if prolonged could lead to acinar atrophy.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant disease caused by TNFRSF1A mutations, encoding tumour necrosis factor receptor 1 (TNFRl). TRAPS is characterized by recurrent fever episodes and systemic inflammation. The pathophysiology of TRAPS is unclear; one hypothesis is that mutant receptor misfolding leads to intracellular retention in the endoplasmic reticulum (ER) causing a proinflammatory unfolded protein response (UPR). A branch of the UPR involves IREla activation, leading to splicing of the transcription factor X-box binding protein 1 (sXBPl) in response to Toll-like receptor (TLR) ligation. Furthermore, TNF and Il-6 have been identified as additional transcriptional targets of sXBPl. Activated UPR is associated with reactive oxygen species (ROS) production. This study aimed to investigate the hypothesis that ER stress, caused by retention of mutant TNFRl, facilitates activation of inflammatory pathways. Results presented show that TRAPS patients displayed increased sXBPl mRNA transcript, as well as increased levels of protein kinase•like ER kinase (PERK) and phosphorylated-PERK, compared to healthy controls (HC). However, other UPR-associated transcripts didn't show evidence of upregulation, indicating a non-classical UPR. TRAPS patients' monocytes had increased basal ROS levels that were not accompanied by an increase in the antioxidant defences. When stimulated with lipopolysaccharide (LPS), TRAPS patients' PBMC demonstrated upregulated IL-6 secretion and sXBPl transcript; however, sXBPl upregulation was only seen in the patients and not in HC. Both of these responses were reduced with antioxidant co-treatment demonstrating dependence on ROS. These results indicate that high ROS levels and sXBPl in TRAPS patients may predispose patients to hyper-inflammatory responses to pathogenic stimuli. Although a full UPR was not evident it is possible that the intracellular accumulation of TNFRI is precipitating both of these events. Despite the ongoing identification of new hereditary periodic fever genes, there are still many patients with undiagnosed fever syndromes. This project also investigated the genetic basis of disease in a number of patients with a TRAPS-like phenotype by Caspase 1 (CASP1) screening and exome sequencing. However, these studies were not able to identify any disease-associated mutations but did reveal a novel 4bp deletion SNP in fR aminopeptidase 1 (ERAP1) in one family.

Brown Norway (BN) rats treated with mercuric chloride (HgCh) develop an auto immune syndrome characterized by the production of a variety of IgG autoantibodies, a marked increase in total serum IgE concentration and vasculitis. These features have some similarities to the Churg-Strauss syndrome in humans. The increase in IgE concentration is associated with increased interleukin-4 (IL-4), thus suggesting the involvement of the Th2 subset of CD4+ T cells. However, direct evidence of CD4+ Th2 cell involvement in this syndrome in the rat is lacking, as a cell surface marker of rat CD4+ Th2 cells has not been found to date. ST2L, a member of the IL-IR gene family has been reported as a stable selective marker of CD4+ Th2 cells in mice and has been proposed as a marker of Th2 cells. Equivalent data of the rat homologue, Fit-I, on rat CD4+ Th2 cells is not currently available. A short 20-amino acid sequence of Fit-l peptide was used to generate a monoclonal antibody against Fit-I. Characterization of the anti-Fit-l monoclonal antibody showed that the IgM antibody generated mainly binds to a population of cells eo-expressing CD45RA, a small sub-population of CD4+ in BN rats. Fit-l expression on CD8+ T cells was not clear. A small comparative study using anti-mouse ST2 polyclonal antibody also showed predominant expression of STL on rat CD45RA + cells and a small subpopulation of CD4+ and CD8+ T cells. The comparative pattern of ST2L and Fit-l expression observed endorses the notion that ST2L and Fit-l as homolgoues. However; the phenotype of rat cells expressing ST2L did not mirror the findings in mice i.e., ST2L expression on CD4+ Th2 cells. In vivo administration of the monoclonal antibody was found to selectively suppress serum levels of IgE antibody in HgCh-treated BN rats. An ELISpot assay was used to demonstrate IL-4 production at the protein level in HgCh-treated rats and the reactivity of anti-Fit-l mAb against IL-4-producing cells in vitro through a complement-dependent cytotoxic mechanism. The results suggest that there is some evidence that cells expressing Fit-l protein and secrete IL-4 may have a role in HgCh-induced IgE in particular and pathogenesis of HgCh-induced auto immune syndrome in BN rats in general, although further work is required to fully characterize the phenotype and cytokine profile of CD45RA+ Fit-l+ CD4+ Fit-l+ T cells. In conclusion, the monoclonal antibody raised against Fit-l is unlikely to be of use as a selective marker of CD4+ Th2 cells but preliminary evidence indicates that in vivo administration of anti-Fit-l mAb may possibly prevent IL-33 binding with ILIRLI on CD45RA+ Fit-l+, CD4+ Fit-l+ T cells and IL-4 producing cells thus blocking signalling cascade which in turn selectively inhibits HgCh-induced IgE antibody production. Perhaps further studies in the future will reveal the role of IL-33/Fit-1 complexin the pathogenesis of HgCh-induced autoimmune syndrome in BN rats.

