[Expression, purification and investigation of sap2 Candida albican's activity in pichia pasturis yeast system]

The secreted aspartic proteinases [Sap2] of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap2 enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein. C. albicans Sap2 gene was amplified by PCR with sticky ends, EcoR1 and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap2 gene was cloned into pGAPZ?A expression vector. The construct was transformed into P. pasturis yeast; the Sap2 gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap2 protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed. In this study, we successfully amplified C. albicans Sap2 gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap2 protein was obtained after 96 h, at 30 C. Expression of Sap2 gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap2 protein had maximum activity in an acidic pH

[Study on co-administration of GM-CSF, IL-15 on the enhancement of the immunogenicity of HIV-1 P24-Nef expression vector in BALB/c mouse model]

Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct

[Comparative study of cytotoxic effect of enterohemorrhagic escherichia coli verotoxin1 on vero and raji cell lines]

Verotoxin is a member of Shiga toxin family. This family contains AB protein toxins with an enzymatic [A] and a binding [B] compartment. Cells that have receptor [Gb3] are sensitive to cytotoxic effects of toxin. It has been shown that various tumor cells have Gb3 receptor and are selectively sensitive to apoptotic effect of verotoxin. Studies on tumor cell lines and laboratory animals have shown antineoplastic and antiangiogenesis effects of this toxin. The aim of this study was comparison of cytotoxic effect of verotoxin 1 on two cell lines: Vero [gold standard for evaluation of cytotoxic effect of Verotoxin] and Raji [a cell of a cultured line of lymphoblastoid cells derived from a Human Burkitt's lymphoma patients]. The toxigenic strain was cultured and the production of toxin was evaluated by reverse passive latex agglutination test. Verotoxin 1 was purified by affinity chromatography. Vero and Raji cell were treated with serial dilutions of toxin, and viability was evaluated by MTT test. Our result indicated that Verotoxin has cytotoxic effect on Raji cell lines. This effect is directly related to toxin concentration. Differences on cytotoxicity of toxin on Raji cells at 1:4-1:128 dilutions in relation to cytotoxicity of toxin on Vero cells at the same dilutions were considered statistically significant [P<0.05]. But difference of cytotoxicity of toxin at higher dilutions was not significant. Our results revealed that Verotoxin has cytotoxicity on Vero and Raji cells, and this effect on Vero cells is more than Raji cells [P<0.05]

Flowcytometric analysis of tumor associated macrophages in invasive ductal carcinoma of breast

Invasive ductal carcinoma is the most common type of breast cancer in Iran. Impaired immune responses occur frequently in cancer patients, but the mechanisms of the induced immune defects remain poorly understood. It is believed that infiltrated immune cells, especially macrophages, may provide help for tumor cell growth and metastasis. To analyze the status of tumor associated macrophages [TAM] by immunophenotyping method. Twenty-three women suffering from breast cancer were examined; nineteen of them were confirmed histologically to have invasive ductal carcinoma. Tumor cell suspensions from biopsy specimens and peripheral blood mononuclear cells from patients and matched controls were processed for analysis by flow cytometry. No significant changes in the percentages of intra-tumor leukocytes and macrophages in the different stages of tumor were observed. There were no significant differences in the percentages of leukocytes [CD45[+], monocytes [CD45[+]/CD14[+] and activated mono-cytes [CD14[+]/HLA-DR[+] and CD14[+]/CD16[+] in the peripheral blood of patients and controls. The results of this study indicated that human breast cancer contain substantial, although variable numbers of macrophages, however, the activation status of these macrophages remain to be elucidated

Investigation of NK cell population in peripheral blood and tumor lesions of patients with breast cancer

Alteration in peripheral blood lymphocytes [PBLs] is usually investigated to provide an evidence of the host immune responses to tumor antigens. The tumor infiltrating NK cells interact most closely with the tumor cells and more accurately reflect tumor host interactions. To analyze peripheral blood and tumor associated Natural Killer [NK] cells in patients with breast cancer by immunophenotyping. Twenty women suffering from breast cancer were examined; 12 of them were confirmed histologically to be invasive ductal carcinoma. PBL and tissue samples from patients and matched control group were processed for analysis by flow cytometry. Results of PBL analysis indicated a significant [P<0.05] increase in both the total number and activated NK cells in invasive ducal carcinoma patients compared to normal controls. No significant differences were noticed in the percent of NK cells and their activation marker expression in intra tumor lesion of the invasive ductal carcinoma and other tumors compared to benign lesions, however a decrease in the total NK number and activated NK cells was observed with progression of the tumor. Data of this investigation conclude that the total and activated NK cell number increase in peripheral blood of patients with breast cancer. The relationship between peripheral blood and intratumor NK cells needs more clarification, however, a decrease in intratumor NK cell number and their activation status occurs with tumor progression