A new scope for orlistat: Effect of approved anti-obesity drug against experimental microsporidiosis.

As the current therapies for intestinal microsporidiosis are either inconsistent in their efficacies or hampered by several adverse effects, alternative antimicrosporidial agents are being sought. The present study is the first that was designed to evaluate the potency of orlistat, an approved anti-obesity drug, against intestinal microsporidiosis caused by both Enterocytozoon bieneusi and Encephalitozoon intestinalis. Results were assessed through studying fecal and intestinal spore load, intestinal histopathological changes, viability, and infectivity of spores from treated animals. Results showed that orlistat has promising antimicrosporidia potential, with better results in E. intestinalis than E. bieneusi. The animals that received orlistat showed statistically significant decrease in the fecal and intestinal spore load, when compared to the corresponding control infected nontreated mice. The results were insignificant compared to fumagillin and albendazole. Light microscopic examination of stained intestinal sections revealed amelioration of the pathological changes and decreased inflammatory cells detected in the control infected nontreated mice. Spores encountered from stool of orlistat-treated E. bieneusi and E. intestinalis mice showed low viability and significant reduction of infectivity versus their control. Thus, considering the results of the present work, orlistat proved its effectiveness against the intestinal microsporidial infection.

Encephalitozoon: Tissue Culture, Cryopreservation, and Murine Infection.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

[Evaluation of the reproductive potential of Encephalitozoon intestinalis in four different cell line]. / Encephalitozoon intestinalis'in dört farkli hücre hattinda üreme potansiyelinin degerlendirilmesi.

Microsporidia are parasites that can cause infections in many vertebrate and invertebrate organisms and produce small spores resistant to environmental conditions. As they are obligate intracellular parasites, axenic cultures cannot be performed. The aim of this study was to investigate the reproductive potential of the parasite in human colon epidermal adenocarcinoma (Caco-2), human monocytic (U937), African green monkey renal epithelial (VERO) and human kidney epithelial (HEK-293) cell lines of tissue and organs where the parasite is located by following the culture of the parasites and the amount of spores for six weeks. RPMI-1640 medium was used for the cultivation of U937 cells, while DMEM was used for other cell lines and the immature U937 cells were stimulated with Phorbol-12-Myristate-13-Acetate before infection. All of the host cell groups were infected with freshly collected Encephalitozoon intestinalis spores in ratio 1:30 and free spores in the culture media were removed after overnight incubation at 37°C under 5% CO2 condition for parasite invasion. The first release of the spores from the infected cells was observed and recorded by following for six weeks. Furthermore, the spore density released from each cell groups was evaluated by measuring the parasite load by Thoma cell counting chamber and quantified by real-time PCR. As a result of the study, it was observed that four cell lines could be infected by E.intestinalis and the spore production can be maintained for six weeks. It was observed that the monolayer macrophages and CaCo-2 cells, started to be detached from the culture flasks in few days following the parasite invasion, thus decreasing the number of host cells. After 1-2 weeks, HEK-293 cells were also detached from the surface, thus negatively affected the pure spore production by contaminating the media with dead host cell suspension. Spores started to appear in VERO cell media at the end of the second week after initial infection, while it took longer time for other cells to start releasing spores. Over the course of six weeks, the VERO cell line had the highest spore-producing potential among the other cell lines. In conclusion, this study compared the potential for reproduction of E.intestinalis in three human cell lines and monkey originated VERO cell line. This study demonstrated that cells derived from the tissues or organs where Microsporidia species causes disseminated infections could be infected by the parasitic spores in vitro. Additionally, the parasite can survive and propagate longer than six weeks. The authors believe that the results of this study will contribute to the further studies related to the parasite in the area of genetics, pharmacology, biochemistry, immunology and eradication studies.

Immune response to Encephalitozoon infection review.

The microsporidia are emerging agents of infectious disease in both immunocompromised and immunocompetent mammals. Recently, there has been an increased interest in studying the immunobiology of microsporidiosis. This paper discusses the humoral and cell-mediated immune responses to Encephalitozoon spp. The T-cell-mediated responses appear to be most important in conferring resistance. This has become evident by the lethal effects of microsporidiosis in T-cell-deficient hosts. However, much still needs to be learned about the immunobiology of microsporidiosis regarding the specific T-cell responses and the cytokines that provide protective immunity and facilitate the macrophage-mediated killing of microsporidia. Such information will become important in developing immunotherapeutic strategies to control microsporidiosis in the future.

Interferon-gamma-mediated activation of enterocytes in immunological control of Encephalitozoon intestinalis infection.

The microsporidian Encephalitozoon intestinalis develops within intestinal epithelial cells (enterocytes) and is an important opportunistic diarrhoeal pathogen associated with AIDS. Little is known about the protective immune response against the parasite although in mice IFN-gamma is involved and is required to prevent dissemination of the infection to other organs. The present study was designed to establish a suitable short-term in vitro culture technique for E. intestinalis that would enable studies of the role of cytokines such as IFN-gamma in the effector phase of immunity. Encephalitozoon intestinalis reproduced considerably better in the murine enterocyte cell line CMT-93 than in the three human enterocyte cell lines Caco-2, HT29 and HCT-8. Treatment of CMT-93 cells with IFN-gamma significantly reduced parasite reproduction in a dose- and time-dependent manner. IFN-gamma also inhibited development of the parasite in Caco-2 cells. Neither production of NO nor Fe deprivation appeared to be involved in IFN-gamma-mediated parasite killing. However studies suggested that tryptophan catabolism by indoleamine 2,3-dioxygenase played an important part in inactivation of E. intestinalis.

In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines.

Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10(4) to 1 x 10(8) spores/mL) of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10(4) spores/mL for vero cell line compared to 1 x 10(5) spores/mL for SIRC cell line and 1 x 10(7) spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.

