Effect of interleukin-22 on immunogenicity of DNA vaccine encoding TSA gene of leishmania major in BALB/c mice

Background and purpose: Previous Research shows the use of plasmids containing genes TSA to be useful as vaccines for Leishmania major. Recently, the role of interleukin-22 (IL-22) in tissue repair has been demonstrated. In this research, the effect of IL-22 on encoding TSA gene of Leishmania major in BALB/c mice was assessed

The Effect of Vitamin D3 Alone and Mixed With IFN-γ on Tachyzoites of Toxoplasma gondii (RH Strain) Proliferation and Nitric Oxide (NO) Production in Infected Macrophages of BALB/C Mice

Introduction: Toxoplasma gondii is an obligatory interacelullar parasite that infects nucleated cells in its intermediate hosts. The aim of the present study was to determine the effect of vitamin D3 on the multiplication of T. gondii in peritoneal macrophage of Balb/c mice and nitric oxide production by macrophages. Methods: According to usage of vitamin D3 (one dose or seven doses) and INFγ in vitro and in vivo, this study was divided into four experiments. In all experiments, the macrophages were col­lected from peritoneum and cultured in RPMI-1640. Then the supernatants were collected after 24 h and their nitric oxide was measure. After 96 h, the macrophages were collected and stained and the number of tachyzoites was measured. Results: The first experiment (the mice were infected with tachyzoites and after 2 h, got one dose vita­min D3 intraperitonealy) showed the best results. The mean of tachyzoites per macrophages was 2.37, and mean ± SD of nitric oxide was 187.8 ± 9.Discussion: High-level production of nitric oxide may be related to the only one injection of vita­min D3. The injection in long time might suppress the immune system

Expression of Plasmid Encoded GRA4 Gene of Toxoplasma gondii RH Strain in CHO Eukaryotic Cells

Background: Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from Toxoplasma gondii. Methods: We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium- phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper. Results: Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM+ serum in western blot analysis. Conclusion: pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model

Diagnosis of liver and pulmonary Hydatid cyst by Dot-ELISA, IFA and IHA tests

History and Objectives: Although some serological tests are not highly sensitive in diagnosis of pulmonary hydatid cyst however Dot-ELISA is introduced as a simple, sensitive and a very efficient means of pulmonary hydatid cyst diagnosis. In the present study, we compared the validity of IHA, IFA and Dot-ELISA techniques in diagnosis of anti-echinococcal antibodies caused bu Echinococcus granulosis in cystic liver and lung hydatid disease. Materials and Methods: 244 sera of which 30 samples from hydatid patients, 104 sera from patients suffering a disease other than hydatidosis and 110 sera from healthy individuals were collected mainly from Tehran's hospitals. The hydatid patients were confirmed by surgery and consisted of 14, 10, 6 patients with liver, lung and liver-lung hydatid cysts respectively. The sera were tested by IHA, IFA and Dot-ELISA techniques. Sensitivity, specificity, predictive values, false positivity and false negativity of each test were evaluated in diagnosis of liver and lung hydatid cyst. Results: The results indicated that the sensitivities of IHA test for liver, lung and liver-lung cysts were 78.7, 60 and 100 respectively, while the sensitivities of IFA in detecting liver lung and liver-lung cysts were 93, 80 and 100 respectively. In this regard, the Dot-ELISA showed the best results whereas its sensitivities for liver, lung and liver-lung cysts were 100 each. Conclusion: These results indicate that detection of anti-echinococcal antibodies in human sera is superior by Dot-ELISA compared to IHA and IFA tests. Hence, the application of this test in medical laboratories for hydatid cyst diagnosis can be recommended

In Vitro Effect of Folic Acid and Cobalamin (Vitamin B12) on Adhesion and Growth of Giardia lamblia

Giardia lamblia is one of the most common intestinal protozoan parasites infecting human in the world. The goal of this study was searching for in-vitro effect of folic acid and cobalamin on adhesion and growth of G. lamblia as two important mechanisms in the pathogenesis in TYI-S-33 medium. G. lamblia trophozoites were obtained by in- vitro excystation procedure. Three groups of Giardia trophozoites were analyzed: control group, G.lamblia was cultured in TYI-S-33 without any vitamin, 2nd group with 0.1 µg/ml vitamin B12 or folic acid, and 3rd group with 0.5 µg/ml of vitamin B12 or folic acid. All culture media tubes incubated at 37 ºC. After 2 h of incubation, the adherence into borosilicate culture tubes, and after 24 h the growth of trophozoites were measured .The results showed that in vitamin B12 groups, the growth was increased significantly (P≤ 0.05) but the adherence decreased significantly (P≤ 0.05). Folic acid inhibited the growth rate significantly (P≤ 0.05), but it increased adherence in axenic culture significantly (P≤ 0.05). The results showed that vitamin B12 and folic acid altogether might reduce pathogenesis of G. lamblia by reducing adherence and growth, respectively

The Effect of Alkanna tincturia and Peganum harmala Extracts on Leishmania major (MRHO/IR/75/ER) in vitro

