seyed mohammad moazzeni

Selecting appropriate antigens, suitable delivery vehicles and proper immune adjuvants are important items in vaccine designing. According to the previous studies, Carcinoembryonic antigen (CEA) and Adenoviral vector are considered as proper antigen and reliable delivery vehicle for cancer vaccines, respectively. Directing antigens to DCs with antibody single-chain fragment variable (scFv) against to DEC-205 (scDEC) can play the role of adjuvant. The aim of this study was to produce a construct encoding CEA targeted to DCs by scDEC in Adenovirus vector platform.

scDEC were cloned into p-track-CEA-GFP. Linearized p-track-scDEC-CEA-GFP and pAdenoVator ∆E1/E3 were co-transformed into BJ5183 cells. Resulting pAd scDEC-CEA-GFP was transfected into HEK293 cells to produce Adenoviruses (Ad scDEC-CEA-GFP). The presence of scDEC-CEA gene in the recombinant virus and expression of CEA protein was detected by PCR and electrochemiluminescence tests, respectively. Flow cytometry tests were used to confirm the attachment of scDEC-CEA protein to DCs. Adenovirus infectivity in cells other than HEK293 was confirmed by transduction of Ad scDEC-CEA-GFP into L929 cells.

PCR test showed the presence of scDEC-CEA gene in adenovirus genome. Electrochemilu-minescence and flow cytometry analysis confirmed expression of CEA protein and attachment of scDEC-CEA protein to DCs, respectively. GFP expression in L929 cells indicated Ad scDEC-CEA-GFP could effectively infect cells other than HEK293.

As construction of a suitable construct for immunotherapy is a major goal of many research groups, the Ad scDEC-CEA-GFP engineered in this study, which properly targets DCs, can be applied in future in vivo cancer immunotherapy investigations in mice.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Matrix metalloproteinases (MMP)-9 facilitates the migration of T-cells to central nervous system (CNS), while tissue inhibitor of metalloproteinases-1(TIMP-1) inhibits the function of MMP-9. This study aimed to determine the appropriate treatment option for multiple sclerosis (MS). Forty-three relapsing-remitting MS (RRMS) patients were randomly divided into two groups of 22 (group A, placebo) and 21 (group B, Saffron pill) individuals. Serum samples were collected from patients’ blood before using the Saffron pills/placebo pills and then after 12 months. The serum level of MMP-9 and its inhibitor, as well as TIMP-1, were measured by ELISA kits. MMP-9 serum levels noticeably decreased in patients with MS following 12 months of treatment with Saffron pills (p=0.006) while the changes were not significant before and after 12 months of treatment with placebo pills. Although the levels of TIMP-1 increased significantly after one year treating with Saffron pills (p=0.0002), a considerable difference was not observed before and after taking the placebo pills. The study finding revealed that 12-months treatment with Saffron could have a significant role in reducing the serum level of MMP-9 and increasing the serum level of TIMP-1 in RRMS patients. Therefore, modulating the serum levels of MMP-9 as an important regulator of T cell trafficking to the CNS might be a promising strategy in the treatment of MS patients.

The goal in studies concerning biomarkers in autoimmune diseases is finding a marker which fluctuates in correlation with the disease’s severity and settles within normal borders after effective treatment. This marker would later be used as an efficient tool in diagnosis and analysis of medicine clout. It seems the most cogent biomarkers are those measurable in serum or plasma. MS is a neurological disease common within the young adult population with a predictable course, often leading to life-immersing disability. The prognosis, however difficult and limited, is currently possible via diagnostic tests (brain MRI) or clinical information (severity and degrees of disability).  Many studies have been conducted in an effort to detect an adequate biomarker. It is still often overlooked that a reliable biomarker should both be clinically applicable and functional is prognosis; as this is what could lead to timely treatment.

