Identification of the flotillin-1/2 heterocomplex as a target of autoantibodies in bona fide multiple sclerosis.

BACKGROUND: Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). METHODS: The target antigen was identified by histo-immunoprecipitation using the patients' sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. RESULTS: Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. CONCLUSIONS: Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1-2% of patients with bona fide MS.

Prevalence of serum anti-neuronal autoantibodies in patients admitted to acute psychiatric care.

BACKGROUND: Autoimmune encephalitis associated with anti-neuronal antibodies may be challenging to distinguish from primary psychiatric disorders. The significance of anti-neuronal antibodies in psychiatric patients without clear evidence of autoimmune encephalitis is unknown. We investigated the serum prevalence of six anti-neuronal autoantibodies in a cohort of unselected patients admitted to acute psychiatric care. METHOD: Serum was drawn from 925 patients admitted to acute psychiatric in-patient care. Psychiatric diagnoses were set according to International Classification of Diseases (ICD)-10 criteria. Antibody analysis was performed with an indirect immunofluorescence test for N-methyl d-aspartate receptor (NMDAR) antibodies and five other anti-neuronal autoantibodies of the immunoglobulin (Ig) classes IgA, IgG and IgM isotype. RESULTS: Anti-neuronal autoantibodies were found in 11.6% of patients: NMDAR antibodies in 7.6%, contactin-associated protein-like 2 (CASPR2) antibodies in 2.5%, glutamic acid decarboxylase-65 (GAD65) antibodies in 1.9%, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antibodies in 0.1%. Leucine-rich glioma-inactivated protein-1 (LGI1) and γ-aminobutyric acid B (GABAB) receptor antibodies were not detected. NMDAR antibodies of class IgG were present in five patients only (0.5%). NMDAR antibodies of all Ig classes were equally prevalent in patients with and without psychosis. There were no significant differences in antibody prevalence in the different diagnostic categories, except for a higher odds ratio of being NMDAR antibody positive for patients without a specific psychiatric diagnosis. CONCLUSIONS: NMDAR IgG autoantibodies, which are known to be strongly associated with anti-NMDAR encephalitis, were rarely found. CASPR2 and GAD65 antibodies were more frequently encountered in the present study than previously reported. Further research on the clinical significance of anti-neuronal autoantibodies in patients with acute psychiatric symptoms is needed.

Neurological disorders in patients with bullous pemphigoid: clinical and experimental investigations.

BACKGROUND: Previous studies have shown that patients with bullous pemphigoid (BP) are more likely to have neurological diseases (ND). OBJECTIVES: To compare clinical findings in BP patients with and without ND and to investigate BP180 autoantibody binding in different neuronal tissues of mammalians. METHODS: Our database was searched for clinical findings of in-patients with the definitive diagnosis of BP. Moreover, brain tissue of mammalians was treated with serum of BP patients with elevated BP180 autoantibodies using biochip mosaics. RESULTS: Of 85/161 (52.8%) patients had a history of at least one ND (BP+ND). BP180 (P = 0.018), eosinophils (P = 0.043) and patients' accommodation in nursing homes (P < 0.0001) remained in the logistic regression model as significant independent predictors for the presence of ND in patients with BP. Subgroup analysis of community-dwelling BP patients revealed 25/93 (26.9%) patients with ND. In this population, the presence of ND also significantly correlated with BP180 (r = 0.26; P = 0.0003) and eosinophils (r = 0.19; P = 0.0087). In the animal model, no BP180-specific immunofluorescence could be detected. CONCLUSIONS: Our data support results of previous studies detecting significantly increased frequency of ND in BP patients. We have shown that raised BP180 titres and blood eosinophils are independent predictors for the presence of ND in BP patients. However, our experimental data do not support previous results indicating that specific binding of BP180 antibodies in neuronal tissue plays a pathogenetic role in ND.

Anti-neuronal anti-bodies in patients with early psychosis.

It may be challenging to distinguish autoimmune encephalitis associated with anti-neuronal autoantibodies from primary psychiatric disorders. Here, serum was drawn from patients with a first-episode psychosis (n=70) or a clinical high-risk for psychosis (n=6) and controls (n=34). We investigated the serum prevalence of 24 anti-neuronal autoantibodies: IgG antibodies for anti-N-methyl-d-aspartate-type glutamate receptor (anti-NMDAR), glutamate and γ-aminobutyric acid alpha and beta receptors (GABA-a, GABA-b), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), glycine receptor (GlyR), metabotropic glutamate receptor 1 and 5 (mGluR1, mGluR5), anti-Tr/Delta/notch-like epidermal growth factor-related receptor (DNER), contactin-associated protein-like 2 (CASPR2), myelin oligodendrocyte glycoprotein (MOG), glutamic acid decarboxylase-65 (GAD65), collapsin response mediator protein 5/crossveinless-2 (CV2), aquaporin-4 (AQP4), anti-dipeptidyl-peptidase-like protein-6 (DPPX), type 1 anti-neuronal nuclear antibody (ANNA-1, Hu), Ri, Yo, IgLON5, Ma2, zinc finger protein 4 (ZIC4), Rho GTPase-activating protein 26, amphiphysin, and recoverin, as well as IgA and IgM for dopamine-2-receptor (DRD2). Anti-NMDA IgG antibodies were positive with serum titer 1:320 in one patient with a clinical high risk for psychosis. He did not receive a diagnosis of encephalitis after comprehensive neurological evaluation. All other antineuronal autoantibodies were negative and there were no additional findings with immunohistochemistry of brain issues.

High prevalence and functional effects of serum antineuronal antibodies in patients with gastrointestinal disorders.

BACKGROUND: Antineuronal antibodies can be associated with both gastrointestinal (GI) and brain disorders. For example, antibodies against the potassium channel subunit dipeptidyl-peptidase-like protein-6 (DPPX) bind to neurons in the central nervous system (CNS) and myenteric plexus and cause encephalitis, commonly preceded by severe unspecific GI symptoms. We therefore investigated the prevalence of antineuronal antibodies indicative of treatable autoimmune CNS etiologies in GI patients. METHODS: Serum samples of 107 patients (Crohn's disease n = 42, ulcerative colitis n = 16, irritable bowel syndrome n = 13, others n = 36) and 44 healthy controls were screened for anti-DPPX and further antineuronal antibodies using immunofluorescence on rat brain and intestine and cell-based assays. Functional effects of high-titer reactive sera were assessed in organ bath and Ussing chamber experiments and compared to non-reactive patient sera. KEY RESULTS: Twenty-one of 107 patients (19.6%) had antibodies against the enteric nervous system, and 22 (20.6%) had anti-CNS antibodies, thus significantly exceeding frequencies in healthy controls (4.5% each). Screening on cell-based assays excluded established antienteric antibodies. Antibody-positive sera were not associated with motility effects in organ bath experiments. However, they induced significant, tetrodotoxin (TTX)-insensitive secretion in Ussing chambers compared to antibody-negative sera. CONCLUSIONS & INFERENCES: Antineuronal antibodies were significantly more frequent in GI patients and associated with functional effects on bowel secretion. Future studies will determine whether such antibodies indicate patients who might benefit from additional antibody-directed therapies. However, well-characterized encephalitis-related autoantibodies such as against DPPX were not detected, underlining their rarity in routine cohorts.