Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.Peer reviewe

Lipid microdomain-dependent macropinocytosis determines compartmentation of Afipia felis.

Phagocytic compartments are specialized endocytic organelles and usually mature along the degradative pathway into phagolysosomes. The rare human pathogen Afipia felis localizes to a compartment that is different from canonical phagocytic compartments. Here, we present evidence that internalization of Afipia by macrophages and unusual phagosome development are considerably decreased by attachment of cholera toxin B subunit to macrophage ganglioside GM1 or by extraction or oxidation of plasma membrane cholesterol. Amiloride (an inhibitor of Na(+)/H(+) exchanger and macropinocytosis) strongly inhibited uptake of A. felis at a late step, i.e. the closure of macropinocytic structures rather than the production of membrane ruffles. Ultrastructural evidence showed that A. felis was taken up by macrophages via macropinocytosis. In contrast, A. felis opsonized with a monoclonal IgG antibody was ingested by a zipper-like mechanism, resulting in normal phagosome maturation. Hence, while the preferred path of A. felis uptake is dependent on the integrity of lipid microdomains and on macropinocytosis, and while this uptake leads to an unusual phagosome and to intracellular survival of A. felis, those bacteria that enter using Fcgamma receptors are delivered to a late endocytic compartment

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8(+) T-cell responses

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8(+) T-cell responses against transgene expressing cells, we created WAP-T-NP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-T-NP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/ NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-T-NP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-T-NP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-T-NP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-T-NP mice. LCMV infection of WAP-T-NP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-T-NP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these 'endogenous' NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-T-NP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-T-NP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients

The N-Terminal Domain of NLRC5 Confers Transcriptional Activity for MHC Class I and II Gene Expression

Ag presentation to CD4(+) and CD8(+) T cells depends on MHC class II and MHC class I molecules, respectively. One important regulatory factor of this process is the transcriptional regulation of MHC gene expression. It is well established that MHC class II transcription relies on the NLR protein CIITA. Recently, another NLR protein, NLRC5, was shown to drive MHC class I expression. The molecular mechanisms of the function of NLRC5 however remain largely elusive. In this study, we present a detailed functional study of the domains of NLRC5 revealing that the N-terminal domain of human NLRC5 has intrinsic transcriptional activity. Domain swapping experiments between NLRC5 and CIITA showed that this domain contributes to MHC class I and MHC class II gene expression with a bias for activation of MHC class I promoters. Delivery of this construct by adeno-associated viral vectors upregulated MHC class I and MHC class II expression in human cells and enhanced lysis of melanoma cells by CD8(+) cytotoxic T cells in vitro. Taken together, this work provides novel insight into the function of NLRC5 and CIITA in MHC gene regulation

Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function

Mutations in the acid sphingomyelinase (aSMase) coding gene sphingomyelin phosphodiesterase 1 (SMPD1) cause Niemann-Pick disease (NPD) type A and B. Sphingomyelin storage in cells of the mononuclear phagocyte system cause hepatosplenomegaly and severe neurodegeneration in the brain of NPD patients. However, the effects of aSMase deficiency on retinal structure and microglial behavior have not been addressed in detail yet. Here, we demonstrate that retinas of aSMase(-/-) mice did not display overt neuronal degeneration but showed significantly reduced scotopic and photopic responses in electroretinography. In vivo fundus imaging of aSMase(-/-) mice showed many hyperreflective spots and staining for the retinal microglia marker Iba1 revealed massive proliferation of retinal microglia that had significantly enlarged somata. Nile red staining detected prominent phospholipid inclusions in microglia and lipid analysis showed significantly increased sphingomyelin levels in retinas of aSMase(-/-) mice. In conclusion, the aSMase-deficient mouse is the first example in which microglial lipid inclusions are directly related to a loss of retinal function. (C) 2015 Elsevier Inc. All rights reserved

Emergence of AnnexinV(pos) CD31(neg) CD42b(low/neg) extracellular vesicles in plasma of humans at extreme altitude

Background Hypobaric hypoxia has been reported to cause endothelial cell and platelet dysfunction implicated in the formation of microvascular lesions, and in its extremes may contribute to vascular leakage in high altitude pulmonary edema or blood brain barrier disruption leading to cerebral micro-hemorrhage (MH). Platelet function in the development of microvascular lesions remained ill defined, and is still incompletely understood. In this study platelet-and endothelial cell-derived extracellular vesicles (PEV and EEV, respectively) and cell adhesion molecules were characterized in plasma samples of members of a high altitude expedition to delineate the contribution of platelets and endothelial cells to hypobaric hypoxiainduced vascular dysfunction. Methods and findings In this observational study, platelet and endothelial cell-derived extracellular vesicles were analysed by flow-cytometry in plasma samples from 39 mountaineers participating in a medical research climbing expedition to Himlung Himal, Nepal, 7,050m asl. Megakaryocyte/platelet-derived AnnexinV(pos), PECAM-1 (CD31) and glycoprotein-1b (GP1b, CD42b) positive extracellular vesicles (PEV) constituted the predominant fraction of EV in plasma samples up to 6,050m asl. Exposure to an altitude of 7,050m led to a marked decline of CD31(pos) CD42(neg) EEV as well as of CD31(pos) CD42b(pos) PEV at the same time giving rise to a quantitatively prevailing CD31(neg) CD42b(low/neg) subpopulation of AnnexinV(pos) EV. An almost hundredfold increase in the numbers of this previously unrecognized population of CD31 neg CD42b(low/neg) EV was observed in all participants reaching 7,050m asl. Conclusions The emergence of CD31(neg) CD42b(low/neg) EV was observed in all participants and thus represents an early hypoxic marker at extreme altitude. Since CD31 and CD42b are required for platelet-endothelial cell interactions, these hypobaric hypoxia-dependent quantitative and phenotypic changes of AnnexinV(pos) EV subpopulations may serve as early and sensitive indicators of compromised vascular homeostasis

Toll-Like Receptor 2, Toll-Like Receptor 4, Myeloid Differentiation Response Gene 88, and Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing Interferon-gamma (TRIF) Selectively Regulate Susceptibility of PO106-125-Induced Murine Experimental Autoimmune Neuritis

The functional relevance of the innate immune system has not yet been dissected in PO106-125-induced murine experimental autoimmune neuritis. Therefore, the role of Toll-like receptor (TLR) 2, TLR4, myeloid differentiation response gene 88, and ToLL IL-1 receptor domain-containing adaptor-inducing interferon-i (TRIF), factors critically involved in the TLR signaling pathway, was studied in experimental autoimmune neuritis. In the absence of TLR2, TLR4, myeloid differentiation response gene 88, or TRIF, the clinical course was significantly attenuated compared to wild-type mice. This could be attributed to impaired NE-kappa B activation, as shown by the absence of nuclear translocation of ReIA with a decreased expression of IL-6, IL 12p40, and IL-17A. Remarkably, PO106-125-immunized TLR2(0/0) mice exhibited a delayed recovery as compared to TLR4(0/0) mice, which was because of an impaired T helper cell 2 polarization. Immunized TLR2(0/0) mice' were unable to induce OX40 and OX4OL by matrix metalloproteinase-2 on spLenic dendritic cells. Subsequently, M2 polarization was impaired and macrophages were unable to sufficiently induce T regulatory cells (T-regs). Thus, in the recovery phase, Tregs were significantly increased in TLR4(0/0) mice as compared to wild-type mice, whereas T-regs in immunized TLR2(0/0) mice were only slightly increased. Our data highlight the relevance of innate immunity and, especially, the tight interaction between the innate and the adaptive immune system, which should be considered for therapeutic approaches of autoimmune diseases

Dual role of B cells with accelerated onset but reduced disease activity in P0(106-125)-induced experimental autoimmune neuritis of IgH(0/0) mice

The role of B cells in autoimmune-mediated diseases of the peripheral nervous system was studied in experimental autoimmune neuritis (EAN) in B cell deficient IgH(0/0) C57BL/6J mice having been immunized with P0(106-125) peptide. Compared to coisogenic IgH(+/+) mice, onset of EAN was accelerated [100% disease incidence at day 9 post immunization (p.i.) vs. day 15 p.i.]. At day 9 p.i., numbers of P0(106-125)-specific interferon (IFN)-gamma-producing CD4(+) T cells were increased, while IL-10 mRNA and production were decreased in IgH(0/0) mice. Beyond day 9 p.i., declining disease activity and a significant reduction of maximal disease activity were correlated with significantly reduced numbers of IFN-gamma-producing CD4(+) T cells in IgH(0/0) mice as compared with IgH(+/+) mice. Correspondingly, neuropathology demonstrated only mild axonal damage, while demyelination and dying back axonopathy with spinal cord motor neuron apoptosis were absent. Thus, depending on the stage of EAN, B cells play a dual, i.e. suppressive and enhancing, role during induction and at height of EAN, respectively. The combined interaction of B cells as well as CD4(+) and CD8(+) T cells is required for the development of EAN