Samaneh Arab; Masoumeh Motamedi; Jamshid Hadjati
Abstract
Background: Dendritic cells (DCs) contribute essentially to the outset and course of immune responses. So in patients with malignancy, there have been considerable interests in use of these cells in different interventions. Objective: To evaluate the impact of Leishmania major’s components ...
Read More
Background: Dendritic cells (DCs) contribute essentially to the outset and course of immune responses. So in patients with malignancy, there have been considerable interests in use of these cells in different interventions. Objective: To evaluate the impact of Leishmania major’s components on DC maturation and their use as a therapeutic agent against tumor cells. Methods: The cancer model was induced by injection of WEHI-164 cells (BALB/c derived fibrosarcoma cell line) subcutaneously in the right flank of animals. Bone-marrow derived DCs (BMDCs) were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. After 5 days, tumor lysate, Leishmania major’s lysate, and Lipopolysaccharide (LPS) were added to the culture and incubated for 2 days. IL-12 production in DCs was measured by ELISA. For Immunotherapy, Mice received DCs subcutaneously around the tumor site. Two weeks after DCs injection, cytotoxicity assay and infiltration of CD8+ lymphocytes were evaluated. Results: Our results showed that immunotherapy with dendritic cells exposed to Leishmania extract led to producing a higher amount of IL-12, compare to the control group. A considerable increment in specific cytotoxic T cells activity, diminished tumor growth rate and improved survival of immunized animals were seen. Conclusion: This study indicates that the use of Leishmania major extract, as well as LPS, can enhance the efficiency of DC-based vaccines and provides a basis for the use of Leishmania major in DC-targeted clinical therapies.
Yousef Nikmanesh; Mohammad Hossein Karimi; Ramin Yaghobi; Sayed Mahdi Marashi; Mahmood Mahmoudi; Ali Moravej; Shohreh Shahmahmoodi
Marek Fol; Magdalena Kowalewicz-Kulbat; El ż bieta Ograczyk; Marcin W ł odarczyk; Krzysztof Krawczyk
Volume 13, Issue 2 , June 2016, , Pages 132-140
Abstract
Background: The immunomagnetic separation technique is the basis of monocyte isolation and further generation of monocyte-derived dendritic cells. Objective: To compare the efficiency of monocyte positive and negative separation, concentration of beads, and their impact on generated dendritic cells. ...
Read More
Background: The immunomagnetic separation technique is the basis of monocyte isolation and further generation of monocyte-derived dendritic cells. Objective: To compare the efficiency of monocyte positive and negative separation, concentration of beads, and their impact on generated dendritic cells. Methods: Monocytes were obtained using monoclonal antibody-coated magnetic beads followed the Ficoll-Paque gradient separation of mononuclear cell fraction from the peripheral blood of 6 healthy volunteers. CD14 expression was analyzed by flow cytometry. Results: The percentage of MDDCs generated from monocytes obtained by positive and negative selection was comparable (51.8 ± 15.0 and 46.7 ± 3.4, respectively; p=0.885). The median values for the number of MDDCs obtained from monocytes after positive selection (3.9 × 106) and for MDDCs obtained from monocytes after negative selection (3.1 × 106) were comparable (p=0.194). The use of the recommended or half of the amount of beads for both types of separation had no significant influence on the percentage of isolated cells. Conclusions: Both types of magnetic separation including recommended and reduced concentrations of beads did not affect the yield and the purity of monocytes and their surface CD14 expression. However, DCs originated from the “positively” separated monocytes had noticeable higher expression of CD80.
Yang Li; Lu Zhang; Shouyu Wang; Peng Shi; Wei Qu
Volume 11, Issue 3 , September 2014, , Pages 166-176
Abstract
Background: Fusion of dendritic cells (DCs) with melanoma cells could reinforce the antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach. Objective: To define the ...
Read More
Background: Fusion of dendritic cells (DCs) with melanoma cells could reinforce the antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach. Objective: To define the dosage of the stimulating factors as well as the induction condition for the optimal DCs preparation and cell fusion. Methods: DCs were generated from murine bone marrow cells, and cultured with four different concentrations of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were confirmed to be mature by detecting the expression of MHC-II, CD11c, CD80, and CD83 by flowcytometry. DCs-melanoma fusion cells were generated using polyethylene glycols (PEG) with different molecular weights and the fusion efficiency was detected by fluorescence-activated cell sorter (FACS). Results: The largest quantity of DCs was found when cells were cultured with 1000 U/ ml of GM-CSF and 500 U/ml of IL-4 (1.69 ± 0.04 ×10 6 ml-1, p<0.001 when compared with the other three groups). The expression levels of MHC-II and CD83 on day 7 after incubation were significantly lower than those on day 3 (MHC-II: p<0.001; CD83: p<0.001). The efficiency of cell fusion under induction of PEG-3000 was significantly higher than that of PEG-4000 (15.4 ± 0.56% vs. 11.1 ± 0.45%, p<0.001). Conclusions: The largest quantity for mature DCs was stimulated with 1000 U/ml of GM-CSF and 500 U/ml of IL-4 and the highest fusion efficiency was under induction of PEG-3000.
Nowruz Delirezh; Ehsan Shojaeefar
Volume 9, Issue 2 , June 2012, , Pages 98-108
Abstract
Background: Generation of an effective dendritic cell (DC) based cancer vaccine depends on appropriate differentiation of monocytes in vitro. Objective: To compare the effects of monocyte separation methods, flask adherence (Flask-DC) and magnetic activated cell sorting (MACS-DC), on phenotypic and functional ...
Read More
Background: Generation of an effective dendritic cell (DC) based cancer vaccine depends on appropriate differentiation of monocytes in vitro. Objective: To compare the effects of monocyte separation methods, flask adherence (Flask-DC) and magnetic activated cell sorting (MACS-DC), on phenotypic and functional characteristics of resultant DCs. Methods: DCs from healthy volunteers were generated from plastic adherence and MACS isolated monocytes in the presence of GM-CSF and IL-4 as well as TNF-α and monocyte conditioned medium (MCM) in 7 day cultures. Mature DCs were then subjected to phenotypic analysis using anti-CD14, anti-CD83 and HLA-DR monoclonal antibodies. Functional and cytokine release assays were carried out using [3H] thymidine uptake test and commercially available ELISA kits for the determination of IL-12, IL-10, IFN-γ and IL-4, respectively. Results: We found that MACS-DCs were more homogenous and the yield and viability were fairly higher than Flask-DCs. MACS-DCs expressed higher levels of CD83 and HLA-DR as well as CD14 compared to the Flask-DCs. Induction of T cell proliferative responses were higher in Flask-DCs and also they elicited higher levels of IL- 12: IL-10 and IFN-γ: IL-4 ratios in cytokine generation assays. Conclusion: MACS method was superior for mass production of viable homogenous and fully mature DCs but their cytokine profile had the potential to polarize the immune system toward Th2 type immune response.
Samaneh Arab; Masoumeh Motamedi; Nematollah Khansari; Seied Mohammad Moazzeni; Zahra Gheflati; Jamshid Hadjati
Volume 3, Issue 3 , September 2006, , Pages 99-105
Abstract
Background: 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. Objectives: To investigate the effect of tumor antigen ...
Read More
Background: 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. Objectives: To investigate the effect of tumor antigen pulsed DCs in the presence of CpG-1826 in treatment of a murine model of cancer. Methods: 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. Results: 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. Conclusion: 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.