Search

Copyright of Journal of Polytechnic is the property of Journal of Polytechnic and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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.

Mononuclear phogocytic cells and epithelial cells are effective during the initiation and regulation of the innate immune response. They have an active role in mucosal immune response both mechanically and by interaction with other cells with cytokine release. Defensins are microbicidal peptides that are expressed in various cells and are thought to be effective in the first line defense against pathogens. IL-12 and IL-10, showing proinflamatory and antiinflamatory activities, respectively, are actors of the cellular immunity and limit the infection of the host without causing immunopathology. The aim of this study was to observe the differences in the release of IL-12 and IL-10 and the expression of human beta-defensin-3 (hBD-3) inCaco-2 (human colon epidermal adenocarcinoma cell) and THP-1 (human leukemia monocytic cell) cell lines cultured alone or in co-culture, by the stimulation of Toxoplasma gondii tachyzoites either in direct contact with the cells or separated by an insert filter from the cells. Twenty-four hours after the addition of RH strain tachyzoites to the cells, the supernatants were collected from the experiment wells, and commercial ELISA kits (Invitrogen) were used according to the manufacturers instructions to measure IL-12 and IL-10 levels. HBD-3 expression of cells collected from the experiment wells afterfour and 24 hourswere analyzed by using real time PCR. For this procedure, complementary c-DNA was obtained (Transcriptor High Fidelity cDNA Synthesis Kit, Roche Diagnostics, Germany)after the extraction of RNA with a commercial kit (High pure RNA isolation kit, Roche Diagnostics, Germany). IL-12 was higher than IL-10 in all experiment wells. IL-12 was induced more in the co-culture wells where Caco-2 and THP-1 cells were challenged together, than the wells in which the cells infected with T.gondii tachyzoites alone. No differences in respect to cytokine response were observed between the cells with which tachyzoites were in contact and the cells which were separated from the parasites with an insert. In hBD-3 experiments, Caco-2 and THP-1 cells interacted in co-culture wells when infected with tachyzoites and displayed a higher level of hBD-3 expression than the condition when they were infected alone. This study showed that, IL-12 release and hBD-3 expression, which play a role in innate immunity, are greater when various antigens of T.gondii interacted with stimulated mononuclear cell and epithelial cells.

Epithelial cells and macrophages contribute to innate immune responses by cell to cell interactions and releasing proinflammatory mediators. In this study, we aimed to illuminate the underlying mechanisms of contribution of macrophages and epithelial cells in the struggle against pathogens. Therefore, Streptococcus pyogenes and Escherichia coli activated Caco-2 cells and THP-1 macrophage-like cells were investigated for their bactericidal activities and nitric oxide (NO) production when they were alone, in contact with eachother and when their contact was blocked by continuing exposure of soluble mediators. Caco-2 epithelial cells and THP-1 macrophage cells were used as effector cells and S. pyogenes or E. coli as target cells and were incubated at an effector cell/target cell ratio of 1/1 in duplication in 24-well plates. After 5 hours incubation, supernatants were collected from each well for growth inhibition assay and were inoculated onto blood agar plates for S. pyogenes and eosin methylene blue agar plates for E. coli. Following overnight incubation at 37 degrees C, bactericidal effect rate was calculated by counting the colony forming units. The supernatants were also collected after 5 and 24 hours incubation for measurement of NO production by using Griess reagent. Bactericidal effect of Caco-2 cells alone against S. pyogenes and E. coli were found 21.9% and 36.2%, when seperated from THP-1 cells via an insert was 31.8% and 30.5%, and when cell-cell contact was established with THP-1 cells was 24.4% and 55.7%, respectively. Bactericidal effect of THP-1 cells alone against S. pyogenes and E. coli was 27.7% and 63.9%, when seperated from Caco-2 cells via an insert was %27.5 and 43.6%, and when cell-cell contact was established with Caco-2 cells was 24.4% and 55.7%, respectively. As a result, we found that Caco-2 epithelial cells and THP-1 macrophage cells had an antibacterial effect against S. pyogenes and E. coli (p < 0.05), and this effect was higher in macrophage cells than epithelial cells. NO levels in epithelial and/or macrophage cell culture supernatants collected after exposure to S. pyogenes and E. coli were significantly higher for S. pyogenes at 5 hours incubation and for E. coli at 24 hours incubation (p < 0.05). Morever, it can be concluded that macrophages played a more active role than epithelial cells in bactericidal effect and NO response. Besides, epithelial cells and macrophages activated each other more when they were in contact than when they were alone or when their contact was blocked.