Search

Celiac disease is an intestinal disorder that develops as a result of interplay between genetic and environmental factors. HLA genes along with non-HLA genes predispose to the disease. Linkage studies have failed to identify chromosomal regions other than the HLA region which have major effects, indicating the existence of multiple non-HLA predisposing genes with modest effects. Association studies have shown that CTLA4 or a closely located gene is one of these genes. The primary HLA association in the majority of celiac disease patients is with DQ2 (DQA1*05/DQB1*02) and in the minority of patients with DQ8 (DQA1*0301/DQB1*0302). Gluten reactive CD4+ T cells can be isolated from small intestinal biopsies of celiac patients but not from controls. DQ2 or DQ8, but not other HLA molecules carried by patients, present peptides to these T cells. A number of distinct T cell gluten epitopes exist, most of them posttranslationally modified by deamidation. DQ2 and DQ8 bind the epitopes such that the glutamic acid residues created by deamidation are accommodated in pockets that have a preference for negatively charged side chains. There is evidence that deamidation in vivo is mediated by the enzyme tissue transglutaminase (tTG). Overall, the results point to control of the immune response to gluten by intestinal T cells restricted by the DQ2 or DQ8 molecules. This is likely to be a critical checkpoint for the development of celiac disease and could explain the dominant genetic role of HLA in this disorder. The products of the other predisposing genes may participate in pathway(s) that lead(s) to lesion formation. The minor genetic effects of the non-HLA genes could indicate a lack of critical checkpoints along these pathways, or that there are several pathways leading to the lesion formation. [ABSTRACT FROM AUTHOR]

Antisynthetase syndrome (ASSD) is a rare clinical condition that is characterized by the occurrence of a classic clinical triad, encompassing myositis, arthritis, and interstitial lung disease (ILD), along with specific autoantibodies that are addressed to different aminoacyl tRNA synthetases (ARS). Until now, it has been unknown whether the presence of a different ARS might affect the clinical presentation, evolution, and outcome of ASSD. In this study, we retrospectively recorded the time of onset, characteristics, clustering of triad findings, and survival of 828 ASSD patients (593 anti-Jo1, 95 anti-PL7, 84 anti-PL12, 38 anti-EJ, and 18 anti-OJ), referring to AENEAS (American and European NEtwork of Antisynthetase Syndrome) collaborative group's cohort. Comparisons were performed first between all ARS cases and then, in the case of significance, while using anti-Jo1 positive patients as the reference group. The characteristics of triad findings were similar and the onset mainly began with a single triad finding in all groups despite some differences in overall prevalence. The "ex-novo" occurrence of triad findings was only reduced in the anti-PL12-positive cohort, however, it occurred in a clinically relevant percentage of patients (30%). Moreover, survival was not influenced by the underlying anti-aminoacyl tRNA synthetase antibodies' positivity, which confirmed that antisynthetase syndrome is a heterogeneous condition and that antibody specificity only partially influences the clinical presentation and evolution of this condition.

Objectives: To evaluate if the timing of appearance with respect to disease onset may influence the arthritis presentation pattern in antisynthetase syndrome (ASSD)., Methods: The patients were selected from a retrospective large international cohort of ASSD patients regularly followed-up in centres referring to AENEAS collaborative group. Patients were eligible if they had an antisynthetase antibody testing positive in at least two determinations along with arthritis occurring either at ASSD onset (Group 1) or during the course of the disease (Group 2)., Results: 445 (70%; 334 females, 110 males, 1 transsexual) out of the 636 ASSD we collected had arthritis, in the majority of cases (367, 83%) from disease onset (Group 1). Patients belonging to Group 1 with respect to Group 2 had an arthritis more commonly polyarticular and symmetrical (p=0.015), IgM-Rheumatoid factor positive (p=0.035), erosions at hands and feet plain x-rays (p=0.036) and more commonly satisfying the 1987 revised classification criteria for rheumatoid arthritis (RA) (p=0.004). Features such as Raynaud's phenomenon, mechanic's hands and fever (e.g. accompanying findings) were more frequently reported in Group 2 (p=0.005)., Conclusions: In ASSD, the timing of appearance with respect to disease onset influences arthritis characteristics. In particular, RA features are more common when arthritis occurs from ASSD onset, suggesting an overlap between RA and ASSD in these patients. When arthritis appears during the follow-up, it is very close to a connective tissue disease-related arthritis. Also, the different prevalence of accompanying features between these two groups is in line with this possibility.

Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-benzoyl-L-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The k ( cat ) and K ( m ) values of hPAD4-mediated deimination of N-α-benzoyl-L-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-benzoyl-L-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA.

