Carol L. Sabourin

Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

Alterations in Inflammatory Cytokine Gene Expression in Sulfur Mustard-Exposed Mouse Skin

Cutaneous exposure to sulfur mustard (bis(2‐chloroethyl) sulfide, HD), a chemical warfare agent, produces a delayed inflammatory skin response and severe tissue injury. Despite defined roles of inflammatory cytokines produced or released in response to skin‐damaging chemicals, in vivo cytokine responses associated with HD‐induced skin pathogenesis are not well understood. Additionally, there is little information on the in vivo temporal sequence of gene expression of cytokines postexposure to HD. The goal of these studies was to identify in vivo molecular biomarkers of HD skin injury within 24 hours after HD challenge. Gene expression of interleukin 1β (IL‐1β), granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin 6 (IL‐6), and interleukin 1α (IL‐1α) in the mouse ear vesicant model was examined by quantitative reverse transcription–polymerase chain reaction (RT‐PCR). An increase in IL‐1β mRNA levels was first observed at 3 hours. IL‐1β, GM‐CSF, and IL‐6 mRNA levels were dramatically increased at 6–24 hours postexposure. IL‐1α mRNA levels were not increased following HD exposure. Immunohistochemical studies demonstrated that IL‐1β and IL‐6 protein was produced at multiple sites within the ear, including epithelial cells, inflammatory cells, hair follicles, sebaceous glands, the dermal microvasculature, smooth muscle, and the dermal connective tissue. An increase in the intensity of staining for IL‐1β and IL‐6 was observed in localized areas at 6 hours and was evident in multiple areas at 24 hours. Positive staining for GM‐CSF immunoreactive protein was localized to the inflammatory cells within the dermis. The number of immunostaining cells was increased as early as 1 hour following HD exposure. These studies document an early increase in the in vivo expression of inflammatory cytokines following cutaneous HD exposure. An understanding of the in vivo cytokine patterns following HD skin exposure may lead to defining the pathogenic mechanisms of HD injury and the development of pharmacological countermeasures.

The Role of CC10 in Pulmonary Carcinogenesis: From a Marker to Tumor Suppression

Abstract: CC10 is infrequently expressed in human non‐small cell lung cancers (NSCLCs), despite being abundantly produced by progenitor cells for normal and neoplastic epithelium. Many abnormalities in the surrounding lung associated with field carcinogenesis, which reflect prolonged exposure to such carcinogens as tobacco smoke, also revealed altered expression of CC10. Exposure of hamsters and mice to the tobacco‐specific carcinogen NNK led to reduced CC10 expression, which was partially reversible. Overexpression of CC10 in immortalized bronchial epithelial cells delayed the induction of anchorage‐independent growth in response to NNK. The data suggest that downregulation of CC10 contributes to carcinogenesis because CC10 antagonizes the neoplastic phenotype.

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques

ABSTRACT A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r2 = 0.89 for log10-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4+ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.

Antigen-specific Vγ2Vδ2 T effector cells confer homeostatic protection against pneumonic plaque lesions

The possibility that Vγ2Vδ2 T effector cells can confer protection against pulmonary infectious diseases has not been tested. We have recently demonstrated that single-dose (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment can induce prolonged accumulation of Vγ2Vδ2 T effector cells in lungs. Here, we show that a delayed HMBPP/IL-2 administration after inhalational Yersinia pestis infection induced marked expansion of Vγ2Vδ2 T cells but failed to control extracellular plague bacterial replication/infection. Surprisingly, despite the absence of infection control, expansion of Vγ2Vδ2 T cells after HMBPP/IL-2 treatment led to the attenuation of inhalation plague lesions in lungs. Consistently, HMBPP-activated Vγ2Vδ2 T cells accumulated and localized in pulmonary interstitials surrounding small blood vessels and airway mucosa in the lung tissues with no or mild plague lesions. These infiltrating Vγ2Vδ2 T cells produced FGF-7, a homeostatic mediator against tissue damages. In contrast, control macaques treated with glucose plus IL-2 or glucose alone exhibited severe hemorrhages and necrosis in most lung lobes, with no or very few Vγ2Vδ2 T cells detectable in lung tissues. The findings are consist with the paradigm that circulating Vγ2Vδ2 T cells can traffic to lungs for homeostatic protection against tissue damages in infection.

Alterations in transforming growth factor‐α and epidermal growth factor receptor expression during rat esophageal tumorigenesis

Transforming growth factor‐α (TGF‐α) stimulates cell proliferation through interaction with its receptor, the epidermal growth factor receptor (EGFR), by activating its tyrosine kinase activities. The simultaneous overexpression of TGF‐α and EGFR by tumor cells is thought to trigger the autocrine growth pathway, leading to uncontrolled proliferation. To examine their roles in rat esophageal tumorigenesis induced by the chemical carcinogen N‐nitrosomethylbenzylamine (NMBA), TGF‐α and EGFR expression was evaluated in normal rat esophageal epithelium, in NMBA‐induced preneoplastic lesions, and in papillomas by quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), in situ hybridization, and immunohistochemical analyses. Compared with the levels in normal epithelium, the TGF‐α and EGFR mRNA levels in esophageal papillomas were 3.6 and 1.9 times higher, respectively. In the preneoplastic epithelium, although a trend of increased TGF‐α and EGFR mRNA levels was observed, collectively there were no significant differences between preneoplastic and normal samples by RT‐PCR analysis. In situ hybridization and immunohistochemical staining showed increased levels of TGF‐α and EGFR mRNA and protein products in papillomas and in pronounced hyperplastic and dysplastic lesions. TGF‐α and EGFR expression correlated with each other and with the expression of proliferating cell nuclear antigen, a marker for cell proliferation. These results suggest that disregulation of TGF‐α and EGFR expression may contribute to autonomous cell growth and may play an important role in rat esophageal tumorigenesis induced by NMBA.

