Heterologous Expression of Human IFNγ and Anti-IL17 Antibody in Leishmania tarentolae Promastigote.

BACKGROUND: Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function. METHODS: In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH-hIFNγ-antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47 pg/mL. RESULTS: Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH-hIFNγ-antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite. CONCLUSIONS: The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

Investigating the Effect of Satureja khuzestanica Essential oil on MDR1 Gene Expression in Leishmania major.

BACKGROUND: Cutaneous leishmaniasis is among the neglected diseases in the world. Pentavalent antimonial compounds are considered the first-line treatment for this disease. However, using alternative natural products has received great attention due to the side effects of chemical drugs and drug resistance of the Leishmania parasite. The present study aims to investigate the effect of Satureja khuzestanica essential oil (SKEO) on MDR1 gene expression. METHODS: In this study, standard strains of Leishmania major promastigotes were exposed to 5, 10, 15, and 20 µg/ml of SKEO. MDR1 gene expression of parasites exposed to essential oil was evaluated using real-time PCR. GAPDH was employed as the housekeeping gene for internal control. RESULTS: Despite the increase, no statistically significant difference was observed in the relative expression of the MDR1 gene between the control group and the groups containing 5, 10, and 20 µg/ml of SKEO (P > 0.05). The relative expression of the MDR1 gene significantly increased in the group containing 15 µg/ml of essential oil compared to the control one (P < 0.05). CONCLUSION: This study showed that the use of essential oil of Satureja khuzestanica plant can have an increasing effect on the expression of MDR1 gene of Leishmania promastigotes, which is the best case if Satureja khuzestanica essential oil reduces the expression of MDR1 gene. So it seems that the use of essential oil of Satoria plant is effective in controlling Leishmania parasite, but its concentrations induce drug resistance. As a result, concentrations of essential oil should be used that have a controlling effect on the growth and proliferation of Leishmania parasite and also have the least effect on the induction of MDR1 gene expression.

Toxoplasma gondii vaccine candidates: a concise review.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. It has been shown that the severity of symptoms depends on the functioning of the host immune system. Although T. gondii infection typically does not lead to severe disease in healthy people and after infection, it induces a stable immunity, but it can contribute to severe and even lethal Toxoplasmosis in immunocompromised individuals (AIDS, bone marrow transplant and neoplasia). The antigens that have been proposed to be used in vaccine candidate in various studies include surface antigens and secretory excretions that have been synthesized and evaluated in different studies. In some studies, secretory antigens play an important role in stimulating the host immune response. Various antigens such as SAG, GRA, ROP, ROM, and MAG have been from different strains of T. gondii have been synthesized and their protective effects have been evaluated in animal models in different vaccine platforms including recombinant antigens, nanoparticles, and DNA vaccine. Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and Google Scholar were searched for articles published up to 2020.The current review article focuses on recent studies on the use and usefulness of recombinant antigens, nanoparticles, and DNA vaccines.

Identification and differentiation of Fasciola hepatica and F. gigantica using multiplex PCR technique.

We aimed to present an alternate method instead of PCR-RFLP and also develop an optimized method for rapid, time-saving and affordable molecular-based approach to discriminate species of liver fluke, Fasciola hepatica and F. gigantica. Seventy-six samples of F. hepatica and 28 F. gigantica were collected from the slaughterhouses of endemic regions in Iran. Following a comprehensive analysis of the mitochondrial complete sequences of both F. hepatica and F. gigantica, the extracted DNAs from all samples were used as templates in multiplex PCR reactions containing two sets of primers specific for cytochrome c oxidase I (cox I) gene of both species. In a parallel experiment, PCR-RFLP was performed for each sample using internal transcribed spacer (ITS1) sequence. Furthermore, following a PCR amplification for cox I gene, the amplicons were purified for sequencing. To assess the validity of the multiplex PCR approach, the obtained data from the multiplex PCR and PCR-RFLP experiments were compared with each other. By sequence analysis of 104 samples, 76 and 28 samples were identified as F. hepatica and F. gigantica, respectively. Results revealed 100% and 92% of accuracy as for multiplex PCR and PCR-RFLP. The designed multiplex PCR strategy offers a valid alternative approach to the conventional methods with distinctive features including convenience, cost-effectiveness, time-saving (3 hours from sampling to obtain final results) and high efficacy.

Bioinformatics analysis for the purpose of designing a novel multi-epitope DNA vaccine against Leishmania major.