Objectives: Mixed connective tissue disease (MCTD) is an immune-mediated, systemic disorder of unknown aetiology. As the epidemiology of the disease is largely unknown, the authors performed a nationwide cross-sectional retrospective study to assess the prevalence and incidence of MCTD in Norway., Methods: Every adult patient (≥ 18 years) with MCTD seen at one of the departments of rheumatology was reviewed for inclusion. Only patients who satisfied the following four criteria were included: clinical diagnosis of MCTD verified by a rheumatologist; positive serum anti-ribonucleoprotein antibody test; fulfilment of at least one of three of following criteria sets: the modified Sharp's criteria, the criteria of Alarcón-Segovia and Villareal and those of Kasukawa; and exclusion of other connective tissue diseases., Results: The four inclusion criteria were fulfilled by 147 adult Caucasian patients. The female to male ratio was 3.3 and the mean age at diagnosis of adult-onset MCTD was 37.9 years (95% CI 35.3 to 40.4 years). At the end of 2008, the point prevalence of living adult MCTD patients in Norway was 3.8 (95% CI 3.2 to 4.4) per 100,000 adults. The incidence of adult-onset MCTD in Norway during the period from 1996 to 2005 was 2.1 (95% CI 1.7 to 2.5) per million per year., Conclusions: MCTD has a female predominance and the incidence and prevalence of MCTD is low, and lower than reported figures for polymyositis, dermatomyositis, systemic sclerosis and systemic lupus erythematosus. The prevalence estimates were similar across the three criteria sets of MCTD.

IL-17-producing T cells (Th17 cells) are believed to contribute to local inflammation and joint damage in rheumatoid arthritis (RA). Limited data exist on Th17 cells located within the inflamed synovial tissue (ST) of patients with RA. Here, we aimed to generate polyclonal T cell lines (TCLs) from the RA ST and assess their cytokine production, including the effects of exogenous IL-15 on IL-17 production in vitro. For five patients with RA, polyclonal TCLs were established from ST obtained by joint surgery. Synovial TCLs were expanded and stimulated by anti-CD3/CD28 microbeads and exogenous cytokines. Cytokine production was assessed by culture supernatant analyses and intracellular flow cytometry, and TCLs were sorted based on their surface expression of CCR6. In addition to IL-17, we detected IL-6, IL-10, IFN-γ and TNF-α in the synovial TCL culture supernatants. Exogenous IL-15 increased the production of IL-17 as well as the other cytokines except IFN-γ. For IL-17, this effect was more pronounced after prolonged culture times. Intracellular flow cytometry confirmed the presence of IL-17+ and IL-17+ IFN-γ+ CD4+ T cells in the TCLs. IL-17+ and IL-17+ IFN-γ+ T cells were enriched in the CD4+ CCR6+ population. In conclusion, Th17 cells can be detected after polyclonal expansion and stimulation of RA synovial TCLs generated by joint surgery. The Th17 cells from the RA ST were enriched in the CD4+ CCR6+ population, and they were sensitive to exogenous IL-15. Th17 cells present within the synovial compartment may contribute to the RA pathogenesis and local joint damage., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Objective: Interstitial lung disease (ILD) is the major determinant of morbidity and mortality in the anti-synthetase syndrome (ASS). Here we have retrospectively assessed 11 ASS patients with ILD treated with the anti-CD20 mAB rituximab at our tertiary referral hospital., Methods: Data on clinical and laboratory parameters, lung imaging by high-resolution CT thorax and pulmonary function tests were collected from patient examinations done up to 6 months before rituximab was initiated, and at 3 and 6 months post-treatment., Results: All the 11 ASS patients had severe and progressive ILD and most of them had previously failed on cyclophosphamide and/or other immuno-modulating agents. Rituximab appeared to stabilize and/or improve the ILD in 7 of 11 ASS patients during the first 6 months after treatment. The rituximab treatment appeared to decrease the serum level of anti-Jo-1 antibodies, but the decrease was most often modest. One patient developed a fatal infection 3 months after the last infusion with rituximab. In the other ASS patients, the treatment was well tolerated., Conclusions: This retrospective case series indicates a short-term beneficial effect of rituximab in ASS. Prospective, controlled studies are needed to validate this finding and further assess safety issues.

The T-cell specific adapter protein (TSAd) encoded by the SH2D2A gene is up-regulated in activated human CD4+ T-cells in a cAMP-dependent manner. Expression of SH2D2A is important for proper activation of T-cells. Here, we show that SH2D2A expression is regulated both at the transcriptional and translational level. cAMP signaling alone induces TSAd-mRNA expression but fails to induce increased TSAd protein levels. By contrast, TCR engagement provides signals for both TSAd transcription and translation. We further show that cAMP signaling can prime T-cells for a more prompt expression of TSAd protein upon TCR stimulation. Our study thus points to a novel mechanism for how cAMP signaling may modulate T-cell activation through transcriptional priming of resting cells.

