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1.  S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors In Vitro and In Vivo 
PLoS ONE  2015;10(2):e0115828.
Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.
PMCID: PMC4338059  PMID: 25706559
2.  Genomic-Based High Throughput Screening Identifies Small Molecules That Differentially Inhibit the Antiviral and Immunomodulatory Effects of IFN-α 
Molecular Medicine  2008;14(7-8):374-382.
Multiple lines of evidence suggest that inhibition of Type I Interferons, including IFN-α, may provide a therapeutic benefit for autoimmune diseases. Using a chemical genomics approach integrated with cellular and in vivo assays, we screened a small compound library to identify modulators of IFN-α biological effects. A genomic fingerprint was developed from both ex vivo patient genomic information and in vitro gene modulation from IFN-α cell-based stimulation. A high throughput genomic-based screen then was applied to prioritize 268 small molecule inhibitors targeting 41 different intracellular signaling pathways. Active compounds were profiled further for their ability to inhibit the activation and differentiation of human monocytes using disease-related stimuli. Inhibitors targeting NF-κB or Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling emerged as “dissociated inhibitors” because they did not modulate IFN-α anti-viral effects against HSV-1 but potently inhibited other immune-related functions. This work describes a novel strategy to identify small molecule inhibitors for the treatment of autoimmune disorders.
PMCID: PMC2376640  PMID: 18475307
3.  Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells  
Infection and Immunity  2002;70(11):6147-6157.
Organisms within the Hardjo serovar of Leptospira species are harbored in cattle throughout the world, causing abortion in pregnant animals as well as being shed in the urine, thereby providing sources of zoonotic infection for humans. We recently showed that sterile immunity in vaccinated cattle is associated with induction of a type 1 (Th1) cell-mediated immune response. Here naïve and previously vaccinated pregnant cattle were challenged with a virulent strain of serovar Hardjo and subsequently evaluated for expression of a type 1 immune response. Lymphocytes that responded in a recall response to antigen by undergoing blast transformation were evident in cultures of peripheral blood mononuclear cells (PBMC) from vaccinated cattle throughout the postchallenge test period while those from naïve cattle were evident at one time point only. Nevertheless, beginning at 2 weeks after challenge, gamma interferon (IFN-γ) was measured in supernatants of antigen-stimulated PBMC cultures from nonvaccinated animals although the amount produced was always less than that in cultures of PBMC from vaccinated animals. IFN-γ+ cells were also evident in antigen-stimulated cultures of PBMC from vaccinated but not from nonvaccinated animals throughout the postchallenge period. The IFN-γ+ cells included CD4+ and WC1+ γδ T cells, and a similar proportion of these two subpopulations were found among the dividing cells in antigen-stimulated cultures as ascertained by carboxyfluorescein succinimidyl ester loading. Finally, while naïve and vaccinated animals had similar levels of antigen-specific immunoglobulin G1 (IgG1) following challenge, vaccinated animals had twofold-more IgG2. In conclusion, while infection may induce a type 1 response we suggest that it is too weak to prevent establishment of chronic infection.
PMCID: PMC130359  PMID: 12379692
4.  Protective Killed Leptospira borgpetersenii Vaccine Induces Potent Th1 Immunity Comprising Responses by CD4 and γδ T Lymphocytes 
Infection and Immunity  2001;69(12):7550-7558.
Leptospira borgpetersenii serovar hardjo is the most common cause of bovine leptospirosis and also causes zoonotic infections of humans. A protective killed vaccine against serovar hardjo was shown to induce strong antigen-specific proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinated cattle by 2 months after the first dose of vaccine. This response was absent from nonvaccinated control cattle. The mean response peaked by 2 months after completion of the two-dose vaccination regimen, and substantial proliferation was measured in in vitro cultures throughout the 7 months of the study period. Variations in magnitude of the response occurred among the vaccinated animals, but by 7 months postvaccination there was a substantial antigen-specific response with PBMC from all vaccinated animals. Up to one-third of the PBMC from vaccinated animals produced gamma interferon (IFN-γ) after 7 days in culture with antigen, as ascertained by flow cytometric analysis, and significant levels of IFN-γ were measured in culture supernatants by enzyme-linked immunosorbent assay. Two-color immunofluorescence revealed that one-third of the IFN-γ-producing cells were γδ T cells, with the remaining cells being CD4+ T cells. The significance of this study is the very potent Th1-type immune response induced and sustained following vaccination with a killed bacterial vaccine adjuvanted with aluminum hydroxide and the involvement of γδ T cells in the response. Moreover, induction of this Th1-type cellular immune response is associated with the protection afforded by the bovine leptospiral vaccine against L. borgpetersenii serovar hardjo.
PMCID: PMC98846  PMID: 11705932

Results 1-4 (4)