"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. −/− mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.innate immunity | interferon and antiviral response | single-cell analysis | NF-κB signaling | IRF3 signaling F ollowing virus infection, eukaryotic cells respond by the activation of innate immunity in a cell type-and strain-specific manner. The early host innate response includes activation of the transcription factors IRF3 and NF-κB, which induce transcription of discrete and overlapping sets of genes, including those encoding type I IFNs and viral stress-induced genes (vSIGs) (1, 2). Following IFN secretion and binding to cognate receptors, there is feed-forward amplification of expression of IFN and several hundred IFN-stimulated genes (ISGs), resulting in the establishment of an antiviral state. Such early responses to the presence of viral particles are spatially distinct from those that occur in adjacent bystander cells lacking direct exposure to virus. The early host innate immune responses to virus generally have been measured using averaged signals from "bulk" populations of cells or whole tissues. Such analyses cannot reveal hierarchical responses in individual infected or bystander cells. Here, we used a single-cell analytic strategy to examine the diversity in the early host antiviral innate immune response to rotavirus (RV) in suckling mice.RVs cause severe dehydrating diarrhea in the young of many mammalian species, resulting in more than 400,000 deaths of children annually (3). Homologous RV (RV derived from the infected host species) replicates efficiently within mature absorptive villous enterocytes of the small intestine but in the immune competent host, does not replicate efficiently in crypts or colon or at extraintestinal systemic locations (4). RV has evolved mechanisms to effectively block IRF3 and NF-κB-dependent IFN induction, as well as feedback-dependent amplification stages of the innate antiviral response (5). Specifically, the RV nonstructural protein NSP1 mediates degradation of IRF3 and/or NF-κB regulatory factor β-TrCP, depending on the viral strain and host cell involved (6, 7). In addi...

Summary To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses can be used as a discovery tool.

Summary Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naïve and memory cells by a T cell receptor (TCR)-independent process. Single cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

The female reproductive tract (FRT) is the most common site of infection during HIV transmission to women, but viral remodeling complicates characterization of cells targeted for infection. Here, we report extensive phenotypic analyses of HIV-infected endometrial cells by CyTOF, and use a ‘nearest neighbor’ bioinformatics approach to trace cells to their original pre-infection phenotypes. Like in blood, HIV preferentially targets memory CD4+ T cells in the endometrium, but these cells exhibit unique phenotypes and sustain much higher levels of infection. Genital cell remodeling by HIV includes downregulating TCR complex components and modulating chemokine receptor expression to promote dissemination of infected cells to lymphoid follicles. HIV also upregulates the anti-apoptotic protein BIRC5, which when blocked promotes death of infected endometrial cells. These results suggest that HIV remodels genital T cells to prolong viability and promote viral dissemination and that interfering with these processes might reduce the likelihood of systemic viral spread.

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BACKGROUND: Apha-2 agonists are combined with local anesthetics to extend the duration of regional anesthesia. We evaluated the effect of combining dexmedetomidine with levobupivacine with respect to duration of motor and sensory block and duration of analgesia. METHODS: Sixty patients scheduled for elective forearm and hand surgery were divided into two equal groups in a randomized double blind fashion. The patients received brachial plexus block via supraclavicular route with the help of nerve stimulator. In group L (n=30) 35cc of levobupivacaine with 1ml of isotonic saline and in group LD (n=30) 35cc of levobupivacine with 1 ml of (100 microgram) of dexmedetomidine was given. Duration of motor and sensory block and time to first rescue analgesia were recorded. Data analysis was done by SPSS version 16.0 [SPSS Inc ILLINOIS, USA, 2008]. Categorical variables were analyzed using Pearson"s Chi-square test. Normally distributed numerical variables were analyzed using unpaired "t" test. Skewed numerical variables within the group were analyzed using Man-Whitney "U" test. All tests were two tailed. Statistical significance was defined as P<0.05. RESULTS: Sensory and motor block durations were longer in group LD as compared to L (P<0.01). Duration of analgesia was significantly longer in group LD as compared to group L (p<0.05). CONCLUSION: Dexmedetomidine added to levobupivacaine in supraclavicular brachial plexus block prolongs the duration of block and the duration of postoperative analgesia.

Our study validates the reliability of Wieneke scoring system in predicting malignancy in pediatric ACTs. It is simple and easy to use and therefore useful in day-to-day practice.

We study the large sample behaviour of a convex clustering framework, which minimizes the sample within cluster sum of squares under an l 1 fusion constraint on the cluster centroids. This recently proposed approach has been gaining in popularity; however, its asymptotic properties have remained mostly unknown. Our analysis is based on a novel representation of the sample clustering procedure as a sequence of cluster splits determined by a sequence of maximization problems. We use this representation to provide a simple and intuitive formulation for the population clustering procedure. We then demonstrate that the sample procedure consistently estimates its population analogue and we derive the corresponding rates of convergence. The proof conducts a careful simultaneous analysis of a collection of M-estimation problems, whose cardinality grows together with the sample size. On the basis of the new perspectives gained from the asymptotic investigation, we propose a key post-processing modification of the original clustering framework. We show, both theoretically and empirically, that the resulting approach can be successfully used to estimate the number of clusters in the population. Using simulated data, we compare the proposed method with existing number-of-clusters and modality assessment approaches and obtain encouraging results. We also demonstrate the applicability of our clustering method to the detection of cellular subpopulations in a single-cell virology study.