Clearly, inflammatory and antiviral properties are delicately balanced during the innate immune response to HIV at mucosal sites

tched between WT and PKC-h2/2 mice. All mice were maintained under specific pathogen-free conditions. FACS sorting of NKT cells and in vitro stimulation Pooled splenocytes and intrahepatic lymphocytes from batches of two WT and two PKC-h2/2 mice were stained with anti-CD3 antibody and CD1d-PBS157 tetramer as above, and the NKT cells sorted by BD FACSAria III. The purified NKT cells were cultured in 96 well plates with 200 ml/well T cell culture medium with or without 100 ng/ml OCH as previously described. After overnight stimulation, cytokine levels in supernatants were assayed with BD Flex set, and cells were harvested for IL4 and IFNc intracellular staining as above. Antibodies and tetramers Monoclonal antibodies CD3, NK1.1, CD44, CD24, CD45.1, CD45.2, IL4, INFc, FasL were purchased from eBioscience. CD40L, Trail and DR5 were purchased from Biolegend. The PE-CD1d-PBS157 tetramer was provided by the tetramer core facility at the National Institutes of Health. Statistical analysis Prism software was used for all statistical analyses. Unpaired students t tests were used to compare experimental groups. A P value of less than 0.05 was considered statistically significant. ConA and OCH treatment ConA was dissolved in pyrogen-free phosphate-buffered saline, and intravenously injected into wild-type and PKC-h2/2 mice via the tail vein. Mouse survival was monitored for 56 h after injection. OCH, a specific antigen of NKT cells, was injected intravenously via the tail vein into both WT and PKC-h2/2 mice. One hour after injection, mice were bled and their sera collected. Mice were then sacrificed for collection of intrahepatic lymphocytes as previously described. Results PKC-h2/2 mice are resistant to ConA-induced hepatitis To determine the function of PKC-h in liver injury, we used an acute hepatitis murine model that depends on ConA-mediated activation of NKT cells. WT and PKC-h2/2 mice, age and sex matched, were treated with 25 mg/kg ConA, and their survival rate was determined. Consistent with “1635054 previous results, this dosage of ConA was lethal for WT mice, whereas all PKC-h2/2 mice survived. Because damaged liver releases aspartate transaminase and alanine transaminase, we measured levels of both enzymes in serum of ConA-treated mice to assess liver damage. Prior to ConA treatment, AST and ALT levels were both very low, and there were no obvious differences between WT and PKC-h2/2 mice. After ConA treatment, although elevated, AST and ALT levels of PKC-h2/2 mice were significantly lower than those of WT mice, suggesting there was less liver damage in the absence of PKC-h. Because ConA treatment stimulates the production of inflammatory cytokines that are critical mediators for liver injury, we also measured serum cytokines after ConA treatment. Levels of the inflammatory cytokines IL-6, IFNc and monocyte chemotactic protein-1 were significantly lower in PKC-h2/2 than WT mice at 1 h, 2 h and 6 h 2 February 2012 | Volume 7 | Issue 2 | e31174 Serum cytokine and ALT/AST assay JW-55 Collected sera were 1:10 diluted with the diluents provided by the “2991807 mouse inflammation CBA kit. Inflammatory cytokines were measured using the mouse inflammation CBA kit. Mouse IL-4 was assayed with the flex set of cytokine assay beads. Mouse serum ALT and AST were assayed with the enzymatic assay kit. Mouse serum Osteopontin were assayed with Osteipontin mouse ELISA Kit. Surface and intracellular staining Cells were incubated with antibody cocktail in staining buffer. Cell PK