Leishmaniasis is one of the main infectious diseases worldwide. In the midst of all the different forms of the disease, Cutaneous Leishmania (CL) has the highest incidence in the world. Many trial vaccines have been developed with the purpose of generating long-term cell-mediated immunity to Leishmania(L) major. As there is not any multi-epitope DNA vaccine with high efficacy against L.major, the aim of this study is to design a new multi-epitope DNA vaccine in order to have effective control upon this infectious disease through the immune bioinformatics. The L.major antigens: Gp63, LACK, TSA, LmSTI1and KMP11 were selected to design a multi-epitope DNA vaccine. The initial structure of the DNA vaccine was designed, benefiting from Gen Bank's website information. Epitopes of MHC-I antigens were predicted through the Immune Epitope Database (IEDB), and the selected epitopes were used to make vaccines construct along with linkers. New multi-epitope vaccine including 459 nucleic acids designed, and inserted between BamH1 and HindIII restriction sites of pCDNA3.1 mammalian expression vector. 12 epitopes among the chosen antigens were selected by two servers (IEDB and ANTIGEN). They had high stability and high antigenic power. Physicochemical features of vaccine measured by ProtParam server, and this structure was thermostable and hydrophilic. it's a suitable model to study on the animal and human phases. The designed vaccine is expected to be an effective candidate through development of (CL) vaccines. However, the effectiveness of this vaccine should also evaluate in vivo model.

Diagnosis of Toxoplasma Infection in Allogenic Pre HCTSP Patients Using Molecular Methods.

Background: We aimed to estimate the incidence of Toxoplasma infection in T. gondiiseropositive patients under allogeneic hematopoietic stem cell transplantation (HSCT). Methods: The present research was a prospective study on 54 whole blood samples of allogeneic HSCT recipients, who were referring to bone narrow transplantation centers affiliated to Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2018. All patients were IgG positive against T. gondii. Results: Overall, 54 Toxoplasma positive pre-HCTSP patients were enrolled. 53.7% (n= 29) were male, also 1.9% (n=1) had germ-line type of the disease. The Multiple myeloma patients had higher age in comparison with other disease, but pairwise comparison showed the difference of age between Multiple myeloma patients were statistically significant with Acute lymphoblastic leukemia, Acute myeloblastic leukemia and Huntington's disease (P< 0.05). The results of PCR assay showed 5.6% (n= 3) of the patients were infected with Toxoplasma. Conclusion: PCR method has detected considerable incidence of Toxoplasma infection for monitoring HSCT recipients at risk for toxoplasmosis, and many patients who showed the incidence of toxoplasmosis had previous infections with the Toxoplasma parasite.

Methylation and Polymorphism in CDH1 Gene Promoter Among Patients with Diffuse Gastric Cancer.

Background: The promoter methylation and single nucleotide polymorphisms (SNPs) affect the transcription activity of cancer-related genes in several cancers including diffuse gastric cancer (DGC). Here we aimed to evaluate the promoter methylation status and the rs16260 at the promoter region of the CDH1 gene in DGC. Methods: This case-control study was performed of 48 formalin-fixed paraffin-embedded (FFPE) blocks of DGC patients and 41 fresh frozen tissue samples of healthy individuals. Methylation status was evaluated using methylation-specific polymerase chain reaction (PCR) and the rs16260 at the promoter region of the CDH1 gene was assessed using PCR and sequencing method. Results: The occurrence of methylation at the promoter region of the CDH1 gene in DGC patients was significantly higher than control samples (P < 0.0001). The methylated status was significantly associated with the poor differentiated histological type of DGC (P = 0.0428). The frequency of AC genotype and the A allele in DGC patients was significantly higher than the control subjects (P = 0.006 and 0.003, respectively). Conclusions: Here we showed that methylation at the CDH1 promoter may contribute to the DGC development, and also the AC genotype was associated with the risk of DGC.

Gastrointestinal parasites in immunocompromised patients; A comparative cross-sectional study.