Mesenchymal stem cells (MSCs), due to their immunomodulatory functions, are an ideal candidate for the treatment of immune-related diseases. Recurrent spontaneous abortion (RSA) is one of the most common complications of pregnancy which in many cases is related to the immune system disorders. Our previous study has shown that the abortion rate was decreased following the syngeneic MSCs therapy in abortion-prone mice. In this study, the therapeutic effect of syngeneic, allogeneic, and xenogeneic MSCs was compared in a mouse model of RSA. In this experimental study, MSCs were isolated from adipose tissue (ASCs) of CBA/J and BALB/c mice and human. After characterization, ASCs were injected (IP) at day 4 of gestation to female CBA/J mice following their mating with DBA/2 male mice. In the control group, phosphate-buffered saline (PBS) was injected and CBA/J×BALB/c mating was also used as the normal pregnancy control. On day 14.5 of pregnancy, embryo resorption rate was determined. The abortion rate significantly decreased following the ASCs therapy from syngeneic (6.31%), allogeneic (6.54%), and xenogeneic group (12.36%) compared to ASCs non-treated group (34.4%). There was no statistical difference between ASCs treated groups, however syngeneic and allogeneic ASCs reduced the abortion rate more efficiently than xenogeneic ASC. The abortion rate was significantly decreased following the intraperitoneal administration of ASCs from various donated sources in abortion-prone mice. These results indicated that the immunogenicity of allogeneic and xenogeneic ASCs is not a contradictory problem for their therapeutic effects on RSA.

Type-1 diabetes (T1D) is an autoimmune disease in which T lymphocytes destroy insulin-producing β-cells. Control of self-reactive T lymphocytes and recovery of diabetic injury is the end point of T1D.

To investigate generation of tolerogenic dendritic cells (tolDCs) as an innovative method of diabetes therapy.

Lentivirus vector production was achieved by GIPZ mouse CD40 shRNA, psPAX2 and pMD2G plasmids DNA. Purified bone marrow derived DCs were treated with CD40 shRNA, and expression of CD40 and mRNA level were evaluated by flow cytometry and Real-Time PCR, respectively. CD40 knock-down DCs were injected into STZ-induced diabetic mice. Blood glucose; glucose tolerance test and weight were analyzed in different groups.

Mice treated with CD40 shRNA transfected DCs showed considerable differences in blood glucose, glucose tolerance, and weight compared to other groups. Also cytokine assays indicated an increase in IL-13 production in the CD40 shRNA group.

In streptozotocin-induced diabetic mice model, administration of tolerogenic dendritic cells could improve diabetic parameters.

The B-cell CD20 antigen is one of the most reliable surface targets in immunotherapy of B lymphoma. In this project, we studied the production and characterization of a new monoclonal antibody against chimeric human CD20 extra loops (hCD20 exl). The results showed that clone C12H, IgG2/k isotype reacted with the antigen in ELISA and immunoblot. The Kd value was found to be 2×10-9M and flow cytometry results showed that 99.9% and 99.7% of the Daudi and Raji cells respectively were stained with C12H monoclonal antibody (mab) but not with Jurkat cell lines. It also effectively competed with Rituximab, thus, the staining of the Daudi and Raji cell lines was reduced to 55.9% and 40.5% of cells respectively. Based on the high affinity reaction of C12H mab and appropriate reactivity of C12H mab with the native antigen on the surface of Raji and Daudi cells in flow cytometry, it was concluded that development and evaluation of C12H mab could be a beneficial candidate for further application in genetically engineered monoclonal antibody.

