Chanaka Mendis

Genetic variations in peripheral blood mononuclear cells in piglets used as an animal model for staphylococcal enterotoxin exposures.

We have used piglets as an animal model for studying the toxic effects of staphylococcal enterotoxins (SEs). Piglets are easy to handle, easy to carry out vital measurements, inexpensive, and more importantly, express remarkably similar pathological symptoms and responses to SE intoxication as humans at comparable doses. Microarray analyses are used to study the effect of many infections on gene expression profile in peripheral blood mononuclear cells. This high throughput application offers detailed depiction of alteration at the molecular levels. When using high throughput gene expression analysis, there is a high possibility of finding genes that vary normally in the tissues under study. It is necessary to verify genes that are normally differentially expressed between piglets. To evaluate the normal physiological variation in gene expression in vivo in piglets, we used cDNA microarray to measure gene expression levels in peripheral blood mononuclear cells from 10 normal Yorkshire piglets. We used analysis of variance to determine genes that showed statistically significant variations across piglets. Out of 1185 genes, 19 (1.6%) genes revealed statistically significant variance between RNA samples. Some of these varying genes are involved in stress response, immune response, and transcription. This study facilitates the characterization of gene expression base line needed for meaningful interpretation of microarray data.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells

BackgroundEffective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs.MethodsTo detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays.ResultsWe found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin.ConclusionHost gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.

Effect of 5-lipoxygenase inhibitor MK591 on early molecular and signaling events induced by staphylococcal enterotoxin B in human peripheral blood mononuclear cells

Staphylococcal enterotoxin B (SEB) has been the focus of a number of studies due to its ability to promote septic shock and a massive impact on the human immune system. Even though symptoms and pathology associated with SEB is well known, early molecular events that lead to lethality are still poorly understood. Our approach was to utilize SEB induced human peripheral blood mononuclear cells (PBMCs) as a prototype module to further investigate the complexity of signaling cascades that may ultimately lead to lethal shock. Our study revealed the activation of multiple divergent intracellular pathways within minutes of SEB induction including components that interconnect investigated pathways. A series of performed inhibitor studies identified a specific inhibitor of 5‐LO (MK591), which has the ability to block JNK, MAPK, p38kinase and 5‐LO signaling‐cascades and drastically reducing the activity of pro‐inflammatory cytokine TNF‐α. Further evaluation of MK591 utilizing cell proliferation assays in PBMCs, human proximal tubule cells and in vivo studies (monkey) showed a decrease in cell proliferation. The inhibitory effect of MK591 was reconfirmed at a genetic level through the utilization of a set of SEB specific genes. Signaling activities, inhibitor studies, cellular analysis and gene expression analysis in unison illustrated the significance of pathway interconnectors such as 5‐LO as well as inhibiting such inter‐connectors (using MK591) in SEB induced human PBMCs.