Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4.

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

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

Granulocyte Macrophage Colony-Stimulating Factor May Modulate the Post-transcription Pathway of Interleukin-6 Expression in Prostate Carcinoma Cells

Interleukin-6 (IL-6) can stimulate a variety of tumors including prostatic carcinoma. Research has recently shown that IL-6 may act to stimulate the progression of prostatic cancer. To date, little research has been performed to better understand the nature of granulocyte macrophage colony-stimulating factor (GM-CSF) and the expression of IL-6. The aim of this study was to evaluate the effects of GM-CSF on the expression of IL-6 in prostate cancer-3 (PC-3) cells. The bone-derived PC-3 cell line was used in this study. Reverse transcription polymerase chain reaction (RT-PCR) and real- time PCR were performed to detect IL-6 mRNA expression. The IL-6 protein was measured by enzyme-linked immunosorbent assay (ELISA) after treatment with hGM-CSF. Our data indicated that IL-6 mRNA expression did not increase after treatment with hGM-CSF in comparison to the control group. However, the expression of IL-6 protein was increased compared to the control group. GM-CSF may modulate the post-transcription pathway of IL-6 expression in prostate carcinoma cells. Our data suggest that GM-CSF may have a role in IL-6-mediated development of prostate cancer.

The effects of fluconazole and cytokines on human mononuclear cells

Candida infections are common infections and fluconazole is one of the most frequently administered antifungal agents in their treatment. The resistance developed against antifungal agents has necessitated the improvement of new treatments. This study focuses on the investigation of the effect of fluconazole and cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) on chemokine production and anticandidal activity of human monocytes. In the study it was observed that GM-CSF caused an increase in candidacidal activity of monocytes. Anticandidal activity of GM-CSF + IFN-gamma combination was not found to be more effective than GM-CSF or IFN-gamma alone. The presence of cytokine and fluconazole caused an increase in the levels of CCL3 and CCL4 chemokines. Accordingly, it was considered that chemokines could contribute to the efficacy of fluconazole in C. albicans infections. Besides, in order to strengthen the immune system some cytokines might be used in addition to antifungal agents for the treatment.

Synergistic activation of p70S6 kinase associated with stem cell factor in MO7e cells

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.

Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: interim analysis

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.

Defects in the differentiation and function of bone marrow-derived dendritic cells in non-obese diabetic mice

Due to their high immunostimulatory ability as well as the critical role they play in the maintenance of self-tolerance, dendritic cells have been implicated in the pathogenesis of autoimmune diseases. The non-obese diabetic (NOD) mouse is an animal model of autoimmune type 1 diabetes, in which pancreatic beta cells are selectively destroyed mainly by T cell-mediated immune responses. To elucidate initiation mechanisms of beta cell-specific autoimmunity, we attempted to generate bone marrow-derived dendritic cells from NOD mice. However, our results showed low proliferative response of NOD bone marrow cells and some defects in the differentiation into the myeloid dendritic cells. NOD dendritic cells showed lower expressions of MHC class II, B7-1, B7-2 and CD40, compared with C57BL/6 dendritic cells. In mixed lymphocyte reactions, stimulatory activities of NOD dendritic cells were also weak. Treatment with LPS, INF-gamma and anti-CD40 stimulated NOD dendritic cells to produce IL-12p70. The amount of IL-12, however, appeared to be lower than that of C57BL/6. Results of the present study indicated that there may be some defects in the development of NOD dendritic cells in the bone marrow, which might have an impact on the breakdown of self tolerance.

Ex vivo generation of functional dendritic cells from mobilized CD34+ hematopoietic stem cells

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.

