Autoantibodies and prediction of reproductive failure

PROBLEM: To determine which autoantibodies are associated with reproductive failure. METHOD OF STUDY: Sera from 269 patients with autoimmune disease and/or reproductive failure were analyzed for anti-phospholipid (aPL), anti-annexin-V, anti-lactoferrin, anti-thyroglobulin, anti-thyroid peroxidase, anti-prothrombin, anti-nuclear, and anti-saccharomycetes cerevisiae antibodies (ASCA), by enzyme-linked immunosorbent assay. Patients were classified as: recurrent pregnancy loss (RPL), infertility, and autoimmune diseases. The results were compared with those of 120 healthy volunteers. RESULTS: In autoimmune diseases, the prevalence of anti-prothrombin, anti-annexin, anti-phospholipid and anti-nuclear antibodies was significantly higher than in the control group, OR 11.0 [CI, 3.5-35.2], 33 [CI, 7.2-174.2], 13 [CI, 1.4-309.7], and 16.1 [CI 2.4-122], respectively. In infertility, the antibodies with significantly higher levels than controls were: aPL OR, 5.11 [CI 1.2-25.4], and anti-prothrombin antibodies, OR, 5.15 [CI, 2.1-12.7]. In RPL, ASCA, anti-prothrombin and aPL were more prevalent than in controls, OR 3.9 [CI, 1.5-10.6], 5.4 [CI, 2.4-12.5] and 4.8[CI, 1.2-22.2] for each antibody, respectively. Anti-prothrombin antibodies and aPL were more significantly associated with late pregnancy losses than early losses. CONCLUSION: ASCA antibodies have not previously been described in RPL. Nor are anti-prothrombin antibodies usually assessed in infertility or RPL. If these results are confirmed in further studies, these antibodies might be assessed routinely in reproductive failure.     

Identification of a peptide mimicking the binding pattern of an antiphospholipid antibody

Our objective was to characterize monoclonal antiphospholipid antibodies (APL) and identify disease-associated antigens in patients with the antiphospholipid syndrome (APS). We used the monoclonal antibody HL-5B, derived from a patient with APS suffering from multiple ischemic events, to screen a 12-mer peptide phage display library (New England Biolabs, London, England). The identified phage clones were sequenced and the derived consensus peptide was synthesized. The peptide was used to perform competitive inhibition experiments for their ability to inhibit the binding of the monoclonal antibody and of serum antibodies to cardiolipin and phosphatidylserine. Additionally patients and control sera were screened for their binding reactivities to this peptide. Using this 12-mer phage display library the peptide APHKHKASLSIY as consensus peptide for the monoclonal antiphospholipid antibody HL-5B could be identified. In competitive inhibition studies we showed that this peptide is able to inhibit the binding of HL-5B to cardiolipin and phosphatidylserine and furthermore another antiphospholipid antibody used as control was also inhibited in its binding to phospholipids. Using 21 sera from APS patients 67% showed a binding to the peptide in a specific ELISA above the cutoff level, generated with sera from 20 healthy controls. Out of the reactive patients' sera we used two exemplarily to perform inhibition studies. Both sera could be inhibited more than 40% in their binding to cardiolipin in a commercially available antiphospholipid antibody assay (Aescu.diagnostics, Wendelsheim, Germany). The identified peptide APHKHKASLSIY simulates the antigenic structure recognized from a subpopulation of serum antiphospholipid antibodies. This might indicate that the diversity of the antiphospholipid antibodies is limited and only few epitopes or few common structures are responsible for the development of those antibodies. Tests using these epitopes will strongly improve laboratory diagnosis of the APS.     

A peptide that shares similarity with bacterial antigens reverses thrombogenic properties of antiphospholipid antibodies in vivo

OBJECTIVE: The factors causing production of antiphospholipid (aPL) antibodies remain unidentified. Recently, studies have shown that aPL and anti-beta2Glycoprotein I (anti-beta2GPI) antibodies with pathogenic properties can be generated with peptides from bacterial and viral origin, that mimic regions of beta2GPI. These data suggest a molecular mimicry between bacterial/viral antigens and self-proteins. In this study we examined the ability of a synthetic peptide (named peptide A, NTLKTPRVGGC) that shares similarity with common bacterial antigens, to reverse aPL-mediated thrombosis in mice in vivo. Peptide A is also found in region I/II of beta2GPI. A scrambled form of peptide A (named scA, GTKGCPNVRLT) was used as a control. METHODS AND RESULTS: Sera from 29 patients with APS bound to peptide A but not to peptide scA by ELISA in a dose-dependent fashion. Cardiolipin (CL) liposomes inhibited the binding of IgG-APS by ELISA to peptide A by 35% and to CL by 56%. The inhibition of binding to cardiolipin and to peptide A was enhanced by addition of beta2GPI to the liposomes. CL/peptide A liposomes but not peptide A alone inhibited the binding of IgG-APS to peptide A. beta2GPI alone did not inhibit binding of IgG-APS to peptide A, to beta2GPI or to CL. For the in vivo experiments, CD1 mice in groups of 20 were injected with affinity purified aPL antibodies or with control IgG-NHS twice intraperitoneally. Seventy hours after the first injection, and 30 min before the surgical procedure (induction of experimental thrombus) mice were infused i.v. in each group with either peptide A or with peptide scA. The femoral vein of the anesthetized mice were dissected to examine the dynamics of an induced thrombus in treated and control mice. The mean aCL titer of mice injected with aPL was 60 GPL units. Mice treated with aPL and infused with peptide scA produced significantly larger thrombi when compared to mice treated with IgG-NHS and peptide scA (2466+/-462 microm2 vs 772.5+/-626.4 microm2). Treatment with peptide A significantly decreased thrombus size in mice injected with aPL antibodies (1063+/-890 microm2 compared to 2466+/-462 microm2). CONCLUSION: The data indicates that a synthetic peptide that shares similarity with common bacterial antigens and with regions of beta2GPI is capable to inhibit thrombogenic properties of aPL in mice. This may have important implications in designing new modalities of prevention and/or treatment of thrombosis in APS.     

Atypical hydrops in keratoconus

To report the clinical presentation, progress and management of atypical acute hydrops. A retrospective case study of three patients with keratoconus, two of whom had previously undergone penetrating keratoplasty. The patients underwent full ophthalmological examination and digital slit-lamp imaging of the cornea throughout the course of the condition. The two patients who had previously undergone keratoplasty had spontaneous hydrops primarily affecting the host bed but in one case extended to the graft inferiorly; however, in the third patient it was traumatic in origin. The Descemet’s tear affected the host rim in only one patient, which resolved spontaneously. In another patient, the hydrops was related to an internal dehiscence of the graft–host junction and had to be managed by an endothelial transplant covering the dehisced graft–host junction. In the third patient, hydrops secondary to trauma was also associated with acute haemops. Progression of keratoconus post keratoplasty can occur exclusively in the recipient bed leading to acute hydrops in the host sparing the transplanted cornea. The progressive thinning and ectasia of the recipient bed can also result in internal graft–host dehiscence leading to chronic oedema. Rapid entry of aqueous or blood cells into the corneal stroma following acute rupture of the Descemet’s membrane suggests that the abnormal stroma of the eye with keratoconus may have an important role to play in the pathogenesis of acute hydrops/haemops.     

The putative protective role of hepatitis B virus (HBV) infection from autoimmune disorders

BACKGROUND: The etiology of autoimmune diseases is not fully clarified and the mechanisms underlying their initiation and progression are still obscure. It is becoming clear that in a genetic susceptible individual an environmental trigger such as infectious agent in general and viruses in particular could initiate the development of an autoimmune disease. Hepatitis B virus (HBV) is notorious in its association with diverse autoimmune diseases. Therefore, we aimed to determine the presence of hepatitis B core antibody (HBcAb), a seromarker for past or present infection with HBV, in a large number of sera collected from patients with different autoimmune diseases. METHODS: A cohort of 675 sera samples of 5 different autoimmune diseases and healthy donors were screened for evidence of a prior infection with HBV. All samples were tested for hepatitis B core antibody (IgG) using the Monolisa anti-HBc PLUS commercial kit (Bio-Rad, Hercules, San Francisco, USA). RESULTS: Lower percentage of HBcAb was found in sera of the autoimmune diseases when compared to normal controls. Fifteen (10.7%) from 140 normal controls were found positive for the presence of HBcAb. Two (2%) out of 98 multiple sclerosis (MS) sera were positive for the presence of HBcAb (OR: 0.17, 95%CI: 0.03-0.77, p=0.01), 3 (2.5%) out of 117 systemic lupus erythematosus (SLE) sera (OR: 0.2, 95%CI: 0.06-0.77, p=0.01), 4 (4.5%) out of 89 type 1 diabetes (T1D), 5 (6.1%) from 82 Sjogren's syndrome (SS) sera and 12 (8%) from 149 rheumatoid arthritis (RA) sera were positive for the presence of HBcAb. CONCLUSIONS: Our data divulge an unexpected low percentage of antibodies to HBcAg in patients with SLE, MS and T1D in comparison to healthy matched donors. This finding may raise a protective role to HBV in some autoimmune diseases i.e. hygiene theory.     

Geographical Differences in Autoantibodies and Anti-infectious Agents Antibodies Among Healthy Adults

Much is known about the geoepidemiology of defined autoimmune diseases (AD); however, there is currently limited data regarding the prevalence of autoantibodies among healthy populations of different geographical areas. The aim of this study was to evaluate a large profile of autoantibodies in healthy adults from distinct global regions as well as the prevalence of anti-infectious agents antibodies in those regions. Sera samples from 557 healthy donors were obtained at six centers located in different countries (i.e., Italy, Netherlands, Israel, Mexico, Columbia, Papua New Guinea (Kitavans)). Sera were tested for the presence of antinuclear antibodies (ANA) and autoantibodies associated with thrombophilia, vasculitis, and gastrointestinal (GI) disease. Sera samples were also screened for antibodies against infectious agents (i.e., EBV, CMV, HBV, Helicobacter pylori, Treponema pallidum, and Toxoplasma gondii). Tests were performed using the BioPlex 2200 or ELISA kits (Bio-Rad Laboratories, USA). We found a significant gradient of ANA positivity among the groups: 45% of Columbians, 38% of Kitavans, 26% of Mexicans, 12% of Italians, 12% of Dutch, and 11% of Israelis were ANA positive. Geographical differences were also observed regarding the prevalence of specific autoantibodies, namely ANA: anti-dsDNA, chromatin, SmRNP, Ro/SSA, La/SSB, Scl70; GI associated: antigliadin; and thrombophilia-associated: anti-??2GP1 and prothrombin. Additionally, significant differences were observed regarding serological markers of all infectious agents screened. The observed variance between healthy ethno-geographical distinct populations in prevalence of autoantibodies may represent different genetic or environmental (e.g., prior exposure to infection) influences. Thus may illuminate possible causes of geoepidemiological differences in AD.