Protein Autoantigens Research Articles

Screening for Potential Drug Targets by Comprehensive Identification of Disease-specific Antigens Incorporated into Immune Complexes in Patients with Immunological Diseases

Our immune system resembles an intelligent security system, which continually monitors for foreign invaders (infectious diseases); however, in some cases, this system recognizes healthy parts as something harmful or foreign and then attacks them (autoimmune diseases). The defining characteristics of an autoimmune disease are the existence of T- and B-cell autoreactivity against self proteins (autoantigens). In addition to autoimmune diseases, aberrant host proteins that occur during a certain state of diseases (e.g., cancer) can be recognized as an autoantigen. Immune complexes (ICs) are produced during an immune response and may reflect some aspects of an ongoing immune response. Therefore, the identity of antigens incorporated into ICs provides the information that in the future may aid in the development of diagnosis and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and this information might be more relevant than information on free antigens. We developed a novel proteomic strategy (immune complexome analysis) in which ICs are separated from serum, followed by direct tryptic digestion and nano-liquid chromatography-tandem mass spectrometry for the identification and profiling of antigens in circulating ICs. We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, anti-neutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases and cancers. In this review, we mainly discuss the results for autoimmune diseases.

Auto-Antigens Targeted By Auto-Antibodies IgG on the Bone Marrow Hematopoietic Cells of the Patients with Immuno-Related Pancytopenia

Objective: To screen and identify the auto-antigens targeted by auto-antibodies IgG on the bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP).Background: Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression.. Our previous studies have demonstrated that DC2 increased in the bone marrow of IRP patients, which promote Th0 cells to polarize to Th2 cells and cause the over-function of B lymphocytes.These B cells produce auto-antibodies that mediated BM destruction.. But the antigens that induced DC2 elevated are not clear at present, our group found several target antigens by western blot method before, including G protein coupled receptor (GPCR) 156 variants, P chain of red blood cells with band three proteins, lactoferrin and WD repeat sequences of proteins.Autoimmune diseases often have several or even dozens of target antigens, so this experiment adopts the method of cDNA expression library of serological analysis (SEREX) to sceen antigens targeted by IRP autoantibodies and further verify their antigenicity.Materials and Methods: We established the recombinant cDNA expression libraries derived from K562 cells. We performed immunoblotting analysis of this library with sera from patients and normal healthy controls. The presumptive phage expressed autoantigen proteins were initially proposed based on the differential affinity to the sera antibodies of patients versus normal healthy controls,the plagues which were positive with IRP serum but negative with normal control serum were positive screened plagues. The insert fragment of each positive plague was sequenced. After the gene sequence confirmation, the sequence of FTL gene, TRMT112 gene and TRAPPC gene were amplified by polymerase chain reaction (PCR) then inserted into Peasy-E1 vector separately. Each recombinant plasmid was transformed into E.coli BL-21 and was identified by PCR and sequencing. After gene identification, the right recombinant plasmid was transformed into E.coli BL21 and the bacteria was induced by isopropyl-β-d-thiogalactoside (IPTG) to express. The expected protein was identified by Western blot and purified by ProteinIso Ni-NTA Resin. Then we coated ELISA plate with purified FTL protein and EPOR protein respectively, and detected the titer of the serum antibody of IRP patients and normal controls by indirect ELISA. Finally, the clinical index of 121 IRP patients were analysed.Results: 1.We established the recombinant cDNA expression libraries derived from K562 cells successfully. The library has good capacity, ligation efficiency and recombination efficiency. 2. We screend 11 positive plagues by immunoblotting, the seven of them were sequenced and blasted successfully. 3.3 fusion protein was expressed and confirmed by Western blot. The purified protein was obtained by ProteinIso Ni-NTA Resin. 4. The titer of antibody against the FTL and EPOR proteins was higher in untreated IRP than in recovered IRP patients and in normal control.Conclusion: We screened 7 proteins that maybe autoantigens of IRP by SEREX. Furthermore, we expressed and purified 3 fusion proteins. Finally we identified the antigenicity of the FTL and epor proteins. DisclosuresNo relevant conflicts of interest to declare.

