Shane C Burgess
Publications
PMID: 14733737;Abstract:
Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Marek's disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.
PMID: 16480010;Abstract:
Background and aim of the study: Cardiovascular risk factors are believed to play a role in the pathogenesis of aortic valve disease. In the present study the hypothesis was proposed that elevated pressure would cause a change in the expression of prototypical pro-inflammatory genes. Hence, the expression of MCP-1, osteopontin (OPN), VCAM-1, GM-CSF and PAI-1 was examined using semi-quantitative realtime RT-PCR. Methods: Porcine aortic valve interstitial cells at passage 1 were exposed to constant pressures of 100, 140, or 170 mmHg or cyclic pressures of 80-120, 120-160, or 150-190 mmHg for 2 h. Static cultures at atmospheric pressure served as controls. Total RNA from pooled experiments was isolated for analysis of gene expression. Single tube primer-mediated RT-PCR was performed directly on the RNA. Results: Cell s responded differently to constant and cyclic pressure. The most notable response was the expression of OPN, which was significantly up-regulated under steady conditions but down-regulated under cyclic conditions. The opposite was true in VCAM-1 expression, which was significantly downregulated at 170 mmHg static pressure, but up-regulated at 140 and 170 mmHg mean cyclic pressure. There was no clear proportional correlation between pressure magnitude and expression of MCP-1, GM-CSF, or PAI-1. However, elevated cyclic pressure caused a proportional increase in VCAM-1 expression and a proportional decrease in OPN expression. Conclusion: Elevated cyclic pressure is a potent sti mulus for the up-regulation of VCAM-1 expression and the down-regulation of OPN expression. This demonstrates an association between hypertension and aortic valve stenosis and calcification. The regulation of the chemotactic genes MCP-1 and GM-CSF is not correlated to a change in compressive forces. © Copyright by ICR Publishers 2006.
PMID: 19540544;Abstract:
Marek's disease virus (MDV), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. In the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to MDV infection were studied using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). Comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. In total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (FDR) ≤ 0.05 and fold change ≥ 2) or qualitatively differentially expressed at least once during different sampling points. Overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin-proteasome protein degradation (UPP), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. Notably, early stages of the disease were characterized by changes in the UPP, and antigen presentation. Furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. The present findings provide a basis for further studies aimed at elucidation of the role of these proteins in MDV interactions with its host. © 2009 Elsevier Inc. All rights reserved.
PMID: 15109053;Abstract:
The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the ~"sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.
PMID: 19615758;Abstract:
T cells from the spleens of B19/B19 and B21/B21 chickens infected with MDV JM-16 strain were fractionated by flow cytometry at 4, 10, 21 days post infection (d.p.i.). The expression of cytokine and viral genes (meq and glycoprotein B (gB)) was measured by real-time RT-PCR. It was determined that CD4+ and CD8+ T cells had both become infected with Marek's disease virus (MDV) in both chicken lines. There was significantly higher expression of meq in CD4+ T cells compared to CD8+ T cells at 10 and 21 d.p.i. Furthermore, at 10 and 21 d.p.i., there was a tendency for higher expression of meq in both T cell subsets of B19 chickens compared to those of B21 chickens. There were temporal changes in the expression of cytokines, interferon (IFN)-γ, interleukin (IL)-18, IL-6, and IL-10, in various T cell subsets. Among these changes, there was an increase in IL-10 expression in both T cell subsets at different time points, especially in the susceptible line at 10 and 21 d.p.i. Our results indicate that cytokines could be differentially induced by MDV in CD4+ and CD8+ T cell subsets and that IL-10 may play a role in the modulation of immune response to MDV. However, an association between cytokine gene expression in T cell subsets and resistance or susceptibility to MD was not established. © 2009 Elsevier B.V. All rights reserved.