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.

Wound repair of the oral mucosa. Immunohistological and 3H-thymidine-autoradiographic observations.

The migratory and proliferative capacity of oral epithelium was studied after induction of subepithelial suction blisters in palatal rat mucosa. FITC-coupled lectins and their affinities to epithelial cells at the wound margin were analyzed by immunofluorescence microscopy. Immunocytochemical distribution of 67 K-keratin polypeptides in different epithelial layers were studied using specific antisera against keratin proteins. By means of 3H-thymidine-autoradiography the proliferation of epithelial basal cells and their migration at the wound edge was quantified by evaluation of the labelling index. Double labelling was performed, combining the autoradiographic and the keratin staining technique. It was found that the subepithelial suction blister showed rapid repair (epithelial regeneration after 3-4 days). Peanut Agglutinin (PNA) showed a selective affinity to the basal 2-3 layers of oral rat mucosa. No modifications of lectin affinities were found in epithelial wound repair. PNA-positive keratinocytes were demonstrated in the migratory epithelial tongue. The selective presence of 67 K-keratin (67 K) in the suprabasal epithelial cell layers was interpreted as an indication of the differentiation process in rat oral mucosa. The 3H-thymidine-labelling index of basal cells increased significantly, beginning at the wound edge after 24 h. The peak of the labelling index curve was located in the vicinity of the wound after 96 h. Double labelling technique revealed 67 K-negative and 3H-thymidine-positive basal cells as well as 67 K-positive and 3H-thymidine-negative cells at the migration front. These observations support findings that migrating epithelial cells in oral wound healing derive both from undifferentiated (str. basale) and from differentiated (inferior str. spinosum) cell compartments.