The complete mitochondrial genome of the foodborne parasitic pathogen Cyclospora cayetanensis

Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes

A Pilot Study Identifying Brain-Targeting Adaptive Immunity in Pediatric Extracorporeal Membrane Oxygenation Patients with Acquired Brain Injury

OBJECTIVES: Extracorporeal membrane oxygenation provides short-term cardiopulmonary life support, but is associated with peripheral innate inflammation, disruptions in cerebral autoregulation, and acquired brain injury. We tested the hypothesis that extracorporeal membrane oxygenation also induces CNS-directed adaptive immune responses which may exacerbate extracorporeal membrane oxygenation-associated brain injury. DESIGN: A single center prospective observational study. SETTING: Pediatric and cardiac ICUs at a single tertiary care, academic center. PATIENTS: Twenty pediatric extracorporeal membrane oxygenation patients (0-14 yr; 13 females, 7 males) and five nonextracorporeal membrane oxygenation Pediatric Logistic Organ Dysfunction score matched patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Venous blood samples were collected from the extracorporeal membrane oxygenation circuit at day 1 (10-23 hr), day 3, and day 7 of extracorporeal membrane oxygenation. Flow cytometry quantified circulating innate and adaptive immune cells, and CNS-directed autoreactivity was detected using an in vitro recall response assay. Disruption of cerebral autoregulation was determined using continuous bedside near-infrared spectroscopy and acquired brain injury confirmed by MRI. Extracorporeal membrane oxygenation patients with acquired brain injury (n = 9) presented with a 10-fold increase in interleukin-8 over extracorporeal membrane oxygenation patients without brain injury (p \u3c 0.01). Furthermore, brain injury within extracorporeal membrane oxygenation patients potentiated an inflammatory phenotype in adaptive immune cells and selective autoreactivity to brain peptides in circulating B cell and cytotoxic T cell populations. Correlation analysis revealed a significant relationship between adaptive immune responses of extracorporeal membrane oxygenation patients with acquired brain injury and loss of cerebral autoregulation. CONCLUSIONS: We show that pediatric extracorporeal membrane oxygenation patients with acquired brain injury exhibit an induction of pro-inflammatory cell signaling, a robust activation of adaptive immune cells, and CNS-targeting adaptive immune responses. As these patients experience developmental delays for years after extracorporeal membrane oxygenation, it is critical to identify and characterize adaptive immune cell mechanisms that target the developing CNS

B Cells Migrate into Remote Brain Areas and Support Neurogenesis and Functional Recovery after Focal Stroke in Mice

Lymphocytes infiltrate the stroke core and penumbra and often exacerbate cellular injury. B cells, however, are lymphocytes that do not contribute to acute pathology but can support recovery. B cell adoptive transfer to mice reduced infarct volumes 3 and 7 d after transient middle cerebral artery occlusion (tMCAo), independent of changing immune populations in recipient mice. Testing a direct neurotrophic effect, B cells cocultured with mixed cortical cells protected neurons and maintained dendritic arborization after oxygen-glucose deprivation. Whole-brain volumetric serial two-photon tomography (STPT) and a custom-developed image analysis pipeline visualized and quantified poststroke B cell diapedesis throughout the brain, including remote areas supporting functional recovery. Stroke induced significant bilateral B cell diapedesis into remote brain regions regulating motor and cognitive functions and neurogenesis (e.g., dentate gyrus, hypothalamus, olfactory areas, cerebellum) in the whole-brain datasets. To confirm a mechanistic role for B cells in functional recovery, rituximab was given to human CD20+ (hCD20+) transgenic mice to continuously deplete hCD20+-expressing B cells following tMCAo. These mice experienced delayed motor recovery, impaired spatial memory, and increased anxiety through 8 wk poststroke compared to wild type (WT) littermates also receiving rituximab. B cell depletion reduced stroke-induced hippocampal neurogenesis and cell survival. Thus, B cell diapedesis occurred in areas remote to the infarct that mediated motor and cognitive recovery. Understanding the role of B cells in neuronal health and disease-based plasticity is critical for developing effective immune-based therapies for protection against diseases that involve recruitment of peripheral immune cells into the injured brain

Proceedings of the 13th annual conference of INEBRIA

CITATION: Watson, R., et al. 2016. Proceedings of the 13th annual conference of INEBRIA. Addiction Science & Clinical Practice, 11:13, doi:10.1186/s13722-016-0062-9.The original publication is available at https://ascpjournal.biomedcentral.comENGLISH SUMMARY : Meeting abstracts.https://ascpjournal.biomedcentral.com/articles/10.1186/s13722-016-0062-9Publisher's versio

Neuroantigen-Specific Autoregulatory CD8+ T Cells Inhibit Autoimmune Demyelination through Modulation of Dendritic Cell Function

