Cross-reactive Autoantibodies Research Articles

Abstract 14677: Surgical Thymectomy for Autoimmune Encephalitis With CRMP-5 and AMPAR Antibodies Induced by SARS-CoV-2 Infection

Introduction: SARS-CoV-2 has been linked to development of autoimmune diseases. We describe a case of a patient with untreated thymoma who developed 2 cross-reactive autoantibodies between thymus and brain: collapsing response mediator protein-5 (CRMP-5) and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptor (AMPAR), triggered by SARS-CoV-2, inducing encephalitis and coma. Emergency thymectomy reduced anti-CRMP-5 and anti-AMPAR antibodies and restored patient neurologic function within 3 months. Description of Case: A 31-year-old male with a known history of thymoma with myasthenia gravis presented with a 1-day history of memory loss, hallucinations, ataxia, involuntary movements, and tachypnea. Rapid nasal swab COVID-19 test was positive. Over several hours, the patient became unresponsive, requiring urgent intubation for airway protection. Brain computed tomography and magnetic resonance imaging with gadolinium showed no abnormalities. Cerebrospinal fluid (CSF) analysis revealed leukocytosis with lymphocytic predominance. Four days later, CSF and serum were tested for and demonstrated high levels of CRMP-5 and AMPAR antibodies, consistent with autoimmune encephalitis. Complete robotic thymectomy was performed. Pathologic analysis revealed type B2 thymoma with negative resection margins. Three months after surgical intervention, antibody titers normalized, the patient regained consciousness, and had no neurologic deficits. Discussion: Surgical thymectomy effectively treats autoimmune encephalitis in patients with thymoma and positive CRMP-5 and AMPAR antibodies in the setting of SARS-CoV-2 infection. This rare condition is typically seen in patients with thymoma or small cell lung cancer who experience acute viral infection. Although autoantibodies to CRMP-5 and AMPAR are known to be induced by viral infection, this is the first time it has been reported for SARS-CoV-2. Timely surgical intervention is necessary to reverse obtundation and acute encephalitis by removing the cross reactive epitopes in the thymus gland. Future studies are needed to explore the molecular mechanism of SARS-CoV-2 induced immune dysfunction and autoimmunity involving cross-reactive epitopes in this deadly syndrome.

Requirements for anti-aquaporin 5 autoantibody production in a mouse model.

Several oral bacteria, including Prevotella melaninogenica (Pm), have aquaporin (AQP) proteins homologous to human AQP5, a major water channel protein targeted in Sjogren's syndrome. This study aimed to understand the antigenic characteristics that induce autoantibodies against an AQP5 "E" epitope (AQP5E) in a mouse model using C57BL/6 mice. Immunization with a PmE-L peptide derived from Pm AQP, which contains amino acid mismatches both at the B- and T-cell epitopes, efficiently induced anti-AQP5E autoantibodies accompanied by increased germinal center (GC) B and follicular helper T cells in the draining lymph nodes. However, PmE, a peptide lacking a T-cell epitope, and AQP5E-L, an AQP5-derived self-peptide, hardly induced either anti-AQP5E autoantibodies or GC responses. Surprisingly, OTII-AQP5E, a peptide that replaced the self T-cell epitope of AQP5E-L with an ovalbumin-derived foreign T-cell epitope, was not any better than AQP5E-L in the induction of anti-AQP5E autoantibodies and GC response, despite the substantial expansion of CD4+ T cells and production of anti-OTII-AQP5E antibodies. The complex of biotinylated PmE-L peptide and highly immunogenic streptavidin (SA) induced a strong extrafollicular B-cell response skewed toward the expansion of SA-specific B cells. However, the expansion of AQP5E-specific GC B cells was limited, resulting in the inefficient induction of anti-AQP5E autoantibodies. Collectively, our results have demonstrated that anti-AQP5E autoantibody production is only allowed when foreign B- and T-cell epitopes drive a strong GC response of AQP5E-specific B cells for affinity maturation. This study helps explain why cross-reactive anti-AQP5 autoantibodies are not produced during the immune response to Pm in most healthy people.

