STAT3 is overactivated in gastric cancer stem-like cells

Objective: Gastric cancer [GC] is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcrip-tion-3 [STAT3] signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell [ESCs] pluripotency. Here, we have investi-gated the activation status of STAT3 in GC stem-like cells [GCSLCs]

Materials and Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, characterized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation

Results: Spheroid cells showed higher potential for spheroid formation than the parental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition [EMT] related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated [P<0.05], but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel [DTX] when compared with parental cells [P<0.05] according to the MTS assay. Although immunostaining and Western blotting showed expression of the STAT3 protein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 [p-STAT3] in spheroid structures relative to the parent cells according to flow cytometry analysis [P<0.05]

Conclusion: The present findings point to STAT3 over activation in GCSLCs. Complementary experiments are required to extend the role of STAT3 in stemness features and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy

[Construction of polyurethane polymeric-based nano-carriers for curcumin in cancer therapy]

Objective: In order to improve the water solubility and bioavailability of curcumin in cancer therapy, we prepared and tested a novel waterborne cationic polyurethane [PU] as a nano-carrier for curcumin loading [CU-PU]. We studied the effect of this prepared nano-drug on melanoma [F10B16] and fibroblasts cells [L929]

Methods: Morphology, size and cell internalization ability of the prepared nanoparticles were analyzed by zetasizer, SEM, AFM and fluorescent microscopy, respectively. We anticipated that curcumin was loaded in the hydrophobic core of the PU carrier. Next, the suitable dose and therapeutic effects of CU-PU for both skin cancer and normal cell lines were evaluated by the MTT assay and real-time PCR

Results: The average diameters and polydispersity of the nanoparticles were 62.37 +/- 1.7 nm and 0.080 +/- 2.1 at 25 C, respectively. The drug encapsulation efficiency was 87 +/- 0.2%. The morphological analysis confirmed both a spherical shape and good dispersion without remarkable aggregation. The MTT assay results showed that the IC50 at 24 hours was 36.2 microM, whereas it was 25.4 microM at 48 hours. Real-time PCR results indicated that the CU-PU significantly decreased mRNA expressions of VEGF, Bcl-2, MMP-9 and COX-2 genes. An increase in mRNA expression of the BAX gene was also observed

Conclusion: Our result provided acceptable evidence for cell proliferation inhibition and the apoptotic effect of CU-PU on skin cancer cells. There were no adverse effects detected for normal cells

Th1 immune response induction by biogenic selenium nanoparticles in mice with breast cancer: preliminary vaccine model

Background: tumor associated antigens can be viably used to enhance host immune response

Objectives: the immunomodulatory effect of biogenic selenium nanoparticles [SeNPs] was compared between treated and untreated mice with crude antigens of 4T1 cells

Materials and Methods: female inbred BALB/c mice [60] were injected by cancinogenic 4T1 cells causing breast cancer. After 10 days, all tumor bearing mice were divided into 4 groups. Group 1 was daily provided oral PBS and injected by the same buffer after tumor induction and was considered as control. Group 2 received only 100 [micro]g/day SeNPs as an oral supplement for 30 days. Group 3 was only injected with 4T1 cells crude antigens with nil supplementation of SeNPs. Group 4 animals were supplemented 100 [micro]g/day SeNPs for 30 days and simultaneously injected with crude antigens of 4T1 cells. All antigens or PBS injections were introduced at 7, 14 and 28 days following tumor induction. Oral PBS and SeNPs supplementation initiated from the first day of tumor induction and continued up to 30 days. During tumor growth, animal weights and survival rates were monitored and at the end of the study the concentrations of different cytokines and DTH responses were measured

Results: data clearly showed that the levels of cellular immunomodulatory components [granzyme B, IL-12, IFN-[lambada], and IL-2] significantly increased [P < 0.05] in mice treated with both SeNPs and crude antigens of 4T1 cells in comparison to the other groups. In contrast, the levels of TGF-[beta] in these mice decreased

Conclusions: although SeNPs showed a noticeable boosting effect for the immune response in mice bearing tumor exposed to crude antigens of 4T1 cells, further complementary studies seem to be inevitable

Ginger extract reduces the expression ofIL-17 and IL-23 in the seraand centralnervous system of EAEMice

Background: IL-17/IL-23 axis plays an important role in the pathogenesis of severalautoimmune diseases such as experimental autoimmune encephalomyelitis [EAE] andmultiple sclerosis [MS]. The immunomodulatory properties of ginger are reported in previous studies

