Anti-microsporidial effect of thymoquinone on Encephalitozoon intestinalis infection in vitro

Objective: To evaluate the anti-microsporidial effects of the active component of Nigella sativa seeds, thymoquinone, against Encephalitozoon intestinalis using an in vitro model.Methods: Anti-microsporidial effect of thymoquinone against Encephalitozoon intestinalis was evaluated by using various concentrations of thymoquinone (0, 1, 5, 10, 15, 20, 30, 35, and 40 mu M) and sterile dimethyl sulfoxide. Real time PCR was used to evaluate the inhibitory effects of thymoquinone on the life cycle of Encephalitozoon intestinalis.Results: The cytotoxic effect of thymoquinone on HEK293 cell line was observed with 30, 35, and 40 mu M concentrations of thymoquinone after 24, 48, and 72 hours of incubation. It was observed that 10, 15, 20, and 30 mu M concentrations of thymoquinone decreased the spore density compared with the control; however, it was significant only at 30 mu M.Conclusions: Thymoquinone shows potent anti-microsporidial effects against Encephalitozoon intestinalis in the in vitro model; however, the toxic concentrations of thymoquinone are also toxic to the host cells

THE EFFECT OF ENCEPHALITOZOON INTESTINALIS ON OXIDATIVE STRESS AND CYTOKINE LEVELS IN U937 CELLS: AN IN VITRO STUDY

This in vitro study was performed to investigate the changes in nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) levels in human monocytic (U937) cells infected with Encephalitozoon intestinalis. E. intestinalis was first cultured in the African green monkey renal epithelial (Vero) cells to obtain sufficient amount of parasite for the study. U937 human macrophage cell line and E. intestinalis 50506 (ATCC) strain were used in the study. U937 macrophages were infected with 5x106 spores after stimulation with Phorbol 12-Myristate 13-Acetate (PMA) and uninfected U937 cells were used as control. Culture media were collected at the 6th, 12th, and 24th hour after infection to determine NO, MDA, TAC, TNF-α and IL-10 levels. NO levels significantly increased at the 6th, 12th and 24th hour. There was no significant difference in MDA and TAC levels at the 6th and 12th hour, but a significant increase in MDA, and a significant decrease in TAC was observed at the 24th hour. TNF-α levels did not differ in all sampling times, and IL-10 level decreased only at the 6th hour. In conclusion, E. intestinalis caused oxidative stress by increasing the levels of oxidants (NO and MDA) and by suppressing TAC level without no significant changes in cytokine levels in U937 cell line

Evaluation of the Reproductive Potential of Encephalitozoon intestinalis in Four Different Cell Line

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 intestinails spores in ratio 1:30 and free spores in the culture media were removed after overnight incubation at 37 degrees 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

The effect of Encephalitozoon intestinalis on oxidative stress and cytokine levels in U937 cells*

Aim:Microsporidae,which can accommodated in many vertebrate and invertebrate organisms, are compulsoryintracellular parasites that form spores. Encephalitozoonintestinalis (E.intestinalis) from the group of Microsporidae causelife-threatening diarrhea in immunosuppressed patients as well as; enteritis,small intestinal perforation, cholangitis, cholecystitis, nephritis,bronchitis, sinusitis, rhinitis, keratoconjunctivitis and disseminatedinfections. In this study, nitric oxide (NO), malondialdehyde (MDA), totalantioxidant capacity (TAC), tumor necrosis factor-α (TNF-α) and interleukin-10(IL-10) levels were determined in U937 cells infected with E. intestinalis.Materialand Method: In the study, U937 human macrophage cell lineand E. intestinalis 50506 (ATCC)strain were used. U937 cells were stimulated with Phorbol 12-Myristate13-Acetate (PMA) and then infected with the parasite. Nitric oxide, MDA, TAC,TNF-α and IL-10 levels were determined in cell media collected at 6th,12th and 24th hours after infection.Results:Comparedto the control group, NO levels at all time points and MDA levels at 24thhours were significantly increased, in the infected group. Total antioxidant capacity did not differ between the control andinfected groups at the 6th and 12th hours, while thevalues of the infected group decreased at 24th hour statistically. Therewas no statistically significant difference in TNF-α values in both groups andat all time points. In the infected group compared to the control group, IL-10levels showed a significant reduction only at 6th hours.Conclusion: It was concluded that there was an increase in oxidantlevels, a decrease in antioxidant levels, and no significant changes incytokine levels in U937 cells infected withE. intestinalis.&nbsp;Key words: Antioxidant, cytokines, Encephalitozoonintestinalis, oxidative stress.&nbsp;</p

Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation

Microsporidia are obligate intracellular parasitic protozoa infecting the wide variety of hosts and are commonly known as a cause of chronic diarrhea particularly in immunocompromised individuals. Molecular-based tests have high sensitivity and specificity in disease diagnosis. However, these tests' performance relies on the isolation of DNA in a good concentration. The standard procedures of commercial DNA extraction kits are usually insufficient for this purpose due to the tough walls of spores. This study aimed to test the significance of pretreatments by glass beads and freeze-thawing processes in DNA isolation from microsporidia spores. The parasite was cultured in growing Vero cells and seven serial dilutions were prepared from the collected spores. DNA purification was performed according to different tissue kits and stool kit procedures with and without any pretreatment. Concentration of isolated DNA samples were evaluated by real-time PCR. As a result of this study, the detectable amount of spores is minimum 10 spores in each 100 mu l sample according to the different tissue kits' standard protocols. However, according to the DNA stool mini kit, the detectable amount of spores was found to be 1,000 spores/100 mu l of stool sample when pretreated with both the freeze-thawing and glass beads methods. In conclusion, the current study demonstrated that further pretreatments are an essential process for DNA extraction from the stool specimens in order to avoid possible false negativity in the diagnosis of microsporidiosis