GA4GH: International policies and standards for data sharing across genomic research and healthcare.

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits

Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain

Abstract Background Neuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus. Methods The identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the Theiler's murine encephalomyelitis virus. Behavioral consequences of immune cell depletion were assessed by Morris water maze. Results Inflammatory monocytes, defined as CD45hiCD11b++F4/80+Gr1+1A8-, and neutrophils, defined as CD45hiCD11b+++F4/80-Gr1+1A8+, were found in the brain at 12 h after infection. Flow cytometry of brain-infiltrating leukocytes collected from LysM: GFP reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. Microscopy of sections from infected LysM:GFP mice showed that infiltrating cells were concentrated in the hippocampal formation. Immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. Immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. Immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the Morris water maze. Conclusions Neutrophils and inflammatory monocytes rapidly and robustly responded to Theiler's virus infection by infiltrating the brain. Inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. Depletion of inflammatory monocytes and neutrophils with the Gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.</p

Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model

Increased astrocyte activation colocalizes with CNS vascular permeability post-induction of PIFS in perforin^−/− mice reconstituted with perforin competent CD8 T cells.

<p>Representative confocal microscopic images illustrating astrocyte expression of GFAP in (A) E7 peptide-treated (n = 4) and (B) VP2_121–130 peptide-treated (n = 6) perforin^−/− mice reconstituted with perforin competent CD8 T cells. Mice treated with (B) VP2_121–130 peptide displayed increased astrocyte activation, as measured by heightened GFAP expression, in areas of CNS vascular permeability when compared to (A) mock E7 peptide-treated negative control mice. CNS permeability is determined by leakage of intravenously administered FITC-albumin.</p

CD8 T cells as sole perforin wielding source are sufficient to induce CNS vascular permeability.

<p>The extent of CNS vascular permeability is illustrated by raw image and 3D transparency rendering of gadolinium-enhancing areas from T1-weighted MRI scans in mice administered (A,C) mock E7 control peptide or (B,D) VP2_121–130 peptide. MRI images are separated to show both methods of CD8+ T cell selection (A,B) negative sort or (C,D) positive sort to isolate and transfer CD8+ cells. (E) Quantification of the 3D volume of vascular leakage from all mice (both negative and positive sort) revealed that VP2_121–130 peptide-treated mice having CD8 T cells as the sole perforin-expressing cell type have a significantly more amount of gadolinium leakage (p<0.05) (n = <b>5,9</b> per group). (F) A significant increase in CNS vascular permeability detectable by intravenously injected rhodamine dextran accumulation in brain homogenates was observed in VP2_121–130 peptide-treated animals (n = 6) compared to mock E7 peptide-treated animals (n = 4) (p<0.05). Results are depicted as mean ± SEM.</p

Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.

<p>(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin^−/− mice followed by induction of PIFS. C57BL/6 perforin^−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 10⁷ GFP+ CD8+ splenocytes (n = 10) or 10⁷ Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2_121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.</p

Disorganization of tight junction proteins and increased CNS vascular permeability can be induced in perforin^−/− mice reconstituted with perforin competent CD8 T cells.

<p>Confocal microscopic images from a representative (A) mock E7 peptide-treated C57BL/6 perforin^−/− mouse reconstituted with perforin competent CD8 T cells depict a preservation of vascular integrity and intact tight junction proteins claudin-5 and occludin. (B) VP2_121–130 peptide-treated C57BL/6 perforin^−/− mice reconstituted with perforin competent CD8 T cells display degradation of tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability as seen through FITC-albumin leakage. (C) FITC intensity in brain tissue from two representative confocal microscopic images of each animal was quantified using ImageJ software. VP2_121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin in brain parenchyma when compared to E7 peptide-treated mice (n = 4) (p<0.05).</p