A Biodegradable Nanoparticle Platform for the Induction of Antigen-Specific Immune Tolerance for Treatment of Autoimmune Disease

Targeted immune tolerance is a coveted therapy for the treatment of a variety of autoimmune diseases, as current treatment options often involve nonspecific immunosuppression. Intravenous (iv) infusion of apoptotic syngeneic splenocytes linked with peptide or protein autoantigens using ethylene carbodiimide (ECDI) has been demonstrated to be an effective method for inducing peripheral, antigen-specific tolerance for treatment of autoimmune disease. Here, we show the ability of biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles to function as a safe, cost-effective, and highly efficient alternative to cellular carriers for the induction of antigen-specific T cell tolerance. We describe the formulation of tolerogenic PLG particles and demonstrate that administration of myelin antigen-coupled particles both prevented and treated relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a CD4 T cell-mediated mouse model of multiple sclerosis (MS). PLG particles made on-site with surfactant modifications surpass the efficacy of commercially available particles in their ability to couple peptide and to prevent disease induction. Most importantly, myelin antigen-coupled PLG nanoparticles are able to significantly ameliorate ongoing disease and subsequent relapses when administered at onset or at peak of acute disease, and minimize epitope spreading when administered during disease remission. Therapeutic treatment results in significantly reduced CNS infiltration of encephalitogenic Th1 (IFN-γ) and Th17 (IL-17a) cells as well as inflammatory monocytes/macrophages. Together, these data describe a platform for antigen display that is safe, low-cost, and highly effective at inducing antigen-specific T cell tolerance. The development of such a platform carries broad implications for the treatment of a variety of immune-mediated diseases.

ANCA disease patients with increased expression of autoantigen genes produce an alternative PR3 transcript and synthesize autoantigen proteins

ANCA disease patients with increased expression of autoantigen genes produce an alternative PR3 transcript and synthesize autoantigen proteins

Antigen and substrate withdrawal in the management of autoimmune thrombotic disorders

AbstractPrevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia, antiphospholipid syndrome and thrombotic thrombocytopenic purpura, include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 in heparin-induced thrombocytopenia and β2-glycoprotein-I (β2GPI) in antiphospholipid syndrome. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In thrombotic thrombocytopenic purpura, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, that is, enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor. A corollary of these concepts is that disrupting platelet factor 4 and β2GPI conformation (or ultralarge multimer of von Willebrand factor oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.

Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.

Comparative autoantibody profiling before and after appearance of malignance: Identification of anti-cathepsin D autoantibody as a promising diagnostic marker for lung cancer

Cancer patients frequently develop autoantibodies. To test the hypothesis that the appearance of autoantibodies precedes the clinical diagnosis of cancer, we applied an immunoproteomic approach to compare autoantibody profiles before and after appearance of malignances. Proteins from A549 cells, a lung adenocarcinoma cell line, were separated by two dimensional electrophoresis and then immunoblotted with serum samples from 8 individuals who were eventually diagnosed with lung cancer. Compared with autoantibody profiles from 3 years prior to the appearance of malignances, 21 immunoreactive spots newly appeared or presented with stronger staining intensity when clinical diagnoses were made. Among them, 10 matched spots on 2-DE gels were identified by mass spectrometry analysis as 5 proteins. With immunoprecipitation analysis, the antigenicity of protein cathepsin D was confirmed, and notably, in lung cancer sera, the occurrences of autoantibodies against the specific forms of cathepsin D differed significantly from the control groups (p<0.05). Our findings suggest that harnessing immunity may have utility for early cancer marker discovery, and that comparing autoantibodies to specific forms of cathepsin D may be a promising early marker of lung cancer.

Bioinformatics-Based Predictions of Peptide Binding to Disease-Associated HLA Proteins Suggest Explanation for Shared Autoimmunity

Aim This study was designed to examine the immunogenetic basis for shared autoimmunity, resulting in autoantigen presentation that leads to the production of two or more disease-specific autoantibodies. Methods A bioinformatics approach based on peptide binding predictions to disease-associated HLA determinants has been developed and tested here using 11 disease associations between autoimmune systemic and mucocutaneous blistering disorders. Various HLAs associated with antigens within a given “disease model” (set of HLA class II and protein sequences known to be associated with a specific autoimmune disease) were tested and ranked against the antigenic proteins, first with proteins they are known to associate with and then with proteins known to be implicated in a second disease model. In every case binding predictions were compared for different proteins binding to the same HLA. Subsequently, disease-related autoantigens have been tested for their binding affinity against each disease-specific HLA class II protein. Results For a single HLA haplotype, several binders have been generated from a related autoantigen with the variable binding score. In most cases, the binding score corresponding to the interactions between the autoantigen-derived epitope and the HLA associated with one disease was similar or lower than the interactions between the epitope from proteins associated with the second disease and the same HLA. Notably, there was no compelling promiscuity in peptide binding to each of the HLA molecules, in spite of the promiscuous nature of HLA class II binding. Conclusions The data suggest that, in susceptible individuals, shared autoimmunity might be initiated by two types of HLA/peptide interaction; first between an autoantigen-derived epitope and its disease-associated HLA molecules, and second, between a different peptide of the same autoantigen and HLA proteins specific for the second disease.

