BackgroundJapanese encephalitis virus (JEV) is a neurotropic flavivirus causing mortality and morbidity in humans. Severe Japanese encephalitis cases display strong inflammatory responses in the central nervous system and an accumulation of viral particles in specific brain regions. Microglia cells are the unique brain-resident immune cell population with potent migratory functions and have been proposed to act as a viral reservoir for JEV. Animal models suggest that the targeting of microglia by JEV is partially responsible for inflammatory reactions in the brain. Nevertheless, the interactions between human microglia and JEV are poorly documented.MethodsUsing human primary microglia and a new model of human blood monocyte-derived microglia, the present study explores the interaction between human microglia and JEV as well as the role of these cells in viral transmission to susceptible cells. To achieve this work, vaccine-containing inactivated JEV and two live JEV strains were applied on human microglia.ResultsLive JEV was non-cytopathogenic to human microglia but increased levels of CCL2, CXCL9 and CXCL10 in such cultures. Furthermore, human microglia up-regulated the expression of the fraktalkine receptor CX3CR1 upon exposure to both JEV vaccine and live JEV. Although JEV vaccine enhanced MHC class II on all microglia, live JEV enhanced MHC class II mainly on CX3CR1+ microglia cells. Importantly, human microglia supported JEV replication, but infectivity was only transmitted to neighbouring cells in a contact-dependent manner.ConclusionOur findings suggest that human microglia may be a source of neuronal infection and sustain JEV brain pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0675-3) contains supplementary material, which is available to authorized users.

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of gd T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that gd T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-g. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme-and granulysin-mediated innate immune mechanism exerted by gd T cells to kill late-stage blood-residing P. falciparum.

In many kidney diseases, the original insult primarily involves the glomerulus and may then pass onto the tubulointerstitium. Several hypotheses link glomerular disease to tubular injury; perhaps the foremost hypothesis involves chronic tubular hypoxia. The reported effects of hypoxia and consecutive stabilization of hypoxia-inducible factors (HIFs), however, are controversial. Hypoxia induces interstitial fibrosis but also has beneficial effects on renal disease progression when HIF is activated pharmacologically. To analyze the impact of HIF on tubulointerstitial disease development in primary glomerular disease, transgenic von Hippel Lindau (VHL)-knockout mice were generated and null expression was induced before the onset of autoimmune IgG-mediated anti-glomerular basement membrane glomerulonephritis (GN). Tubular VHL knockout and, thus, local HIF-α stabilization increased renal production of vascular endothelial growth factor, tumor growth factor-β(1), and platelet-derived growth factor-B, resulting in augmented formation of capillaries and interstitial matrix, and conversion of fibroblasts to myofibroblasts. Within the glomerular disease, VHL knockout reduced the glomerular damage and attenuated tubulointerstitial injury. Likewise, proteinuria, plasma urea concentration, and tubulointerstitial matrix were decreased in VHL knockout with GN. These findings shown that tubular HIF-α stabilization in glomerular disease is beneficial for disease outcome. In comparison with VHL knockout alone, GN is a much stronger activator of fibrosis such that stimuli other than hypoxia may be considered important for renal disease progression.

Malaria affects the poorer regions of the world and is of tremendous health and economic burden for developing countries. Extracellular vesicles (EVs) are small vesicles released by almost any cells in the human body, including malaria infected red blood cells. Recent evidence shows that EVs might contribute to the pathogenesis of malaria. In addition, EVs hold considerable value in biomarker discovery. However, there are still significant gaps in our understanding of EV biology. So far most of our knowledge about EVs in malaria comes from in vitro work. More field studies are required to gain insight into their contribution to the disease and pathogenesis under physiological conditions. However, to perform research on EVs in low-income regions might be challenging due to the lack of appropriate equipment to isolate EVs. Therefore, there is a need to develop and validate EV extraction protocols applicable to poorly equipped laboratories. We established and validated two protocols for EV isolation from cell culture supernatants, rodent and human plasma. We compared polyethylene glycol (PEG) and salting out (SA) with sodium acetate for precipitation of EVs. We then characterized the EVs by Transmission Electron Microscopy (TEM), Western Blot, Size-exclusion chromatography (SEC), bead-based flow cytometry and protein quantification. Both protocols resulted in efficient purification of EVs without the need of expensive material or ultracentrifugation. Furthermore, the procedure is easily scalable to work with large and small sample volumes. Here, we propose that both of our approaches can be used in resource limited countries, therefore further helping to close the gap in knowledge of EVs during malaria.

Receptor‐mediated endocytosis is a pivotal function of the renal proximal tubule (PT) to reabsorb proteins from the ultra filtrate. PT dysfunction occurring in nephrotic syndrome results in an impairment of this process. The regulation of endocytosis and signaling pathways involved remain unknown. We aimed to identify whether low (0.1mg/ml) vs. high albumin concentration (10mg/ml) affect endocytosis and to uncover responsible signaling pathways. OK‐cells incubated with 0.1mg/ml albumin significantly augmented receptor‐mediated endocytosis at 1h and 4h. In contrary, incubating OKC with 10mg/ml albumin significantly diminished endocytosis after 30 min till 4h. Analyses of the expression pattern and phosphorylation status of the kinases including PI3K, Akt, TORC1/‐2, WNK1/OSR1 revealed that the WNK1‐induced pathway shows strongest changes upon low vs. high albumin concentrations. Transient transfection of OKC using WNK1, KD‐K233M‐WNK1, OSR1 and KD‐D164A‐OSR1 strongly inhibited endocytosis to 2.8±1.1*; 6.7±1.0*; 2.8±0.5*; and 7.4±2.9*; *P<0.05, respectively, suggesting a regulation by protein‐protein interaction. In rat kidney, IHC of WNK1 and OSR1 demonstrated a strong expression in the BBM of PT, supporting a role in endocytosis. We conclude that WNK1 and OSR1 are the main kinases activated upon high protein concentrations leading to consequent impairment of receptor‐mediated endocytosis.