Objective: To evaluate the effects of ginger extract on the expressionof IL-17 and IL-23 in a model of EAE

Methods: EAE was induced in C57BL/6 miceby immunization with myelin oligodendroglial glycoprotein and then treated with PBSor ginger extracts, from day +3 to +30. At day 31, mice were scarificed and theexpression of IL-17 and IL-23 mRNA in spinal cord were determined by using realtime-PCR. The serum levels of cytokines were measured by ELISA

Results: ThemRNA expression of IL-17, IL-23 P19 and IL-23 P40 in CNS and serum levels of IL-17 and IL-23 were significantly higher in PBS-treated EAE mice than non-EAE group[p<0.003, p<0.001, p<0.001, p<0.05 and p<0.01, respectively]. In 200 mg/kg gingertreatedEAE mice the mRNA expression of IL-17, P19 and P40 in CNS and serum IL-23 levels were significantly decreased as compared to PBS-treated EAE mice [p<0.05,p<0.001, p<0.001 and p<0.05, respectively]. Moreover, 300 mg/kg ginger-treated EAEgroup had significantly lower expression of IL-17, P19 and P40 in CNS and lowerserum IL-17 and IL-23 levels than PBS-treated EAE group [p<0.02, p<0.001, p<0.001,p<0.03 and p<0.004, respectively]

Conclusion: Ginger extract reduces the expressionof IL-17 and IL-23 in EAE mice. The therapeutic potential of ginger for treatment ofMS could be considered in further studies

Curcumin-loaded chitosan tripolyphosphate nanoparticles as a safe, natural and effective antibiotic inhibits the infection of Staphylococcus aureus and Pseudomonas aeruginosa in vivo

Curcumin as a yellow natural compound extracted from turmeric root is known it as an antibacterial agent. One of the nanoparticles ability is to decrease the defects of usual drug delivery systems. Chitosan is a low toxic, biodegradable, biocompatible and safe polymer which is used in production of nanoparticles. Nanoparticles like chitosan-tripolyphosphate [TPP] are able to increase antibacterial properties of curcumin. Curcumin-loaded chitosan-TPP nanoparticles containing chitosan, curcumin and TPP salt were synthesized by ionotropic gelation methods. First, the skin of anesthetized mice was inoculated with staphylococcus aureus and pseudomonas aeruginosa suspension. Then the infected mice were treated with curcumin-loaded chitosan-TPP nanoparticles for 3 days. Following that, antibacterial characteristics of the mice treated with curcumin-loaded chitosan- TPP nanoparticles were evaluated by bacterial culture of these mice. Our results showed the size of 160 +/- 10 nm and the charge of +7 +/- 2 mV in curcumin-loaded chitosan-TPP nanoparticles. These nanoparticles were also spiral shape. The encapsulation efficiency of curcumin in chitosan-TPP nanoparticles was 75 +/- 2%. Bacterial culture showed that curcumin-loaded chitosan-TPP nanoparticles inhibited staphylococcus aureus and pseudomonas aeruginosa growth. Our study demonstrated that curcumin-loaded chitosan-TPP nanoparticles can be utilized as a potent agent in treatment of Staphylococcus aureus and Pseudomonas aeruginosa infections

In-vitro evaluation of antileishmanial activity and toxicity of artemether with focus on its apoptotic effect

Artemisinin and its derivatives are very important new class of antimalarial drugs. One of the most important artemisinin derivatives is artemether. The antiparasitic activity of artemether as a derivative of artemisinin is related to endoperoxide bridge in its structure. The aim of this study was the evaluation of antileishmanial effect of artemether, with more focus on its apoptotic effect. In this study we used artemether in concentration of 5, 10, 25, 50 and 100 microg/mL for promastigote assay, promastigote proliferation measurements by MTT assay, detection of apoptotic cells by Flow cytometry analysis and DNA ladder assay. The application of artemether, promastigote IC[50] was measured as 25 microg/mL. The percentage of apoptotic promastigotes by using 25 microg/mL of artemether was 42.28. The results of present study showed that artemether has inhibition effect on intracellular and extracellular growth of Leishmania major. Promastigotes of Leishmania major undergo apoptosis after exposure to artemether

Evaluation of the number of CD4[+] CD25[+] FoxP3[+] Treg cells in normal mice exposed to AFB1 and treated with aged garlic extract