Expression of the somatostatin receptor subtype-2 gene predicts response of human pancreatic cancer to octreotide

BACKGROUND Somatostatin inhibits proliferation of many solid tumors. The current study examines whether inhibition of the growth of pancreatic cancer by the somatostatin analog, octreotide, requires tumor expression of somatostatin receptors. METHODS We studied five human pancreatic cancer cell lines, Capan-1, Capan-2, CAV, MIA PaCa-2, and Panc-1. Solid tumors were established in nude mice (n = 20/cell line) by flank injection of tumor cells. Subcutaneous octreotide (500 micrograms/kg/day) was administered by osmotic pumps to 10 of the animals in each group, and the other 10 received control infusions of saline solution. On day 36, the tumors were excised and weighed. Plasma levels of the putative trophic peptides cholecystokinin, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin were assessed by radioimmunoassay. Each of the five cell lines was assayed for the presence of cell surface somatostatin receptors by using whole cell competitive binding assays with 125I-somatostatin. Expression of the somatostatin receptor subtype-2 (SSR2) gene was determined with reverse transcriptase-polymerase chain reactions. Southern blot hybridization was used to assess the presence of the SSR2 gene. RESULTS Octreotide inhibited tumor growth in the MIA PaCa-2 group (512 +/- 75 mg control versus 285 +/- 71 mg treated; p < 0.05) but had no significant effect on tumor weight in the other four cell lines. Plasma levels of cholecystokinin, epidermal growth factor, insulin-like growth factor-1, and insulin were not altered by chronic octreotide infusion. Cell surface somatostatin receptors and SSR2 gene expression were detected only in the MIA PaCa-2 tumors. The gene for the SSR2 receptor was found in all five tumor lines. CONCLUSIONS Octreotide-mediated inhibition of pancreatic cancer growth is dependent on expression of somatostatin receptors. The expression of somatostatin receptors should be considered in the design and interpretation of clinical trials with somatostatin analogs for treatment of pancreatic cancer.

Conserved host response to highly pathogenic avian influenza virus infection in human cell culture, mouse and macaque model systems.

BackgroundUnderstanding host response to influenza virus infection will facilitate development of better diagnoses and therapeutic interventions. Several different experimental models have been used as a proxy for human infection, including cell cultures derived from human cells, mice, and non-human primates. Each of these systems has been studied extensively in isolation, but little effort has been directed toward systematically characterizing the conservation of host response on a global level beyond known immune signaling cascades.ResultsIn the present study, we employed a multivariate modeling approach to characterize and compare the transcriptional regulatory networks between these three model systems after infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Using this approach we identified functions and pathways that display similar behavior and/or regulation including the well-studied impact on the interferon response and the inflammasome. Our results also suggest a primary response role for airway epithelial cells in initiating hypercytokinemia, which is thought to contribute to the pathogenesis of H5N1 viruses. We further demonstrate that we can use a transcriptional regulatory model from the human cell culture data to make highly accurate predictions about the behavior of important components of the innate immune system in tissues from whole organisms.ConclusionsThis is the first demonstration of a global regulatory network modeling conserved host response between in vitro and in vivo models.

Macaque Proteome Response to Highly Pathogenic Avian Influenza and 1918 Reassortant Influenza Virus Infections

ABSTRACT The host proteome response and molecular mechanisms that drive disease in vivo during infection by a human isolate of the highly pathogenic avian influenza virus (HPAI) and 1918 pandemic influenza virus remain poorly understood. This study presents a comprehensive characterization of the proteome response in cynomolgus macaque (Macaca fascicularis) lung tissue over 7 days of infection with HPAI (the most virulent), a reassortant virus containing 1918 hemagglutinin and neuraminidase surface proteins (intermediate virulence), or a human seasonal strain (least virulent). A high-sensitivity two-dimensional liquid chromatography-tandem mass spectroscopy strategy and functional network analysis were implemented to gain insight into response pathways activated in macaques during influenza virus infection. A macaque protein database was assembled and used in the identification of 35,239 unique peptide sequences corresponding to approximately 4,259 proteins. Quantitative analysis identified an increase in expression of 400 proteins during viral infection. The abundance levels of a subset of these 400 proteins produced strong correlations with disease progression observed in the macaques, distinguishing a “core” response to viral infection from a “high” response specific to severe disease. Proteome expression profiles revealed distinct temporal response kinetics between viral strains, with HPAI inducing the most rapid response. While proteins involved in the immune response, metabolism, and transport were increased rapidly in the lung by HPAI, the other viruses produced a delayed response, characterized by an increase in proteins involved in oxidative phosphorylation, RNA processing, and translation. Proteomic results were integrated with previous genomic and pathological analysis to characterize the dynamic nature of the influenza virus infection process.