Anti-Phospholipid Antibodies and Coronavirus Disease 2019: Vaccination Does Not Trigger Early Autoantibody Production in Healthcare Workers.

A molecular mimicry between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins supports the possibility that autoimmunity takes place during coronavirus disease 2019 (COVID-19) contributing to tissue damage. For example, anti-phospholipid antibodies (aPL) have been reported in COVID-19 as a result of such mimicry and thought to contribute to the immunothrombosis characteristic of the disease. Consistently, active immunization with the virus spike protein may elicit the production of cross-reactive autoantibodies, including aPL. We prospectively looked at the aPL production in healthcare workers vaccinated with RNA- (BNT162b2, n. 100) or adenovirus-based vaccines (ChAdOx1, n. 50). Anti-cardiolipin, anti-beta2 glycoprotein I, anti-phosphatidylserine/prothrombin immunoglobulin G (IgG), IgA, and IgM before and after vaccination were investigated. Anti-platelet factor 4 immunoglobulins were also investigated as autoantibodies associated with COVID-19 vaccination. Additional organ (anti-thyroid) and non-organ (anti-nuclear) autoantibodies and IgG against human proteome were tested as further post-vaccination autoimmunity markers. The antibodies were tested one or three months after the first injection of ChAdOx1 and BNT162b2, respectively; a 12-month clinical follow-up was also performed. Vaccination occasionally induced low titers of aPL and other autoantibodies but did not affect the titer of pre-existing autoantibodies. No significant reactivities against a microarray of approximately 20,000 human proteins were found in a subgroup of ChAdOx1-vaccinees. Consistently, we did not record any clinical manifestation theoretically associated with an underlying autoimmune disorder. The data obtained after the vaccination (two doses for the RNA-based and one dose for the adenovirus-based vaccines), and the clinical follow-up are not supporting the occurrence of an early autoimmune response in this cohort of healthcare workers.

The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

Cross-reactive autoantibodies in RA.

Cross-reactive autoantibodies in RA.

Hepatic Campylobacter jejuni infection in patients with Castleman-Kojima disease (idiopathic multicentric Castleman disease with thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome).

Castleman-Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD-TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD-TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD-TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD-TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD-TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD-TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD-TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross-reactive autoantibodies.

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.

Autoantibody cross-reactivity with a microbial protein from a prevalent human gut commensal in antiphospholipid syndrome

Abstract Given the vast antigenic source of the gut microbiota, we hypothesized that human gut commensal bacteria induce and sustain autoreactivity via cross-reactivity. As a paradigm, we studied the antiphospholipid syndrome (APS), which is a serious autoimmune clotting disorder of unknown etiology but with a well-defined major autoantigen, b2-glycoprotein I (b2GPI). Infectious triggers are implicated in transient autoantibody production, but the persistent stimuli for anti-b2GPI antibodies remain unknown. To this end, we characterized b2GPI-specific monoclonal autoantibody reactivity to an in silico identified candidate commensal, Roseburia intestinalis (R.int), that is abundant in human stool from APS patients as detected by a species-specific PCR. Using an APS-derived monoclonal antibody, P1-117, that binds to the major B cell epitope in domain I of b2GPI (RGGMR), we tested for cross-reactivity to anaerobically cultured R.int and its candidate cross-reactive R.int protein (WP_006857340.1). P1-117 bound significantly to R.int lysates whereas a control antibody specific for another epitope within b2GPI did not. Further, P1-117 displayed significant binding to the recombinantly expressed R.int cross-reactive candidate protein. BALB/cJ mice immunized with whole heat-killed R.int displayed significant cross-reactive splenocytes and low titers of cross-reactive IgG autoantibodies. In conclusion, we present here in vitro and in vivo studies supporting cross-reactivity of pathogenic b2GPI-specific autoantibody with a common gut commensal. This study provides a concept for persistently colonizing gut commensals as chronic cross-reactive triggers in genetically susceptible autoimmune hosts such as APS patients.

Association of anti-peptidyl arginine deiminase antibodies with radiographic severity of rheumatoid arthritis in African Americans.

BackgroundEvidence suggests that the presence of peptidyl arginine deiminase type 4 (PAD4) antibodies is associated with radiographic-severity rheumatoid arthritis (RA) among Caucasian patients. The presence of anti-PAD4 antibodies that were cross-reactivity against PAD3 was associated with more aggressive erosive disease (compared with the presence of anti-PAD4 antibodies without anti-PAD3 crossreactivity) in Caucasian RA patients. The objectives of this study were to determine the prevalence of serum anti-PAD4 and anti-PAD4/PAD3 cross-reactive autoantibodies in African Americans with RA and whether these antibodies associate with radiographic severity and radiographic progression.MethodsSerum anti-PAD4 and anti-PAD4/PAD3 antibodies were measured by immunoprecipitation, and the temporal trends in titers were analyzed. We compared total radiographic scores among anti-PAD4-positive, anti-PAD4/PAD3-positive, and anti-PAD4-negative patients and used a zero-inflated negative binomial model to determine associations between radiographic severity and antibody status. Logistic regression was used to analyze radiographic progression.ResultsOf 192 African-American patients with RA, 73 % were anti-citrullinated peptide/protein antibody (ACPA)-positive, 46 out of 192 (24 %) of whom had serum anti-PAD4 antibodies. Median (interquartile range) total Sharp van der Heijde radiographic scores were 2 (1–97.5) in ACPA-positive patients and 0 (0–3) in ACPA-negative patients (P < 0.001). Of the 46 anti-PAD4-positive patients, 20 had anti-PAD4 antibodies that cross-reacted with PAD3. In patients with early RA, anti-PAD4 and anti-PAD4/PAD3 antibody titers increased over time (P = 0.006, P = 0.001, respectively). Median (interquartile range) total radiographic scores were higher for anti-PAD4-positive than for anti-PAD4-negative patients (3 (1–115) versus 2 (0–11), respectively; P = 0.005). Median (interquartile range) total radiographic score for anti-PAD4/PAD3-positive patients was 76 (3–117) (P < 0.001) versus anti-PAD4-negative patients. Only anti-PAD4/PAD3 antibodies associated with radiographic severity (incidence rate ratio = 2.81; 95 % confidence interval 1.23, 6.43).ConclusionThis analysis suggests that autoantibodies against PAD4 and PAD3 proteins may serve as biomarkers for identifying African-American patients with RA and higher radiographic severity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1126-7) contains supplementary material, which is available to authorized users.

The production of cross-reactive autoantibodies that bind to bovine serum albumin in mice administered reducing sugars by subcutaneous injection.

IntroductionIn a previous study, we identified the formation of cross-reactive autoantibodies that bound to bovine serum albumin (BSA) in a D-galactose-induced aging mouse model.Aim of the studyIn this study, we investigated the effect of other reducing sugars (namely, glucose and fructose) on the formation of autoantibodies. The effects of concentration and route of administration on the formation of autoantibodies were examined in detail.Material and methodsThree concentrations (100, 500, and 1,000 mg/kg) of reducing sugars were tested. The effects of different routes of administration (subcutaneous, oral, and intraperitoneal) on the formation of autoantibodies were also analysed. The immunoreactivities of serum samples from mice treated with reducing sugars were analysed by an enzyme-linked immunosorbent assay (ELISA) using BSA or mouse serum albumin antigens (MSA).ResultsRepeated subcutaneous administration of all reducing sugars lead to autoantibody formation in a concentration-dependent manner. However, these autoantibodies did not cross-react with MSA, and simultaneous treatment of aminoguanidine with reducing sugars did not show any inhibitory effects on the formation of autoantibodies. No autoantibodies were detected after oral or intraperitoneal administration of reducing sugars. Immunohistochemistry data showed that the target antigen(s) of the autoantibodies were present only in the skin tissue of mice treated with reducing sugars.ConclusionsOur results show that administration of reducing sugars by subcutaneous injection leads to the formation of autoantibodies that cross-react with BSA; the formation and target antigen(s) of the autoantibodies may originate from within the skin tissue treated with the reducing sugars.

Crossreactive autoantibodies directed against cutaneous and joint antigens are present in psoriatic arthritis.