Macrophages play a crucial role in appropriate embryo implantation and maintenance of pregnancy. This study was done to evaluate the frequency and distribution of uterine (local changes) and spleen (systemic changes) macrophages on days 4.5 and 7 of NMRI mouse pregnancy which are simultaneous with beginning of implantation and complete establishment of embryo in the uterus. The precise day of pregnancy was determined through vaginal plug detection. Blood was collected from pregnant mice on days 4.5 and 7 of gestation and serum 17- β estradiol and progesterone concentrations were measured using the ELISA method. The frequency and localization of macrophages in different regions of uterus and spleen were also investigated by immunohistochemistry staining using anti F4/80 antibody. The results of this study showed that frequency of uterine macrophages has decreased on day 7 in comparison to day 4.5 of gestation. Macrophages are distributed throughout the whole uterus on day 4.5 while their distribution was restricted to myometrium and stroma beneath the circular muscles on day 7 of pregnancy. The frequency of spleen macrophages as a representative of their systemic changes was not different in studied groups. Serum 17-β estradiol and progesterone concentrations increased significantly on day 7 compared to day 4.5 of pregnancy. Considering the changes in 17-β estradiol and progesterone concentrations and their indirect effects on macrophages recruitment through regulation of M-CSF and GM-CSF secretion by uterine stromal cells, the differences in frequency and distribution of macrophages in different stages of pregnancy could be explained. While the macrophages chemotactic factors and homing receptors and their sources in spleen are different from uterus and don’t response to ovarian hormones variations. Regarding the role of macrophages in embryo implantation and regulation of feto-maternal immune responses, it seems that the changes in their frequency and localization can be helpful for a successful pregnancy.

Human Wharton’s Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease.Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit.IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells.Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

One of the common causes of recurrent spontaneous abortion is interference of immunological factors. Administration of cyclosporine, as an immunosuppressive drug, has reportedly reduc the rate of abortion in abortion-prone model of mice CBA/J mated DBA/2 male mice. On the other hand, asymmetric antibodies and TGF-β1 have been considered as an important factor in successful pregnancy outcomes. Intravaginal administration of TGF-β has also been reported to reduce the resorption rate in the same mouse model. In this study, the immunomodulatory effects of cyclosporine on serum level of asymmetric antibodies (AAbs) and T cell growth factor beta (TGF-β1) were investigated. Female CBA/J mice were mated with male DBA/2. The pregnant females were divided into two groups: 1- test group; received intraperitoneal injection of 1mg/kg cyclosporine (n=7), 2- control group; received intraperitoneal injection of Phosphate Buffered Saline (PBS) (n=7). Both groups received treatments on the 5th day of pregnancy. Serum samples were collected on the 13.5th day of pregnancy, when the percentage of abortion was also calculated. The percentage of AAbs and abortion rate were significantly reduced in cyclosporine group (P<0.05, for both variables), while the serum levels of TGF-β1 did not show significant difference between groups (P>0.05). Our study showed that despite the significant reduction in percentage of AAbs and no significant difference in serum level of TGF-β1, the abortion rate was markedly reduced in cyclosporine group. So other mechanisms of immunomodulation and reduction of abortion rate except asymmetric antibodies and TGF-β1 should be involved in this mouse model following cyclosporine administration.

Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. [AGA1]. The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. [AGA2] The final concentration of purified protein was 500 µg/ml.

Dendritic cells (DCs) are potent antigen presenting cells for triggering of the immune reaction post transplantation. These cells are centrally involved in the initiation of T cell-dependent immune responses. To compare the level of DC maturation and function in liver transplant recipients with healthy controls. In this study, twelve peripheral blood samples were selected from six liver transplant patients and six healthy controls. After the generation of DCs from monocytes, expression levels and mean fluorescent intensity (MFI) of several DC maturation markers were evaluated using flowcytometry. Secretion of IL-6, IL-12 and IL-23 proinflammatory cytokines was determined using ELISA. Gene expressions of TLR-2, TLR-4 and IL-23 were analyzed using real-time PCR. DC expression markers including CD83 (p=0.007) and CD86 (p=0.02), as well as secretion of IL-6 (p=0.02) and IL-12 (p=0.007) by DCs were significantly increased in liver transplant patients compared with healthy controls. The MFI of CD86 (p=0.009) and HLA-DR (p= 0.005) expression on DCs was also higher in patients. The expression of TLR-2 transcripts in DCs of patients was higher than that of the controls (p=0.03). Based on these findings, increased frequency of DCs expressing CD83 and CD86, higher expression of CD86, HLA-DR, and TLR-2 as well as elevated secretion of proinflammatory cytokines in DCs of liver transplant recipient's point to the more mature phenotype and active function of DCs in patients compared with controls.

