Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems

Materials and Methods: To express LmSTI1 in the methylotrophic yeast Pichia pastoris [P. pastoris], the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively

Results: The expression level was 0.2% of total soluble proteins

Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization

Comparative analysis of CD4+ and CD8+ T Cells in tumor tissues, lymph nodes and the peripheral blood from patients with breast cancer

CD4+ and CD8+ T cells are the main types of lymphocytes in cell-mediated immunity and play a central role in the induction of efficient immune responses against tumors. The frequencies of T cell subtypes in the peripheral blood and tumor tissues, and draining lymph nodes [dLN] can be considered as useful markers for evaluation of the immune system in cancers. In this study, the frequencies of CD4+ and CD8+ T cells in blood, tumor tissues, and dLN samples of breast cancer patients were compared with each other and with similar tissues from normal individuals. Immunophenotyping was carried out by flow cytometry and the expression levels of CXCL10, granzyme B, and mammaglobin were evaluated by real-time PCR In the peripheral blood, there were no differences in the T cell subsets between the patients and the normal individuals. The frequency of CD8+ T cells was significantly higher in tumor tissue than normal breast tissues while granzyme B expression was similar. Based on mammaglobin expression levels, dLN have been classified into micro- and macro-metastatic dLN. We found significantly lower frequency of CD4+ in macro-metastatic dLN than micro-metastatic dLN. CD8+ frequency was similar in both dLN; however, granzyme B expression was higher in micro-metastatic ones. There was not any significant difference in CXCL10 expression between the two types of dLN. Based on our results, although the tumor does not affect the systemic immunity, tumoral cells affect the local immune system in the tumoral tissues and the metastatic dLN.

Study of 5 HT[3] and 5 HT[4] receptors expression in HT29 cell line and human colon adenocarcinoma tissues

Serotonin [5HT] has been shown to be a mitogenic factor in several carcinomas. Its mitogenic effect is elicited through a wide range of 5HT receptor subtypes. In this study, the effects of 5HT, 5HT[3] [1-phenylbiguanide hydrochloride] and 5HT[4] [cisapride] agonists in promoting the growth of the HT29 cell line and the growth-inhibition effect of the 5HT[3] receptor antagonist [Y-25130 hydrochloride] and 5HT4 receptor antagonist [RS 23597-190] were investigated. The expressions of 5HT[3] and 5HT[4] receptors in human colon cancer tissues and the HT29 cell line were studied. The growth-promoting and growth-inhibition effects of 5-HT, 5HT[3] and 5HT[4] agonists and antagonists on the HT29 cell line were studied using MTT assay. Receptor expression has been demonstrated by western blotting. The results showed that 5HT, 5HT[3], and 5HT[4] agonists caused significant proliferation of HT29 cells. 5HT[3] and 5HT[4] receptor antagonists had an inhibitory effect on the growth of these cells. Western blot analysis gave bands from colon tissue extracts and the HT29 cell line. The results indicate which 5HT[3] and 5HT[4] receptors are significantly expressed in both colon cancer tissue and the HT29 cell line. Expression for the 5HT[3] receptor is more potent. Furthermore, 5HT plays a mitogenic role in colon cancer cells and antagonists of 5HT[3], and 5HT[4] receptors can inhibit cancer cell growth

Immune responses of mice immunized with active recombinant shiga toxin and its derivatives

Bacterial protein toxins have been exploited as therapeutic agents and as vaccines. An issue of deserving interest is development of new generations of vaccines and immune adjuvants. In this study an active assembled recombinant Shiga toxin of Escherichia coli [rStx1] and its derivatives, recombinant A and B subunits [Stx1-A and Stx1-B], were used to immunize mice. The elicited antibody responses were compared with and without using adjuvant. Protection against intraperitoneal lethal dose of rStx challenge was observed by immunization with sublethal dose of rStx1, rStx-A and rStx-B subunits. The immunological studies on toxin subunits can be used for immunization against systemic shiga toxin mediated disease and also subunits as a vector for antigen presentation in immunotherapeutic approaches. In our experiment, while stimulation of the immune system by A and B subunits were different, both subunits produced neutralizing antibodies. Regarding B subunit the amount of specific IgG1/IgG2a antibody ratio was higher than A subunit. In addition B subunit stimulated proliferation of immune cells with IFNê production the same as rStx1, suggesting that B subunit can be used as an immunomodulator to stimulate the immune response in conjunction with other recombinant proteins

