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Effective subunit vaccines require the incorporation of adjuvants that stimulate cells of the innate immune system to generate protective adaptive immune responses. Pattern recognition receptor agonists are a growing class of potential adjuvants that can shape the character of the immune response to subunit vaccines by directing the polarization of CD4 T cell differentiation to various functional subsets. In the current study, we applied a high-throughput in vitro screen to assess murine CD4 T cell polarization by a panel of pattern recognition receptor agonists. This identified lipopeptides with TLR2 agonist activity as exceptional Th1-polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists with Ag in mice replicated the findings from in vitro screening by promoting strong Th1 polarization. In contrast, TLR2 agonists inhibited priming of Th1 responses when administered cutaneously in mice. This route-specific suppression was associated with infiltrating CCR2+ cells in the skin-draining lymph nodes and was not uniquely dependent on any of the well characterized subsets of dendritic cells known to reside in the skin. We further demonstrated that priming of CD4 T cells to generate Th1 effectors following immunization with the Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain, a lipoprotein-rich bacterium recognized by TLR2, was dependent on the immunization route, with significantly greater Th1 responses with i.v. compared with intradermal administration of BCG. A more complete understanding of route-dependent TLR2 responses may be critical for informed design of novel subunit vaccines and for improvement of BCG and other vaccines based on live-attenuated organisms. [ABSTRACT FROM AUTHOR]

Abstract: Interferon gamma (IFNγ) is important for immune resistance to herpes simplex virus (HSV) infection. To examine the influence of IFNγ on the development of HSV-specific immune responses and test for IFNγ-independent adaptive immune mechanisms of protection, IFNγ-deficient mice (IFNγ−/−) were immunized with thymidine kinase-deficient HSV-2 (HSV-2 333tk−). HSV-specific cellular and humoral responses were elicited in immunized IFNγ−/− mice resulting in increased resistance relative to non-immune C57BL/6J (B6) mice following challenge with fully virulent HSV-2. CD8+ T cells from IFNγ−/− mice displayed cytotoxic activity and secreted TNFα. HSV-specific CD4+ T cells from immunized IFNγ−/− mice secreted IL-4, TNFα, and IL-17, but unlike T cells from HSV-immune B6 mice, could not clear virus from genital tissue following adoptive transfer. HSV-immune IFNγ−/− mice produced predominantly IgG1 HSV-specific antibodies while immune B6 mice produced predominantly IgG2c antibodies. Transfer of equivalent amounts of HSV-specific antibodies from either strain to naïve mice imparted equivalent early resistance against infection of the genital epithelia. However, protection against neurological symptoms mediated by immune B6 antibodies was superior late in infection. Taken together, these results demonstrate that the limited resistance of HSV-immune IFNγ−/− mice to HSV-2 infection resulted from the action of HSV-specific Ab rather than IFNγ-independent effector functions of T cells. Further, protection against neurological manifestations of HSV-2 infection was superior in mice receiving Ab from immune B6 mice suggesting that Ab-mediated protective mechanisms involving IFNγ-induced IgG subclasses were more effective once virus had spread to neural tissues. [Copyright &y& Elsevier]

In primary infection, CD8+ T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4+ T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4+ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4+ T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8+ T-cell-deficient and CD8+ T-cell-depleted mice, suggesting that CD4+ T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4+ T cells resolved neural infection, CD8+ T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4+ T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism. [ABSTRACT FROM AUTHOR]

The article presents a study conducted by General Motors (GM), concerning the importance of the automotive occupational health services and its economic value to its employees and to the company as well. GM has spearheaded the investigation of their occupational health services within the company's several plants and want to make a comparison of health services between the plants. Through investigation, the firm will be able to formulate decisions in their business operation concerning the services offered in clinics in different plants of the firm. There are several measures which have conducted by GM particularly in surveying the clinics of the firms various automotive plants and through this, the company is able to manage its clinics, staff and its responsibilities.