Aflatoxin B[1] [AFB[1]] suppresses the immune system. To decrease such sup-pressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previous studies demonstrated that aged garlic [dry garlic bulbs preserved in the freezer for six months at -20°C] have increased immunostimulator fractions and reduced immunosuppressor fractions. This study focuses on the immunosuppressor activity of AFB[1] and immunostimulator activity of aged garlic extract [AGE] through the evaluation of CD4[+] CD25[+] FoxP[+] regulator cell [Treg] counts and the pattern of cytokine production in Balb/c normal mice. In this experimental research, AFB[1] was separated from Aspergillus flavus [PTCC 5004] by HPLC and AGE prepared using the Mantis method. The Delayed-Type Hypersensitivity [DTH] test was carried out to determinate the effectiveness of different doses of AGE and AFB[1], which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB.[1] on the number of Treg cell in 4 groups: 10 microl/kg/day of AFB [1], and AGE diluents were administered for 4 consecutive days to group 1. AFB[1], 2. control, 3. AGE + AFB[1] and 4. AGE via intraperitoneal [IP] route, respectively. Mice were sacrificed and splenocytes harvested and the percentage of splenic Treg cells was measured by flow cytometry analysis. The ELISA method was utilized to measure Cytokine production. The findings reveal that AGE increased the level of IFN-gamma and IL-4 cytokines produced by splenocytes stimulated by specific tumor antigen and decreased the number of Treg cells in the spleen [p<0.05]. AFB[1] increased the number Treg cells in the spleen and decreased cytokine production [p<0.05]. In groups 2 [control] and 4 [AGE] the number of Treg cells decreased [p value<0.05] whereas in groups 1 and 3 the number of Treg cells increased [p<0.05]. This study indicated that AGE is able to alter the cytokine production in normal mice into a Th[1] protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB.[1] is able to alter the cytokine production into a Th[2] protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB[1]

Purified protein fraction of garlic extract modulates cellular immune response against breast transplanted tumors in BALB/c mice model

Garlic [Allium sativum] has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration. In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4[+] and CD8[+] cells were assessed by flow cytometry. Un-paired student's t test using the SPSS program was applied for all statistical analyses. Garlic extract included different type of proteins with different molecular weight. One of protein's fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of -10-13 kDa and different isoelectric points [pI]. These molecules augmented the delayed type hypersensitivity [DTH] response compared to the control group. Intratumor injection of the fraction provoked a significant increase in the CD8[+] subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8[+] T-lymphocytes in treated animals. The data confirms that protein fractions purified from fresh garlic bulbs augment CD8[+] T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These findings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment

Sclareol reduces CD4+ CD25+ FoxP3+ t-reg cells in a breast cancer model in vivo

Sclareol is a phytochemical used in people's diet in Southeast Asia. To investigate the immunotherapeutic effectiveness of Sclareol against breast cancer by direct intraperitoneal injection. Sclareol was isolated and purified from Salvia sclarea. Effect of Sclareol on cell growth inhibition was evaluated by MTT assay. Intraperitoneally injected Sclareol effects on reducing the tumor volume and shifting the cytokine profile were investigated. We also assessed if intraperitoneally injected Sclareol could improve the outcome of cancer therapy through suppressing the regulatory T cells. The results confirmed a significant decrease in the tumor size. Furthermore, a significant decrease in the level of IL-4 and an increase in the level of IFN-gamma were noticed in the intraperitoneally injected Sclareol group [p<0.05]. It was also observed that the splenocytes of treated animals significantly increase in cell proliferation assay. Moreover, measurements of splenic T regulatory cell indicated that intraperitoneally injected Sclareol significantly decreased the number of splenic T regulatory cell. Our results suggest that Sclareol, by reducing T-reg cells frequency and also tumor size can enhance the effect of cancer therapy as an immunostimulant

Heat shock proteins enriched-promastigotes of leishmania major inducing Th2 immune response in BALB/c mice

Heat shock proteins [HSP] are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen [SLA] was determined. BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10[6] stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks. No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-gamma, IL-4, TGF-beta, IgG1 and IgG2b were increased in both groups, IFN-gamma was significantly higher in SLA group and IgG2a in HSP-enriched SLA. These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection

effect of artemether-induced apoptosis in promastigotes of leishmania major [MRHO/IR/75/ER] under in-vitro conditions

