IMMUNE RESPONSES TO MICROSPORIDIA

Tulane University Of Louisiana, New Orleans LA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Enterocytozoon bieneusi is the most prevalent microsporidian infecting humans, yet virtually no studies have been published about the immune responses to this pathogen. Nonhuman primates are natural hosts of E. bieneusi infections, and na[unreadable]ve and immune-compromised rhesus macaques exhibit similar signs of disease (eg. diarrhea and wasting) as reported in immune-compromised infected humans. E. bieneusi infections are being reported more frequently in immune-competent humans, and 35-40% of the immune-competent pigtail macaques, rhesus macaques, and baboons housed outdoors at the Tulane National Primate Research Center were detected with E. bieneusi spores in feces by histochemistry and PCR. To begin to study the immune responses to this microsporidian, a preliminary microarray analysis was performed. Duodenal and jejunal intestinal cells from immune-competent E. bieneusi-infected and non-infected rhesus macaques (with and without diarrhea) were incubated with E. bieneusi spores or medium for 24 hours. Total RNA was extracted to generate Cy-labeled cDNA samples using the Low RNA Input Linear Amplification Kit (Agilent Technologies Inc., CA). Labeled cDNA was hybridized overnight to the 44,000 element 60mer oligonucleotide based rhesus macaque microarray printed in a 4x44k format (Agilent Technologies Inc., CA), which can interrogate the transcription of over 18,000 unique macaque genes at once. After hybridization and washing, slides were evaluated in a dual-confocal continuous microarray scanner (GenePix 4000B) using GenePix Pro version 6.1 as the image acquisition and extraction software. Microarray data were based on duplicate measurements. Pathway analyses were performed by uploading significant data sets into Ingenuity Pathways Analysis algorithm. Among the pathways and cytokines related to immune responses that were significantly perturbed (P 0.05) in the E. bieneusi-infected monkey lymphocytes with diarrhea and the uninfected monkeys were those related to inflammatory disease, hepatic disease, and interferon signalling (e.g. TNF-alpha, IFN-gamma). These pathways were not significantly perturbed in intestinal cells of infected animals that were not exhibiting signs of diarrhea, suggesting that healthy immune-competent animals are able to regulate inflammatory pathways in response to E. bieneusi infection.