Role of interleukin-1B and granulocyte-macrophage colony stimulating factor in chemotherapy induced feppile neutropenia in children with hematologic malignancies

Cytokines might have a role in the etiopathogenesis of hematologic malignancies. Serum levels of interleukin-1B [IL-1B] and granulocyte- macrophage colony stimulating factor [GM-CSF] were evaluated in 48 febrile neutropenic children undergoing cytoreductive chemotherapy for treatment of some hematologic malignancies [20 patients with acute lymphoblastic leukemia [ALL], 16 patients with acute non-lymphoblastic leukemia [ANLL], and 12 patients with non-Hodgkin's lymphoma [NHL]. They had significantly high mean serum value of IL-1B as compared with 20 healthy, sex and age matched children as controls [p <0.001]. In addition, they had significantly low mean serum value of GM-CSF in comparison with the control group [p <0.001]. Although, higher mean serum values of IL-1B were found in patients' groups with high grade fever, that with positive blood culture and that with mild to moderate neutropenia, yet, they were not significant when compared with patients' groups with low grade fever, that with negative blood culture and that with severe neutropenia, respectively. Thus, from this study, it is clear that febrile neutrophic children with hematologic malignancies under chemotherapy had high serum levels of IL-1B and low levels of GM-CSF

Biological therapies in bone marrow transplantation.

Biological materials are increasingly being utilized in bone marrow transplants. Alpha interferon is given post autotransplants, in an attempt to eradicate residual cancer cells. Colony stimulating factors (granulocyte macrophage colony stimulating factor: GM-CSF and granulocyte colony stimulating factor: G-CSF) are widely used after bone marrow transplants to accelerate bone marrow recovery. Their use has been associated with fewer infections, shorter hospital stay and lower procedural costs. Both these factors benefit some patients with graft failure as well. The recent demonstration that both GM-CSF and G-CSF can mobilize large numbers of haematopoietic stem cells into the peripheral blood has resulted in the widespread use of peripheral blood stem cell transplantation as an alternative to autologous bone marrow transplantation. Identification of tumour cells in the mobilized peripheral blood stem cells, however, has raised some concern.

Differential responses of CD34-positive acute myelogenous leukemic blasts to the costimulating effects of stem cell factor with GM-CSF and/or IL-3

Stem cell factor (SCF), a c-kit ligand, has a preferential effect on the proliferation of several classes of immature hematopoietic progenitor cells in combination with GM-CSF or IL-3. To analyze the costimulatory role of SCF in leukemic growth, we investigated the effect of SCF in the presence of GM-CSF and/or IL-3 on isolated CD34-positive (CD34+) leukemic blasts from 15 patients with acute myelogenous leukemia (AML). Cultures of CD34+ cells from normal bone marrow were used as controls. When the proliferation of CD34+ AML blasts in the presence of GM-CSF and/or IL-3 were evaluated in vitro for the effects of SCF, two patterns emerged. In one pattern, CD34+ AML blasts responded with a significant increase in DNA synthesis and/or colony formation when SCF was used with GM-CSF and/or IL-3 relative to the growth with SCF alone; This result is consistent with those CD34+ bone marrow cells from normal donors. Six patients (40%) were included in this category. The addition of SCF as a single factor resulted in colony formation in all six of these cases. In the other pattern, nine of the patients (60%) had CD34+ leukemic cells whose growth with SCF plus either GM-CSF, IL-3, or GM-CSF+IL-3, was not significantly different from the growth noted in the presence of SCF alone. Among them seven cases that did not form colonies in response to SCF alone, and one case showing autocrine, background growth were included. In the six cases in which the costimulating effects of SCF were documented, CD34+ c-kit+ blasts comprised 50.5 +/- 18.7% of the CD34+ leukemic blasts-higher than 21.8 +/- 19.4% of cases in which the costimulating effect of SCF was not documented. In the cases showing high c-kit antigen expression(> or = 40%), SCF had a costimulatory effect in 71% (5/7) of the patients. In conclusion, our data indicate that CD34+ leukemic blasts from a good proportion of patients with AML did not respond to the costimulating effects of SCF in the presence of GM-CSF adn/or IL-3, in contrast to those CD34+ bone marrow cells from normal donors. The possible use of SCF for acute leukemia must await further cytogenetic and molecular studies, which should clarify the preferential costimulating role of SCF in normal hematopoiesis.