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Repetitive mild traumatic brain injuries (mTBI) disrupt central nervous system (CNS) barriers, the erosion of which has been linked to long-term neurodegenerative and psychiatric conditions. Although much attention has been devoted to CNS vasculature following mTBI, little is known about the glia limitans superficialis - a barrier of surface-associated astrocytes that helps protect the CNS parenchyma and maintain homeostasis. Here, we identify the glia limitans superficialis as a crucial barrier surface whose breakdown after acute repeat mTBI facilitates increased cell death and recruitment of peripheral myelomonocytic cells. Using intravital microscopy, we show that brain resident microglia fortify this structure after a single mTBI yet fail to do so following secondary injury, which triggers massive recruitment of myelomonocytic cells from the periphery that contribute to further destruction of the glia limitans superficialis but not cortical cell death. We demonstrate instead that reactive oxygen species (ROS) generated in response to repeat head injury are largely responsible for enhanced cortical cell death, and therapeutic administration of the antioxidant, glutathione, markedly reduces this cell death, preserves the glia limitans, and prevents myelomonocytic cells from entering the brain parenchyma. Collectively, our findings underscore the importance of preserving the glia limitans superficialis after brain injury and offer a therapeutic means to protect this structure and the underlying cortex.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome., We discovered vascular malformations due to failure of developmental vascular regression in patients with EPAS1 gain-of-function mutation syndrome and the corresponding transgenic mouse model.

We document here that infection of prediabetic mice with a virus expressing an H-2Kb-restricted mimic ligand to a self epitope present on beta cells accelerates the development of autoimmune diabetes. Immunization with the mimic ligand expanded autoreactive T cell populations, which was followed by their trafficking to the islets, as visualized in situ by tetramer staining. In contrast, the mimic ligand did not generate sufficient autoreactive T cells in naive mice to initiate disease. Diabetes acceleration did not occur in H-2Kb-deficient mice or in mice tolerized to the mimic ligand. Thus, arenavirus-expressed mimics of self antigens accelerate a previously established autoimmune process. Sequential heterologous viral infections might therefore act in concert to precipitate clinical autoimmune disease, even if single exposure to a viral mimic does not always cause sufficient tissue destruction.

DCs play a pivotal role in bringing forth innate and adaptive immune responses. Viruses can specifically target DCs, rendering them ineffective in stimulating T cells, which can ultimately lead to immunosuppression. In the present study we have identified several potential mechanisms by which lymphocytic choriomeningitis virus (LCMV) induces immunosuppression in its natural murine host. The immunosuppressive LCMV variant clone 13 (Cl 13) infects DCs and interferes with their maturation and antigen-presenting capacity as evidenced by a significant reduction in the surface expression of MHC class I, MHC class II, CD40, CD80, and CD86 molecules. Additionally, Cl 13 infects hematopoietic progenitor cells both in vivo and in vitro, impairing their development. One mechanism by which hematopoietic progenitors are developmentally impaired is through the Cl 13-induced production of IFN-alpha and IFN-beta (IFN-alpha/beta). Mice deficient in the receptor for IFN-alpha/beta show a normal differentiation of progenitors into DCs despite viral infection. Thus, a virus can evolve a strategy to boost its survival by preventing the maturation of DCs from infected progenitor cells and by reducing the expression of antigen-presenting and costimulatory molecules on developed DCs.

To detect and characterize autoreactive T cells in diabetes-prone NOD mice, we have developed a multimeric MHC reagent with high affinity for the BDC-2.5 T cell receptor, which is reactive against a pancreatic autoantigen. A distinct population of T cells is detected in NOD mice that recognizes the same MHC/peptide target. These T cells are positively selected in the thymus at a surprisingly high frequency and exported to the periphery. They are activated specifically in the pancreatic LNs, demonstrating an autoimmune specificity that recapitulates that of the BDC-2.5 cell. These phenomena are also observed in mouse lines that share with NOD the H-2 g7 MHC haplotype but carry diabetes-resistance background genes. Thus, a susceptible haplotype at the MHC seems to be the only element required for the selection and emergence of autoreactive T cells, without requiring other diabetogenic loci from the NOD genome.