Adenosine Blockage in Tumor Microenvironment and Improvement of Cancer Immunotherapy

Immunotherapy has been introduced into cancer treatment methods, but different problems have restricted the efficacy of these protocols in clinical trials such as the presence of various immunomodulatory factors in the tumor microenvironment. Adenosine is an immunosuppressive metabolite produced by the tumor to promote growth, invasion, metastasis, and immune evasion. Many studies about adenosine and its metabolism in cancer have heightened interest in pursuing this treatment approach. It seems that targeting the adenosine pathway in combination with immunotherapy may lead to efficient antitumor response. In this review, we provide information on the roles of both adenosine and CD73 in the immune system and tumor development. We also describe recent studies about combination therapy with both purinergic inhibitors and other immunotherapeutic methods.

Construction and production of Foxp3- Fc [IgG] DNA vaccine/fusion protein

Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell [DC] based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc[IgG] can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I [cross priming]

Methods: DNA construct containing fragment C [Fc] portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli [E. coli] strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining

Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification

Conclusion: Due to successful expression of Foxp3-Fc [IgG], it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means

[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

In vitro cytotoxicity of four calcium silicate-based endodontic cements on human monocytes, a colorimetric MTT assay

OBJECTIVES: This study was performed to evaluate the cytotoxicity of four calcium silicate-based endodontic cements at different storage times after mixing. MATERIALS AND METHODS: Capillary tubes were filled with Biodentine (Septodont), Calcium Enriched Mixture (CEM cement, BioniqueDent), Tech Biosealer Endo (Tech Biosealer) and ProRoot MTA (Dentsply Tulsa Dental). Empty tubes and tubes containing Dycal were used as negative and positive control groups respectively. Filled capillary tubes were kept in 0.2 mL microtubes and incubated at 37degrees C. Each material was divided into 3 groups for testing at intervals of 24 hr, 7 day and 28 day after mixing. Human monocytes were isolated from peripheral blood mononuclear cells and cocultered with 24 hr, 7 day and 28 day samples of different materials for 24 and 48 hr. Cell viability was evaluated using an MTT assay. RESULTS: In all groups, the viability of monocytes significantly improved with increasing storage time regardless of the incubation time (p < 0.001). After 24 hr of incubation, there was no significant difference between the materials regarding monocyte viability. However, at 48 hr of incubation, ProRoot MTA and Biodentine were less cytotoxic than CEM cement and Biosealer (p < 0.01). CONCLUSIONS: Biodentine and ProRoot MTA had similar biocompatibility. Mixing ProRoot MTA with PBS in place of distilled water had no effect on its biocompatibility. Biosealer and CEM cement after 48 hr of incubation were significantly more cytotoxic to on monocyte cells compared to ProRoot MTA and Biodentine.

Effects of mustard gas on immune system of exposed Iranian people: a review of conducted studies

Exposure to high dosages of sulfur mustard [SM] can cause bone marrow depression, immune system suppression, impairment of the immune functions, and eventually results in diseases due to secondary immune disorders. In this article, we have studied the effects of this poison on Iranian veterans by analysis of related published studies. In a systematic search, the effects of SM gas on Iranian victims were reviewed. We used known international medical databases such as ISI, Medline, Scopus and Iranian databases such as Iranmedex and Irandoc. About 350 published articles were assessed. Among them, 43 articles were related to immunologic field. No special evaluation was conducted on the quality of the reviewed manuscripts and the credit of journal was considered sufficient. In accomplished studies conducted on Iranian people, both cellular and humoral immunity were affected. The reported changes were as follows: increasing the number of inflammatory cells in chronic phase which indicates ongoing active alveolitis, neutrophils [in chronic bronchitis], eosinophils, CRP titer, RF titer, IgG [especially in asthmatic patterns] ,IgM, Ig E, IL-6, TFG-beta1target protein in bronchoalveolar lavage fluid, and decreasing the number of leukocytes, lymphocytes, natural killer cells [NKCs], IL-8and IL-6 in blood. Eventually, in reported changes, chemo taxis factors, plasma opsonins and nitroblue tetrazolium [NBT] test were normal. In sever and prolonged exposure to mustard gas, the immune system would be suppressed. Therefore, the victims should be monitored for infections and even cancers

Effect of protein and DNA components of toxoplasma gondii on IL-12 production from dendritic cells and T cell proliferation

The aim of the present study was to investigate the effect of protein and DNA components of Toxoplasma gondii on maturation of dendritic cells and their efficiency in IL-12 production and proliferation of T cells. for DC generation, Bone marrow cells were cultured in the presence of GM- CSF and IL-4 for 5 days. Tumor lysate and protein or DNA components of Toxoplasma gondii were added to the culture media and incubated for another 2 days. LPS was added as control for DC maturation. Proliferation of T cells were determined by MLR and IL-1 production was measured by ELISA kit. Maturation of dendritic cell were determined by flowcytometry. DCs treatment with protein components of Toxoplasma gondii caused a significant increase in IL- 1 2 production and proliferation of T cells [P<0.001]. Different compositions of microbial body like protein and DNA components of Toxoplasma gondii can cause augmentation of antigen presentation capacity of DC and their IL-12 production capability. Among these components the protein was more effective as compared to DNA