"nBackground: Cutaneous leishmaniasis is an important health problem caused by Leishmania spp. As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. In the present study, inhibitory and kill­ing effects of Peganum harmala and Alkana tinctoria extracts on amastigotes and promastigotes forms of Leishmania were evaluated in-vitro."nMethods: The seeds of Peganum harmala, Stems and roots of Alkanna tictoria were collected and crude extrac­tion carried out. In this experimental study,  Leishmania major promastigotes were cultured in RPMI-1640 with 10% FBS at 22-26°C, and infected macrophages with amastigotes   were cultured in RPMI-1640 with 10% FBS at 37°C in 5% CO2. Then the extracts of each plant were added to cultivated parasites and incubated for 3 days. Promastigote and amastigote assay was carried out using   counting assay based on growth inhibition."nResults: The results indicated that both extractions can inhibit the growth of promastigotes, and in concentra­tions of 40µg/ml of P. harmala , 200µg/ml of A. tincturia, and 20 µg/ml of equal combination of P. hamala and A. tincturia are Inhibitory Concentration (IC50) for parasites growth. By adding these concentra­tions of the extracts to the infected macrophages in the culture, their effects were separately eva­lu­ated. The mean of amastigotes number in macrophages in the culture with P. harmala, A. ticturia, combina­tion and control groups were 0.7, 0.7, 0.6, 2.3 amastigotes per macrophage, respectively."nConclusion: By this method, inhibition of intracellular and extracellular growth of L. major was demon­strated suggesting that, plant drugs with efficacy and safe products can be applied as new treatment for cutane­ous leishmaniasis

Differential Genomics Output and Susceptibility of Iranian Patients with Unilocular Hydatidose

Background: The objective of this study was to investigate HLA-DRB1*and DQB1* allelic polymorphisms in Iranian patients with hydatidose. This is the first survey dealing with the correlation between HLA-DRB1* and DQB1* al-leles and cystic echinococcosis in Iranian patients. Methods: The study was carried out on 56 patients with confirmed cystic echi-nococcosis and 30 apparently healthy individuals living in Arak- Markazi Prov-ince by HLA-DRB1 and DQB1 typing through PCR-SSP method. The first step was to identify the patients and blood sampling. DNA was prepared from whole blood and PCR-SSP with 31 primer mixes for per sample was used. PCR reaction mixtures were loaded in agarose gels and bands were observed under UV illumination and gel document after electrophoresis. Analysis of results was carried out with specific softwares and frequency and interpretation tables for calculation of P-value in χ2 test were provided via Fisher΄s exact test. Signifi-cant samples were analyzed by logistic regression and odds-ratios were calculat-ed. Results: A statistically significant positive association was found between HLA-DQB1*03 and the resistance to cystic echinococcosis (P<0.02) (odds-ratio=2.87). Conclusion: Immunogenetic susceptibility to unilocular hydatidose varies ac-cording to the HLA antigens in Arak, Markazi Province, and DQB1*03 mole-cules are associated with the level of immune response to parasite antigens

Characterization and expression of GRA7 gene of Toxoplasma gondii RH strain in eukaryotic pcDNA3 plasmid

Background: Toxoplasmosis, as a widespread and important disease, can cause severe complications in the congenital and HIV cases. Vaccination is one of the preventive approaches in humans and domestic animals. The GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by the infected host cells , has been considered as a candidate to produce a vaccine . Materials and Methods: In this experimental study, the inserted plasmid containing GRA7 gene was extracted from TOP10 bacteria and digested with the BamH1 and EcoR1 enzymes. The isolated gene was inserted into the pcDNA3 plasmid . The cloning was confirmed by the PCR and sequencing. The protein expression was confirmed by the SDS-PAGE and Western blotting. Results: The results showed that the GRA7 gene was cloned into the pcDNA3 plasmid. The isolated gene cloned in pcDNA3 was confirmed by PCR and showed the 733bp band. The pcGRA7 plasmid expressed in the CHO cells showed the 29 kDa band using the SDS-PAGE and Western blotting. Conclusion: Considering that cloning of GRA7 gene has been done in the pcDNA3 plasmid and transformed in the eukaryotic cells, it can be used for the DNA vaccine researches

Determination and cloning of the gene encoding EG95 protein in Iranian isolate of Echinococcus granulosus

Background: Echinococcus granulosus is a cestode parasite that causes cystic hydatid disease in humans worldwide. The gene encoding EG95 protein may be a good candidate to design a DNA vaccine to prevent the disease. Considering the importance of EG95 gene and the scarceness of research on it in Iran, this study was carried out to determine and clone the gene encoding EG95 from Iranian isolate of E. granulosus. Materials and Methods: At the first stage, protoscoleces was isolated from hydatid cyst fluid and then RNA was extracted from protoscoleces and after performing RT-PCR, the amplified cDNA samples were detected by gel electrophoresis. In next stage, the obtained gene was cloned in pTZ57R/T vector. Two methods were used for conformation of cloning: colony PCR amplification and digestion with the EcoRI and XhoI restriction enzymes. Finally, the cloned EG95 gene in pTZ57R/T vector was sequenced.Results: Homological comparison of sequences showed that cDNA of EG95 in Iranian isolate of E. granulosus had 492 bp and was different from the standard strain of EG95 reported from New Zealand and Australia (X90928.1). Moreover, cloning of EG95 gene in pTZ57R/T plasmid was confirmed by digestion of this plasmid with the restriction enzymes. Conclusion: The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies