Autoantigen In Disease Research Articles

Aberrant overexpression of the autoantigen protein vimentin promotes Th17 cell differentiation and autoimmune arthritis via activation of STAT3 signaling

Vimentin contributes to the positioning and function of organelles, cell migration, adhesion, and division. However, secreted vimentin accumulates on the cell surface (Mor-Vaknin et al., 2003; Ramos et al., 2020 [1,2]) where it acts as a coreceptor for viral infection and as an autoantigen in inflammatory and autoimmune diseases. The roles of vimentin in Th17 cells were examined in mice with knockdown of vimentin. We also examined whether STAT3 is required for vimentin expression.Vimentin expression was significantly increased in Th17 cells through STAT3 activation, and vimentin+ IL-17+ T cells were markedly increased in the joint and spleen tissues of CIA mice. The arthritis score and expression levels of proinflammatory cytokines were significantly decreased in CIA mice treated with vimentin shRNA vector.In this study, we demonstrated that vimentin is significantly expressed in Th17 cells through STAT3 activation. Our results provide new insights into the role of vimentin in Th17 cells and the complex pathogenesis of RA.

Role of Human Leukocyte Antigen Class II in Antibody-Mediated Skin Disorders.

HLA class II molecules are key factors determining susceptibility to autoimmune disorders, and their role in immune-mediated skin conditions such as psoriasis has been extensively investigated. However, there is currently little understanding of their role in antibody-mediated skin diseases such as autoimmune blistering disorders. We researched the available literature using PubMed to narratively review the current knowledge on HLA associations in antibody-mediated blistering skin pathologies. Our results summarized the risk alleles that are identified in the literature, together with certain known protective alleles: in the pemphigus group, alleles HLA-DQB1*0503 and HLA-DRB1*0402 are most commonly associated with disease; in the pemphigoid group, the most studied allele is HLA-DQB1*0301; in epidermolysis bullosa acquisita, few genetic studies are available; in dermatitis herpetiformis, the association with haplotypes HLA-DQ2 and HLA-DQ8 is strongly established; finally, in linear IgA bullous disease, specific HLA alleles may be responsible for pediatric presentations. Our current pathogenic understanding of this group of disorders assigns a key role to predisposing HLA class II alleles that are able to bind disease autoantigens and therefore stimulate antigen-specific autoreactive T cells. The latter engage B lymphocytes that will produce pathogenic autoantibodies. The distribution of HLA alleles and their disease associations are variable across demographics, and an in-depth pathogenetic understanding is needed to support associations between HLA alleles and disease phenotypes. Additionally, in a personalized medicine approach, the identification of HLA alleles associated with the risk of disease may become clinically relevant in identifying susceptible subjects that should avoid exposure to known triggers, such as medication, when possible.

Tuning peptide specificity for T cell tolerance in Type 1 diabetes

Abstract Type 1 Diabetes (T1D) is a classical T-cell mediated autoimmune disease, and fundamental data have implicated insulin as the dominant autoantigen in T1D disease. In the NOD mouse model of T1D, notable studies have found that mice lacking native insulin expression but with an altered insulin sequence to maintain blood glucose levels are protected from insulitis and diabetes. Growing evidence also indicates how the insulin peptide is bound within MHC Class II (peptide register) is essential in determining the strength of interactions and recognition of autoreactive T-cells. We have uncovered a unique peptide binding characterized by the dominant insulin epitope InsB:9–23. The majority of InsB:9–23-specific CD4 T-cells in the periphery recognize insulin bound in the unusual register 3, and knocking in this single amino acid variation into just one copy of the insulin gene in NOD mice, confers protection against T1D. In addition, these mice were protected from T1D, even when treated with checkpoint inhibitor blockade, unlike their wild-type littermates. Introducing this superagonist epitope into the NOD mouse has allowed us to address several key questions surrounding pathways of peptide generation, presentation by MHC molecules, and recognition by auto-reactive pathogenic T cells. In addition, we have been able to model how central and peripheral tolerance mechanisms can be altered when major epitopes in the Insulin gene are mutated, allowing us to explore means of dominant tolerance to understand the potential for translation into treatments for T1D. Supported by grants from NIH (R01 DK133443-01A)

TRIM21 Expression as a Prognostic Biomarker for Progression-Free Survival in HNSCC

Patients with head and neck squamous cell carcinoma (HNSCC) continue to have a rather poor prognosis. Treatment-related comorbidities have negative impacts on their quality of life. TRIM21 is a cytosolic E3 ubiquitin ligase that was initially described as an autoantigen in autoimmune diseases and later associated with the intracellular antiviral response. Here, we investigated the role of TRIM21 as a biomarker candidate for HNSCC in predicting tumor progression and patient survival. We analyzed TRIM21 expression and its association with clinical-pathological parameters in our HNSCC cohort using immunohistochemistry. Our HNSCC cohort included samples from 419 patients consisting of primary tumors (n = 337), lymph node metastases (n = 156), recurrent tumors (n = 54) and distant metastases (n = 16). We found that cytoplasmic TRIM21 expression was associated with the infiltration of immune cells into primary tumors. In addition, TRIM21 expression was significantly higher in primary tumors than in lymph node metastases, and increased TRIM21 expression was correlated with shorter progression-free survival in HNSCC patients. These results suggest that TRIM21 could be a new biomarker for progression-free survival.

Regulatory T cells in psoriatic arthritis: an IL-17A-producing, Foxp3intCD161 + RORγt + ICOS + phenotype, that associates with the presence of ADAMTSL5 autoantibodies

In psoriatic arthritis (PsA), predisposing class I HLA alleles, the presence of synovial clonally proliferated CD8 + T cells and autoantibodies all point towards the loss of immune tolerance. However, the key mechanisms that lead to immune dysregulation are not fully understood. In other types of inflammatory arthritis, T regulatory cell (Treg) dysfunction and plasticity at sites of inflammation were suggested to negatively affect peripheral tolerance. We here addressed if Treg variances associate with psoriatic disease. We collected clinical data, sera and peripheral blood mononuclear cells from 13 healthy controls, 21 psoriasis and 21 PsA patients. In addition, we obtained synovial fluid mononuclear cells from 6 PsA patients. We studied characteristics of CD4 + CD25 + CD127loFoxp3 + Tregs by flow cytometry and used ELISA to quantify antibodies against ADAMTSL5, a recently discovered autoantigen in psoriatic disease. In comparison with their circulating counterparts, Tregs from inflamed joints express increased levels of ICOS, CTLA-4 and TIGIT. Furthermore, synovial fluid-derived Tregs have a distinct phenotype, characterized by IL-17A production and upregulation of CD161 and RORγt. We identified a subset of Tregs with intermediate Foxp3 expression as the major cytokine producer. Furthermore, ICOS + Tregs associate with PsA disease activity as measured by PASDAS. Lastly, we observed that presence of the Foxp3int Tregs associates with an increased abundance of anti-ADAMTSL5 autoantibodies. Tregs derived from the inflammatory environment of inflamed PsA joints exhibit a distinct phenotype, which associates with loss of peripheral immune tolerance in psoriatic disease.

Synthesis of single-chain antibody fragment fused to the elastin-like polypeptide in Nicotiana benthamiana and its application in affinity precipitation of difficult to produce proteins.

Plants are economical and sustainable factories for the production of recombinant proteins. Currently, numerous proteins produced using different plant-based systems with applications as cosmetic and tissue culture ingredients, research and diagnostic reagents, and industrial enzymes are marketed worldwide. In this study, we aimed to demonstrate the usefulness of a plant-based system to synthesize a single-chain antibody (scFv)-elastin-like polypeptide (ELP) fusion to be applied as an affinity precipitation reagent of the difficult to produce recombinant proteins. We used the human tissue transglutaminase (TG2), the main celiac disease autoantigen, as a proof of concept. We cloned a TG2-specific scFv and fused it to a short hydrophobic ELP tag. The anti-TG2-scFv-ELP was produced in Nicotiana benthamiana and was efficiently recovered by an inverse transition cycling procedure improved by coaggregation with bacteria-made free ELP. Finally, the scFv-ELP was used to purify both plant-synthesized human TG2 and also Caco-2-TG2. In conclusion, this study showed for the first time the usefulness of a plant-based expression system to produce an antibody-ELP fusion designed for the purification of low-yield proteins.

Vimentin as a Multifaceted Player and Potential Therapeutic Target in Viral Infections.

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection—a missing link for ACPA production

ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007...

E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2

The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-Å crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called "linchpin" residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.

A Modifying Autoantigen in Graves' Disease.

The TSH receptor (TSHR) is the major autoantigen in Graves' disease (GD). Bioinformatic analyses predict the existence of several human TSHR isoforms from alternative splicing, which can lead to the coexpression of multiple receptor forms. The most abundant of these is TSHRv1.3. In silico modeling of TSHRv1.3 demonstrated the structural integrity of this truncated receptor isoform and its potential binding of TSH. Tissue profiling revealed wide expression of TSHRv1.3, with a predominant presence in thyroid, bone marrow, thymus, and adipose tissue. To gain insight into the role of this v1.3 receptor isoform in thyroid pathophysiology, we cloned the entire open reading frame into a mammalian expression vector. Immunoprecipitation studies demonstrated that both TSHR-stimulating antibody and human TSH could bind v1.3. Furthermore, TSHRv1.3 inhibited the stimulatory effect of TSH and TSHR-Ab MS-1 antibody on TSHR-induced cAMP generation in a dose-dependent manner. To confirm the antigenicity of v1.3, we used a peptide ELISA against two different epitopes. Of 13 GD samples, 11 (84.6%) were positive for a carboxy terminal peptide and 10 (76.9%) were positive with a junction region peptide. To demonstrate that intracellular v1.3 could serve as an autoantigen and modulate disease, we used double-transfected Chinese hamster ovary cells that expressed both green fluorescent protein (GFP)-tagged TSHRv1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR monoclonal antibody and observed the culture supernatant contained v1.3-GFP protein, demonstrating the release of the intracellular receptor variant by this mechanism.

Glycosylation of thyroid-stimulating hormone receptor.

Thyroid-stimulating hormone receptor (TSHR) is a typical membrane receptor with 7-transmembrane helix domain (7TMR), coupled to the G protein. The mature receptor, present in the cell membrane, is composed of the A subunit comprising a large extracellular domain, and the B subunit, which consists of a short extracellular fragment anchored in the cell membrane and an intracellular part. The TSH receptor is subject to numerous post-translational modifications that determine its final structure and significantly affect its activity. One of them is glycosylation. TSHR is abundantly N-glycosylated, due to the presence of six N-glycosylation sites in the extracellular domain (Asn77, Asn99, Asn113, Asn177, Asn198, Asn302), mostly evolutionarily conserved. N-glycans constitute 30-40% of the receptor molecular weight. The glycans are necessary for the receptor trafficking to the plasma membrane and binding of TSH to the receptor. Fucosylated and sialylated N-oligosaccharides were found on TSHR molecules. The increased sialylation of TSHR glycans correlates positively with the receptor binding ability and prolongs the time of receptor incorporation into the cell membrane. TSHR is the main autoantigen in Graves' disease (GD), one of the thyroid autoimmune diseases. One hypothesis assumes that the higher N-glycosylation of THSR in human compared to animals influences the breaking of autotolerance and GD development. N-oligosaccharides are the important part of THSR molecule, necessary for the proper functioning of receptors and probably involved in thyroid autoimmunity in GD.

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

The insulin-like growth factor-I receptor and its role in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves' disease, remains a disfiguring and potentially blinding condition. Here, the author reviews the role of insulin-like growth factor-I receptor pathway in TAO and how it might be therapeutically targeted. The recent literature is reviewed. TAO involves reactivity of orbital connective tissues and their remodeling. While many of the details concerning the pathogenesis of TAO remain to be determined, several insights have come to light recently. Among them is the apparent involvement of IGF-IR. This receptor protein, a membrane-spanning tyrosine kinase receptor can form both physical and functional complexes with the thyrotropin receptor (TSHR). This is notable because TSHR is the established primary autoantigen in Graves' disease. IGF-IR activity is critical to signaling downstream from both IGF-IR and TSHR. In addition, antibodies against IGF-IR have been detected in patients with Graves' disease and in rodent models of TAO. Evidence has been put forward that these antibodies may act directly on IGF-IR, perhaps in some manner activating the receptor. These experimental observations have led to the development of a novel therapy for active TAO, utilizing a monoclonal anti-IGF-IR inhibitory antibody which had been produced originally as treatment for cancer. The agent, teprotumumab was recently evaluated in aclinical trial and found to be highly effective and relatively well-tolerated. It is currently undergoing assessment in a follow-up trial. Should the current study yield similarly encouraging results, it is possible that teprotumumab will emerge as a paradigm-shifting medical therapy for TAO.