Evaluation is central to Labov and Waletzky’s (1997) narrative model; without it stories lack any ‘point’. But in narratives elicited from witnesses and suspects at the start of police interviews, evaluation is markedly sparse or absent. This paper examines the presence of three kinds of evaluation in the interview’s questioning phase: interviewer evaluation, elicited evaluation and interviewee evaluation, focusing on discourse markers and evaluative patterns and frameworks that reveal how evaluation is carried out in a range of question and response speech acts (Stenström 1984) and looking at the marking of stance by interviewers in relation to the evidential value of the elicited detail. It shows how interviewers and interviewees change their stance and footing, moving from interrogator to therapist and from cooperation to non-cooperation. Drawing on pragmatic principles from conversational analysis of institutional interaction (Drew and Heritage 1992) and from appraisal theory (Martin 2000), analysis reveals features of contested and collaborative evaluation, marked in turns that reveal concessive and adversative positions. Conclusions point to the function of evaluative frames as important features of interviewer activity, suggesting that these function to achieve a change of state in suspect and witness knowledge and in the evidential value of information. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)–based and interleukin-2 (IL-2)–based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II–specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2–producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential. [ABSTRACT FROM AUTHOR]

The effect of human immunodeficiency virus (HIV) infection and high-level HIV replication on the function of monocytes was investigated. HIV-positive patients had elevated levels of spontaneous production of some or all of the monocyte proinflammatory cytokines measured (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha [TNF-α]) compared to uninfected controls. In patients on therapy with high frequencies of monocytes producing proinflammatory cytokines, this frequency was diminished in the context of viremia during an interruption of therapy. Diminished production of proinflammatory cytokines during viremia was restored by culture with autologous CD4+ T cells or monocytes from an on-therapy time point or lipopolysaccharide (LPS). Microarray analysis demonstrated that diminished monocyte production of proinflammatory cytokines was correlated with elevated type I interferon-stimulated gene transcripts. The addition of exogenous alpha 2A interferon diminished the spontaneous production of IL-1β, IL-6, and TNF-α but did not affect responses to LPS, recapitulating the changes observed for HIV-viremic patients. These results suggest that monocyte function is diminished during high-level HIV viremia and that this effect is mediated by chronic stimulation by type I interferons. This effect on monocytes during viremia may play a role in diminished innate or adaptive immune system functions in HIV-infected patients. In addition, the restoration of these functions may also play a role in some immune reconstitution syndromes observed during initiation of therapy. [ABSTRACT FROM AUTHOR]

Discusses the role of virus-specific T-cell function in induction and maintenance of T-cell responses in experimental animals. Observation of T cells in infected patients; Role of diminished responses in the loss of control of replication; Evaluation of the effects of viremia.

Analysis of Ag-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4+ T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4+ T cells induced by Mycobacterium bovis bacillus Calmette-Guerin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from bacillus Calmette-Guerin-vaccinated mice and show that highquality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4+ CD154+ cells was distinct from that of CD154" cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts. [ABSTRACT FROM AUTHOR]

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors. [ABSTRACT FROM AUTHOR]

Abstract: Macrophages encounter flaviviruses early after injection by arthropod vectors. Using in vivo imaging of mice inoculated with firefly luciferase-expressing single-cycle flavivirus particles (FLUC-SCFV), we examined the initial dissemination of virus particles in the presence or absence of lymph node (LN)-resident macrophages. Higher luciferase activity, indicating higher SCFV gene expression, was detected in the footpad of macrophage-depleted mice after 24h post infection (hpi). Moreover, FLUC-SCFV particles disseminated to the spleen within 14hpi in macrophage-depleted, but not control mice. Although macrophages presented SCFV to naïve T cells in vitro, depletion of subcapsular sinus (SCS) macrophages did not alter the magnitude or effector function of the WNV-specific CD8+ T cell response. Together, these results indicate that SCS macrophages play a role in limiting the dissemination of SCFV early in infection but are not required for the generation of a polyfunctional WNV-specific CD8+ T cell response in the draining LN. [Copyright &y& Elsevier]

