ObjectivesThe objective of this research project has been to determine whether mammalian neuraminidase 3 (Neu3) is required for the onset and progression of colitis resulting from recurrent nonlethal gastric infections of a foodborne bacterial pathogen. Findings from the applicant’s laboratory1) previously developed a mouse model of recurrent human food poisoning using Salmonella enterica Typhimurium (ST), a common endemic source of infection in the human population. We previously demonstrated the requirement for induced neuraminidase activity coincident with the induction of enterocyte Neu3 in a TLR4-dependent manner coincident with desialylated Intestinal Alkaline Phosphatase (IAP). Desialyation of IAP caused IAP endocytosis and IAP deficiency in the intestinal lumen, resulting in LPS toxicity and inflammatory damage to the colon, with disease markers similar to human Ulcerative Colitis. The overall objective of this research has been to investigate the direct role of mammalian Neu3 in the onset and progression of colitis caused by recurrent ST infections in this model of human food poisoning. The determination of Neu3 involvement is critical to understanding disease pathogenesis and the potential to inform future therapeutic development. MethodsGermline mutations for study in the mouse were crossed at least 6 generations into the C57BL/6J genomic background prior to analyses and housed in approved specific pathogen-free vivaria. Use of littermates as controls is accomplished to conclusively link genotype to phenotype. Mice lacking Neu3 have been previously engineered and reported2). Neu3 deficiency is well tolerated without overt disease signs evident during the first 6 months of adult life. Animals of both sexes were studied in order to detect any differential effects, which we did not observe. All mice studied were between 2 and 14 months of age. Animal cohorts were administered monthly gastric ST infections over 6 months of life beginning at 8 weeks of age, or phosphate-buffered saline (PBS) as a control. This was done as previously published and under careful monitoring and supervision with appropriate institutional animal and biohazard training certification. Morbidity with death as an endpoint is a potential outcome in this research, and protocols including this endpoint have been approved by the Institutional Animal Care and Use Committee (IACUC). Animals received daily maintenance by trained technicians, and veterinary care as required. Studies measured the onset and severity of intestinal inflammation with mice weighed biweekly and assessed for disease symptoms, including the presence of diarrhea, fecal blood, reduced colon length, inflammation, epithelial barrier function, and rectal prolapse. Intestinal epithelial barrier function was measured at 20 weeks of age prior to the fourth infection. The expression and abundance of neuraminidases was investigated by nucleotide-based RNA analyses and antibody-based protein abundance measurements of duodenal epithelium samples as well as in situ immunohistology of duodenal tissue sections. Neuraminidase activity was measured using commercially available fluorometric substrates. IAP activity and abundance was similarly measured in the small intestine and colon as well as in intestinal contents. Glycosylation status of IAP was assessed using lectin-based techniques. The applicant’s laboratory has previous experience with these methodologies evident from publications using these and other mouse models of disease1,3). These studies of Neu3-null mice were in fact identical in methodologies used to those published that first implicated Neu3 in the pathogenesis of colitis1).Biostatistical calculations were undertaken in design and interpretation of animal strain and population-based comparative data. Power calculations were performed for all experimental approaches to ensure sufficient numbers of animals were studied to achieve statistical significance in comparative analyses. Results with different populations were assessed by t distribution (student’s t test or paired t test) and ANOVA with post-hoc or regression analysis when appropriate. Survival differences were 74Environmental and pathogenic triggers of intestinal inflammatory diseaseJamey D. Marth1-3 Won Ho Yang, 1,3 Douglas M. Heithoff, 1-3 Peter V. Aziz, 4 Markus Sperandio, 5 Victor Nizet, 1,3 Michael J. Mahan, and 1-3,6Jamey D. Marth 1Center for Nanomedicine; 2SBP Medical Discovery Institute; 3 Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara; 4 Walter Brendel Center for Experimental Medicine, Ludwig Maximilian University, Munich, Germany; 5 Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, Dentistry and Pharmaceutical Sciences
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