Gordon J. Leitch

In Vitro Culture, Ultrastructure, Antigenic, and Molecular Characterization of Encephalitozoon cuniculi Isolated from Urine and Sputum Samples from a Spanish Patient with AIDS

ABSTRACT In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.

Apical Spore Phagocytosis Is Not a Significant Route of Infection of Differentiated Enterocytes by Encephalitozoon intestinalis

ABSTRACT Encephalitozoon intestinalis is a microsporidian species that infects the intestinal mucosal epithelium, primarily in immunodeficient individuals. The present study employed undifferentiated and differentiated human colonic carcinoma cell lines to determine if this parasite species infected polarized epithelial cells by spore phagocytosis or by impalement with the deployed spore polar tube. Apical surface spore attachment differed between cell lines such that SW480>HT-29>Caco-2>HCT-8, with attachment being greater to undifferentiated Caco-2 cells than differentiated cells and greater to partially differentiated HCT-8 cells than differentiated HCT-8 cells. Attachment was inhibited by chondroitin sulfate A, suggesting that it was mediated by host cell sulfated glycoaminoglycans. Infection rates 3 days postinfection paralleled spore attachment in the various cell lines. The undifferentiated cell line SW480 and undifferentiated Caco-2 and HCT-8 cells exhibited modest spore phagocytosis while the more differentiated cell line HT29 and differentiated Caco-2 and HCT-8 cells did not. All cell lines were impaled by the polar tubes of germinating spores. When normalized to the number of spores attached to the apical membrane, such impalement was greatest in the more differentiated Caco-2 and HCT-8 cells. The host cell apical surface influenced parasite spore germination, as in populations of large undifferentiated Caco-2 cells to which >3 spores had attached, the frequency distribution of the percentages of spores germinated per cell was bimodal, indicating that the surface of some cells favored germination, while others did not. This study suggests that phagocytosis is not a biologically significant mode of infection in differentiated intestinal epithelial cells.

Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS.

Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patients serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patients serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.

Role of P Glycoprotein in the Course and Treatment of Encephalitozoon Microsporidiosis

ABSTRACT Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of theEncephalitozoon microsporidia, E. cuniculi,E. hellem, and E. intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.

Adenovirus masquerading as microsporidia.

Enterocytozoon bieneusi is a microsporidian that causes a severe, debilitating, chronic diarrhea in some patients with the acquired immunodeficiency syndrome. Specific diagnosis of E. bieneusi currently requires an invasive biopsy procedure and time-consuming preparation of specimens for electron microscopy. Our attempts to establish an in vitro culture system using mammalian cell cultures inoculated with duodenal aspirates, biopsy, or both, from 2 infected patients resulted in inadvertent coculture of an adenovirus and E. bieneusi. The adenovirus-infected cells deceptively appeared to contain spores of microsporidia based on light microscopic examination. Transmission electron microscopy revealed only a few microsporidia, but numerous cells infected with an adenovirus that was subsequently identified as adenovirus type 8. We believe that adenovirus infections prevented the cultured cells from supporting the proliferation of E. bieneusi and ultimately destroyed the cell cultures.

Some Ion Exchange Properties of a Myelin Extract from Bovine Optic Nerve.

Summary The water content of pellets of a myelin extract of bovine optic nerve decreased with increasing salt concentration of the medium in which they were spun down. This water content was always less when Ca was the medium cation than when either equiequivalent Na or K was present. The pellet water cation concentration was always greater than that of the medium for the above 3 cation species. It is concluded that the extracted material carried a net negative charge. Extract Ca could not be completely exchanged by Li; Na, K, Rb, Cs, or Mg. At higher salt concentrations the ease of exchange of Ca by monovalent cations was in the same sequence as their hydrated diameters, the smallest counterions exchanging most readily.