Stopping Type 1 Diabetes: Attempts to Prevent or Cure Type 1 Diabetes in Man

Type 1 diabetes—or, more accurately, type 1A diabetes—is thought to arise from selective immunologically mediated destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans with consequent insulin deficiency (1). This occurs in individuals in whom genetic susceptibility outweighs genetic protection and is probably initiated by environmental factors not yet clearly defined. The disease arises via a cellular-mediated immune process, presumably a specific reaction to one or more β-cell proteins (autoantigens). There is consequent progressive impairment of β-cell function and apparent decline in β-cell mass. A secondary humoral immune response is characterized by the appearance of autoantibodies that serve as markers of the immune damage to β-cells. This insidious type 1 diabetes disease process generally evolves over a variable period of years (Fig. 1). The decline in β-cell function—and presumably in mass—is evidenced metabolically by loss of first-phase insulin response to an intravenous glucose challenge and later by the appearance of impairment in glycemic regulation, which is manifested as dysglycemia either as impaired glucose tolerance, impaired fasting glucose, or “indeterminate” glucose levels (values >200 mg/dl [11.1 mmol/l] at 30, 60, or 90 min during an oral glucose tolerance test). Ultimately, the clinical syndrome of type 1 diabetes becomes evident when the majority of β-cell function has been lost and presumably most β-cells have been destroyed; at this juncture, frank hyperglycemia supervenes. Although that broad sequence can be articulated, there are still gaps in many of the details. Further understanding of the nature of the disease process will facilitate the design of intervention strategies aimed at abrogating β-cell destruction and ultimately at prophylaxis of type 1 diabetes. FIG. 1. Progression of the type 1 diabetes disease process. This is a cellular autoimmune process occurring in individuals with a genetic predisposition to the disease, presumably triggered by some environmental factor. Humoral antibodies indicate …

A novel immunotherapeutic approach to melanoma-bearing hosts with protein-transduction domain-containing immunogenic foreign antigens

Active immunotherapy is accomplished by two critical factors; (1) induction of strong antigen-specific immune responses, and (2) abundant expression of antigen-epitopes on target cells. Previously, we have shown that the nona-arginine protein-transduction domain (R9-PTD) induced efficient protein-antigen transduction to a variety of cell types in vitro and in vivo. We have also demonstrated that intradermal (i.d.) injections of R9-PTD-containing immunogenic foreign antigens (rR9-OVA) induced dual immunological effects: the induction of OVA-specific Tc1- and Th1-dominant immune responses, and the induction of CTL-mediated immune responses at the injection area by expressing OVA-epitopes. We investigated and compared the antitumor effects by intratumoral (i.t.) injections of rR9-containing natural tumor-associated autoantigen (TAA) and other rR9-containing proteins, including rR9-OVA, into B16 melanoma. Our results clearly demonstrate that multiple i.t. injections of rR9-OVA, but not rR9-containing TAA, elicited strong antitumor effects in B16-bearing mice, and resulted in complete tumor regression in some (50%) animals. These antitumor effects were abrogated by depletion of CD8(+) T cells, and SIINFEKL-specific CTL lysed rR9-OVA-treated B16 cells in vitro. I.t. injections with rR9-OVA altered the proportion of CD4(+) T cell subsets in B16-bearing mice. Interestingly, B16-tumor growth at the untreated site was also reduced following multiple injections of rR9-OVA at the other B16 tumor site. Finally, we confirmed that mice that had rejected B16 tumors by i.t. injections of rR9-OVA subsequently acquired CTL activities to Trp2-epitope/B16 cells. Multiple i.t. injections of rR9-PTD-containing immunogenic foreign Ags elicit strong antitumor effects, and thereby may provide important clinical benefits to melanoma patients.

Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 μL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 μL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms.

Novel antigens in type 1 diabetes: The importance of ZnT8

The presence of circulating islet cell autoantibodies distinguishes type 1A diabetes (T1D) from other diabetic syndromes and determination of autoantigen genes and proteins is instrumental in understanding T1D as a clinical entity and in investigating the pathogenesis of the disease. ZnT8 was recently defined as a candidate autoantigen based on a -bioinformatics analysis focused on discovery of beta-cell-specific proteins associated with the regulatory pathway of secretion. The native molecule does not lend itself easily to solution-phase autoantibody assays, but ligands based on the predicted domain structure and molecular modeling have led to robust diagnostic procedures showing high specificities and sensitivities that complement current T1D autoantibody assays and add to the predictive value of their measurement. The incorporation of genetic and structural epitope analysis into ZnT8A determinations adds a further dimension to its diagnostic value and understanding of its role in the autoimmune disease process.

Antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis

Multiple sclerosis (MS) is a chronic relapsing disease of the central nervous system (CNS) in which immune processes are believed to play a major role. To date, there is no reliable method by which to characterize the immune processes and their changes associated with different forms of MS and disease progression. We performed antigen microarray analysis to characterize patterns of antibody reactivity in MS serum against a panel of CNS protein and lipid autoantigens and heat shock proteins. Informatic analysis consisted of a training set that was validated on a blinded test set. The results were further validated on an independent cohort of relapsing-remitting (RRMS) samples. We found unique autoantibody patterns that distinguished RRMS, secondary progressive (SPMS), and primary progressive (PPMS) MS from both healthy controls and other neurologic or autoimmune driven diseases including Alzheimer's disease, adrenoleukodystropy, and lupus erythematosus. RRMS was characterized by autoantibodies to heat shock proteins that were not observed in PPMS or SPMS. In addition, RRMS, SPMS, and PPMS were characterized by unique patterns of reactivity to CNS antigens. Furthermore, we examined sera from patients with different immunopathologic patterns of MS as determined by brain biopsy, and we identified unique antibody patterns to lipids and CNS-derived peptides that were linked to each type of pathology. The demonstration of unique serum immune signatures linked to different stages and pathologic processes in MS provides an avenue to monitor MS and to characterize immunopathogenic mechanisms and therapeutic targets in the disease.

Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on gp41 and Lacks Reactivity against Self-Antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

T Cell Repertoire Diversity Is Required for Relapses in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis

Comparison of TCRalphabeta repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT(-/-)) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRalphabeta repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT(+/-) and TdT(-/-) mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Valpha-Jalpha and Vbeta-Jbeta rearrangements in both strains. However, whereas TdT(+/+) and TdT(+/-) mice undergo successive EAE relapses, TdT(-/-) mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT(+/-) mice, new public Valpha-Jalpha and Vbeta-Jbeta rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4(+) T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRalphabeta repertoire diversification contributes to autoimmune progression.

Reconstruction of a pathway of antigen processing and class II MHC peptide capture

Endocytosed antigens are proteolytically processed and small amounts of peptides captured by class II MHC molecules. The details of antigen proteolysis, peptide capture and how destruction of T-cell epitopes is avoided are incompletely understood. Using the tetanus toxin antigen, we show that the introduction of 3-6 cleavage sites is sufficient to trigger a partially unfolded conformation able to bind to class II MHC molecules. The known locations of T-cell epitopes and protease cleavage sites predict that large domains of processed antigen (8-35 kDa) are captured under these conditions. Remarkably, when antigen is bound to the B-cell antigen receptor (BCR), processing can trigger a concerted 'hand-over' reaction whereby BCR-associated processed antigen is captured by neighbouring class II MHC molecules. Early capture of minimally processed antigen and confinement of the processing and class II MHC loading reaction to the membrane plane may improve the likelihood of T-cell epitope survival in the class II MHC pathway and may help explain the reciprocal relationships observed between B- and T-cell epitopes in many protein antigens and autoantigens.

Prediction and Prevention of Type 1 Diabetes: Progress, Problems, and Prospects

Type 1 diabetes mellitus (T1D) arises from selective immunologically mediated destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans with consequent insulin deficiency. This occurs in genetically susceptible individuals and is a cellular-mediated process, presumably a specific reaction to one or more beta-cell proteins (autoantigens), although probably initiated by some environmental factor(s). There is consequent progressive impairment of beta-cell function and decline in beta-cell mass. A secondary humoral immune response is characterized by the appearance of autoantibodies that serve as markers of the immune damage to beta-cells. This insidious T1D disease process evolves over a period of years. The decline in beta-cell function and mass is evidenced metabolically by loss of first-phase insulin response to an intravenous glucose challenge, and later by the appearance of impairment in glycemic regulation, manifested as dysglycemia--usually as impaired glucose tolerance, but occasionally as impaired fasting glucose. Ultimately, the clinical syndrome of T1D becomes evident when the majority of beta-cells have been destroyed and frank hyperglycemia supervenes. Given this sequence of events, for which it is possible to envision intervention to interdict the process, it is not surprising that much research effort has been expended to identify individuals at risk of the disease.

IgG subclass distribution of autoantibodies in pediatric opsoclonus–myoclonus syndrome

Opsoclonus–myoclonus syndrome (OMS) in children is a rare disorder including a severe eye movement disturbance, myoclonia, ataxia and often developmental retardation. Both OMS forms, idiopathic or neuroblastoma-associated (paraneoplastic), have been suspected to be autoimmune. Recently, autoantibodies have been found in OMS sera. We here show that autoantibodies in OMS, both intracellular and surface binding, belong mainly to the IgG3 subclass, although the total serum IgG3 level is normal. These results support the autoimmune hypothesis and point to a protein autoantigen as antigenic target.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Deletion of the gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen results in a mild metabolic phenotype

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (-14%) and female (-11%) G6pc2 (-/-) mice, while female G6pc2 (-/-) mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible.