Autoantibodies Against the Complement Regulator Factor H in the Serum of Patients With Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by pathogenic, complement-activating autoantibodies against the main water channel in the CNS, aquaporin 4 (AQP4). NMOSD is frequently associated with additional autoantibodies and antibody-mediated diseases. Because the alternative pathway amplifies complement activation, our aim was to evaluate the presence of autoantibodies against the alternative pathway C3 convertase, its components C3b and factor B, and the complement regulator factor H (FH) in NMOSD. Four out of 45 AQP4-seropositive NMOSD patients (~9%) had FH autoantibodies in serum and none had antibodies to C3b, factor B and C3bBb. The FH autoantibody titers were low in three and high in one of the patients, and the avidity indexes were low. FH-IgG complexes were detected in the purified IgG fractions by Western blot. The autoantibodies bound to FH domains 19-20, and also recognized the homologous FH-related protein 1 (FHR-1), similar to FH autoantibodies associated with atypical hemolytic uremic syndrome (aHUS). However, in contrast to the majority of autoantibody-positive aHUS patients, these four NMOSD patients did not lack FHR-1. Analysis of autoantibody binding to FH19-20 mutants and linear synthetic peptides of the C-terminal FH and FHR-1 domains, as well as reduced FH, revealed differences in the exact binding sites of the autoantibodies. Importantly, all four autoantibodies inhibited C3b binding to FH. In conclusion, our results demonstrate that FH autoantibodies are not uncommon in NMOSD and suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage in NMOSD.

The Role of GnRH Receptor Autoantibodies in Polycystic Ovary Syndrome

Objective Is polycystic ovary syndrome (PCOS) associated with activating autoantibodies (AAb) to the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR)? Design and Methods We retrospectively screened sera from 40 patients with PCOS and 14 normal controls (NCs) with regular menses using enzyme-linked immunosorbent assay (ELISA) for the presence of GnRHR-ECL2-AAb. We obtained similar data from 40 non-PCOS ovulatory but infertile patients as a control group (OIC) of interest. We analyzed GnRHR-ECL2-AAb activity in purified immunoglobulin (Ig)G using a cell-based GnRHR bioassay. Results The mean ELISA value in the PCOS group was markedly higher than the NC (P = .000036) and the OIC (P = .0028) groups. IgG from a sample of 5 PCOS subjects, in contrast to a sample of 5 OIC subjects, demonstrated a dose-dependent increase in GnRHR-stimulating activity qualitatively similar to the acute action of the natural ligand GnRH and the synthetic agonist leuprolide. The GnRHR antagonist cetrorelix significantly suppressed (P < .01) the elevated GnRHR activity induced by IgG from 7 PCOS patients while the IgG activity level from 7 OIC subjects was unchanged. Five other OIC subjects had relatively high ELISA values at or above the 95% confidence limits. On further study, 3 had normal or low activity while 2 had elevated IgG-induced GnRHR activity. One suppressed with cetrorelix while the other did not. The copresence of PCOS IgG increased the responsiveness to GnRH and shifted the dosage response curve to the left (P < .01). Conclusions GnRHR-ECL2-AAb are significantly elevated in patients with PCOS compared with NCs. Their presence raises important etiological, diagnostic, and therapeutic implications.

SUN-LB5 GnRHR ECL-2 Epitopes Targeted by Activating Autoantibodies in Polycystic Ovary Syndrome

Background: Activating autoantibodies (AAb) are directed to the gonadotropin releasing hormone receptor second extracellular loop (GnRHR-ECL2) and are pathogenic when induced in rats. We previously reported GnRHR-ECL2-AAb were elevated in sera from patients with PCOS (Rotterdam criteria) compared to ovulatory infertile controls (OIC). Methods:Human studies: ELISA detection of GnRHR Abs used a synthetic h-GnRHR-ECL2 28 mer peptide (LifeTein) as the target antigen. We assayed AAb activity in GnRHR transfected cells using a GeneBLAzer FRET assay (Invitrogen). ELISA AAb epitope locations on the ECL2 were identified on a minipin plate (pins 1-11 containing sequential 2 aa offsetting octapeptides, Mimotope, Inc) using sera from 30 PCOS subjects, 33 OIC and 18 normal controls (NC). Results:Human sera: An ELISA assay for GnRHR-ECL2-AAbs in the PCOS group was markedly higher than the NC group (P<0.0001) and the OIC subjects (P<0.003). The minipin data demonstrated one or more positive OD peaks on pins 4 (20%), 5 (47%) and 8 (47%) which shared L-aa sequences FSQC or CSFSQ. OIC had only 5 subjects with peaks at minipins 4 or 5 and NC had only 3 lower peaks and one with higher OD values over all minipins. GnRHR-AAb Specific Activity (SA) was estimated by measuring serum activity before and after suppression of AAb sensitive activity by addition of retro inverso D-aa (RID) peptides. These were specifically designed to mimic and decoy the AAb L-aa epitope sequences of pins 5 and 8). SA was measured in 10 selected PCOS and 10 OIC subjects who had positive ELISA values. The baseline activity in the PCOS group was significantly higher than OIC (P < 0.01) and dropped 50% with preincubation with peak 5 RID and 25% with peak 8 RID. The addition of both peak 5 and peak 8 RID suppressed the PCOS group activity to OIC levels (P > 0.2). There was no significant change in activity in the OIC subjects by the addition of peaks 5 + 8 RID peptides. Conclusion: These PCOS GnRHR-AAb data confirm the presence of significant activation of the GnRHR by AAb targeted to specific epitope(s) proximate to the disulfide GnRHR-ECL2 linkage to the nearby ECL1. These data are compatible with a pathophysiological role for GnRHR-AAb in PCOS and may provide both diagnostic and therapeutic opportunities.

β1-Adrenergic and M2 Muscarinic Autoantibodies and Thyroid Hormone Facilitate Induction of Atrial Fibrillation in Male Rabbits

Activating autoantibodies to the β1-adrenergic and M2 muscarinic receptors are present in a very high percentage of patients with Graves' disease and atrial fibrillation (AF). The objective of this study was to develop a reproducible animal model and thereby to examine the impact of these endocrine-like autoantibodies alone and with thyroid hormone on induction of thyroid-associated atrial tachyarrhythmias. Five New Zealand white rabbits were coimmunized with peptides from the second extracellular loops of the β1-adrenergic and M2 muscarinic receptors to produce both sympathomimetic and parasympathomimetic antibodies. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization and subsequent treatment with thyroid hormone. Antibody expression facilitated the induction of sustained sinus, junctional and atrial tachycardias, but not AF. Addition of excessive thyroid hormone resulted in induced sustained AF in all animals. AF induction was blocked acutely by the neutralization of these antibodies with immunogenic peptides despite continued hyperthyroidism. The measured atrial effective refractory period as one parameter of AF propensity shortened significantly after immunization and was acutely reversed by peptide neutralization. No further decrease in the effective refractory period was observed after the addition of thyroid hormone, suggesting other cardiac effects of thyroid hormone may contribute to its role in AF induction. This study demonstrates autonomic autoantibodies and thyroid hormone potentiate the vulnerability of the heart to AF, which can be reversed by decoy peptide therapy. These data help fulfill Witebsky's postulates for an increased autoimmune/endocrine basis for Graves' hyperthyroidism and AF.