BackgroundPsoriatic arthritis (PsA) is a chronic inflammatory disease of unknown origin, characterized by erosions and new bone formation. Diagnosis of PsA is mainly clinical and there are no biomarkers available. Moreover in PsA autoantibodies have not been described so far. Indeed an autoimmune origin has been suggested but never proven. Aim of the study was to investigate the possible presence of autoantibodies typically associated with PsA.MethodsWe used pooled IgG immunoglobulins derived from 30 patients with PsA to screen a random peptide library in order to identify disease relevant autoantigen peptides.ResultsAmong the selected peptides, one was recognised by nearly all the patients’ sera. The identified peptide (PsA peptide: TNRRGRGSPGAL) shows sequence similarities with skin autoantigens, such as fibrillin 3, a constituent of actin microfibrils, desmocollin 3, a constituent of the desmosomes and keratin 78, a component of epithelial cytoskeleton. Interestingly the PsA peptide shares homology with the nebulin-related anchoring protein (N-RAP), a protein localized in the enthesis (point of insertion of a tendon or ligament to the bone), which represents the first affected site during early PsA. Antibodies affinity purified against the PsA peptide recognize fibrillin, desmocollin, keratin and N-RAP. Moreover antibodies directed against the PsA peptide are detectable in 85% of PsA patients. Such antibodies are not present in healthy donors and are present in 13/100 patients with seroposive rheumatoid arthritis (RA). In seronegative RA these antibodies are detectable only in 3/100 patients.ConclusionsOur results indicate that PsA is characterized by the presence of serum autoantibodies crossreacting with an epitope shared by skin and joint antigens.

The Effect of Inhibitory Antibodies on Acute Treatment of Bleeding Episodes with OΒΙ-1, an Antihemophilic Factor VIII (recombinant), Porcine Sequence, in Patients with Acquired Hemophilia Α

▪Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder, caused by auto-antibodies that inhibit the activity of endogenous human factor VIII (hFVIII). In most cases, the inhibition is governed by non-linear non-saturable (Type II) kinetics, resulting in incomplete inhibition and thus, underestimation of the FVIII inhibitor titer. A highly pure, B-domain deleted, antihemophilic FVIII (recombinant), porcine sequence (OBI-1) that is less susceptible to the inhibitory activity of anti-hFVIII antibodies has been developed as a replacement therapy to manage bleeding episodes in AHA patients.In one study of patients with AHA, the presence of pre-treatment anti-porcine FVIII (pFVIII) antibodies was reported not to correlate with the efficacy of a plasma-derived pFVIII1. However, cross-reactivity of anti-hFVIII antibodies with the recombinant pFVIII OBI-1 or de novodevelopment of anti-pFVIII antibodies could impair efficacy of OBI-1 treatment in subjects treated with pFVIII replacement therapy.This phase 2/3 open-label clinical trial investigated the efficacy and safety of OBI-1 in the treatment of serious bleeds in 28 adults with AHA. The primary efficacy endpoint was assessed at 24 hours (e.g., effective, partially effective). Subjects received an initial dose of OBI-1 (200 U/kg); additional doses were based on the subject’s target FVIII levels, anti-pFVIII titer, and clinical factors. This evaluation aims to address potential concerns regarding anti-pFVIII antibodies and their effect on the response to OBI-1 treatment in AHA patients.Overall 10/28 subjects (36%) had detectable anti-pFVIII inhibitors (range 0.8-29 BU) prior to treatment, presumably due to cross-reactive anti-hFVIII auto-antibodies also neutralizing pFVIII. The rise in FVIII activity level immediately after the first OBI-1 infusion was lower in subjects with baseline anti-pFVIII inhibitors ≥5 BU/mL (median rise: 29%, n = 4) than in subjects without detectable or with low anti-pFVIII inhibitor levels (<5 BU/mL, median rise: 96%; n=6). However, with tailored dosing all 10 subjects achieved clinically relevant FVIII activity levels. Furthermore, all had a positive response to treatment at or before 24 hours.All subjects received immunosuppressive therapy to treat their underlying autoimmune disease during the study. After treatment, 2 subjects with baseline anti-pFVIII inhibitors had an increase in anti-pFVIII titer (from 3 to 6 BU and from 15 to 183 BU); 8/10 subjects had a decline in anti-pFVIII titer to non-detectable levels. Five subjects did not have baseline anti-pFVIII but developed antibodies de novo (range 0.6-166 BU at final assessment). These de novo titers were first detected between 8 and 85 days after treatment initiation while the median duration of exposure to OBI-1 was 7 days. As 2 of these subjects had a concomitant increase in anti-hFVIII antibody titers, the de novoanti-pFVIII titers were possibly due to either, i) an anamnestic reaction to pFVIII that resulted in an increase in cross-reactive anti-hFVIII inhibitors, or ii) an immunogenic response to pFVIII yielding anti-pFVIII antibodies cross-reacting with hFVIII. Another 2/5 subjects had a concurrent decrease in anti-hFVIII antibody titers, suggesting an immunogenic response to pFVIII. These data indicate that immunosuppressive therapy used to treat auto-antibodies may not have the same suppressive effect on allo-antibodies.The presence of anti-pFVIII antibodies prior to treatment appears to have had an effect on the immediate FVIII activity level rise after the first dose, particularly if the titer was greater than 5 BU. However, this early low FVIII activity level could be overcome by a tailored dosing regimen, guided by measuring FVIII activity levels. Consistent with previous reports with a plasma-derived pFVIII1, our findings indicate a lack of correlation between baseline anti-pFVIII antibody titers and efficacy of OBI-1. OBI-1 replacement therapy facilitates objective and successful assessment of efficacy independently of inhibitors through monitoring of FVIII activity levels.1Morrison et al. (1993), Blood. 81:1513 DisclosuresFarin:Baxter Healthcare: Employment. Novack:Baxter Healthcare: Employment. Mo:Baxter Healthcare: Employment. Bourgeois:Baxter BioScience: Employment. Horling:Baxter BioScience: Employment. Ewenstein:Baxer Healthcare: Employment.

