Cellular vacuolization caused by overexpression of the PIKfyve-binding deficient Vac14L156R is rescued by starvation and inhibition of vacuolar-ATPase.

Phosphoinositides (PI) and converting enzymes are crucial determinants of organelle identity and morphology. One important endolysosomal specific PI is PI(3,5)P2, generated by the PIKfyve kinase, which orchestrates in combination with Vac14 and Fig4. Dysfunction of this complex leads to large intracellular vacuoles in various cell types and is linked to neurological diseases. Here, we characterize the vacuolization phenotype caused by overexpression of the PIKfyve binding deficient mutant Vac14L156R in podocytes, which represent specialized cells of the kidney. Vacuolization of podocytes, which was associated with strong maturation defects in the endolysosomal system, could be completely rescued by starvation or treatment of cells with the v-ATPase inhibitor Bafilomycin A1. Moreover, we elucidated a strong and reversible de-vacuolization effect of the cholesterol export inhibitor U18666A, which was accompanied by increased basification of the lysosomal pH values. Taken together, our data give new hints to potential therapeutic targets in the treatment of disease linked to intracellular vacuolization.

The anti-obesity drug orlistat reveals anti-viral activity.

The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.

Serodiagnosis of Anti-glomerular Basement Membrane Disease Using a Newly Developed Chemiluminescence Immunoassay.

Circulating autoantibodies directed against the kidney glomerular basement membrane (GBM) antigens are important markers in the diagnosis and monitoring of autoimmune glomerulonephritides, including the classic Goodpasture's syndrome. Rapid and reliable diagnostic tools for the detection of anti-GBM autoantibodies are crucial as anti-GBM disease can progress rapidly and, if too late or incorrectly diagnosed, can have serious, even fatal consequences. The performance of the newly developed standardized chemiluminescence immunoassay (ChLIA) was evaluated in comparison with the established Anti-GBM ELISA (IgG) (EUROIMMUN). For the assessment of its diagnostic performance, sera from 67 clinically characterized anti-GBM disease patients and 221 disease controls were analyzed. The clinical sensitivity of the Anti-GBM ChLIA (IgG) reached 100% at a specificity of 98.6%. The Anti-GBM ELISA (IgG) performance was less sensitive (89.6%) without any positive findings in the control group, indicating a specificity of 100%. Both methods were homogeneous (κ = 0.901). The Anti-GBM ChLIA (IgG) represents a promising alternative tool for accurate anti-GBM assessment in routine diagnostic settings with the advantage of rapid turnaround time and fully automated random-access processing.

A New Chemiluminescence Immunoassay for Phospholipase A2 Receptor 1 Autoantibodies Allows Early Identification of Autoantibody Recurrence in Patients With Membranous Nephropathy.

BACKGROUND: Circulating autoantibodies against the M-type phospholipase A2 receptor 1 (PLA2R1) are important biomarkers in membranous nephropathy (MN), supporting the diagnosis and the clinical monitoring of patients. Standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) and enzyme-linked immunosorbent assay (ELISA) are widely established for the detection of anti-PLA2R1 autoantibodies (PLA2R1-ab). The RC-IFA provides higher sensitivity than the ELISA, but lacks exact graduated quantification of antibody levels. In this study, we evaluated the diagnostic performance of a novel PLA2R1-ab immunoassay based on chemiluminescence (ChLIA) by comparing it to RC-IFA and ELISA in samples from patients with MN with different diagnostic scenarios. METHODS: Serum samples from patients with biopsy-proven MN and disease controls were analyzed for PLA2R1-ab by ChLIA, ELISA, and RC-IFA. RESULTS: The ChLIA demonstrated almost perfect agreement with RC-IFA for the identification of patients with PLA2R1-associated MN, while additionally allowing fine-graduated quantification of PLA2R1-ab levels. In patients with a relapse of MN, the ChLIA allowed an earlier detection of PLA2R1-ab recurrence by at least 3 months in 63% of cases compared with the ELISA. CONCLUSIONS: The PLA2R1-ab ChLIA had the same excellent diagnostic performance as the RC-IFA and outperformed the ELISA in the diagnosis of MN and the early identification of relapses. It thus presents a favorable tool for accurate PLA2R1-ab assessment in routine diagnostic settings, while enabling fast processing and fully automated random-access implementation.

The clinically licensed antifungal drug itraconazole inhibits influenza virus in vitro and in vivo.

Influenza A virus (IAV) is a common pathogen of respiratory disease. The IAV-induced seasonal epidemics and the sporadic pandemics are associated with high morbidity and mortality. Therefore, effective protection and therapy for IAV infections is an important challenge in countering this public health threat. Because vaccinations only protect against known circulating strains, and the currently available antivirals pose the risk of resistance formation, drugs targeting host cell factors needed for viral replication offer a promising therapeutic approach. In this study, we describe the use of the antifungal therapeutics posaconazole and itraconazole in the therapy of IAV. We show that both drugs efficiently inhibit the propagation of IAV in the cell culture model without being cytotoxic. The mode of action is probably based on several targets and includes both a priming of the interferon response and the induced imbalance of cellular cholesterol. The antiviral effect of itraconazole could be confirmed in the mouse model, where the administration of itraconazole led to a drastic reduction in mortality and a significant increase in the survival rate. Thus, our data indicate a promising therapeutic potential of at least itraconazole in influenza therapy.

Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus.

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.IMPORTANCE With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L per se as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.