[Identification and characterization of cancer stem cells in mouse malignant melanoma]

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 subpopulations 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

Expression of activation-induced cytidine deaminase gene in B lymphocytes of patients with common variable immunodeficiency

Common variable immunodeficiency [CVID] is a heterogeneous disorder characterized by reduced serum level of IgG, IgA or IgM and recurrent bacterial infections. Class switch recombination [CSR] as a critical process in immunoglobulin production is defective in a group of CVID patients. Activation-induced cytidine deaminase [AID] protein is an important molecule involving CSR process. The aim of this study was to investigate the AID gene mRNA production in a group of CVID patients indicating possible role of this molecule in this disorder. Peripheral blood mononuclear cells [PBMC] of 29 CVID patients and 21 healthy controls were isolated and stimulated by CD40L and IL-4 to induce AID gene expression. After 5 days AID gene mRNA production was investigated by real time polymerase chain reaction. AID gene was expressed in all of the studied patients. However the mean density of extracted AID mRNA showed higher level in CVID patients [230.95 +/- 103.04 ng/ml] rather than controls [210.00 +/- 44.72 ng/ml; P=0.5]. CVID cases with lower level of AID had decreased total level of IgE [P=0.04] and stimulated IgE production [P=0.02]; while cases with increased level of AID presented higher level of IgA [P=0.04] and numbers of B cells [P=0.02] and autoimmune disease [P=0.02]. Different levels of AID gene expression may have important roles in dysregulation of immune system and final clinical presentation in CVID patients. Therefore investigating the expression of AID gene can help in classifying CVID patients

study of AID gene expression in B lymphocytes from CVID patients

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 A/L 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 patients' 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

Spleen and liver dendritic cells differ in their tolerogenic and cytokine induction potential

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[5]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-8 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

[Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector]

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

[ effect of ovulation induction on the distribution of uterus natural killer T [NKT] cell population at seventh day of mouse pregnancy]

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

[Induction of specific T-cell response following in vivo treatment of dendritic cells by 1,25-dihydroxycholecalciferol]

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 [IFN gamma/IL-4] ratio by ELISA technique. Statistical analysis of the obtained results showed reduced expression of maturation markers and co-stimulatory molecules by cholecalciferol treated DCs. The specific T-cell stimulation potential of treated DCs as well as the induced IFN gamma/IL-4 ratio was 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

Calcitonin gene-related peptide effects on phenotype and IL-12 production of monocyte-derived dendritic cells in rheumatoid arthritis patients

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 [CDRP], as a multifunctional neuropeptide. DCs 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. Our 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. Decreased CD83, CD86, and HLA-DR expression and reduced IL-12 production by CGRP were found in the RA patients' 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

Immediate exposure to TNF-alpha activates Dendritic cells derived from non-purified cord blood mononuclear cells

Tumor necrosis factor alpha [TNF-alpha] is a primary mediator of immune regulation and might be required in the early stages of DC development from CD34[+] cells. However, details of optimal timing of exposure to TNF-alpha in DC development process in monocytes or non-purified hematopoitic cells are still lacking and clear benefits of this approach to the development of DCs remain to be validated. To evaluate the effect of early and late exposure to TNF-alpha on DC development from non-purified cord blood mononuclear cells. To define the effects of early exposure to TNF-alpha on cord blood mononuclear cells, we cultured UCB-MNC in the presence of SCF, Flt3L, GM-CSF and IL-4 for 14 days and matured them for an extra 4 days. TNF-alpha was added on day 0, 7 and 14 in TNF-alpha [+] group, and only on day 14 in TNF-alpha [-] group where it was used only as a maturation factor. Immediate exposure to TNF-alpha was shown to: [1] enhance the survival of cells in the first week of culture; [2] produce mature DCs with higher maturation markers [CD80, CD83, CD86 and HLA-DR]; and [3] increase secretion of IL-12 by mature DCs. In contrast, delayed exposure to TNF-alpha stimulate mature DCs with less purity producing a high level of IL-10 and a low level of IL-12. We developed a simple, easy and cost effective method to generate DCs from non-fractionating mononuclear cells in this study. Also we confirm the presence of a large number of functional DCs under inflammatory conditions, where local concentrations of TNF-alpha were high

effects of candida albicans cell wall protein fraction on dendritic cell maturation

