CRYPTOSPORIDIUM-PARVUM IS CYTOPATHIC FOR CULTURED HUMAN BILIARY EPITHELIA VIA AN APOPTOTIC MECHANISM

While the clinical features of sclerosing cholangitis secondary to opp ortunistic infections of the biliary tree in patients with acquired im munodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosyoridium parvum associated with th is syndrome actually cause disease are obscure. We established an in v itro model of biliary cryptosporidiosis employing a human biliary epit helial cell line. Using morphological and biochemical techniques, we e xamined the interaction of C. parvum with cultured human cholangiocyte s, When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C, parvum oocysts tha t had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The in fectious process was both plasma membrane domain- and cell-specific, b ecause no attachment or invasion occurred when the basolateral membran e of cholangiocytes was exposed to the parasite, or when a human hepat ocyte cell line (HepG2) was used. Time-lapse video microscopy and scan ning electron microscopy (SEM) showed that sporozoite attachment was r apid, involved extensive cholangiocyte membrane ruffling, and culminat ed in parasite penetration into a tight-fitting vacuole formed by inva gination of the plasma membrane similar to those found in naturally oc curring infection in vivo. Transmission electron microscopy (TEM) show ed that C, parvum organisms formed parasitophorus vacuoles and were ab le to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects we re noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morpho logically and biochemically beginning within hours after exposure to t he organism. The novel finding of specific cytopathic invasion of bili ary epithelia by C, parvum may be relevant to the pathogenesis and pos sible therapy of the secondary sclerosing cholangitis seen in AIDS pat ients with biliary cryptosporidiosis.