Association of the tumor necrosis factor -308 A/G promoter polymorphism with Tourette syndrome.

Several lines of evidence suggest that certain subtypes of obsessive-compulsive and tic disorders might be paediatric manifestations of post-streptococcal autoimmunity caused by cross-reactive autoantibodies. As tumor necrosis factor (TNF) is known to play a seminal role in coordinating the humoral immune response, TNF gene polymorphisms have been proposed as genetic risk factors both in obsessive-compulsive disorder (OCD) and Tourette syndrome (TS). The aim of this study was to investigate two TNF promoter polymorphisms (-238 A/G: rs361525 and -308 A/G: rs1800629) on the genetic susceptibility to OCD and TS in a child psychiatric sample (102 patients with OCD and 117 patients with TS). In the case-control set-up, the genotype and allele frequencies were compared to a control group from the general population (n = 405). As a control child psychiatric sample, 194 children with attention-deficit hyperactivity disorder were also genotyped. Our results revealed that the TNF -308 G-allele was more frequent in children with TS compared to controls (90.2% vs 84.8%, P = 0.037). For confirmation of this genetic association, a family-based analysis, the transmission disequilibrium test was used, which showed preferential transmission of the G-allele to patients with TS (nominal P-value 0.011). Moreover, this allele was also transmitted more frequently to children with tic symptoms (nominal P-value 0.039). No association was found between OCD or obsessive-compulsive symptoms and the studied TNF polymorphisms. Based on these findings, the TNF -308 G-allele can be associated with Tourette syndrome, highlighting the potential pathophysiological role of TNF dysregulation.

A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)

Abstract One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. Anti-EBNA-1 antibody obtained from mice from all three strains was observed to cross-react with dsDNA. However, the frequency of mice producing cross-reactive antibody was greater and the amount of cross-reactive antibody was higher in BAFF than wild type mice. This suggests that a viral protein such as EBNA-1 may trigger the development of antibodies that cross-react with dsDNA and that genetic susceptibility to lupus may enhance the development of cross-reactive antibodies upon viral exposure.