Leishmaniasis is one of the significant causes of morbidity and mortality in several countries. It is an important problem in endemic areas such as Iran. The goal in treatment of leishmaniasis is to reduce the disease period and leave no evidence of any remaining scars or lesions. A derivative of artemisinin is artemether. Scientists believe that the strong action of artemether against parasites is due to the presence of an endoperoxide bridge. Due to problems in the treatment of Leishmania major, in this research we have studied the effect of artemether on Leishmania major under in vitro conditions. Parasites were cultured in NNN and RPMI, after which artemether at concentrations of 5, 10, 25, 50 and 100 microg/ml were used for the promastigote assay. Apoptosis was detected by flow cytometry and DNA ladder assay. The inhibitory concentration [IC50] of artemether was determined to be 25 microg/ml. The percentage of apoptotic promastigotes at 25 imcrog/ml of artemether was 42.28. The results of DNA fragmentation show that exposure of Leishmania major promastigote cells to 25 microg/ml of artemether lead to DNA fragmentation. We have proven the effect of artemether on apoptosis of Leishmania major by flow cytometry and the DNA ladder assay

[Cloning, expression, purification and antigenicity of recombinant UreB332-HpaA fusion protein from Helicobacter pylori]

Helicobacter pylori is a widely distributed Gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in developing a H. pylori vaccine is growing up rapidly. The aim of this study was to construct a recombinant vector containing fusion genes encoding a fragment of B subunit from Helicobacter pylori [H. pylori] urease [UreB332] and Helicobacter pylori adhesion A [HpaA] and expressed it in E. coli BL21, as well as determining its antigenicity as a vaccine candidate of H. pylori. The target genes encoding UreB332 and HpaA amplified from standard H. pylori chromosome by PCR, digested by restricted endonuclease enzyme and inserted into the prokaryotic expression vector pET28a [+] which was digested by corresponding restricted endonuclease enzyme. The target fusion protein was expressed in the BL21 [DE3] E. coli. Furthermore, UreB332-HpaA antigenicity was studied by western blotting after Ni-NTA agarose resin purification. Enzyme digestion analysis, PCR and sequencing showed that the target genes were inserted correctly into the recombinant vector. The fusion protein UreB332-HpaA was recognized by the rabbit anti H. pylori polyclonal antibody and the human sera infected with H. pylori. Our results in addition to favorable properties of HpaA and UreB antigens, support the application of rUreB332-HpaA fusion protein, as a good candidate for the development of H. pylori vaccine

[Evaluation of immune responses from immunization of HIV-1 P24-Nef fusion peptide vaccine with monophosphoryl lipid A [MPL] in BALB/c mice]

Several vaccines against HIV have been investigated but none has been approved as an effective HIV vaccine. An approach that could induce stronger immune response against the pathogen is utilizing a multi-epitopic vaccine. This strategy was used in the design of several vaccines and resulted in improved immune responses. In this study a multi-epitopic fusion peptide including parts of HIV-1 Nef and P24 as a vaccine candidate was injected into mice and immune humoral responses measured with total antibody and IgG sub-classes using ELISA. Also measurement of cellular immune responses through evaluation of spleen cells proliferation response using MTT and cytotoxicity by LDH were performed. Finally, the cytokine pattern of IFN-gamma and IL-4 were also determined with ELISA. The results indicate that candidate vaccine stimulated mouse splenic lymphocyte proliferation response and also induced strong cytotoxicity responses. Analysis of humoral immune response has shown that the candidate vaccine has induced specific antibody production mainly of the IgG2a sub-class. Also cytokine pattern evaluation has shown that IFN-gamma secretion was dominant. The use of immunogen and conserved epitopes from P24 and Nef induced strong humoral and cellular immune responses and this construct could be candidate for further studies in animal models

Candida albicans structural and secreted proteins modulate CD4/CD8 ratio in tumor infiltrating lymphocytes of spontaneous adenocarcinoma bearing mice