Recent evidences suggest that tumors arise from a small subpopulation of cells، the cancer stem cells (CSCs) or tumor initiating cells. CSCs are able to resist the conventional methods of cancer therapy due to existence of ABC transporters on their surface. This leads to CSC resistance and maintenance resulting in post-treatment relapse and metastasis. Therefore، precise identification and characterization of these cells as a target for new therapeutic regimens is the goal of numerous studies. This study، with the intent to design a new method of immunotherapy for targeting cancer stem cells in mouse malignant melanoma، initially characterized the cancer stem cells in this malignancy. In order to identify the CSCs we induced a melanoma tumor using the B16F10 cell line in C57BL/6 mice. The tumor bulk was dissociated by an enzymatic method and homogeneous tumor cells were sorted using anti-CD44 and anti-CD24 antibodies. The sorted tumor cell subpopulations were compared according to their ability to form cell spheres in serum free medium (SFM). We determined the tumor formation ability of all cell subpopulations by transplanting serial dilutions of B16-F10 and all sorted cells sub-populations into C57/BL6 mice. The results showed that although all separated cell subpopulations and B16-F10 cells formed non-adherent spheroids in SFM in the presence of B-27، but the CD24+ cells presents a significantly higher ability to produce spheroids. The B16F10 cell line، CD44+CD24- and CD44-CD24- cells showed equal potencies in tumor induction (1 in 21730 cells). The CD44-CD24+ cells tumor induction potency was 1 in 17426 and this ability for the double positive cells (CD44+CD24+) was 1 in 11295. Collectively،the double positive (CD24+CD44+) cells were more potent in both spheroid formation and tumorogenicity. Hence they might be the CSC population of mouse melanoma.

Abstract The present study aimed to investigate the immunomodulatory activity of molecules secreted by decidual cells on dendritic cells (DCs) function in abortion-prone compared with non-abortion-prone mice. The decidual cell supernatants (DS) were obtained from abortion and non-abortion mouse models. DCs were purified from CBA/J mice spleens and treated with antigen and DS. Treated DCs were injected into mice palms. After 5 days، draining lymph nodes were removed، cultured in the presence of cognate antigen، and proliferation of lymphocyte cells was measured by 3H-thymidin incorporation. Our results showed that immunosuppressive activity of DS from non-abortion-prone mice significantly decrease dendritic cells'' ability to stimulate lymphocytes proliferation compared with DS from abortion-prone mice (Simulation index (SI) of 4. 93 ± 0. 34 versus 11. 84 ± 0. 79). We، also found that DS prepared from non-resorption sites compared with DS from resorption sites in abortion-prone mice had increased immunosuppressive activity on DC function (SI of 7. 31 ± 1. 02 versus 2. 67 ± 0. 49). Due to our results، we concluded that immunomodulatory activity of molecules secreted within decidual tissue is different between abortion-prone and non-abortion-prone mice. Based on the key role of DCs in inducing fetomaternal tolerance، we claimed that these molecules، through modulation of DCs function، play crucial role on pregnancy outcome.

Common variable immunodeficiency (CVID) is one of the most frequent cases of primary immunodeficiency. It is likely that this heterogeneous disease is caused by several distinct genetic disorders. The activation-induced cytidine deaminase (AID) enzyme is involved in class switching, somatic hypermutation (SHM) and processes associated with gene conversion in the germinal center. In order to clarify the possible role of AID in the pathogenesis of CVID, we have studied the AID gene expression in CVID patients. Peripheral blood mononuclear cells (PBMC) from 21 patients and healthy controls were isolated. The isolated cells were stimulated by CD40L and IL-4 to induce AID gene expression. After five days, total RNA from the stimulated cells was extracted and AID gene expression was investigated by RT-PCR. RT-PCR results showed that after stimulation by CD-40L and IL-4, the AID gene was expressed in all of the samples. The control samples were also positive for AID gene mRNA expression. In this investigation we studied the expression of AID gene in CVID patient's B lymphocytes for the first time. Regards to our results which showed that all patients normally expressed the AID gene mRNA and considering that one of the main problems in a number of CVID patients is disorders in phenomena related to the germinal center and complete differentiation of B lymphocytes, it can be concluded that possible defects in other molecules involved in class switching is responsible for this disease. Understanding the various genetic defects responsible for this heterogeneous disease could lead to its division into more homogenous subtypes with distinct therapeutic strategies, so further investigations is recommended.

