Apoptosis as a Potential Target to Arrest and Survival of Hydatid Cyst.

Background: Hydatidosis is a serious and life-threatening disease that may lead to the death of the host if diagnosed and treated improperly. Apoptosis has been investigated as a mechanism of host innate immunity in suppressing parasites and also the survival of cysts in the human body. The present study investigates the process and role of apoptosis caused by a host cell or parasite in hydatid cysts. Materials and Methods: Survey cytotoxic effect and apoptotic mortality of hydatid-treated lymphocytes were investigated. Also, to determine the mechanism of apoptosis in host and parasite, the mean gene expressions of Bcl-2, Bax, Caspase 3 in hydatid-treated lymphocytes, and Fas-L gene in the laminated-germinal layer of fertile and infertile hydatid cysts were evaluated. Results: The viability of fertile and infertile hydatid fluid-treated lymphocytes was significantly different compared with the control group. Flow cytometry also showed apoptotic cells. Bax mean gene expression was significantly different between fertile and infertile treated lymphocytes. However, there was no significant difference in the mean expression of Caspase 3, and Bcl-2 genes in these two groups. Although the expression of the Fas-L gene in infertile cysts was higher than in fertile cysts, the result was not significant. Conclusion: It seems that hydatid cyst fluid may induce apoptosis in lymphocytes so that, hydatid cysts can escape from the immune system and stay alive. On the other hand, the results represent the possible immune path of host apoptosis against the parasite as one of the important routes in infertility of hydatid cysts.

Anti-Toxoplasma gondii antibodies attach to mouse cancer cell lines but not normal mouse lymphocytes.

Toxoplasma gondii (T. gondii) is prevalent intracellular parasite and a cause of worldwide infection in the human population. An inhibitory effect of this parasite on cancer growth has been demonstrated in cell culture and animal models. To determine whether the anticancer activities of T. gondii are associated with host immune response, in the current study the reactivity of anti-T. gondii antiserum with the surface of cancer cell lines was investigated. Anti-T. gondii antibodies were raised in rabbit and the reaction of this antiserum in comparison with other anti-parasite antisera (anti-T. vaginalis, anti-hydatid cyst fluid, anti-protoscolices antigens) with mouse melanoma or breast cancer cells lines was investigated using flow cytometry. Anti-T. gondii antiserum reacted markedly with the surface of mouse melanoma and breast cancer cells, and less so with the normal mouse spleen lymphocytes. Meanwhile, the other anti-parasite antisera did not react strongly with the surface of cancer cells compared with normal mouse spleen lymphocytes. In summary, it has been demonstrated herein that anti-T. gondii antiserum may selectively react with the surface of mouse cancer cells but not with normal mouse spleen lymphocytes. Therefore, further study on anti-Toxoplasma antibodies may be useful for directing the application of selective drug delivery in cancer treatment.

Molecular characterization of Candida dubliniensis and Candida albicans in the oral cavity of drug abusers using duplex polymerase chain reaction.

BACKGROUND AND PURPOSE: Candida dubliniensis is closely related to the most pathogenic and prevalent yeast, namely C. albicans.Candida species can opportunistically overgrow in vulnerable individuals and cause a variety of diseases. The current study aimed to identify and isolate C. dubliniensis species present in the Candida albicans species complex identified in the oral cavity of drug abusers. MATERIALS AND METHODS: This study was conducted on 53 strains of C. albicans species complex, isolated from the oral mucosa of drug abusers in Isfahan, Iran. DNA extraction was accomplished through boiling procedure. Duplex polymerase chain reaction (PCR) was performed to amplify ITS1-5.8S-ITS2 region using four specific primers. Fungal species were identified based on the difference in the size of the bands created in the Agarose gel. RESULTS: Out of the 53 isolates under study, 30 (56.6%) and 14 (26.4%) samples were identified as C. albicans and C. dubliniensis, respectively. In the remaining 9 samples (17%), both types of Candida species were confirmed. CONCLUSION: The findings of the present study revealed the presence of a noticeable amount of C. dubliniensis in the oral cavity of drug abusers. Therefore, the probable presence of this fungus should be considered during the examination of oral infection among this group. To date, no research has directly investigated this issue in Iran.

Locked nucleic acid -anti- let-7a induces apoptosis and necrosis in macrophages infected with Leishmania major.