Immune response following oral immunization with BCG encapsulated in alginate microspheres

Different methods have been used for BCG vaccination. Alginate microspheres are useful in delivery of vaccines to the gastrointestinal tract by oral route. To compare the immune response following oral microencapsulated and subcutaneous [SC] route administration of BCG vaccine in BALB/c mice. Alginate microspheres were produced by an internal emulsification method within olive oil. Four groups of mice were studied, including two groups receiving oral gavages of microencapsulated and free BCG, one receiving SC injection of BCG, and a control group. T cell proliferation, specific anti-BCG total IgG, and IgG subclasses [IgG1 and IgG2a] were compared between groups 5 and 12 weeks after vaccination. The best result was achieved using oral microencapsulated form in comparison with oral BCG alone. Delivery of oral BCG with alginate microspheres is an effective way to induce immune response in BALB/c mice

Characterization of leishmania species and L. major stains in different endemic areas of cutaneous leishmanasis Iran

Both zoonotic and anthroponotic cutaneous leishmaniasis [CL] caused by L. major and L. tropica, respectively, are endemic in different parts of Iran. This study was performed to investigate the new changes in epidemiological pattern of CL, and to identify the species of Leishmania and the strains of L. major isolated from different endemic areas of Iran. Seventy-two isolates from 245 samples collected from different endemic areas of Iran were characterized by monoclonal antibodies [mAb] specific for L. major, L. tropica, and L. infantum. Flow cytometry, isoenzyme analysis and PCR amplification were used for identification. Isoenzyme analysis was carried out by PAGE and cellulose acetate. Eight enzymes including malate dehydrogenase [MDH], malic enzyme [ME], glucose 6-phosphate dehydrogenase [G6PD], glucose phosphate isomerase [GPI], nucleoside hydrolase inosine [NHi], nucleoside hydrolase deoxy inosine [NHd], superoxide dismutase [SOD] and 6-phosphogluconate dehydrogenase [6PGD] were used for isoenzyme analysis. PCR assay was developed using specific primers against kinetoplast DNA. The isolates were compared with reference strains [RS]. Data obtained from different methods showed 45 out of 72 isolates were similar to RS of L. major and 22 similar to L. tropica, and also five samples were identified as Crithidia. Isoenzyme migration pattern of L. major isolates was indistinguishable from each other in six enzymatic systems but differences were observed in profile of two enzymes of SOD and MDH. The data indicate that L. major species are dominant in the studied endemic areas, and different strains of L. major are present in different geographic areas of Iran. Moreover, it seems that enzymatic system is more useful approach than others for characterization of Leishmania species and L. major strains

Function of neutrophils in different phases of chronic myelogenous leukemia

In chronic myelogenous leukemia [CML], the mature granulocytes originate from a stem cell line harboring an abnormal chromosome, therefore it is possible that metabolic-functional abnormalities occur in the morphologically mature cells. In the present study, the phagocytic activity including intracellular killing, nitro blue tetrazolium [NBT] reduction, and phagocytosis were studied in 37 CML patients in different stages of the disease. The results were compared with those of 37 normal controls. Patients' neutrophils display significantly lower intracellular killing [P<0.01], NBT reduction [P<0.01] and phagocytosis [P<0.001] than that of normal controls. Analysis of the results revealed an inverse correlation between phagocytic activity and leukocyte count or percentage of immature cells [r = -0.3, P<0.01]. In conclusion, the results indicate that neutrophils of CML patients have impaired phagocytic activity. This defect is more prominent in patients in blastic phase, whereas patients in remission show normal values