Proteinase 3(PR3) is a human polymorphonuclear leukocyte serine proteinase and is the main target antigen for antineutrophil cytoplasmic antibodies (ANCA) found in Wegener's granulomatosis (WG). Wedevefoped a stable expression system for conformationally intact recombinant PR3 (rPR3) in Chinese hamster ovary cells (CHO-cells). The part of PR3 cDNA that encoded the active form of PR3 was selected by using appropriate primers, and a signal sequence was also added in front ofPR3 cDNA. The signal sequence- PR3 (S-PR3) was cloned into the pME 18 expression vector and the result product was electroporated into E. coli (DH5 a strain). After isolation and purification, the presence of pMEI8-S-PR3 was confirmed by using appropriate restriction endonuclease and agarose gel electrophoresis. The pME18- S-PR3 was electroporated with CHO-cells and the presence of rPR3 was tested in culture medium after 10 days. There was 12 ng/mL rPR3 in culture medium that had activity and was recognized by ANCA in ELISA.

Objective. Dendritic cells (DCs) are derived from bone marrow precursor cells and migrate to lymphoid and non-lymphoid organs via blood. DCs have been shown to play an important role in inducing immunity and/or tolerance. Several DC subgroups with different biological roles have been identified. Myeloid DCs usually induce immune response and tolerance is usually induced by lymphoid DCs. Differences in frequency and ratio of DC subtypes appears to be one of the reasons for the difference in immune response in various lymphoid tissues. Considering the recent applications of different DC subgroups for the induction of immune response or tolerance induction and their use as vaccine for the immunotherapy of tumors or autoimmune diseases. Materials and Methods. In this study, it was tried to investigate the function and phenotype of Splenic DCs, these cells were separated by mechanical and enzymatic methods from mouse spleen and their surface markers using tlowcytometry. To study the ability of separated DCs in immune response induction, their functional capability was investigated by lymphocyte transformation test. Result. Following collagenase digestion of Splenic tissue, using density gradient centrifugation (13% nycodenz) and adherance characteristic of DCs to tissue culture dish, more than 7-9 X 105 DCs were separated from each spleen with purity of 90-95 percent. The phenotypic analysis of these cells showed that at least three DC subpopulations: CD11c+, CD 8α, CD11b+: CD11c+, CD8α+, CD11b-: CD11c+, CD8α+, CD11b+ could be found in spleen. The ratio of lymphoid (CD11c+, CD&i+, CD11b-) and myeloid (CD11c+, CD8α-, CD11b+) DCs subgroups was almost equal in spleen. Lymphocyte transformation test showed that lymphocytes proliferation in response to ova albumin is higher in ova albumin pulsed DCs injected mice compared to control groups (P<0.004). Conclusion. Considering the obtained results it can be said that Splenic DCs can process and present the antigen in vivo and induce antigen specific response in vitro. Similar ratio of lymphoid and myeloid DCs in spleen may be implicated in regulation of and homeostasis of immune response by this organ.

Purpose: Here, we aim to evaluate the antileishmanial activity of compounds with a benzoxazinoid (BX) skeleton, previously synthesized by our group, against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum promastigotes. Methods: Anti-promastigote activity, as well as cytotoxicity, were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assays. The selectivity index (SI) for each compound was calculated using a ratio of the cytotoxicity of compounds and the geometric mean (GM) of antileishmanial concentrations to each species tested. The comparisons between groups were carried out using a t test or analysis of variance (one-way ANOVA). A P value of less than 0. 05 was considered significant. Results: All the compounds tested were active, with IC50 falling between 92 ± 6. 19 μ g/mL and 238± 6. 57 μ g/mL for L. braziliensis, and 89 ± 6. 43 μ g/mL and 188 ± 3. 58 μ g/mL against L. infantum. Bex2, Bex3, Pyr1, Pyr2, and Pyr4 were compounds that showed activity similar to the drug Glucantime® , exhibited low cytotoxicity against Splenic hamster cells (CC50 raging between >400 and 105. 7± 2. 26 μ g/mL) and had favorable selectivity indices (SI 1. 12 to 3. 96). Conclusion: The analogs in question are promising prototypes for the pharmaceutical development of novel, safer and more effective leishmanicidal agents.

Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22. 5-kDa glycoprotein used to treat diseases such as multiple sclerosis (MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells, we cloned hIFNβ-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein. Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3. 1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ 1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques. Results: The hIFNβ 1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ 1apcDNA3. 1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot. Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression. Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The aim of this study was to sub-clone the peroxisomal protein (PEP) cDNA into a mammalian expression vector leading to the formation of a chimeric PEP-cDNA containing the FLAG epitope. The FLAG-PEP recombinant cDNA was constructed using the method of splicing by overlap extension polymerase chain reaction (SOE PCR) and inserted into the pUcD2SRaMCSHyg eukaryotic expression vector. To investigate the intracellular localization of the PEP protein that was linked to the FLAG tandem, the constructed plasmid was used for transient transfection of the Chinese hamster Ovary (CHO) cells. The CHO cells that were transfected with the recombinant plasmid showed peroxisomal localization of FLAG-PEP as was previously shown for catalase.

