Target Of Autoimmune Response Research Articles

Identification of novel antigens contributing to autoimmunity in cardiovascular diseases

In myocarditis and dilated cardiomyopathy (DCM) patients the immune system may play an important role in disease progression. In this study, we aimed to identify new antigens as a target for autoimmune response that might play a crucial role in these diseases. Therefore, a peptide-array was used to investigate antibody binding profiles in patients with autoimmune myocarditis or DCM compared to healthy controls and thus to identify disease relevant antigens. To analyze the pathogenicity of the identified antigens, an experimental autoimmune myocarditis (EAM) model was used. Hereby, 3 peptide sequences, derived from myosin-binding-protein-C (MYBPC) fast-type, RNA-binding-protein 20 (RBM20), and dystrophin, showed pathogenic effects on the myocardium of mice. In summary, 3 potentially cardiopathogenic peptides (MYBPC fast-type, RBM20, dystrophin) were identified. Thus, this study could serve as a basis for future investigations aimed at determining further antigens leading to pathogenic effects on the myocardium of DCM as well as myocarditis patients.

Multiple TRIM proteins are targets of autoimmune response in lupus and Sjogren's syndrome. (HUM7P.308)

Abstract TRIM21 belongs to the large family of tripartite motif containing proteins, and is often targeted by autoantibodies in lupus and Sjogren’s syndrome. Considering the significant protein domain homology between different TRIM proteins, we hypothesized that additional TRIM proteins are targets of autoimmunity. Based on the literature, in this study we investigated autoantibody responses to TRIM38. While 9% of lupus patients (n=149) had anti-TRIM38 antibodies, the incidence in Sjogren’s syndrome patients (n=150) was 12%, and in controls (n=50) it was 4%. With respect to TRIM21, the incidence was 62%, 68%, and 4% respectively. In Sjogren’s syndrome patients, the presence of anti-TRIM38 antibodies was closely associated with the increased severity of dry eye parameters. Epitope mapping studies showed that anti-TRIM21 antibodies reacted with the RING, Coiled coil and PRY-SPRY domains of TRIM21, whereas anti-TRIM38 antibodies reacted only with the Coiled coil and PRY-SPRY domains of TRIM38. All anti-TRIM38 positive patients also had anti-TRIM21 antibodies. Affinity purified anti-TRIM21 antibodies from lupus patients did not immunoprecipitate TRIM38, indicating lack of cross-reactivity at B cell level. However, we observed T cell cross-reactivity between TRIM21298-312 and TRIM38302-316. Our study suggests that immune responses to TRIM proteins can evolve through epitope spreading and contribute towards exacerbating the pathogenesis in autoimmune disorders.

Oxidized Human Beta2-Glycoprotein I: Its Impact on Innate Immune Cells

Beta2-glycoprotein I (β2-GPI), an abundant 50 kDa plasma glycoprotein, is the most common target for antiphospholipid antibodies (aPLs). These autoantibodies are associated with thrombotic events in patients with anti-phospholipid antibody syndrome (APS) and systemic lupus erythematosus (SLE) and are proatherogenic. β2-GPI can also stimulate a vigorous adaptive cellular immune response in these patients. Although much is known about β2-GPI as a cofactor in autoimmune diseases, crucial information is still lacking to clarify why this abundant self plasma protein is the target of autoimmune responses. Throughout the years, a remarkable number of theories have been proposed to explain how the immune system recognises self. On the basis of a large variety of epidemiological, clinical and experimental evidence, it has been suggested that an unfortunate interplay of genetic susceptibility and environmental factors may play an important role in generating an abnormal immune response. Among the environmental factors, oxidative stress is one of the major events causing protein structural modifications, thus inducing the appearance of neo/cryptic epitopes of β2-GPI able to activate the immune system. In particular, oxidized β2-GPI is able to induce phenotypic and functional maturation of dendritic cells which represent the link between innate and adaptive immunity. Chronic activation of autoimmune reactions against this self protein modified by oxidative events may contribute to local and systemic inflammation, thus sustaining endothelial dysfunction in patients with APS, SLE and cardiovascular diseases. The role of oxidative stress in β2-GPI-mediated immune response is described in the light of our research experience and of relevant literature emerging in the field.

Protein microarrays: catching the proteome

After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics platforms give scientists a global view of biological systems. In today's proteome era, efforts are undertaken to adapt microarray technology in order to analyse the expression of a large number of proteins simultaneously and screen entire genomes for proteins that interact with particular factors, catalyse particular reactions, act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses. In this review, we will summarise the current stage of protein microarray technology. We will focus on the latest fields of application for the simultaneous determination of a variety of parameters from a minute amount of sample. Future challenges of this cutting-edge technology will be discussed.

Reduced thymic expression of islet antigen contributes to loss of self-tolerance.

