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

BACKGROUND: Effective 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. METHODS: To 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. RESULTS: We 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. CONCLUSION: Host 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-alpha. 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.

Induction of immunomodulator transcriptional responses by cholera toxin.

Cholera toxin (CT) is the causative agent of cholera, binds to GM1 glycosphingolipids, induces the production of cellular cAMP and is also a very powerful mucosal adjuvant. Although the mechanism of the CT induction of cAMP production is well understood, molecular mechanisms of the adjuvanticity of cholera toxin are yet to be delineated. Here, we examined the interaction of CT with human lymphocytes and monocytes by analyzing the host transcriptional profiles using cDNA arrays. The time courses of the transcriptional activations and repressions of affected genes in lymphocytes and monocytes in response to cholera toxin were determined. CT induced the expression of IL-8 and MIP-1 early in the CT exposure. VEGF, TIMP1, HIF-1alpha, MMP11, hek 8, MCP1, IL-6, GCP 2, urokinase plasminogen activator, and TNF-alpha receptor were upregulated after 4h CT treatment. These genes showed increased expression for 48 h. MRP-14, MRP-8A increased expression after 16 h CT treatment. RT-PCR and real-time PCR using cDNA specific primers confirmed the CT induction and repression of selected genes. The results suggest that immunomodulatory genes were among the genes that were affected the most by CT, and induction of these genes may contribute to the CT adjuvanticity.

Cholera toxin induced novel genes in human lymphocytes and monocytes.

Cholera toxin (CT) is well known as an inducer of the accumulation of cellular cAMP through the ADP-ribosylation of the Gs protein by CT. CT is also one of the most powerful mucosal adjuvants. However, the molecular mechanisms of the CT adjuvanticity are not well understood. Here, the transcriptional responses of cultured human lymphocytes and monocytes in response to CT were analyzed using differential display-PCR. The full complement of cellular mRNA was examined by high resolution polyarylamide gel electrophoresis and sequence analyses of the PCR products of 240 primer sets. Over 100 genes with altered expression were initially identified. The expressions of 65 of these genes were further analyzed and confirmed using custom glass cDNA arrays, RT-PCR and real-time PCR. Immunomodulatory genes such as CD2, HIF1, CXCL2, L-plastin, LILR and IFI30 were affected by CT. In addition, 14 novel genes with previously unknown functions were found to be CT induced. These CT induced gene expression alterations provide more insight in the mechanisms of CT actions. The CT induced gene expressions alterations could contribute to the CT adjuvanticity.

Cholera toxin induced gene expression alterations.

The cholera toxin (CT) is a well-known inducer of cAMP and cAMP regulates gene expression of many genes. However, little is known as to the alterations in gene expression in response to CT. Here the alterations of the expression of 800 selected genes in response to CT were examined using cDNA microarrays. Gene expression alterations in human lymphocytes and monocytes were found after exposure to CT at varying concentrations for different time periods. Over 200 genes showed varying degrees of alterations of expression in CT-treated cells. The CT-induced changes in gene expression were compared by cDNA microarrays under the same conditions to those in response to forskolin, a specific activator of adenylate cyclase, and MDL-12, an irreversible inhibitor of adenylate cyclase. Thirty-five CT-responsive genes were found responded similarly to forskolin but differently to MDL-12. Fourteen CT-responsive genes were affected similarly by MDL-12 but differently by forskolin. Many of these CT responsive genes were involved in immunity, inflammation and oxidative stress. The CT induced responses correlated with those induced by CT subunits. The down regulation of Th1 markers and upregulation of Th2 markers by CT are consistent with the CT induction of Th2 cells.

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.