P165 New relevant associations with vogt-koyanagi-disease (VKH) were found in Mexicans, using NGS technology. A new allele at DPA1 locus was detected in a VKH patient

Aim VKH is a systemic autoimmune disorder affecting tissues containing melanin, mainly the eyes. It is characterized by severe bilateral granulomatous uveitis. VKH is a T cell mediated disease vs. specific auto-antigens relevant in its pathogenesis. We and others showed that a shared epitope in the first DRB1 domain is clearly associated with VKH. DRB1*04:05 and 04:04 are strongly involved in Mexicans and other contributing DRB1 alleles, were also published by us: DRB1*04:05> *04:04 > *04:07 > *01:01 > *01:02. We now explored the whole MHC region, to unravel the complete MHC genetics of VKH. Methods We selected 77 unrelated donors from Mexico City and 49 VKH patients from the APEC. DNA samples were extracted in the Maxwell 16 instrument. For HLA typing, the MIA FORA NGS FLEX HLA Kit (Immucor, Inc) and The Illumina MiSeq, were used. Sequence analysis was performed with the Mia Fora software. Genotypes were assigned at all loci unambiguously, except for DPB1. Allele and haplotype frequencies (AF/HF) were done with the Arlequin, PyPop and Hapl-o-Mat v.1.1 software. Results A hierarchy of Class I/class II associations were found with deep sequencing: DQB1*03:02:01 (p = 0.000007); DQA1*03:01:01(p = 0.0002); C*03:04:01 (p = 0.007); DRB1*04:04:01(p = 0.005); DRB1 *04:07:01 (p = 0.01); DRB1*04:05:01; (p = 0.02); B*48:01:01 (p = 0.04); A*68:01:01(p = 0.02); DPB1 *03:01:01(p = 0.02). The OR was between 2.9 and 7.3. Amerindian (AMI), Hispanic and Asian alleles are evident at all loci. The most frequent haplotype in VKH is of Mexican AMI descent : A*02:01:01 B*39:05:01 C*07:02:01 DRB1*04:07:01

Acute Loss of Apolipoprotein E Triggers an Autoimmune Response That Accelerates Atherosclerosis.

Objective- Dyslipidemia is a component of the metabolic syndrome, an established risk factor for atherosclerotic cardiovascular disease, and is also observed in various autoimmune and chronic inflammatory conditions. However, there are limited opportunities to study the impact of acquired dyslipidemia on cardiovascular and immune pathology. Approach and Results- We designed a model system that allows for the conversion to a state of acute hyperlipidemia in adult life, so that the consequences of such a transition could be observed, through conditionally deleting APOE (apolipoprotein E) in the adult mouse. The transition to hypercholesterolemia was accompanied by adaptive immune responses, including the expansion of T lymphocyte helper cell 1, T follicular helper cell, and T regulatory subsets and the formation of germinal centers. Unlike steady-state Apoe-/- mice, abrupt loss of APOE induced rapid production of antibodies recognizing rheumatoid disease autoantigens. Genetic ablation of the germinal center reduced both autoimmunity and atherosclerosis, indicating that the immune response that follows loss of APOE is independent of atherosclerosis but nevertheless promotes plaque development. Conclusions- Our findings suggest that immune activation in response to hyperlipidemia could contribute to a wide range of inflammatory autoimmune diseases, including atherosclerosis.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.

Reduced expression of TRIM21/Ro52 predicts poor prognosis in diffuse large B-cell lymphoma patients with and without rheumatic disease.

TRIM21 (also known as Ro52) is an autoantigen in rheumatic disease and is predominantly expressed in leucocytes. Overexpression is associated with decreased proliferation, and the TRIM21 gene maps to a tumour suppressor locus. We therefore investigated the expression of TRIM21 in patients with diffuse large B-cell lymphoma (DLBCL) and its potential usefulness as a prognostic biomarker. TRIM21 expression levels were assessed by immunohistochemistry in lymphoma biopsies from three cohorts of patients with DLBCL: 42 patients with rheumatic disease treated with a cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP)-like regimen, 76 CHOP-treated and 196 rituximab-CHOP-treated nonrheumatic patients. Expression was correlated with clinical and biomedical parameters. TRIM21 expression was assessed in relation to lymphocyte proliferation by quantitative PCR and correlated with (3) H-thymidine incorporation and propidium iodine staining. TRIM21 expression levels differed in the lymphomas compared to normal lymphoid tissue, with reduced expression correlating with shorter overall survival in all three cohorts. In the two larger cohorts, progression-free survival was assessed and was also found to correlate with TRIM21 expression. The association was independent of commonly used clinical prognostic scores, lymphoma subtype and several previously reported prognostic biomarkers. In agreement with this clinical observation, we noted an inverse correlation between TRIM21 expression and proliferation of leucocytes invitro. We show that loss of TRIM21 expression is associated with more aggressive lymphoma and increased proliferation, whereas maintenance of TRIM21 expression is associated with better prognosis in patients with DLBCL. Based on our findings, we suggest that TRIM21 should be considered as a novel biomarker for lymphoma characterization and for predicting patient survival.