Type 1 diabetes (T1DM) results from a failure of central and peripheral tolerance to islet cell antigens. ICA69 belongs to a group of molecules expressed predominantly in neuroendocrine tissues (including pancreatic islets), which are targets of autoimmune responses in T1DM. These molecules are also expressed in the thymus and peripheral lymphoid organs by dendritic cells. The aim of the present study was to evaluate possible variation in thymic ICA69 expression, comparing diabetes-resistant controls to T1DM-prone NOD mice. Thymic tissue was retrieved from 3- to 6-week-old female B6, NOD-H2(b), and NOD mice. Paraffin-embedded sections were stained with an ICA69-specific antibody in an immunoperoxidase assay. ICA69 staining of thymic sections from B6 and NOD.H2(b) showed strong and continual staining, yet the sections from the NOD mice showed significantly reduced staining for ICA69. Corroboration of the reduced level of ICA69 in the thymus of NOD mice has been obtained via analysis for the expression of ICA69 versus other candidate autoantigens (glutamic acid decarboxylase 65, glutamic acid decarboxylase 67, and insulin 2) in the thymus. Real-time PCR analysis, using cDNA generated from the thymus, displayed that the expression of GAD65, GAD67, and INS2 were equivalent when comparing NOD at any age to B6, BALB/cJ, and ALR/LtJ. In marked contrast, the level of ICA69 in the thymus of the NOD mice examined was significantly reduced when compared to the controls. In fact, the real-time PCR analysis strongly suggested that ICA69 was not expressed in the thymus of NOD mice. These findings support the hypothesis that the level of thymic ICA69 expression may be of importance in regulating self-tolerance in T1DM.

Recent Developments in Protein Microarray Technology

AbstractFor Abstract see ChemInform Abstract in Full Text.

Recent developments in protein microarray technology.

The sequencing of the human genome and the advent of DNA chips and sophisticated bioinformatics platforms have enabled molecular biologists to take a more global view of biological systems and to analyze naturally occurring genetic variation. Microarrays of antibodies can measure the concentrations of many proteins quickly and simultaneously. Microarrays of genomically encoded proteins allow scientists to screen entire genomes for proteins that interact with particular factors, catalyze particular reactions, or act as substrates for protein-modifying enzymes or as targets of autoimmune responses. The new protein microarray platforms will prove invaluable to basic biological research, and will dramatically accelerate the pace of discovery of drug targets and diagnostic biomarkers.

Autoantibodies in primary Sjögren's syndrome are directed against proteasomal subunits of the alpha and beta type.

The proteasome subunit HC9 (alpha3) has recently been identified as a major target of the humoral autoimmune response in patients with autoimmune myositis and systemic lupus erythematosus. Since B cell hyperreactivity is a common feature of systemic autoimmune diseases, patients with primary Sjögren's syndrome (SS) and other control groups were investigated to evaluate the significance of autoantibodies against the proteasome. Analyses of autoantibodies directed against the 20S proteasome were performed using enzyme-linked immunosorbent assay, immunoblot, and 2-dimensional electrophoresis. Forty-three patients with primary SS, 47 patients with rheumatoid arthritis including 9 with secondary SS, 19 patients with gastrointestinal tumors, and 80 healthy controls were tested for antiproteasome antibodies. Antiproteasome antibodies were detected in 39% of patients (17 of 43) with primary SS. In contrast, only 1 of 47 patients with rheumatoid arthritis showed positive reactivity (P < 0.001). Serum samples from 19 tumor patients (P < 0.003) and 80 healthy controls (P < 0.001) were serologically negative. Moreover, immunoblotting and 2-dimensional analysis of the antiproteasome response revealed a polyspecific recognition pattern in 7 patients with primary SS. Different proteasomal subunits of the alpha and beta type, including subunits that carried the proteolytic active sites, were recognized by the patients' sera. The humoral antiproteasome response in primary SS, in contrast to its secondary form, is characterized by an extensive recognition pattern of several subunits, indicating a polyspecific B cell activation against the 20S proteasome. Moreover, proteolytically active beta-type subunits, which are important for the generation of major histocompatibility complex class I-restricted antigens, appear to be targets of the autoimmune response. The data indicate that the proteasome itself may stand on a cross point of pathways that links mechanisms of the immune defense with features of systemic autoimmunity.

Anti-endometrial autoantibodies in women with a diagnosis of infertility.