<div><p>Experimental autoimmune encephalomyelitis (EAE) is a well-established murine model of multiple sclerosis, an immune-mediated demyelinating disorder of the central nervous system (CNS). We have previously shown that CNS-specific CD8+ T cells (CNS-CD8+) ameliorate EAE, at least in part through modulation of CNS-specific CD4+ T cell responses. In this study, we show that CNS-CD8+ also modulate the function of CD11c+ dendritic cells (DC), but not other APCs such as CD11b+ monocytes or B220+ B cells. DC from mice receiving either myelin oligodendrocyte glycoprotein-specific CD8+ (MOG-CD8+) or proteolipid protein-specific CD8+ (PLP-CD8+) T cells were rendered inefficient in priming T cell responses from naïve CD4+ T cells (OT-II) or supporting recall responses from CNS-specific CD4+ T cells. CNS-CD8+ did not alter DC subset distribution or MHC class II and CD86 expression, suggesting that DC maturation was not affected. However, the cytokine profile of DC from CNS-CD8+ recipients showed lower IL-12 and higher IL-10 production. These functions were not modulated in the absence of immunization with CD8-cognate antigen, suggesting an antigen-specific mechanism likely requiring CNS-CD8-DC interaction. Interestingly, blockade of IL-10 <i>in vitro</i> rescued CD4+ proliferation and <i>in vivo</i> expression of IL-10 was necessary for the suppression of EAE by MOG-CD8+. These studies demonstrate a complex interplay between CNS-specific CD8+ T cells, DC and pathogenic CD4+ T cells, with important implications for therapeutic interventions in this disease.</p></div

Adoptive transfer of neuroantigen-CD8+ T cells inhibits APC function of DC, but not monocytes.

<p>MOG-CD8+ (protected mice; black bars) and OVA-CD8+ (control mice; gray bars) T cells were transferred to naïve mice, followed by immunization with MOG_35–55/CFA. Twelve days post-transfer, CD11c+ (DC) or CD11b+ (monocyte) populations were magnetically isolated from splenocyte preparations and cultured at 1∶20 ratio (APC:CD4) with CD4+ T cells derived from either naïve OT-II mice (top panel) or MOG_35–55/CFA-immunized mice (MOG-CD4+, bottom panel), in the presence (or absence) of corresponding peptide antigens. Cultures were pulsed with ³H-thymidine on day 3 and harvested on day 4 for scintillation counting. Δ counts per minute (ΔCPM, background subtracted) are plotted on the y-axis. Data are representative of 3 independent experiments. (n = 15 per group). **p<0.01; ns = not significant.</p

DC-derived IL-10 is required for modulation of EAE by MOG-CD8+ T cells.

<p>(<b>A</b>) Effect of DC-derived IL-10 on CD4+ T cell proliferation was evaluated using a thymidine-incorporation assay. Magnetically sorted DC from OVA-CD8+ or MOG-CD8+ recipient mice were co-cultured with CD4+ T cells from MOG_35–55/CFA immunized mice. 4 µg/ml of anti-IL-10 antibody or IgG isotype control was added to the indicated cultures. (<b>B</b>) WT MOG-CD8+ and OVA-CD8+ T cells were transferred to either naïve wild-type (left panel) or IL-10−/− (right panel) mice, followed by EAE induction. Mean EAE scores are plotted on the y-axis vs. days post-transfer on the x-axis. Data represent two independent experiments, with 6–8 mice per group (*p<0.05).</p

Immunization with cognate antigen is required for DC modulation.

<p>MOG-CD8+ or OVA-CD8+ T cells were transferred intravenously into naïve mice, followed by either no immunization or OVA/CFA immunization. Seven days post transfer, CD11c+ DC were isolated from spleen and were either (<b>A</b>) used as APCs in ³H-thymidine-based proliferation assay with MOG-CD4+ T cells as responders (y-axis corresponds to ΔCPM), or stimulated at 1×10⁶/ml with 250 ng/ml of LPS, followed by measurement of (<b>B</b>) IL-12 and (<b>C</b>) IL-10 in the supernatants. Representative data of 2 independent experiments are shown (n = 6 per group). ns = not significant.</p

A Logic-Based Formalization of KPIs for Virtual Enterprises

Open innovation is gaining increasing interest as a model to foster innovation through collaboration and knowledge sharing among organizations, especially in the context of Virtual Enterprises (VE). One of the main issues to overcome in such distributed settings is the integration of heterogeneous data, and the need to evaluate common Key Performance Indicators (KPI) capable to measure overall performances of the VE. In this paper we propose a conceptualization of KPIs into an ontology, to provide a common vocabulary to semantically annotate data belonging to different organizations. KPIs are described in terms of dimensions and a mathematical formula. In order to support reasoning services over KPIs formulas we refer to a logic-based formalization in Prolog, where formulas are translated as facts, and several predicates are included to support both mathematical functionalities for formula manipulation and highe-level functions especially suited for VE setup