Candida albicans is one of the most important opportunistic pathogens that suppress immunologic mechanisms of the host. It is speculated that structural and secretory proteins of C. albicans have immunomodulatory effects in cancer. To evaluate the effects of C. albicans structural and secreted proteins on intratumoral CD4/CD8 ratio as well as the survival rate in BALB/c tumor model. Structural and secretory proteins from C. albicans were isolated and examined for their effects on tumor growth and survival of adenocarcinoma bearing mice. The results indicated that in mice treated with C. albicans structural protein, the survival rate significantly decreased compared with the control groups. Also, mice treated with secretory proteins showed a decrease in survival rate but it was not statistically significant [p>0.05]. Investigating the frequency of tumor infiltrated CD4+ and CD8+ T lymphocytes indicated that the percentages of tumor infiltrated CD4+ T lymphocytes in response to structural and secreted proteins were higher compared to the control groups. Our study suggests that C. albicans structural and secreted proteins modulate intratumor T lymphocyte infiltration

Effect of shark liver oil on peritoneal murine macrophages in responses to killed-Candida albicans

Shark Liver Oil [SLO] is an immunomodulator. Macrophages play a key role in host defense against pathogens like fungi. Candida albicans have mechanisms to escape immune system. We determined the effect of killed-Candida on the in vitro viability of macrophages and the effect of SLO on augmentation of this potency. Peritoneal macrophages were separated and cultured [3-105/well]. At first, the effect of killed-Candida [200 cells/well] on macrophage viability was evaluated, using MTT test. Then, MTT was performed on macrophages stimulated with killed-Candida in the presence of SLO. Killed-Candida suppressed the ability of MTT reduction and hence macrophages viability [P=0.026], but addition of SLO [100 mg/ml] significantly enhanced cell viability [P=0.00]. So, SLO could neutralize the inhibitory effect of Candida. Simultaneous with cytotoxic effect of killed-Candida cells on macrophages viability, SLO augment macrophages viability. So, it can be applied in candidiasis as a complement

Effect of invasive aspergillosis infection on the immune responses of Cancer mice

Using a cancer murine model of invasive aspergillosis [IA], we investigated the expression of TLR-2, Dectin-1 and the level of cytokine production by CD4+ T helper cells in different groups of mice [with or without cancer], also, the effect of invasive aspergillosis on the immune response pattern of cancer mice. Patterns of susceptibility and resistance to infection obtained with different groups of mice injected with Aspergillus fumigatus conidia. TLR-2 and Dectin-1 analyzed applying flowcytometry and cytokine production of cultured splenocytes by ELISA method. Cancer mice that challenged with A. fumigatus conidia showed significant increase in TLR-2 and Dectin-1 levels compared with the two other control groups [normal mice challenged with A. fumigatus and non-infected cancer mice]. Moreover, it showed insignificant decrease in IFN-gamma and IL-10 levels and insignificant increase in TNF-alpha level. The data demonstrated remarkable rise in IL-4 level and the mortality of cancer mice that intravenously infected with A. fumigatus. Probably IA causes stimulation in innate immunity and Th2 cells, also some disorganization in cytokine production in CD4+ T helper cells. We hypothesize that concomitance of IA and cancer may change the microenvironment for local or systemic immune responses. Other complementary studies could help supporting our hypothesis

Immunogenicity of a new HIV-1 DNA Construct in a BALB/c mouse model

Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progression. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. In the present study a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. HIV-1 P24-Nef gene was cloned in PCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-gamma evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-gamma [p<0.05]. Cytotoxicity and lymphocyte proliferation responses of mice vaccinated with the candidate vaccine were significantly increased compared to control groups [p<0.05]. HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model

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

CTL responses to a DNA vaccine encoding E7 gene of human papillomavirus type 16 from an Iranian isolate

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among female population worldwide. Specific human papillomaviruses and, most notably, HPV types 16 and 18 are recognized as being causally associated with cervical carcinomas. The early HPV type 16 genes, E6 and E7, directly participate in the in vitro transformation of primary human keratinocytes and represent an excellent target for immune therapy of HPV related disease. The aim of this study was the evaluation of the efficacy of a DNA vaccine containing human papillomaviruse type 16 E7 gene [Iranian isolate] in induction of CTL responses in an animal model. In this study, the expression vector containing HPV type 16 E7 gene was constructed and chosen as a model antigen in the development of a therapeutic DNA vaccine in an animal model. CTL responses, cytokine assay, lymphocyte stimulation test, CD4 and CD8 staining and flowcytometry were done for evaluating of the immune responses. Our findings indicate that the target DNA vaccine can induce an E7-specific CTL response, which is important in the lysis of infected tumor cells, compared to negative control [p < 0.005] after in vivo immunization in the mouse system. The developed vaccine may be promising as an anti-cancer vaccine

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