Introduction: Heavy ions are nucleus of elements of iron, argon, carbon and neon that all carry positive electrical charges. For these particles to be useful in radiotherapy they need to accelerated to high energy by more than thousand mega volts. Also the cosmic environment is considered to be a complicated mixture of highly energetic photons and heavy ions such as iron. Therefore, the health risks to astronauts during long mission should be considered.Materials and Methods: The induction of interphase death was tested on Chinese hamster ovary cells by exposing them to accelerated heavy ions (carbon, neon, argon and iron) of 10-2000 linear energy transfers (LETs). The fraction of cells that underwent interphase death was determined by observing individual cells with time-lapse photography (direct method) as well as by the indirect method of counting cells undergoing interphase death made visible by the addition of caffeine (indirect method).Results: The interphase death due to the exposure to X- rays is increased linearly as the dose exceeds the threshold dose of 10 Gy. Whereas the interphase death increases at a higher rate due to the exposure to high LET heavy ions and no threshold dose was observed. The range of LET values corresponding to the maximum RBE for the interphase death is 120-230 keV/mm. The probability of inducing the interphase death by a single heavy ion traversing through the nucleus is about 0.04-0.08.Discussion and Conclusion: The relative biological effectiveness (RBE) of heavy ions as compared to X- rays as determined at the 50% level of induction is increased with LET. It reached a maximum value at a LET of approximately 230 keV/mm and then decreased with further increase in LET. The range of LET values corresponding to the maximum RBE appears to be narrower for interphase death than for reproductive death.

Background: Human chorionic gonadotropin (hCG) is a member of glycoprotein hormones family consist of two different non-covalently heterodimeric chains: alpha and beta subunits with 92 and 145 amino acids respectively.This hormone plays an important role in human reproduction and physiology especially for maintenance of the corpus luteum during the first months of pregnancy Materials and Methods: The aim of our study was to production of recombinant hCG in Chinese hamster ovary (CHO) cells. In this regard, cDNA encoding alpha and beta subunits were amplified by PCR and after digestion by XhoI/SalI cloned into the pcDNA3.1 (+) expression vector.The recombined plasmids were transformed into the TOP10F/ strains of E.coli. After that, cloning was confirmed by PCR and sequencing methods. Then two gene constructs were linearized by PvuI and subsequently co-transfected into the CHO cells using the electroporation Technique. The transfected cells were examined by PCR Results: Positive cell lines were subjected for expression analysis of recombinant hCG by SDS-PAGE and Western blotting methods Conclusion: Many researches confirm that production of recombinant protein in eukaryotic expression systems such as CHO cells is a reliable method for producing of therapeutic human chorionic gonadotropin.

Introduction: Mammalian reproduction looks like an immunological paradox, because fetal alloantigens encoded by father genes should induce cell meditated immune responses leading to fetal loss. Maternal immune system, in addition to local modulation, undergoes systemic modulations during pregnancy. Dendritic cells (DCs), as professional antigen presenting cells, play a key role in initiation and control of immune response and it seems that functional changes in these cells during pregnancy may contribute to the systemic immune tolerance. To address this issue, in this study we isolated and purified DCs from pregnant mice and evaluated their stimulatory potential to induce proliferative response of allogeneic T cells in unidirectional mixed leukocyte reaction (MLR). Materials and Methods: Following collagenase digestion of Splenic tissue, using density gradient centrifugation (13% Nycodenz) and adherence properties of DCs to the bottom of tissue culture dish, 7×105 DCs were isolated from each spleen with more than 95 percent purity. Allogeneic T cells were isolated by nylon wool column, using their non-adhesive character to nylon wool. After radiation, isolated dendritic cells from pregnant and non-pregnant Balb/ c mice were used in mixed leukocyte culture with C57BL/6 mice T lymphocytes. T lymphocyte proliferation was measured 72 hours by H-thymidine incorporation. Results: 7 ×105 dendritic cells with the purity of >95% were isolated from each spleen. Also the yield of T-lymphocyte from inguinal and Brachial lymph nodes was about 3-5 ×105 with the purity of %85-90 The results showed that there is no statistical difference between stimulatory potential of DCs from pregnant (cpm=33000) and non-pregnant (cpm=35000) mice in induction of allogeneic T-Cell proliferation. Conclusion: These findings can result from low concentration of immune suppressor factors in circulatory system of pregnant mice or due to separation of dendritic cells from pregnancy microenvironment and their maturity in vitro in the absence of the immune suppressor factors.