Abstract: CD8+ T cells are important for resolution of HSV-2 lesions from the female genital epithelium. It is uncertain whether optimal clearance of viruses such as HSV-2 that cause a limited, non-systemic infection solely requires expression of effector functions by infiltrating CD8+ T lymphocytes, or if the clearance rate is reflective of the expression level of critical effector functions. To address this, CD8+ T cells from normal OT-I mice or OT-I mice deficient in IFNγ (IFNγ−/−) or the IFNγ receptor (IFNγR−/−) were activated in vitro in the presence of IFNγ or IL-4 to generate a series of effector populations (Tc1 and Tc2-like respectively) that secreted different levels of IFNγ and expressed different levels of HSV-specific cytolytic function. Compared with Tc1 cells, Tc2-like cells produced the type 2 cytokines IL-4 and IL-5, exhibited decreased IFNγ secretion, diminished proliferation in vitro, and decreased antigen-specific cytolysis in vivo. Clearance of an ovalbumin-expressing HSV-2 strain (HSV-2 tk− OVA) by adoptively transferred Tc2-like cells was delayed relative to Tc1 cell recipients. Because donor Tc2-like cells proliferated in vivo and infiltrated the infected genital epithelium similar to Tc1 cells, the diminished virus clearance by Tc2-like effector cells correlated with reduced expression of critical effector functions. Together, these results suggest that high level expression of protective T cell functions by effector T cells is necessary for optimal clearance of HSV-2 from the genital epithelium. These results have important implications for vaccines designed to elicit CD8+ T cells against viruses such as HSV-2 that infect the genital tract. [Copyright &y& Elsevier]

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated in Uganda from a sentinel monkey in 1947. Mosquito and sentinel animal surveillance studies have demonstrated that ZIKV is endemic to Africa and Southeast Asia, yet reported human cases are rare with <10 cases reported in the literature. In June 2007, an epidemic of fever and rash associated with ZIKV was detected in Yap State, Federated States of Micronesia. We report the genetic and serologic properties of the ZIKV associated with this epidemic. [ABSTRACT FROM AUTHOR]

Abstract: Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times. [Copyright &y& Elsevier]

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7- CD45RA- phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses. [ABSTRACT FROM AUTHOR]

Early clearance of a thymidine kinase-deficient strain of herpes simplex virus type 2 from the female genital tract required T-cell-produced gamma interferon (IFN-γ). Transfer of activated CD8+ T cells to irradiated C57BL/6 mice resulted in rapid virus clearance, but clearance was greatly delayed in recipients deficient in the IFN-γ receptor (IFN-γ R). Early virus clearance was demonstrated in radiation chimeras in which IFN-γ R expression was limited to parenchymal cells, but resolution was significantly delayed in chimeras deficient in IFN-γ R expression and chimeras expressing IFN-γ R only on hematopoietic cells. Together, these results suggest that early IFN-γ-mediated protection was manifested mainly by stimulation of genital parenchymal cells. [ABSTRACT FROM AUTHOR]

Interpretation: During the 1999 West Nile virus outbreak, thousands of symptomless and symptomatic West Nile viral infections probably occurred, with fewer than 1% resulting in severe neurological disease. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) infection has been associated with perturbations of plasmacytoid dendritic cells (PDC), including diminished frequencies in the peripheral blood and reduced production of type I interferons (IFNs) in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV-infected patients compared to uninfected controls. Using a flow cytometric assay to detect IFN-α-producing cells within unseparated peripheral blood mononuclear cells, we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-α production by PDC in vitro in response to CpG A ligands or inactivated HIV particles. The primary cause of diminished IFN- production was reduced responsiveness of PDC to de novo stimulation, not diminished per cell IFN-α production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDC from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene expression. Treatment of hepatitis C virus-infected patients with IFN-α2b and ribavirin for hepatitis C virus infection resulted in a profound suppression of de novo IFN-α production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDC from HIV-1-infected patients may not represent diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo. [ABSTRACT FROM AUTHOR]