BACKGROUND: Protozoan parasites of the genus Leishmania are etiologic agents which are intracellular pathogens of vertebrates and replicate inside infected macrophages. Leishmania have developed complex strategies to reverse host immune responses in favor of it. One of the major species causing cutaneous involvements is Leishmania major. MicroRNAs (miRNA) are non-coding small RNAs encoding 22-nucleotide (nt) long RNAs. miRNAs affect diverse biological processes, including cell cycle, proliferation, differentiation, growth and development, metabolism, aging, apoptosis, gene expression and immune regulation. This study aimed at evaluating apoptosis and necrosis after transfection locked nucleic acid (LNA) inhibitor of let-7a in the human macrophages miRNAs upon infectionwith L. major. MATERIALS AND METHODS: Inhibition of let-7a in macrophages was derived originally from the human monocytes (MDM), using locked nucleic acid (LNA) antagomir. The total cellular RNA was extracted 24 and 48 h post transfection. The levels o Let-7a expression was measured by qPCR Real Time using specific primer. Annexin-V/Propidium Iodide staining method was performed to detect apoptosis and necrosis in the MDM cells. Data were analyzed using the Kruskal-Wallis and Mann-Whitney tests. RESULTS: Let-7a inhibition increased the MDM cells apoptosis and necrosis using flow cytometry method. CONCLUSIONS: The results suggested that inhibition of let-7a could be a new approach in treatment of leishmaniasis.

Identification of Candida species in patients with oral lesion undergoing chemotherapy along with minimum inhibitory concentration to fluconazole.

BACKGROUND: Various species of Candida, especially Candida albicans was known as the most important etiological agent of fungal infections. Oral candidiasis is the most common fungal infection in patients undergoing chemotherapy. The purpose of this study was to identify Candida species from oral lesions of these patients and antifungal susceptibility of the clinical isolates. MATERIALS AND METHODS: Among 385 patients with cancer, 55 (14.3%) showed oral lesions. Oral swabs were performed to identify the yeasts using direct smear and CHROMagar medium. Micro dilution method was prepared in different concentrations of fluconazole and minimum inhibitory concentration and minimum fungicidal concentration of each species were compared. RESULTS: Oral candidiasis confirmed in 36 cases by direct examination and culture. C. albicans and non-albicans represented in 26 (72.2%) and 10 (27.8%) of the isolates, respectively. 76.5% of C. albicans and 23.5% non-albicans isolates were resistant to fluconazole. Data were shown that 62% and 30.7% of resistant strains of C. albicans were found in patient with gastrointestinal cancer and lymphoma respectively. CONCLUSION: Data were shown that C. albicans is the most commonly identified species in oral candidiasis and majority of fluconazole resistant C. albicans were found in patients with gastrointestinal cancer and lymphoma. Therefore, we recommend an alternative drug instead of fluconazole as a first line of treatment for these type of cancers and administration of fluconazole in patients undergoing chemotherapy should be prescribed in accordance with the type of cancer.

IL-4 gene expression in adventitial layer (fibrous layer) of hepatic ovine and bovine hydatid cysts.

Cystic Echinococcosis is a parasitic disease with cosmopolitan distribution caused by the tape worm Echinococcus granulosus. Fibrous layer is developed around the cyst as a host immune response reaction. The aim of this study was to evaluate the rate of IL-4 gene expression in fibrous layer of bovine and ovine hepatic hydatid cysts using quantitative technique of Real-Time PCR. In this descriptive study the samples of hydatid cyst fibrous layer were taken from 6 bovine and 6 ovine hepatic hydatid cysts. Samples of normal liver tissue close to the cyst were also taken as controls. Total RNA from each sample was extracted and then converted to cDNA. Afterward, the rate of IL-4 gene expression for each sample was evaluated using real-time PCR technique. Data were analyzed by REST software (version 2.0.13, 2009). In sheep the rate of IL-4 gene expression in the fibrous layer of hepatic hydatid cysts was 1.98 times more than the rate of IL4 gene expression in control samples, but the difference was not significant (P = 0.561). In cattle the rate of IL-4 gene expression in the fibrous layer of hepatic hydatid cysts was 9.84 times more than that of control samples which was statistically significant (P < 0.001). With high rate of IL4 expression especially in fibrous layer of bovine hydatid cyst, it can be concluded that this interleukin may play an important role in host parasite relationship.

Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

BACKGROUND: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM) and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM). In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). MATERIALS AND METHODS: L. major (MRHO/IR/75/ER) from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN) medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS) 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. RESULTS: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. CONCLUSION: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

Cytokines gene expression in newly diagnosed multiple sclerosis patients.