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

Various experimental models suggest that the cholesterol-lowering drugs statins may also modulate immune responses. Cellular level studies on human disorders are needed, however, to provide a rational basis for clinical testing of statins as immune therapy. Coeliac disease, a chronic small intestinal inflammation driven by HLA-DQ2 restricted mucosal T cells that are specific for ingested wheat gluten peptides, is in many ways ideal for this purpose. In addition, there is a need for alternative treatment to the gluten-free diet in this disorder. Here we have assessed the effects of atorvastatin on gluten-reactive T cells, dendritic cells and the coeliac mucosa by in vitro culture of biopsies. Atorvastatin inhibited gluten-induced proliferation and specific cytokine production of human intestinal gluten-reactive T cell clones and lines. Dendritic cells exposed to atorvastatin displayed a reduced expression of the costimulatory molecule CD83 upon maturation with lipopolysaccharide. Incubation of intestinal biopsy specimens with atorvastatin in vitro, however, did not influence gluten-induced cytokine release. In conclusion, atorvastatin has specific effects on isolated gluten-reactive T cells and dendritic cells, but does not shut down the gluten-induced production of proinflammatory cytokines in intestinal biopsies.

Celiac disease is driven by intestinal T cells responsive to proline-rich gluten peptides that often harbor glutamate residues formed by tissue transglutaminase-mediated glutamine conversion. The disease is strongly associated with the HLA variant DQ2.5 (DQA1*05, DQB1*02), and intestinal gluten-reactive T cells from DQ2.5-positive patients are uniquely restricted by this HLA molecule. In this study, we describe the mapping of two novel T cell epitopes of gamma-gliadin and the experimental identification of the DQ2.5 binding register of these and three other gamma-gliadin epitopes. The new data extend the knowledge base for understanding the binding of gluten peptides to DQ2.5. The alignment of the experimentally determined binding registers of nine gluten epitopes reveal positioning of proline residues in positions P1, P3, P6, and P8 but never in positions P2, P4, P7, and P9. Glutamate residues formed by tissue transglutaminase-mediated deamidation are found in position P1, P4, P6, P7, or P9, but only deamidations in positions P4 and P6, and rarely in P7, seem to be crucial for T cell recognition. The majority of these nine epitopes are recognized by celiac lesion T cells when presented by the related but nonassociated DQ2.2 (DQA1*0201, DQB1*02) molecule. Interestingly, the DQ2.2 presentation for most epitopes is less efficient than presentation by the DQ2.5 molecule, and this is particularly prominent for the alpha-gliadin epitopes. Contrary to previous findings, our data do not show selective presentation of DQ2.5 over DQ2.2 for gluten epitopes that carry proline residues at the P3 position.

Celiac disease is an HLA-DQ2-associated disorder characterized by an intestinal T cell response. The disease-relevant T cells secrete IFN-gamma upon recognition of gluten peptides that have been deamidated in vivo by the enzyme tissue transglutaminase (transglutaminase 2 (TG2)). The celiac intestinal mucosa contains elevated numbers of mast cells, and increased histamine secretion has been reported in celiac patients. This appears paradoxical because histamine typically biases T cell responses in the direction of Th2 instead of the Th1 pattern seen in the celiac lesions. We report that histamine is an excellent substrate for TG2, and it can be efficiently conjugated to gluten peptides through TG2-mediated transamidation. Histamine-peptide conjugates do not exert agonistic effects on histamine receptors, and scavenging of biologically active histamine by gluten peptide conjugation can have physiological implications and may contribute to the mucosal IFN-gamma response in active disease. Interestingly, TG2 is able to hydrolyze the peptide-histamine conjugates when the concentrations of substrates are lowered, thereby releasing deamidated gluten peptides that are stimulatory to T cells.

Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gliadins. Initial studies used gliadin that had been subjected to non-enzymatic deamidation during pepsin/trypsin digestion to enrich for the gliadin-specific T cells in small intestinal celiac biopsies. These T cells recognized synthetic gliadin peptides only after their deamidation in vitro by purified tissue transglutaminase (tTG). However, as these studies used a deamidated antigen for re-stimulation prior to testing for antigen specificity, this raised the possibility that T cells specific for native epitopes had not been expanded in vitro and had thus been overlooked. To address this possibility and to look for more direct evidence that endogenous tTG mediates deamidation of gluten in the celiac lesions, we have here used a minimally deamidated chymotrypsin-digest of gliadin to challenge biopsies and then investigated the specificity of the T cell lines derived from them. Interestingly, these T cell lines only barely responded to the chymotrypsin-digested gliadins, but efficiently recognized the in vitro tTG-treated variants of the same gliadins. Moreover, the addition of a tTG-inhibitor during the gliadin challenge often resulted in T cell lines with abolished or reduced responses to deamidated gliadin. These data demonstrate that DQ2-restricted T cells within adult celiac lesions predominantly recognize deamidated gliadin epitopes that are formed in situ by endogenous tTG.

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

Background/aims: Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis. Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease. However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen. To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar., Methods: The alpha-gliadin and gamma-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced. Genes encoding mature gliadins were expressed in Escherichia coli and tested for recognition by T cells., Results: In total, 16 alpha-gliadin genes with complete open reading frames were sequenced. These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database. Expression of these gliadin genes produced a panel of recombinant alpha-gliadin proteins of purity suitable for use as an antigen for T cell stimulation., Conclusion: This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease.