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Anti-aquaporin 4 antibodies and water permeability

Background: NMO-IgG autoantibody is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggest that NMO-IgG may play a central role in NMO physiopathology. Objective: The aim of this in vitro-based study was to characterize molecular and functional consequences of interaction between NMO-IgG and primary cultures of astrocytes. Methods: Three NMO-IgG positive NMO patients from Neuroimmunology of the Ramos Mejia Hospital, Argentina were selected. As control, we included serum from 3 NMO-IgG negative healthy controls. Primary cultures of rat astrocytes were incubated with heat-inactivated control or NMO patients’ sera (dilution 1/50), for 1 h at 4?C or 12 h at 37?C and then immunofluorescence studies as well as water permeability measurements by fluorescence videomicroscopy were performed. Fluorescence was quantified by densitometric analysis using Image-J software. Results: Immunofluorescence studies showed that cells exposition to NMO patients? sera for 1 h at 4?C (to restrict membrane fluidity) resulted in IgG bind to the plasma membrane in a linear pattern that colocalized with AQP4, while serum IgG from controls did not bind significantly. In contrast, after exposure for 12 h at 37?C, NMO patient’s sera containing AQP4 specific IgG resulted in disappearance of AQP4 fluorescence from the plasma membrane while control sera had no effect on AQP4 expression. Even more, 1 h exposition of astrocytes to NMO-IgG does not change the water permeability, indicating that the water pore is not affected by the binding of IgG, but 12 h of exposition to NMO-IgG clearly induced a significant reduction of the water permeability (time constant of water transport, Tau (s), control vs. NMO-IgG sera 18.04 ?1,04 vs. 34.96 ? 4.45, p < 0.01). Conclusions: NMO patient’s sera IgG has a selective pathogenic effect on cells membranes expressing AQP4. We demonstrated that NMO-IgG binding to astrocytes alters AQP4 expression and decreases water permeability.

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