Menstrual blood stromal stem cells (MBSCs) share some phenotypic and functional similarities with mesenchymal stem cells (MSCs). MSCs are shown to inhibit either the function or generation of different immune cells, including dendritic cells (DCs). However, data regarding MBSCs’ potential effects on immune system cells are elusive. Here, we examine whether MBSCs affect the generation of human monocyte-derived DCs. Menstrual blood samples were collected from apparently healthy women on the second day of their menstrual cycles. The adherent portions were subcultured to omit unwanted cells and obtain MBSCs. Magnetically-isolated peripheral blood monocytes were differentiated towards DCs through treatment with recombinant granulocyte monocyte colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in the presence or absence of MBSCs. After five days, monocyte-derived DCs were analyzed for the expression of surface markers by flow cytometry. IL-6 level was determined in the co-culture supernatants. Co-culture with MBSCs significantly down regulated the expression of DC marker (CD1a) and up regulated the expression of monocyte marker (CD14) on monocyte-derived DCs compared with the control group. IL-6 level was shown to be significantly higher in the supernatant of the monocyte-MBSC co-culture. Collectively, this is the first study to show the inhibitory impacts of MBSCs on the generation of DCs. IL-6 could be viewd as a potential factor mediating this effect. Regarding the known advantages over MSCs, MBSCs could be considered as a promising future candidate for immunomodulatory purposes in the clinical setting.

Enterotoxigenic Escherichia coli bacterium is the most important bacterial agent causing diarrhea. Specific virulence factors, such as enterotoxins and colonization factors, distinguish ETEC from other classes of diarrheagenic E.coli. In this study, heat-labile toxin was purified which could be utilized for anti-toxin assay in GM1 gangelioside receptor-ELISA based method and for identification of ETEC producing toxin.

In this experimental study, bacterial strain producing heat-labile toxin was first cultivated for production and purification of toxin. Then supernatant soluble proteins were precipitated with ammonium sulfate and purified using biochemical methods. Finally, purified protein was dialyzed against Tris 0.02 mM pH 8 and analyzed on gel electrophoresis. GM1 gangelioside receptor-ELISA based method was used for detection and assessment of the purified toxin. Through this method, the effect of anti-recombinant heat-labile toxin B subunit neutralization on heat-labile toxin was investigated.

Toxin purification was revealed by the presence of 12 and 28 KD protein bands. This study demonstrated that anti-recombinant heat-labile toxin B subunit antibody can detect the purified toxin and can inhibit its binding to GM1 receptor up to 80%.

Purification of heat-labile toxin and gangelioside receptor-ELISA assay can be used for accurate detection and epidemiological study of clinical isolates.

The aim of this study was to evaluate and compare the population of Natural killer T lymphocyte (NKT) in the uterus and spleen of the hyperstimulated and control mice at the seventh day of pregnancy. The superovulated and control mice were put individually in a cage with a male mouse. In the next morning they were considered for observing the vaginal plag. The day was assumed as the first day of pregnancy. On the 7th day of pregnancy, the samples were collected from uterus of implantation site, interval site and spleen tissues and 5 micrometer cryosections were prepared. The immunohistochemical reaction was used for CD 161 and CD3 markers and the distribution of NKT cells population were compared with nucleated cells in hyperstimulated and control groups. There were not significant differences between the NKT cell population of the spleen, decidual and myometrial tissue of interval site in the control and hyperstimulated groups. But this population was increased in the hyperstimulated group (2.26 ± 1.43) compared with control (0.79 ± 0.17; P≤0.05) in the decidual of implantation site, moreover there was not any significant difference at myometrial tissue of both groups in implantation site. It seems that ovulation induction could not affect systematically on the population of NKT cells of pregnant mice while it could locally cause an increase in the decidual NKT cells.