CYP21A2 polymorphisms in patients with autoimmune Addison's disease, and linkage disequilibrium to HLA risk alleles.

Steroid 21-hydroxylase, encoded by CYP21A2, is the major autoantigen in autoimmune Addison's disease (AAD). CYP21A2 is located in the region of the HLA complex on chromosome 6p21.3, which harbours several risk alleles for AAD. The objective was to investigate whether CYP21A2 gene variants confer risk of AAD independently of other risk alleles in the HLA loci. DNA samples from 381 Norwegian patients with AAD and 340 healthy controls (HC) previously genotyped for the HLA-A, -B, -DRB1, and -DQB1 and MICA loci were used for genotyping of CYP21A2. Genotyping of CYP21A2 was carried out by direct sequencing. Linkage of CYP21A2 to the HLA loci was assessed using UNPHASED version 3.0.10 and PHASE version 2.1. Heterozygotes of the single-nucleotide polymorphisms (SNPs) rs397515394, rs6467, rs6474, rs76565726 and rs6473 were detected significantly more frequently in AAD patients compared with HC (P<0.005), but all SNPs were in a linkage disequilibrium (LD) with high-risk HLA-DRB1 haplotypes. rs6472C protected against AAD (odds ratio=0.15, 95% CI (0.08-0.30), P=3.8×10(-10)). This SNP was not in an LD with HLA loci (P=0.02), but did not increase protection when considering the effect of HLA-DRB1 alleles. Mutations causing congenital adrenal hyperplasia were found in heterozygosity in <1.5% of the cases in both groups. Genetic variants of CYP21A2 associated to AAD are in LD with the main AAD risk locus HLA-DRB1, and CYP21A2 does not constitute an independent susceptibility locus.

Structural basis for the recognition in an idiotype-anti-idiotype antibody complex related to celiac disease.

Anti-idiotype antibodies have potential therapeutic applications in many fields, including autoimmune diseases. Herein we report the isolation and characterization of AIM2, an anti-idiotype antibody elicited in a mouse model upon expression of the celiac disease-specific autoantibody MB2.8 (directed against the main disease autoantigen type 2 transglutaminase, TG2). To characterize the interaction between the two antibodies, a 3D model of the MB2.8-AIM2 complex has been obtained by molecular docking. Analysis and selection of the different obtained docking solutions was based on the conservation within them of the inter-residue contacts. The selected model is very well representative of the different solutions found and its stability is confirmed by molecular dynamics simulations. Furthermore, the binding mode it adopts is very similar to that observed in most of the experimental structures available for idiotype-anti-idiotype antibody complexes. In the obtained model, AIM2 is directed against the MB2.8 CDR region, especially on its variable light chain. This makes the concurrent formation of the MB2.8-AIM2 complex and of the MB2.8-TG2 complex incompatible, thus explaining the experimentally observed inhibitory effect on the MB2.8 binding to TG2.

Dysregulation of autoantigen genes in ANCA-associated vasculitis involves alternative transcripts and new protein synthesis.

Proteinase 3 (PR3) and myeloperoxidase (MPO) are two major autoantigens in patients with vasculitis with ANCA. The genes encoding these autoantigens are abnormally expressed in peripheral granulocytes of patients with active ANCA-associated vasculitis. This study provides evidence that this transcriptional dysregulation results in a variety of mRNA processing events from the PRTN3 gene locus. In addition to elevated levels of PR3 message, leukocyte RNA from patients contained PR3 transcripts with an alternative 3' untranslated region. Furthermore, we detected usage of an alternative transcription start site within intron 1 of the PRTN3 gene locus that coincided with active disease (odds ratio, 3.3; 95% confidence interval, 1.3 to 8.4; P=0.01). This promoter may be developmentally regulated, because it was active in normal human bone marrow, multiple leukemia cell lines, MCF-7 cells, and subjects after GM-CSF treatment but not subjects with a neutrophil left shift. This transcript, which lacks exon 1 of PRTN3, encodes a 24-kD protein (p24(PR3/MBN)) with a sequence similar to that previously described for myeloblastin. Notably, PR3, p24(PR3/MBN), and MPO were synthesized in cultured neutrophils from patients with active ANCA-associated vasculitis, indicating that increased transcription results in newly synthesized autoantigens in peripheral neutrophils of patients. The synthesis of p24(PR3/MBN) seems to expand the autoantigen repertoire, because immunoblots showed that sera from patients recognized p24(PR3/MBN). These findings emphasize the importance of transcriptional dysregulation of the autoantigen in autoimmune disease.