Multiple Sclerosis (MS) is characterized by multiple areas of inflammation, demyelination and neurodegeneration. Infiltrating Th1 CD4+ T cells secrete proinflammatory cytokines. They stimulate the release of some cytokines, expression of adhesion molecules and these cytokines may cause damage to the myelin sheath and axons. In this study, we analyzed plasma levels and gene expressions of five important cytokines in the new diagnosed MS Patients by ELISA and Real time PCR. PCR amplifications were performed to determine the IL-17, IL-23, IL-10, IL-27 and TGF-ß mRNA expression levels using the SYBR Green PCR Kit. Our results showed significant decrease in IL-10, IL-27 and TGF-ß but there was no significant difference in the IL-17 and IL-23 between patients and healthy controls. Altogether, our results indicated that dysregulation of cytokines, mainly increased expression of pro-inflammatory cytokines and decreased expression of inhibitory cytokines occurred in MS patients. This study may shed light to the probable role of these cytokines in neurodegeneration mechanism and current or future use of cytokines in managing and treatment of multiple sclerosis.

Identification of Leishmania isolates from Varzaneh city, Isfahan province, Iran using nested polymerase chain reaction method.

BACKGROUND: Leishmaniasis, a parasitic disease, is caused by the Leishmania genus, a protozoan parasite transmitted by sand fly arthropods. Cutaneous leishmaniasis (CL) in old world is usually caused by L. major, L. tropica, and L. aethiopica complexes. One of the most important hyper endemic areas of CL in Iran is Isfahan province. Varzaneh is a city in the eastern part of Isfahan province. Due to different biological patterns of parasite strains which are distributed in the region, this study was design to identify Leishmania species from human victims using Kinetoplastid DNA as templates in a molecular PCR method. MATERIALS AND METHODS: Among 186 suspected cases, 50 cases were confirmed positive by direct microscopy after Giemsa staining. Species characterization of the isolates was done using Nested- PCR as a very effective and sensitive tool to reproduce mini circle strands. RESULTS: After Nested-PCR from all 50 cases, 560 bp bands were produced which according to products of reference strains indicate that the infection etiologic agent has been L. major. 22 (44%) of patients were females and 28 (56%) of them were males. Their age ranges were between 7 months and 60 years. CONCLUSION: According to the results of the study and the particular pattern of infection prevalent in the region, genetic studies and identification of Leishmania parasites are very important in the disease control and improvement of regional strategy of therapy protocols.

Identification of leishmania isolates from healing and nonhealing cutaneous leishmaniasis patients using internal transcribed spacer region PCR.

BACKGROUND: Cutaneous Leishmaniasis (CL) is a parasitic disease in most parts of Iran, especially in the Isfahan province. The most common form of CL is a self-healing lesion but in rare situations, infection might develop to non-healing forms. Clinical symptoms and treatment process might be influenced by several agents such as host immune response and parasite strains. In this study, the isolates which caused healing and nonhealing forms of CL in Isfahan were characterized by internal transcribed spacer (ITS) polymerase chain reaction (PCR) technique. OBJECTIVES: The aim of this study was to identify Leishmania species isolated from healing and non-healing CLs using PCR method. PATIENTS AND METHODS: Thirty patients resident in Isfahan province, with healing or non-healing form of CL were entered into this study. After DNA extraction, the identification of Leishmania isolates was done by ITS1-PCR method. RESULTS: Leishmania major was found as the predominant species (100%) in both healing and non-healing forms of CL. CONCLUSIONS: It seems that there is no difference between Leishmania species in healing and non-healing forms of CL. In order to recognize the reason of long lasting lesions in non-healing patients, the study about parasite strains and immune factors at the molecular level mostly in nonhealing patient is recommended.

Toll like receptor 2 and 4 expression in peripheral blood mononuclear cells of multiple sclerosis patients.

BACKGROUND: Multiple sclerosis (MS) is a T cell mediated autoimmune disease with unknown etiology. Appropriate MS therapeutic strategies need thorough understanding of both disease etiology and pathogenesis mechanisms. Ligation of TLR-2 and TLR-4 stimulates the production of several cytokines leading to CNS autoimmunity and neurodegenerative diseases. OBJECTIVE: To find a relationship between MS disability and TLR-2 and TLR-4 expression on mononuclear cells in the blood of MS patients. METHODS: Forty-five new case (NC) MS patients (33 females and 12 males) and 45 age and gender-matched healthy controls (HC) were recruited to the study. PBMCs were prepared and the expressions of TLR-2 and TLR-4 were assessed by flowcytometry technique using appropriate monoclonal antibodies. RESULTS: Our results showed that the expression of TLR-2 and TLR-4 proteins in the patients group was significantly higher than that of healthy controls. TLR-2 but not TLR-4 was correlated with expanded disability status scale (EDSS) scores. CONCLUSION: High expressions of TLR-2 and TLR-4 may represent a state of innate immune activation in patients with MS.