No description available.

Rationnel: la sclérodermie systémique (ScS) est une maladie auto-immune rare caractérisée par une vasculopathie, des phénomènes dysimmunitaires et une fibrose excessive. Objectif: préciser l’implication des lymphocytes B (LB) et des granulocytes basophiles et établir leur rôle pro-fibrosant dans la physiopathologie de la ScS. Méthodes: des analyses phénotypiques et fonctionnelles des LB et des basophiles de patients atteints de ScS suivis dans le service de Médecine Interne de l’hôpital Cochin ont été réalisées par cytométrie en flux. Des co-cultures entre basophiles immatures de lignée KU812F, LB purifiés, et fibroblastes dermiques isolés ont été réalisées afin de préciser les interactions entre ces cellules. Enfin, l’activation des fibroblastes de patients ScS par le transforming growth factor ß (TGF-ß) a été étudiée dans une étude de protéomique. Résultats : une proportion augmentée de LB et de basophiles activés ont été identifiées chez respectivement 90 et 65 patients ScS comparativement à des sujets sains. De plus les LB des patients ScS produisent significativement plus d’interleukine (IL)-6 et de TGF-ß que les LB de sujets sains, et un surnageant capable d’augmenter la prolifération et la production de collagène par les fibroblastes dermiques. De plus, les basophiles de lignée KU812F ont la capacité d’augmenter la sécrétion de TGF-ß et d’IL-6 par les LB de patients ScS et d’augmenter la survie et la prolifération de fibroblastes de patients ScS. L’analyse des protéomes de fibroblastes dermiques de sujets sains et de patients ScS cultivés avec du TGF-ß a confirmé des différences entre les protéomes de patients ScS et de sujets sains, a permis de décrire l’exacerbation protéique provoquée par le TGF-ß, et de proposer la voie de l’epidermal growth factor receptor (EGFR) comme une voie d’intérêt dans la ScS. Conclusion : nous avons ici précisé les rôles des LB et des basophiles dans la ScS. En particulier nous avons démontré leur rôle pro-fibrosant. De plus, nous avons mis en évidence les conséquences protéiques de la sécrétion de TGF-ß sur les fibroblastes de patients ScS. Du point de vue thérapeutique, ces travaux suggèrent l’inhibition de la voie EGFR comme cible potentielle dans la ScS. / Background : systemic sclerosis (SSc) is a rare autoimmune disease characterized by a vasculopathy, an immune dysregulation and fibrosis. Objective : to decipher the roles of B lymphocytes (LB) and basophils in SSc pathophysiology and to characterize their implication in fibrosis mechanisms. Methods : phenotypic and functional analyzes of LB and basophils of patients with ScS followed in Cochin Hospital were performed using flow cytometry. Co-cultures between immature basophils from the KU812F cell line, LB purified from SSc patients, dermal fibroblasts isolated from SSc patients were performed to study cells interactions. Lastly, the effects of TGF-β on fibroblasts were studied in a proteomic study. Results : compared to healthy controls (HC), increased proportions of activated LB and basophils were identified in 90 and 65 SSc patients respectively. Both LB and basophils of SSc patients produced significantly more TGF-β than LB and basophils of HC. Moreover, the supernatant of LB of SSc patients increased fibroblast proliferation and collagen production. KU812F basophils increased the secretion of TGF-β and interleukine (IL)-6 by LB from SSc patients and the survival and the proliferation of SSc fibroblasts. The analysis of dermal fibroblast proteome from HC and SSc patients cultured with TGF-β confirmed differences in the proteomes of SSc patients and HC, allowed the description of the protein exacerbation mediated by TGF-β and pointed toward the epidermal growth factor receptor (EGFR) pathway as a nod of interest in SSc. Conclusion : we have here emphasized the roles of LB and basophils in SSc. Noteworthy; we have showed their capacity to promote fibrosis. We also showed the protein consequences of the TGF-ß abundance on SSc fibroblasts. From a therapeutic point of view, this work suggests the inhibition of the EGFR pathway as a potential target in SSc fibrosis.