Candida albicans is a member of the normal human microflora. C. albicans cell wall is composed of several protein and carbohydrate components which have been shown to play a crucial role in C. albicans interaction with the host immune system. Major components of C. albican cell wall are carbohydrates such as mannans, beta glucans and chitins, and proteins that partially modulate the host immune responses. Dendritic cells [DC], as the most important antigen-presenting cells of the immune system, play a critical role in inducing immune responses against different pathogens. We investigated the effect of the cell wall protein fraction [CPF] of C. albicans on DC maturation. The CPF of C. albicans cells was extracted by a lysis buffer containing sodium dodecyl sulphate, 2-mercaptoethanol and phosphate-buffered saline. The extract was dialyzed and its protein pattern was evaluated by electrophoresis. Dendritic cells were purified from Balb/c mice spleens through a three-step method including mononuclear cell separation, as well as 2-h and overnight cultures. The purified CPF was added at different concentrations to DC. The purity and maturation status of DC were determined by flow cytometry using monoclonal antibodies against CD11c, MHC-II, CD40 and CD86. Treatment of DC with 10 micro g/ml of CPF increased the expression of maturation markers including MHC-II, CD86 and CD40 on DC compared to the control group. In this study we used C. albicans CPF with the molecular weight of 40-45 kDa for pulsing and maturation of dendritic cells. Since according to our results CPF significantly increased the expression of maturation markers on DC, we suggest that CPF may act as an efficient immunomodulator, or may be used as a potential adjuvant to boost the host immune system against infections

14kDa protein molecule isolated from garlic suppresses indoleamine 2, 3-dioxygenase metabolites in mononuclear cells in vitro

A wide range of biological activities of garlic in vitro and in vivo have been verified including its antioxidant, antitumor and anti-inflammatory effects. Indoleamine 2,3-dioxygenase [IDO] is an enzyme widely distributed in mammals and is inducible preferentially by IFN-?. IDO degrades the essential amino acid tryptophan to form N-formyl kynurenine. In the present in vitro study, the modulatory effect of 14kDa molecule isolated from garlic on IDO induction was tested. Cultures of mononuclear cells were exposed to 14kDa garlic fraction. Then, their proliferation responses and IDO metabolites were measured. A significant down-regulatory effect of garlic on IDO activity was found and also the proliferation responses of mononuclear cells increased. If these results are verified in vivo, an explanation will be provided on how garlic may interfere in IDO induction, which paves the way for elucidating its specific therapeutic effect in preventing tumor progress

High production of IL-18 by dendritic cells induced by sera from patients with primary antibody deficiency

Predominantly antibody deficiencies are a category of primary immunodeficiency diseases, which consist of several rare disorders such as common variable immunodeficiency [CVID] and X-linked agammaglobulinemia [XLA]. We evaluated the effects of CVID and XLA patients' sera as a source of microenviromental factors on maturation and function of monocyte-derived DCs. Blood was collected from 10 CVID and 5 XLA patients before immunoglobulin replacement therapy and also from 8 healthy volunteers in order to obtain necessary sera for this study. Monocyte derived DCs were generated from blood cells obtained from healthy volunteers in the presence of GM-CSF, IL-4 and 10% serum concentrations from cases and controls. Immature DCs were incubated with monocyte conditioned medium [MCM] and TNF-alpha in order to generate mature DCs. Interleukin 18 [IL-18] production by CD40L-activated mature DCs was measured after 24 hours of culture in vitro. IL-18 production by DCs generated in the presence of CVID and XLA patients' sera were 6.75 +/- 2.59 and 7.08 +/- 1.75 ng/ml, respectively, which were significantly higher than normal serum conditioned DCs [3.55 +/- 0.68] ng/ml. These results suggest that the sera of patients with predominantly antibody deficiencies may contain soluble factor[s] that can induce a significant increase in IL-18 production by DCs

Comparison of functional competence of umbilical cord and adult peripheral blood dendritic cells in allogenic mixed leukocyte reaction

Dendritic cells [DCs] are the most potent stimulators of primary T cell responses and play a key role in immune reactions after stem cell transplantation. Very little is known about the cord blood [CB] dendritic cells and their potential involvement in the low incidence and lower severity of acute graft-versus-host disease after CB transplantation. The aim of this study was the isolation of cord blood and peripheral blood dendritic cells and comparison of their functional competence and determination of their probable role in graft versus host disease after stem cell transplantation. In this study, fresh peripheral blood DCs [PBDCs] wereenriched as HLA-DR[+] cells, lacking the CDS, CDllb, CD14, CD16, CD19 and CD56, using immunomagnetic bead depletion. For cord blood dendritic cells [CBDCs] enrichment CD34[+] and CD66b[+] cells were needed to be depleted too. Immunomagnetically enriched PB/CB dendritic cells were co-cultured with adult Tlymphocytes and cell proliferation was measured by 3H-thymidine incorporation. Results showed that CBDCs were significantly poor stimulators of the mixed leukocyte reaction as compared with PBDCs [P < 0.05]. The demonstrated impairment of CBDCs function could be of importance in interpretation of the low incidence and milder severity of graft-versus-host disease [GVHD] in umbilical CB transplantation compared with peripheral blood or bone marrow stem cell transplantation