Dendritic cells (DCs) play an important role in induction of cellular immune responses. It seems that DCs that reside in different organs may be distinct in their ability to induce immune responses. This study was done to address the differences between spleen and liver DCs in induction of immune response and/or tolerance. CD11c+ DCs were separated from the liver and spleen of C57BL/6 mice and pulsed with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. 6105 MOG35-55 pulsed spleen or liver DCs were injected in foot pad of different groups of mice. Control groups received unpulsed DCs. After 5 days, the mononuclear cells (MNCs) of the regional lymph nodes were isolated from immunized mice for cytokine assays and lymphocyte transformation test. To study the immunologic or tolerogenic effects of DCs, three weeks after immunization of mice with MOG pulsed liver or spleen DCs, experimental autoimmune encephalomyelitis (EAE) was induced in DC-immunized mice by injection of MOG along with complete Freund's adjuvant. Our results showed that spleen DCs were more potent in stimulating lymph node T cells as illustrated in lymphocyte transformation test. Moreover IL-10 production was higher in mice immunized with liver DCs compared with those immunized with splenic DCs (p=0.017). However, no significant difference in IFN-γ production was observed between two groups. We also found that liver DCs+MOG immunized mice displayed a significantly delayed disease onset compared with spleen DCs+MOG immunized mice and the control groups. The disease score was also milder in liver DCs immunized mice compared with other groups. It seems that the higher IL-10 production induced by the liver DCs may be one of the main factors in down regulation of immune responses in this organ. It can be concluded also that the liver DCs may inhibit the progress of EAE by shifting the cytokines profile.

In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient.

The partial NS3 (pNS3) gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line.

After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively.

This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine.

Enterotoxigenic Escherichia coli is considered as the most important agent of children diarrhea and mortality in developing countries. This bacterium causes 300-600 thousands of deaths in the children under 5 years of age per year. With difficulties in treatment as well as its wide prevalence, designing an effective vaccine against this microorganism is the objective of world Health Organization (WHO). The CfaB protein as immunogen and major subunit of fimberia has a critical role in the bacterial attachment to small intestine epithelium and the produced antibody against this protein can prevent attachment of bacterium to epithelial surface. Hence, this molecule alone or with other virulent factors has been considered by many researchers in vaccine designing. In this study, expression of colonization factor B with the aim of studying the immunogenesity of this protein as a component of vaccine candidate was performed.

cfaB gene was amplified by PCR and cloned into pET28a and its expression was evaluated. Since there was no expression, which was due to presence of rare codon, the cfaB gene was again cloned into pET28a using codon bias in E.coli and subsequently expressed.

Presence of a 20KD band on SDS-PAGE gel indicated the expression of CfaB protein, which was later confirmed by immunoblotting with anti-His tag and anti CfaB antibodies and purified on Ni-NTA column.

Codon optimization and expression in heterologous hosts is a useful approach for obtaining large quantities of recombinant protein.

