Sachin Mani

Functional piglet model for the clinical syndrome and postmortem findings induced by staphylococcal enterotoxin B.

Staphylococcal enterotoxin (SE) B causes serious gastrointestinal illness, and intoxication with this exotoxin can lead to lethal toxic shock syndrome. In order to overcome significant shortcomings of current rodent and nonhuman primate models, we developed a piglet model of lethal SEB intoxication. Fourteen-day-old Yorkshire piglets were given intravenous SEB, observed clinically, and sacrificed at 4, 6, 24, 48, 72, or 96 hrs posttreatment. Clinical signs were biphasic with pyrexia, vomiting, and diarrhea within 4 hrs, followed by terminal hypotension and shock by 96 hrs. Mild lymphoid lesions were identified as early as 24 hrs, with severe lymphadenopathy, splenomegaly, and prominent Peyers patches found by 72 hrs. Widespread edema—most prominent in the mesentery, between loops of spiral colon, and in retroperitoneal connective tissue—was found in animals at 72 hrs. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and brain, circulating lymphoblasts, and lymphocytic portal hepatitis. Preliminary molecular investigation using gene array has uncovered several gene profile changes that may have implications in the pathophysiology leading to irreversible shock. Five genes were selected for further study, and all showed increased mRNA levels subsequent to SEB exposure. The use of this piglet model will continue to elucidate the pathogenesis of SEB intoxication and facilitate the testing of new therapeutic regimens that may better correlate with human lesions.

Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate

Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

The cellular and molecular immune response of the weanling piglet to staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB) is a biothreat agent, etiologic agent of food poisoning, and potent inducer of toxic shock syndrome. This heat-stable exoprotein is thought to act as a superantigen to induce T cell–specific pathology. Most animal models do not accurately map the clinical syndrome of human SEB exposure. Previously, we have demonstrated the utility of the weanling piglet model of SEB intoxication. Here, we analyze gross and histopathologic specimens from lymphoid tissue of these animals. Hematological testing was completed to observe changes in circulating leukocytes. Further, these leukocytes were differentiated and the subsets were subsequently analyzed using flow cytometry. Cytokine mRNA was quantified in lymphoid tissue and peripheral blood cells and compared to actual protein concentration using ELISA. The mRNA expression levels for several cell markers implicated in T and B cell differentiation were quantified and compared to control animals, as were levels for apoptosis-related genes. Lymphadenopathy was constantly seen post mortem. SEB-exposed animals had a leukocytosis which increased linearly over the time course. Monocyte levels increased over time, while lymphocyte levels peaked at 6h and then returned to baseline. Most cytokines had mRNA levels that were upregulated after exposure. Detection of serum cytokine changes was accomplished; however, these patterns did not always follow those seen in the differentially expressed genes. Both pro- and anti-apoptotic genes were differentially expressed in exposed animals. This paper reports, for the first time, the immunological findings in the weanling piglet model of SEB intoxication. From this work it is clear that there is not one absolute cell-mediated pathway contributing to the pathology these animals exhibit as a result of SEB exposure.

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.

Gene expression patterns in human blood cells exposed to common flu-like viruses and arenavirus

Viral infections are the main cause of disease in the world, with the respiratory diseases being the main cause of morbidity. The arenaviruses are zoonotic pathogens transmitted from rodents to humans, and are the cause of flu-like disease that can rapidly develop into fatal hemorrhagic fevers. The respiratory diseases and in particular those produced by arenaviruses, have similar prodromal phases. For this reason it is important to look for differences in gene expression during the early stages of disease and identify specific patterns that diagnose exposure to a specific pathogen. The common flu-like viruses we studied were flu A, PI3, rhinovirus 15, and RSV A2; and LCMV-Arm, LCMV-WE, Mop, Mop29). We compared the effects of different viral infections on gene expression in human PBMC. RNA form these cultures was isolated at 4, 8, and 24 hours after exposure to virus, and cDNAs were hybridized to custom microarrays bearing 9000 humans genes. Gene responses that were altered included the immune, cell adhesion, inflammatory, apoptotic, and antiviral pathways. Correlation of these genes with in vivo exposure provides the opportunity to identify diagnostic and therapeutic markers that may provide early indications of impending severe illness.