Kinetics of Encephalitozoon spp. infection of human macrophages.

Microsporidia are obligate intracellular, eukaryotic parasites that are known to infect a variety of invertebrate and vertebrate species and have been reported to include a broad range of host specificities for various cell types. Although it is clear that some species of microsporidia have the ability to disseminate, causing multiorgan infections, it is not understood how dissemination occurs. One hypothesis suggests that mononuclear phagocytes engulf the pathogen and migrate to various organs while the parasite persists and proliferates. This implies that microsporidia have developed methods by which to escape intracellular degradation and can, instead, use the host as a source of nourishment and a vehicle for dissemination. In our study, we investigated the infection kinetics of 2 Encephalitozoon spp. known to cause disseminated disease in humans. Using fluorescence and scanning electron microscopy, it was determined that spore adherence to the host was rapid (3-6 hr), as was the uptake and organization of internal parasitophorous vacuoles (24 hr). Furthermore, replication was shown to occur within macrophages at 72 hr, as measured by the bromodeoxyuridine proliferation assay, and the production of mature spores occurred in host cells at 120 hr. Parasitic replication could be reduced by pretreatment of macrophages with interferon-gamma and bacterial lipopolysaccharide.

Efficacy of gaseous chlorine dioxide as a sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on produce.

The efficacy of gaseous chlorine dioxide to reduce parasite and bacterial burden in produce was studied. Basil and lettuce leaves were inoculated with Cryptosporidium parvum and Cyclospora cayetanensis oocysts, Encephalitozoon intestinalis spores, and a cocktail of two isolates of nalidixic acid-resistant Escherichia coli O157:H7. The inoculated samples were then treated for 20 min with gaseous chlorine dioxide at 4.1 mg/liter. Cryptosporidium had a 2.6 and 3.31 most-probable-number log reduction in basil and lettuce, respectively. Reduction of Encephalitozoon in basil and lettuce was 3.58 and 4.58 CFU/g respectively. E. coli loads were significantly reduced (2.45 to 3.97 log), whereas Cyclospora sporulation was not affected by this treatment.

New insect system for testing antibiotics.

New and efficient methods to screen antibiotics are needed to counter increased antibiotic resistance in pathogens and the emergence of new diseases. Here we report a new insect model for screening antibiotics in vivo using the grasshopper Romalea microptera. The system is inexpensive, efficient, and flexible, avoids animal-welfare problems, and can be used to test against most major pathogenic groups. We employed this system to test 11 commercial antibiotics against a pathogenic Encephalitozoon species (Microsporidia). Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts, whereas spore counts of grasshoppers fed with albendazole, ampicillin, chloramphenicol, griseofulvin, metronidazole, sulfadimethoxine, or tetracycline were not significantly different from the infected controls. Quinine produced a distinct, but nonsignificant, reduction in spores, and streptomycin a nonsignificant increase in spores. Although 2 antibiotics significantly reduced spore counts, in no case was the pathogen totally eliminated. This study demonstrates the validity of this system as a method to screen antibiotics. It also corroborates the difficulty researchers and physicians have had in treating microsporidia infections, and suggests that quinine and related alkaloid compounds should be further examined as possible therapeutic agents against this group of ubiquitous pathogens. In addition, streptomycin and related compounds should be tested to determine if this widely used antibiotic enhances microsporidiosis.

Antimicrosporidial activities of fumagillin, TNP-470, ovalicin, and ovalicin derivatives in vitro and in vivo.

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.

Apical spore phagocytosis is not a significant route of infection of differentiated enterocytes by Encephalitozoon intestinalis.

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.

Inhibitory activity of human immunodeficiency virus aspartyl protease inhibitors against Encephalitozoon intestinalis evaluated by cell culture-quantitative PCR assay.

Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis.

Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line.

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.

A new monoclonal antibody enzyme-linked immunosorbent assay to measure in vitro multiplication of the microsporidium Encephalitozoon intestinalis.

Microsporidia are obligate intracellular eukaryotic parasites that infect a wide range of hosts, including invertebrates and vertebrates. Microsporidia have emerged as important opportunistic pathogens of humans with the onset of the AIDS pandemic. The potential impact of these infections in human pathology has required the development of antiparasitic strategies, based on the search for molecules having an effect on the development and/or the multiplication of microsporidia. This creates a demand for a simple and reliable in vitro technique for measuring the multiplication of microsporidia. We developed a new monoclonal antibody (MAb) enzyme-linked immunosorbent assay (ELISA) technique and measured the growth of Encephalitozoon intestinalis in an in vitro culturing system using this method. The monoclonal antibody is specific for a coat protein of E. intestinalis sporogonic stages produced in parasitophorous vacuole. An anti-mouse antibody labeled with peroxidase was used as conjugate. This ELISA is a suitable, specific and semiquantitative technique for measuring the spread of E. intestinalis. It is easy to perform and required 5 h from start to end. A good correlation was observed when the ELISA data were compared with the manual microscopic counts of parasitophorous vacuoles obtained after immunofluorescent assay (IFA). Moreover, the ELISA method proved more accurate than the immunofluorescent assay. In summary, the ELISA system described in this study provides a simple reliable assay for measuring the spread of microsporidia in vitro and may prove valuable for the screening of putative interesting antimicrosporidial compounds.

Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA.

A new real-time PCR assay for quantitation of Encephalitozoon intestinalis DNA was developed which used a TaqMan fluorescent probe for specific detection. Serial dilutions of E. intestinalis spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of E. intestinalis DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven E. intestinalis infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10(5) to 10(6) spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of E. intestinalis DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of E. intestinalis burden in clinical specimens.

Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation.

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).