Gastrointestinal parasites (GIPs), including helminths and protozoa species, are a major health problem in many parts of the world. About 3.5 billion people are affected by the parasites worldwide. GIPs are one of the leading causes of death among immunocompromised individuals and can cause serious clinical complications, especially in people with Human Immunodeficiency Virus (HIV)/AIDS, hemodialysis patients, and transplant recipients. This study aimed to compare the prevalence of GIPs among immunocompromised patients and immunocompetent individuals in Lorestan province, West Iran. In the current study, with a sampling of 232 participants (114 hemodialysis, AIDS, and organ transplantation immunocompromised patients and 118 immunocompetent individuals as the control group), demographic characteristics and risk factors for GIPs were collected through a pre-designed questionnaire. Stool samples of patients and the control group were examined for GIPs using different diagnostic methods including direct smear (saline and Lugol's iodine), Ziehl-Neelsen staining, agar-plate culture, and concentration method (formalin ether sedimentation). To evaluate the relative status of the immune system, TCD4+ cells were counted in the blood samples of the subjects by flow cytometry. The results were analyzed using SPSS 21 software, Fisher exact, and chi-square statistical tests. Multivariate modeling of the data was performed using logistic regression. The prevalence of GIPs in immunocompromised patients was more than twice that of immunocompetent individuals in the control group (42.06% vs. 17.79%). The most prevalent parasites identified among immunocompromised patients were Cryptosporidium sp. (27.1%), Blastocystis sp. (16.7%), and Entamoeba coli (14.6%) respectively. Cryptosporidium sp. had the highest frequency among hemodialysis patients (6.49%), AIDS patients (26.92%), and transplant recipients (18.18%) respectively. Patients with AIDS had the highest positive results for Cryptosporidium sp. followed by Microsporidia sp. (23.7%). In immunocompetent individuals, the highest prevalence of GIPs was related to Blastocystis sp and Trichomonas hominis (28.57%). Statistical analysis of the data showed that there was a statistically significant difference between various age groups regarding infection with GIPs so the highest rate of GIPs infection was observed in the age group lower than 50 years (P = 0.035). The statistical difference between the variable of location and infection with GIPs was insignificant but remarkable (P = 0.070). According to the results, it can be concluded that GIP is more common in immunocompromised patients than in immunocompetent individuals with cryptosporidium sp. predominance. Due to the favorable conditions of immunocompromised patients for GIPs and considering them as one of the important sources of parasitic infections and parasite transmission in society, control, prevention, and monitoring of their social behaviors along with health issues are inevitable.

Prevalence and characterization of Blastocystis spp. in central southwest of Iran.

Blastocystis is one of the most common intestinal protozoan parasites of human all over the world. Due to high prevalence and widespread species diversity of this parasite and the possibility of potential transmission of them to human, this study was carried out to investigate the prevalence of Blastocystis and characterize its subtypes (genotypes) in Shahrekord, central southwest of Iran. Microscopic examination showed that 6.4% (55/864) of the subjects were infected with Blastocystis spp. All of positive samples were sequenced successfully. The molecular methods showed that the infection was caused by four subtypes, including ST1, ST2, ST3 and ST7. The most common identified genotype of Blastocystis was ST3 (36.4%). The statistical analysis of data showed that there was no significant correlation between the prevalence of Blastocystis or its subtypes with age, gender, job, level of education, contact with animals, and clinical manifestations of infection in the patients. While the frequency of blastocystosis in this population seems to be less than many parts of Iran, but it can be argued that Blastocystis is still the most common intestinal parasite of human in this region compared with other intestinal parasites and it seems that the transmission of infection has an anthroponotic pattern.

Molecular characterization of Theileria spp. in livestock and the first report on the occurrence of Theileria sp. OT3 in Iran.

Theileria lestoquardi, T. ovis, and T. annulata are recognized as major causative agents for ovine and cattle theileriosis in Iran, respectively. Recently, there have been reports on the presence of Theileria spp. (Theileria sp. OT1, Theileria sp. OT3, and Theileria sp.). In this study, 37 blood samples were collected from sheep and cattle with clinically suspected Theileria infection in the Northwest of Iran. The samples were analyzed using a light microscope. DNA samples were amplified via nested-polymerase chain reaction (PCR) of 18S rRNA gene. The amplicons were digested with HpaII, following restriction fragment length polymorphism (RFLP) and sequenced to reconfirm Theileria species. The microscopic examination indicated that 4 out of 37 (10.8%) blood samples were infected with Theileria spp. Based on the nested PCR-RFLP and sequencing data, 5.4%, 13.5%, and 27% of blood samples were infected with Theileria sp. OT3, T. ovis, and T. annulata, respectively. The pairwise distance matrix of Theileria sp. OT3 showed 99.8-100% identity and 0-0.2% divergence in comparison with the registered sequences. The present study is the first report of Theileria sp. OT3 in Iran. To the evaluate evolution of Theileria spp. and providing resultant genetic data, further research with a larger sample is necessary in the region.