The purpose of this study was to investigate the frequency of anti-endometrial antibodies (AEA) in infertile women. Sera from fertile women (n = 6), and from patients with ovulatory dysfunction (n = 11), tubal obstruction (n = 9) and unexplained infertility (n = 5) were investigated for the presence of anti-endometrial membrane antibodies. We used two human endometrial cancer cell lines and human endometrial cells from gynecological biopsies as an antigenic source for analysis. The immunoenzymatic assay (ELISA) was performed with cultured endometrial cells in monolayers. Immunoblot analysis was performed with these two cell lines. A good correlation between the response with each cell line and with human endometrial cells was obtained, indicating that the antigens analyzed were probably similar. Endometrial antibodies were detectable in a high percentage of women with tubal obstruction (77.8 and 66.7%, respectively) and ovulatory dysfunction (54.5 and 45.5%, respectively). Unexplained infertility showed anti-endometrial immunological response (40 and 60%, respectively). Some endometrial antigens in infertile women are the target for autoimmune response. The serum from a patient with tubal obstruction and ovulatory dysfunction showed two antigens by immunoblot, with molecular weights of 97 and 50 kDa. The presence of anti-endometrial antibodies, detected by ELISA, is associated with infertility, mainly with ovulatory dysfunction and tubal obstruction. Some endometrial antigens may be involved in these two pathologies.

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases.

Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody expression may be induced through antigen-driven mechanisms.

Dissociation of immune responses to the ss‐a (ro) 52‐kd and 60‐kd polypeptides in systemic lupus erythematosus and sjögren's syndrome

The cellular RNA particle SS-A (Ro) is a target of autoimmune response in many patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Recent immunologic, biochemical, and DNA cloning studies have shown that the SS-A particle consists of at least 2 polypeptide components, of 52 kd and 60 kd. Immunodiffusion analysis of 60 sera from patients with primary SS revealed 47 (78%) to be SS-A precipitin positive. Western blotting studies of the sera showed 3 groups of reactivities: 22 (47%) possessed autoantibodies against both the 60-kd and the 52-kd polypeptides, 19 (40%) reacted only with the 52-kd protein, and 6 (13%) were nonreactive in Western blots although positive in immunodiffusion. Fifty-one of 90 SLE sera (57%) were SS-A precipitin positive by immunodiffusion. In Western blots, 24 (47%) possessed antibodies against both the 60-kd and the 52-kd antigens, while 9 (18%) reacted only with the 60-kd protein. Eighteen (35%) were nonreactive by Western blot, although positive by immunodiffusion. Antibody to the 52-kd antigen without concomitant antibody to the 60-kd antigen was seen only in patients with primary SS, whereas antibody to the 60-kd antigen without concomitant antibody to the 52-kd antigen was seen only in SLE patients. Although antibodies to SS-A are detected in both SS and SLE, our findings show that there is dissociation of immune responses to the 2 component antigens of the particle, which may be evidence of different events initiating the autoimmune process in these diseases.

Neuroimmunology. II: Antigenic specificity of the nervous system.

Antigenic specificity of the nervous system refers to a property conveyed by unique cell surface structures that are present on different classes of nervous system tissue. These structures are of major importance for the study of nervous system structure and function, and can play a central role in determining patterns of nervous system injury. Thus, the major classes of nervous system cells are identified by structures that are unique to them: neurons by the presence of tetanus toxin receptors on their surface and oligodendrocytes by the presence of surface galactocerebroside, for example. With the advent of hybridoma technology, a large number of monoclonal antibodies are being identified which have increased by several orders of magnitude the ability to define subclasses of nervous system tissue according to unique antigens. In addition, surface antigens of nervous system tissue may determine the specificity of nervous system injury by (1) functioning as receptors for viruses or (2) being the targets of autoimmune responses. Patterns of viral injury to the nervous system are often extraordinarily selective (e.g., poliovirus tropism for motor neurons), and nervous system viral tropism is due is some instances to the interaction of a virus with a unique surface antigen on neural cells. The specificity of injury in autoimmune disease (such as that against the acetylcholine receptor in myasthenia gravis) likewise must be explained by an immune response against unique antigenic determinants on the tissue being damaged. Some antigens are known to be shared between nervous system and other tissues or between nervous system and infectious agents such as bacteria or viruses. The presence of shared antigenic structures between the nervous system and infectious agents creates the possibility that an immune response generated against a virus may concurrently damage normal nervous system tissue.

IgM-autoantibodies against isologous erythrocytes also react with isologous IgG(Fc).

The mechanisms by which the adaptive immune system of animals discriminate between foreign and self components are not known. It is generally believed that antibodies are produced only in response to foreign agents. However, against this axiom there is mounting evidence that normal animals produce autoantibodies against a variety of self components. In two murine model systems, a high proportion of IgM-producing cells secrete autoantibodies. In one model, the autoantibodies are specific for isologous mouse IgG(Fc) and in the other, the autoantibodies are specific for antigens revealed on the surface of mouse erythrocytes (RBC) by proteolytic enzymes. The function of these two autoimmune responses is a mystery. Here we show that the two responses are related. The lysis of mouse RBC, modified with bromelain (brom) by IgM autoantibodies was completely inhibited by isologous IgG(Fc), and antibodies against mouse IgG(Fc) formed precipitin bands with solubilized membranes from mouse RBC. We conclude that mouse RBC membranes and IgG(Fc) have at least one antigenic determinant in common and that this determinant is the target for autoimmune responses in normal mice.