Blocking antibodies are valuable tools for inhibiting the specific receptor- ligandinteractions. The interaction of co-stimulatory molecules on the antigen presenting cells with their ligands on T cells is an essential step for T cell activation. In the present study, the effect of blocking antibody against CD40 on its T cell stimulatory potential is investigated.The DCs (dendritic cells) were collected from the mice spleens and then cultured in vitro.We used purified rat anti-mice CD40 (Clone HM40-3) (BD USA) as a blocking antibody andthe appropriate titer of the blocking antibody was determined by flow cytometry. The DCs were then treated by antibody and used in MLR assay.The results of these experiments showed that CD40 blockade were associated with theincrease in the of IL-4 secretion, shifting the DCs to stimulate Th2 cytokine production bythe allogenic T cells, while the secretion of IL-12 by DCs decreased. Similarly, the DCs withreduced CD40 expression poorly responded to alloantigen stimulation in the MLR.Collectively, these results emphasize the importance of CD40 pathway in tolerogenicDCs generation and also support the idea that downregulation of CD40 is effective ininhibiting the allostimulatory function.

Objective (s) Recent studies on human indicate that the introduction of therapeutic use of tolerogenic dendritic cell (DC) for chronic inflammatory conditions is imminent. For the purpose of defining CGRP potency in tolerogenic DC production, we investigated the phenotype and IL-12 production of DCs generated from the monocytes of rheumatoid arthritis (RA) patients in the presence of the calcitonin gene-related peptide (CGRP), as a multifunctional neuropeptide.Materials and MethodsDCs were generated from isolated monocytes from four resistant and two early female RA patients using IL-4, GM-CSF, and CGRP at concentrations of 0, 1, and 100 nM. Then, the phenotype of neuropeptide-treated or untreated DCs was determined using flow cytometry and the IL-12 production was measured by ELISA.ResultsOur study showed that, on the last day of the culture, at a concentration of 1 nM CGRP, the mean fluorescence intensity (MFI) for CD80 increased (14.13%) and the MFIs for CD83, CD86, and HLA-DR decreased (14.57%, 5.28%, and 6.88% respectively). Moreover, at 100 nM CGRP concentration, the MFI for CD80 increased (11.10%) and the MFIs for CD83, CD86, and HLA-DR decreased (4.27%, 18.60%, and 19.75% respectively). In addition, our results indicated that the mean concentrations of IL-12 produced at 0, 1, and 100 nm CGRP concentrations measured 13.72±2.41, 11.01±1.61, and 7±1.34 pg/ml respectively. ConclusionDecreased CD83, CD86, and HLA-DR expression and reduced IL-12 production by CGRP were found in the RA patient's monocyte-derived DCs. CD83 is a well-defined DC activation marker. HLA-DR and CD86 are appropriate molecules for inducing an immune response. IL-12 promotes cell-mediated immunity. Therefore we suggest that CGRP may be used as an inducer in the production of tolerogenic DCs.

Dendritic cells (DCs), as the managers of the immune response, have a crucial role in forming the direction and nature of the immune response. Some compounds such as 1,25-dihydroxycholecalciferol affect the function of DCs and can be used to shift the immune functions toward favorite directions. The aim of this study was to investigate the in vivo effects of 1, 25- dihydroxycholecalciferol on DCs surface markers, their potential to induce specific T-cell responses and the cytokines profile. 1, 25-dihidroxycholecolciferol was regularly injected intraperitoneal into C57BL/6 mice. DCs were separated from the spleens of calciferol treated and non-treated mice using magnetic beads. The expression of DCs surface markers was investigated by flow cytometric analysis. The separated cells were pulsed by myelin oligodendrocyte glycoprotein (MOG) and injected subcutaneously into front footpads of syngeneic mice. After five days, the lymphocytes from regional lymph nodes were separated and used for the lymphocyte transformation test (LTT) and determination of the interferon gamma/interleukin 4 (IFNγ/IL-4) ratio by ELISA technique. Statistical analysis of the obtained results showed reduced expression ofmaturation markers and co-stimulatory molecules by cholecalciferol treated DCs. Thespecific T-cell stimulation potential of treated DCs as well as the induced IFNγ/IL-4 ratiowas also down-regulated compared to non-treated cells (p value<0.05). It seems that 1,25-dihydroxycholecolciferol can regulate the DCs function and maturation state in vivo. The T-cell stimulation rate and Th1/Th2 cytokines ratio also changes following interaction with cholecalciferol treated DCs.