Common variants in CLDN2 and MORC4 genes confer disease susceptibility in patients with chronic pancreatitis

A recent Genome-wide Association Study (GWAS) identified association with variants in X-linked CLDN2 and MORC4 and PRSS1-PRSS2 loci with Chronic Pancreatitis (CP) in North American patients of European ancestry. We selected 9 variants from the reported GWAS and replicated the association with CP in Indian patients by genotyping 1807 unrelated Indians of Indo-European ethnicity, including 519 patients with CP and 1288 controls. The etiology of CP was idiopathic in 83.62% and alcoholic in 16.38% of 519 patients. Our study confirmed a significant association of 2 variants in CLDN2 gene (rs4409525—OR 1.71, P = 1.38 x 10-09; rs12008279—OR 1.56, P = 1.53 x 10-04) and 2 variants in MORC4 gene (rs12688220—OR 1.72, P = 9.20 x 10-09; rs6622126—OR 1.75, P = 4.04x10-05) in Indian patients with CP. We also found significant association at PRSS1-PRSS2 locus (OR 0.60; P = 9.92 x 10-06) and SAMD12-TNFRSF11B (OR 0.49, 95% CI [0.31–0.78], P = 0.0027). A variant in the gene MORC4 (rs12688220) showed significant interaction with alcohol (OR for homozygous and heterozygous risk allele -14.62 and 1.51 respectively, P = 0.0068) suggesting gene-environment interaction. A combined analysis of the genes CLDN2 and MORC4 based on an effective risk allele score revealed a higher percentage of individuals homozygous for the risk allele in CP cases with 5.09 fold enhanced risk in individuals with 7 or more effective risk alleles compared with individuals with 3 or less risk alleles (P = 1.88 x 10-14). Genetic variants in CLDN2 and MORC4 genes were associated with CP in Indian patients

Exosomes Derived from M. Bovis BCG Infected Macrophages Activate Antigen-Specific CD4+ and CD8+ T Cells In Vitro and In Vivo

Activation of both CD4+ and CD8+ T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4+ and CD8+ splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4+ and CD8+ T cells. The isolated T cells also produced IFN-γ upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate

Exosomes from BCG infected macrophages induced effector memory T cells <i>in vivo</i>.

<p>Lymphocytes from lungs, MLN and spleens were isolated from mice intranasally injected with exosomes or PBS and cultured in the presence or absence of BCG lysate. Cells were stained with anti-CD4-PE, anti-CD8-PE, antiCD44-PECy5, and antiCD62L-FITC and analyzed by flow cytometry. (A) Expression of effector memory CD4⁺ T cells (CD44^hi and CD62L^low). (B) Percentage of total CD4⁺ T cells which were CD44^hi and CD62L^low. Values are mean±SD of two different experiments. (C) Expression of memory CD8⁺ T cells (CD44^hi and CD62L^low). (B) Percentage of total CD8⁺ T cells which were CD44^hi and CD62L^low. Values are mean±SD of two different experiments. Total lymphocytes were gated using FSC/SSC and intracellular IFN-γ levels were detected on gated CD4⁺ and CD8⁺ lymphocytes. Cells were negative for staining with isotype control antibodies. Exo- exosomes, UI- uninfected. NS-not significant.</p

Activation of BCG sensitized splenic T cells by exosomes from BCG-infected macrophages.

<p>Balb/C mice were infected with BCG (1×10⁶/mouse). Four weeks post-infection, splenocytes were harvested. (A) Splenocytes from BCG infected mice were cultured with BCG or UI exosomes, BCG lysate or media. 12 hr post-treatment, cells were stained with anti-CD4-PE, anti-CD8-PE-Cy5 and anti-IFN-γ-FITC and analyzed by flow cytometry. (B) Splenocytes from BCG infected mice, labeled with 2.5 µM CFSE, were treated with BCG or UI exosome, BCG lysate or media. 72 hr post-treatment, cells were stained with anti-CD4-PE or anti-CD8-PE-Cy5 and analyzed by flow cytometry. (C) Splenocytes from BCG infected mice were cultured with BCG or UI exosomes, BCG lysate or media. 72 hr post-treatment, cells were stained with anti-CD4-PE, anti-CD8-FITC and anti-CD69-PE-Cy5 and analyzed by flow cytometry. Total lymphocytes were gated using FSC/SSC. CFSE, CD69 and intracellular IFN-γ levels were detected on gated CD4⁺ and CD8⁺ lymphocytes. Results are shown as percent positive. Cells were negative for staining with isotype control antibodies. Results are representative of at least 3 independent experiments.</p

APCs are required for optimal T cell stimulation by exosomes.

<p>BMDCs were generated from Balb/C bone marrow cells and CD3⁺ T cells were purified from the spleens of BCG infected mice. Purified CD3⁺ T cells (5×10⁵) were cultured alone or in presence of BMDCs (1×10⁵) with or without BCG exosomes. Cells were cultured for 12 hr and stained with anti-CD4-PE, anti-CD8-PE-Cy5 and anti-IFN-γ-FITC (A) or cultured for 72 hr and stained with anti-CD4-PE, anti-CD8-FITC and anti-CD69-PE-Cy5 (B). Cells were analyzed by flow cytometry. Total lymphocytes were gated using FSC/SSC and intracellular IFN-γ and CD69 levels were detected on gated CD4⁺ and CD8⁺ lymphocytes. Results are shown as percent positive. Cells were negative for staining with isotype control antibodies. Results are representative of at least 2 independent experiments.</p

Exosomes from BCG infected macrophages induce antigen-specific IFN-γ producing T cells.

<p>Lymphocytes from lungs, MLN and spleens were isolated from mice intranasally injected with exosomes or PBS and cultured in the presence or absence of BCG lysate. Cells were stained with anti-CD4-PE, anti-CD8-PE and anti-IFN-γ-FITC and analyzed by flow cytometry. (A) T cells stained for CD4 and intracellular IFN-γ. (B) percentage of total CD4⁺ T cells which expressed IFN-γ. Values are mean±SD of two independent experiments. (C) T cells stained for CD8 and intracellular IFN-γ (D) Percentage total CD8⁺ T cells which expressed IFN-γ. Values are mean±SD of two different experiments. Total lymphocytes were gated using FSC/SSC and intracellular IFN-γ levels were detected on gated CD4⁺ and CD8⁺ lymphocytes. Cells were negative for staining with isotype control antibodies. Exo- exosomes, UI- uninfected. NS-not significant.</p

Taste dysfunction in vestibular schwannomas

Background: Gustatory dysfunction associated with vestibular schwannomas (VS) is a poorly represented clinical presentation. Materials and Methods: One hundred and forty-nine cases operated from 1997 to 2005 where at least six-month follow-up was available were included. All patients were tested for taste sensations using four modalities of standard taste solutions. Apart from the taste sensations, any altered or abnormal taste perceptions were recorded both in the preoperative and postoperative period. Results: After applying the exclusion criteria, the taste dysfunction was studied in 142 patients. The evidence of decreased taste sensation was found in 58 (40.8%) patients prior to surgery. Preoperatively, taste disturbance was found in 29 (37.2%) giant, 28 (45.9%) large and one (33.3%) medium-sized tumors, respectively. There were no significant age or sex-related differences. The postoperative taste disturbances were found in 65 (45.8%) patients. Among patients with anatomically preserved facial nerve, postoperative taste disturbances were found in 55 (42.3%) patients whereas nine (6.9%) patients reported improvement in taste sensations. Conclusions: Taste dysfunction is common following vestibular schwannoma surgery. Patient counseling prior to surgery is necessary to avoid any distress caused by taste dysfunction. Taste dysfunction should be included in the facial nerve functional grading system while assessing outcome

Pre-equilibrium particle emission in alpha induced reactions

696-699Study of the mechanism of pre-equilibrium particle emission in alpha particle induced reactions has been done in the present work. The cross-sections for systems α + 144Sm and α + 154Sm have been calculated using the statistical model code ALICE-91. Significant pre-equilibrium particle emission contribution has been obtained for these systems at higher projectile energy. At higher projectile energies, the pre-equilibrium particle emission has been found to affect predominantly over the equilibrated compound nucleus emissions. The contribution of pre-equilibrium particle emission is found larger for α + 144Sm system than that of α + 154Sm system. The present results indicate that the systems having spherical targets have more contribution of pre-equilibrium particle emission than that of systems with deformed targets. These results suggest that the shape of target (spherical or deformed) also affect the dynamics of pre-equilibrium particle emission at energy above the fusion barrier