Cryptosporidium infection of human intestinal tissues causes increased expression of Osteoprotegerin

Cryptosporidium hominis and parvum primarily infect intestinal epithelial cells, which, in turn, play a key role in activating and communicating with the host immune system. To determinate which genes are regulated during early infection of non-transformed human epithelial cells, human ileal mucosa was removed (from surgical specimens), placed on collagen membranes, and cultured as explants. Explant cultures were infected with C. parvum, C. hominis, or control culture medium. After 24 hrs, RNA was extracted and analyzed using Affmetrix GeneChip microarrays. Among the more prominent genes with regulated expression was Osteoprotegerin (OPG), which was increased in all of the explants at 24 hrs and further up-regulated 1.58 fold by C. parvum and 2.54 fold by C. hominis infection compared with uninfected explants. Using real time PCR, we confirmed a 3.14 and 3.79 fold increase in OPG mRNA after infection with C. parvum and C. hominis respectively. Keywords: gene expression analysis via microarray Ileal tissue, which would otherwise have been discarded, was obtained from 3 individuals undergoing bowel resections. The mucosa was removed mechanically and cutured as explants in medium CMRL-1066 and incubated 3 hrs prior to ex vivo infection. Specimens from each individual were divided into 4 parts. A base lane specimen was preserved in RNAse inhibitor directly. The other 3 specimens were infected with 106 oocysts of C. parvum, C hominis or excystation solution. After 24h, the tissues were place in RNAse inhibitor. Subsequently, RNA was extracted with RNAeasy Mini Kit Quiagen and submitted to Baylor College of Medicine microarray facility. The samples were analyzed for gene expression profiles using the Affymetrix Human Genome U133 2.0 array.