Potential Role of Bacterial Infection in Autoimmune Diseases: A New Aspect of Molecular Mimicry

Molecular mimicry is an attractive mechanism for triggering autoimmunity. In this review, we explore the potential role of evolutionary conserved bacterial proteins in the production of autoantibodies with focus on granulomatosis with polyangiitis (GPA) and rheumatoid arthritis (RA). Seven autoantigens characterized in GPA and RA were BLASTed against a bacterial protein database. Of the seven autoantigens, proteinase 3, type II collagen, binding immunoglobulin protein, glucose-6-phosphate isomerase, α-enolase, and heterogeneous nuclear ribonuclear protein have well-conserved bacterial orthologs. Importantly, those bacterial orthologs are also found in human-associated bacteria. The wide distribution of the highly conserved stress proteins or enzymes among the members of the normal flora and common infectious microorganisms raises a new question on how cross-reactive autoantibodies are not produced during the immune response to these bacteria in most healthy people. Understanding the mechanisms that deselect auto-reactive B cell clones during the germinal center reaction to homologous foreign antigens may provide a novel strategy to treat autoimmune diseases.

Rheumatoid arthritis is caused by aProteusurinary tract infection

Genetic, molecular and biological studies indicate that rheumatoid arthritis (RA), a severe arthritic disorder affecting approximately 1% of the population in developed countries, is caused by an upper urinary tract infection by the microbe, Proteus mirabilis. Elevated levels of specific antibodies against Proteus bacteria have been reported from 16 different countries. The pathogenetic mechanism involves six stages triggered by cross-reactive autoantibodies evoked by Proteus infection. The causative amino acid sequences of Proteus namely, ESRRAL and IRRET, contain arginine doublets which can be acted upon by peptidyl arginine deiminase thereby explaining the early appearance of anti-citrullinated protein antibodies in patients with RA. Consequently, RA patients should be treated early with anti-Proteus antibiotics as well as biological agents to avoid irreversible joint damages.

Inflammation and atherosclerosis: direct versus indirect mechanisms

It is now widely accepted that the development of atherosclerotic lesions involves a chronic inflammatory response that includes both innate and adaptive immune mechanisms. However, it is still unclear precisely what induces the inflammatory response. Furthermore, inflammation within the blood vessel can be divided into direct mechanisms where the primary inflammatory events occur within the intima of the blood vessel and contribute to both the initiation and progression of the plaques and indirect mechanisms where inflammation at nonvascular sites can contribute to the progression of the lesions. The direct mechanisms include lipid deposition and modification, influx of lipoprotein associated factors and microparticles derived from many different cell types, and possibly bacterial and viral infection of vascular cells. Indirect mechanisms derive from inflammation related to autoimmune diseases, smoking, respiratory infection, and pollution exposure, and possibly periodontal disease and gastric infection. The mechanisms include secretion of cytokines and other inflammatory factors into the circulation with subsequent uptake into the plaques, egress and recruitment of activated inflammatory cells, formation of dysfunctional HDL and crossreactive autoantibodies.