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

Listeria monocytogenes activated dendritic cell based vaccine for prevention of experimental tumor in mice

The use of dendritic cells [DCs] as a cellular adjuvant provides a promising approach in immunotherapy of cancer. It has been demonstrated that Listeria monocytogenes activated DCs pulsed ex vivo with tumor antigens trigger a systemic Th1-biased specific immune response and a single dose of this vaccine will cause a considerable anti tumor immunity. The present study was designed to evaluate the ability of multiple doses of tumor antigen-pulsed DCs, matured in the presence of Listeria monocytogenes components in induction of a potent anti-tumor response and the prevention of tumor formation in an experimental model. Bone-marrow derived DCs [BMDCs] were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysates with/without Listeria monocytogenes lysate were added to the culture media for another 2 days. Mice received mature and tumor antigen pulsed dendritic cells subcutaneously in 3 groups. Tumor growth was monitored and two weeks after immunotherapy, cytotoxic activity of CD8+ T cells was evaluated in different groups. According to the findings, repeated doses of vaccine did not lead to a significant increase in the activity of cytotoxic T cells and decreased tumor growth of immunized animals. The current study suggests that increased doses of vaccine do not have sufficient efficiency for prevention of tumor induction. Generation of T regulatory responses upon repeated doses of such vaccines should be considered in future investigations

[Evaluation of different Listeria monocytogenes components on dendritic cells for eliciting TH1 response]

Dendritic cells [DCs] play a critical role in the beginning and in the course of immune responses. By observing. Considering the defect in these cells in patients with tumor malignancy. In recent years there has been considerable interests in correction and use of these cells. In our prvious studies we showed that lysate of Listeria monocytogenese can induce maturation of dendritic cells, and induce TH1 response and increase the survival of tumor in an experimental model. The main objective in the present study is to evaluate the effects of different constituents of Listeria monocytogenes on DCs and their efficiency to induce TH1 response. After preparation of different components of Listeria monocytogenese [lysate of Listeria monocytogenes, protein and nucleic acid components], mouse bone marrow cells were cultured for 5 days in the presence of IL-4 and GM-CSF and treated with different components of Listeria for another 2 days. DCs were evaluated for the expression of Co-stimulatory molecules, MHC Class II molecules and Cytokine secretion. The results showed that, all of the components are able to mature DCs efficiently and induce TH1 response. But dendritic cells matured with protein components shift the response to TH1 more efficiently. Our findings indicated that dendritic cell maturation protein components of Listeria monocytogenese shift the response to TH1 more efficiently and may have beneficial effects in cancer immunotherapy

effects of cured dentin bonding agents on secretion of pro-inflammatory cytokines, IL-1beta and TNF-alpha, by human monocytes

Dentin Bonding Agents [DBA] have been used as root-end filling materials. Present study evaluated the effects of polymerized DBA on secretion of pro-inflammatory cytokines by normal human monocytes. In this study, monocytes were directly isolated from human peripheral blood, and exposed to cured Scotch Bond 1 [single bond] and Prime and Bond for 36 and 72 hours. Secretion of IL-1beta and TNF-alpha in the presence of lipopolysaccaharide was evaluated in supernatants of monocyte culture. DBAs significantly caused reduction of cytokine production by human monocytes after 36 and 72 hours. Prime and bond exposure caused more prominent decrease in TNF-alpha production after 72 hours. We conclude that DBA in polymerized form can alter normal function of human monocytes

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

Dendritic cell maturation with CpG for tumor immunotherapy

Bacterial DNA has immunostimulatory effects on different types of immune cells such as dendritic cells [DCs]. Application of DCs as a cellular adjuvant represents a promising approach in the immunotherapy of infectious disease and cancers. To investigate the effect of tumor antigen pulsed DCs in the presence of CpG-1826 in treatment of a murine model of cancer. WEHI-164 cells [Balb/c derived fibrosarcoma cell line] were injected subcutaneously in the right flank of mice. Bone marrow cells were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysate, CpG-1826, and oligodeoxynucleosides, as control, were added to the culture media and incubated for 2 days. Cytokine production in DCs culture media was measured by ELISA. Then DCs were injected subcutaneously around the tumor site in the right flank of mice. Tumor growth rate was monitored in case and control groups. Two weeks after DCs immunotherapy, cytotoxic assay was conducted using various amounts of effector [splenic T cells] and target cells [WEHI-164 or CT26] for 6 h. Immunotherapy with DCs treated with CpG led to a significant increase in the activity of cytotoxic T cells and decreased tumor growth in immunized mice. In the control group which received DCs without CpG treatment, no change in cytotoxic activity and tumor growth rate was detected. The current study suggests that specific anti tumor immune responses can be induced by DCs matured with CpG and proposes CpG usage in DCs targeted clinical strategies

Effects of gamma irradiation on proliferation and IL-5 production of peripheral blood lymphocytes

Gamma irradiation is routinely used for suppression of lymphocyte function in transfusion and transplantation procedures. In recent years, some investigators focused on the effects of ionizing radiation on special aspects of lymphocyte function and considered the possibility of its clinical application for treatment of some immunological disorders. In this study, we evaluated the effects of five different doses of