No abstract

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1 −/− ) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1 −/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex-that is, instead of a single ubiquitous channel, VRACs could be formed by cell type-or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.bestrophin 1 | volume-regulated anion channel | induced pluripotent stem cell | retinal pigment epithelium | mouse sperm

Key Points• A new technology is presented to assess apparent affinities of FVIII-specific antibodies, differentiated for isotypes and IgG subclasses.• Affinities of FVIII-specific antibodies in patients with FVIII inhibitors are up to 100-fold higher than in patients without inhibitors.Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIIIspecific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses. (Blood. 2015;125(7):1180-1188

Preexisting immunity against adeno-associated virus (AAV) is a major challenge facing AAV gene therapy, resulting in the exclusion of patients from clinical trials. Accordingly, proper assessment of anti-AAV immunity is necessary for understanding clinical data and for product development. Previous studies on anti-AAV prevalence lack method standardization, rendering the assessment of prevalence difficult. Addressing this need, we used clinical assays that were validated according to guidelines for a comprehensive characterization of anti-AAV1,-AAV2,-AAV5, and-AAV8 immunity in large international cohorts of healthy donors and patients with hemophilia B. Here, we report a higher than expected average prevalence for anti-AAV8 ($40%) and anti-AAV5 ($30%) neutralizing antibodies (NAbs), which is supported by strongly correlating anti-AAV IgG antibody titers. A similar anti-AAV8 NAb prevalence was observed in hemophilia B patients. In addition, a high co-prevalence of NAbs against other serotypes makes switching to gene therapy using another serotype difficult. As anti-AAV T cell responses are believed to influence transduction, we characterized anti-AAV T cell responses using interleukin-2 (IL-2) and interferon-g (IFN-g) ELISpot assays, revealing a similar prevalence of IFN-g responses ($20%) against different serotypes that did not correlate with NAbs. These data, along with the long-term stability of NAbs, emphasize the need to develop strategies to circumvent anti-AAV immunity.

We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130 -149) and TMD4 (aa 179 -201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.Bestrophin-1 is encoded by the vitelliform macular dystrophy type 2 (VMD2) 2 gene as a 585-amino acid putative integral transmembrane protein localized to the basolateral aspect of the retinal pigment epithelium (RPE) (1). Disease-related mutations in the gene are responsible for the autosomal dominant Best macular dystrophy (BMD) (2, 3) but have also been associated with a minor proportion of cases presenting with adult vitelliforme macular dystrophy (4), bulls eye maculopathy (5), and autosomal dominant vitreoretinochoroidopathy (6). Thus far, a total of 100 distinct mutations have been identified including 92 in BMD, four in adult vitelliforme macular dystrophy, one in bulls eye maculopathy, and three in autosomal dominant vitreoretinochoroidopathy patients (for further information see BMD mutation data base). Of these, more than 93% are missense mutations, distributed in a non-random fashion across the highly conserved N-terminal half of the protein.Clinically, BMD is characterized by striking yellowish lesions in the macular area eventually leading to a decline in central vision at later stages in disease pathology. The disease manifests in teenage years although with reduced penetrance and considerable variability in phenotypic expression. Histopathologically, aberrant remnants of heterogeneous material including proteins, lipids, and lipofuscin-containing particles are found in the central retina at the level of the RPE. Typically, BMD patients display a characteristic electrooculogram (EOG) abnormality with a greatly reduced light peak/dark trough ratio reflecting RPE dysfunction (7).Accumulating experimental evidence suggests bestrophin-1, but also its highly conserved family members bestrophin-2, bestrophin-3 and bestrophin-4 (8, 9), to be involved in Ca 2ϩ -dependent transport of chloride ions across cellular membranes. It is still unclear whether bes...

The findings imply that microglia/glia activation may be triggering events in the photoreceptor degeneration of retinoschisin-deficient mice. Furthermore, the data point to a role of Erk1/2-Egr1 pathway activation in RS pathogenesis.

Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.