It has been suggested that SIDS may be a patho-physiological response elicited by combinations of microbial products and/or other environmental factors such as environmental tobacco smoke (ETS) at a time when the developing immune system is more vulnerable to the effects of inflammatory mediators. We have developed an animal model to mimic dual infection, using a non-lethal strain of Influenza A virus and a sub-lethal dose of endotoxin. We hypothesized that exposure to Influenza A virus prior to bacterial infection can mimic the pathology of SIDS and alter cytokine-mediated T-helper cell responses. More specifically, we postulate that at critical windows of immune system development exposure to Influenza A virus changes the regulation of the immune response to bacterial products by altering the kinetics of expression and increasing the production of IL-6, IL-10, TNFa and other inflammatory cytokines that normally promote a protective response. The immature immune system is either unable to tolerate these changes and/or unable to adequately coordinate the response resulting in death. We are using this model to examine the relationship between environmental risk factors for sudden unexplained death in infants including age, prone sleeping and exogenous factors such as ETS or mycotoxins. Rat adapted Influenza A virus (RAIV), developed from Influenza A/Port-Chalmers/1/73 (H3N2) virus was used for infectivity in these experiments. Using time points that correspond to critical points in immunological development and the peak age for SIDS in humans (2-6 months), preliminary studies were conducted to ascertain optimal timing between doses and endotoxin levels to model the mortality and pathology of SIDS. These studies established that inoculation with RAIV at 10 days of age followed by endotoxin 0.2 mg/kg 2 days post RAIV caused mortality with the lowest morbidity. These elements became the defining parameters of the model. Mortality only occurred when specific criteria such as timing between infectious insults and developmental age of the pup were met and age was a key risk factor related to mortality in the model. Adult rats were examined following similar challenges and no mortality was observed. To examine the mechanisms underlying this mortality, a series of experiments were conducted to examine pathology and evaluate immune parameters 2-8 hours following endotoxin administration. Immunophenotyping was performed on cells isolated from the spleen, blood and bronchoalveolar lavage fluid. Tissues were examined for histologic evidence of SIDS-like pathology and inflammatory mediator gene expression. Levels of inflammatory mediators in serum and lavage fluid were also measured. The data from these studies suggests that mortality is due to a rapid systemic shock event rather than lung specific damage. Gross pathologic findings such as lung petechiae and liquid blood around the heart on necropsy were consistent with those seen in infants dying of SIDS. Histopathologic lesions including sub-endocardial hemorrhage and mild cortical thymocyte necrosis were found with greater severity and frequency in dually challenged animals. Vascular congestion in the liver and spleen was consistent with endotoxin exposure and with the release of inflammatory cytokines and reactive nitrogen species (RNS) that may have led to circulatory collapse. Endotoxin induced an immediate release of IL-6 and IL-10 in virally infected animals, as compared to age-matched endotoxin only controls. Macrophage numbers in RAIV-inoculated animals were significantly elevated in the spleen at time of death. A statistically significant decrease in serum IFN-gamma levels was observed in animals that received endotoxin and RAIV. Cytokine mRNA expression was dysregulated in the lungs and livers of the RAIV infected animals. We have defined similarities of SIDS pathology in human infants and that seen in this model and examined basic immune factors associat ed with dual challenge. These studies suggest that the developing immune system can be primed to respond in an exaggerated way to a second immune challenge resulting in unexpected death. It is well documented that autoimmune disease susceptibility is largely dependent upon the presence of particular major histocompatibility complex (MHC) genotypes, however, development of disease symptoms also appears to require critical environmental factors, such as bacterial or viral infections or chemical exposure. MRL/lpr mice exhibit severe renal necrosis (glomerulonephritis) similar to that observed in human patients with systemic lupus erythematosus, with females normally presenting circulating autoantibodies against nucleoprotein particles and subsequent renal complement-antibody immune complexes at approximately 14-16 weeks of age vs. males at 20-22 weeks. Neonatal exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to alter thymocyte maturation and significantly impair immune function in adult animals. We hypothesized that prenatal exposure to TCDD may increase or exacerbate post-natal development of autoimmune disease in genetically-prone MRL/lpr mice. We have observed significant, dose-dependent increases in anti-ssDNA, urinary protein, and renal abnormalities in TCDD-exposed female MRL/lpr mice as compared to controls at six, ten, and 12 weeks of age, respectively. Similar results were noted in TCDD-exposed males at eight, ten and 12 weeks of age.