Streptococcus and rheumatic fever

To give an overview of the current hypotheses of the pathogenesis of rheumatic fever and group A streptococcal autoimmune sequelae of the heart valve and brain. Human monoclonal antibodies (mAbs) derived from rheumatic heart disease have provided evidence for crossreactive autoantibodies that target the dominant group A streptococcal epitope of the group A carbohydrate, N-acetyl-beta-D-glucosamine (GlcNAc), and heart valve endothelium, laminin and laminar basement membrane. T cells in peripheral blood and in rheumatic heart valves revealed the presence of T cells crossreactive with streptococcal M protein and cardiac myosin. For initiation of disease, evidence suggests a two-hit hypothesis for antibody attack on the valve endothelium with subsequent extravasation of T cells through activated endothelium into the valve to form granulomatous lesions and Aschoff bodies. Autoantibodies against the group A streptococcal carbohydrate epitope GlcNAc and cardiac myosin and its peptides appear during progression of rheumatic heart disease. However, autoantibodies against collagen that are not crossreactive may form because of the release of collagen from damaged valve or to responses to collagen bound in vitro by certain serotypes of streptococci. In Sydenham chorea, human mAbs derived from disease target the group A carbohydrate epitope GlcNAc and gangliosides and dopamine receptors found on the surface of neuronal cells in the brain. Human mAbs and autoantibodies in Sydenham chorea were found to signal neuronal cells and activate calcium calmodulin-dependent protein kinase II (CaMKII) in neuronal cells and recognize the intracellular protein biomarker tubulin. To summarize, pathogenic mechanisms of crossreactive autoantibodies which target the valve in rheumatic heart disease and the neuronal cell in Sydenham chorea share a common streptococcal epitope GlcNAc and target intracellular biomarkers of disease including cardiac myosin in the myocardium and tubulin, a protein abundant in the brain. However, intracellular antigens are not believed to be the basis for disease. The theme of molecular mimicry in streptococcal autoimmune sequelae is the recognition of targeted intracellular biomarker antigens such as cardiac myosin and brain tubulin, while targeting extracellular membrane antigens such as laminin on the valve surface endothelium or lysoganglioside and dopamine receptors in the brain. Antibody binding to these cell surface antigens may lead to valve damage in rheumatic heart disease or neuropsychiatric behaviors and involuntary movements in Sydenham chorea.

Autoimmunity in Rheumatic Diseases Is Induced by Microbial Infections via Crossreactivity or Molecular Mimicry

A general consensus supports fundamental roles for both genetic and environmental, mainly microbial, factors in the development of autoimmune diseases. One form of autoimmune rheumatic diseases is confined to a group of nonpyogenic conditions which are usually preceded by or associated with either explicit or occult infections. A previous history of clinical pharyngitis, gastroenteritis/urethritis, or tick-borne skin manifestation can be obtained from patients with rheumatic fever, reactive arthritis, or Lyme disease, respectively, whilst, other rheumatic diseases like rheumatoid arthritis (RA), ankylosing spondylitis (AS), and Crohn's disease (CD) are usually lacking such an association with a noticeable microbial infection. A great amount of data supports the notion that RA is most likely caused by Proteus asymptomatic urinary tract infections, whilst AS and CD are caused by subclinical bowel infections with Klebsiella microbes. Molecular mimicry is the main pathogenetic mechanism that can explain these forms of microbe-disease associations, where the causative microbes can initiate the disease with consequent productions of antibacterial and crossreactive autoantibodies which have a great impact in the propagation and the development of these diseases.

Molecular mimicry between virus and host and its implications for dengue disease pathogenesis

Numerous infectious agents may trigger autoimmunity or even result in autoimmune diseases. Several mechanisms have been proposed for pathogen-triggered autoimmunity including molecular mimicry, cryptic antigens, epitope spreading, bystander activation and polyclonal activation. In the case of dengue virus infection which causes serious public health problems, the mechanisms regarding the pathogenesis of dengue hemorrhagic syndrome are not fully resolved. Our previous studies suggest a mechanism of molecular mimicry in which antibodies directed against dengue virus non-structural protein 1 (NS1) cross-react with human platelets and endothelial cells and cause their damage and dysfunction, which may be related to the clinical features of dengue disease. Several cell surface proteins recognized by patient serum samples and anti-NS1 antibodies have been identified. Based on proteomic studies and sequence analysis, the C-terminal region of dengue virus NS1 shows sequence homology with target proteins. In addition, different regions of dengue virus proteins including core, prM, E and NS1 proteins show sequence homology with different coagulatory molecules. As an example, the amino acid sequence 101-106 of E protein (WGNGCG) shows sequence homology with factors XI, X, IX, VII, II (thrombin), plasminogen and tissue plasminogen activator. Furthermore, single chain variable region against NS1 can interfere with fibrin formation, which leads to prolonged thrombin time. We hypothesize that molecular mimicry between dengue virus proteins and coagulatory molecules may induce cross-reactive autoantibodies that can interfere with coagulation activation. A molecular mimicry pathogenesis for dengue disease which involves cross-reactivity of dengue virus with human endothelial cells, platelets and coagulatory molecules is proposed.