Effects Of Granulocyte-macrophage Colony-stimulating Factor Research Articles

A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). Multicenter, randomized, placebo-controlled, double-blinded prospective design. Fourteen Scandinavian fertility clinics. A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. OIR at gestational week 7, with follow-up at week 12 and birth. At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. NCT00565747.

Protection of hyperoxia-induced lung injury by granulocyte-macrophage colony-stimulating factor via RAGE-NF-κB signaling pathway in newborn rats

To explore the effects of granulocyte-macrophage colony-stimulating factor (GMCSF) on hyperoxia exposure lung injury in newborn rats and elucidate its protective mechanism of operating via the signaling pathway of advanced glycation end products (RAGE)-NF-κB. Twenty-four 3-day-old SD rats from 3 litters were randomly divided into 3 groups. They were hyperoxia exposure plus GMCSF group (group A), hyperoxia exposure group (group B) and air exposure group (group C). The rats from groups A and B were placed in a sealed Plexiglas chamber with a minimal in-and-outflow, providing 6 - 7 exchanges per hour of chamber volume and maintaining O(2) levels above 95%. While the rats in group C only were exposed to air simultaneously. The rats in group A received subcutaneous injections of recombinant murine GMCSF (9 µg/kg) during hyperoxia exposure at 24 h, 72 h and 120 h respectively. And the rats in groups B and C received subcutaneous injections of saline vehicle alone at the same time point. Seven days later, all were sacrificed and immunohistochemistry was employed to assess the expression of RAGE in lung tissue. The levels of tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF) and serum samples were detected by ELISA (enzyme-linked immunosorbent assay). The RAGE mRNA and NF-κB mRNA in tissue homogenates were detected by RT-PCR while RAGE and NF-κB by Western blot. Also the values of lung damage score were calculated with microscopic histology. The value of lung damage score in group C, B and A was 0.46 ± 0.20, 3.06 ± 0.33 and 2.31 ± 0.56 respectively, there was significantly difference among three groups (P = 0.000). The expression of RAGE mRNA and protein in three groups were 0.14 ± 0.02, 0.34 ± 0.06, 0.28 ± 0.04 and 0.30 ± 0.04, 0.76 ± 0.11, 0.55 ± 0.08 respectively. There were both significantly differences among three groups (P = 0.000, P = 0.000). The expression of NF-κB mRNA and protein in three groups were 0.41 ± 0.21, 0.90 ± 0.36, 0.69 ± 0.30 and 0.41 ± 0.26, 0.96 ± 0.43, 0.77 ± 0.33 respectively, there were both significantly difference among three groups (P = 0.000, P = 0.017). The level of TNF-α in BALF was 76 ± 10, 224 ± 42 and 143 ± 24 respectively, there was significantly difference among three groups (P = 0.000). All indicators above in group B and group A were significantly more than those in group C (all P < 0.05), while these indicators in group A were lower than those in group B. But there was no difference in the level of TNF-α of serum among three groups (P > 0.05). GMCSF may protect hyperoxia-induced lung injury via down-regulating the signaling pathway of RAGE-NF-κB.

E0091 Potential effect of granulocytemacrophage colonystimulating factor on the arterial remodelling at the reference site after angioplasty

ObjectiveArterial remodelling may also occur at the reference site. Resent studies showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) could facilitate the repair of endothelium and reduce vascular inflammation. To observe the...

Granulocyte-macrophage colony-stimulating factor augments neutrophil killing of Torulopsis glabrata and stimulates neutrophil respiratory burst and degranulation

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the interaction between the fungus Torulopsis glabrata and human neutrophils was examined. Pre-incubation of neutrophils with GM-CSF increased the neutrophil fungal killing. The cytokine also increased the oxygen-dependent respiratory burst in response to opsonized fungi, measured by the lucigenin-dependent chemiluminescence assay and superoxide release. Under the same conditions the cytokine augmented release of constituents from both specific and azurophilic granules. Besides these priming effects, GM-CSF was a weak stimulus of the neutrophil respiratory burst and degranulation. The priming and stimulatory effects of GM-CSF were observed at 10-1000 U/ml with an optimal concentration of 100 U/ml.

Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α) and IL-6 on the production of immunoreactive IL-1 and TNF-α by human monocytes

The effects of GM-CSF, IL-2, IFN-gamma, TNF-alpha and IL-6 on the production of IL-1 (both secreted and cell associated) and TNF-alpha by peripheral blood monocytes were studied. Monocytes were cultured for 20 h in suspension and in serum-free conditions which minimized background stimulation of monokine production. GM-CSF, IL-2 and TNF-alpha directly induced the production of cell-associated IL-1 but little or no IL-1 or TNF-alpha secretion. Combination of GM-CSF with IFN-gamma, IL-2 or TNF-alpha synergistically enhanced IL-1 secretion and had an additive effect on cell-associated IL-1 production. Combination of IL-2 with IFN-gamma or TNF-alpha also synergistically enhanced IL-1 secretion but the effect on cell-associated IL-1 production was less than additive. GM-CSF synergistically enhanced TNF-alpha secretion induced by IFN-gamma but not by lipopolysaccharide. GM-CSF did not enhance TNF-alpha secretion induced by IL-2 or TNF-alpha. In contrast, IL-2 synergistically enhanced TNF-alpha secretion induced by IFN-gamma. These results are discussed in relation to cytokine involvement in rheumatoid arthritis.

Heterogeneity of Human Macrophages in Culture and in Atherosclerotic Plaques

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.

Cross-Inhibition of Interferon-Induced Signals by GM-CSF Through a Block in Stat1 Activation

We investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on biologic signals induced by interferon-alpha (IFN-alpha) and IFN-gamma. In hematopoietic cell lines, IFN-induced signaling was investigated by Western blotting, electrophoretic mobility shift assays (EMSA), flow cytometry, protein-tyrosine phosphatase (PTP) assays, and RT-PCR. GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced Stat1 tyrosine phosphorylation in a time-dependent manner. EMSA showed that GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced IFN-gamma activator sequence (GAS) binding activity. As a consequence, IFN-induced transcription of the early response gene, IFN-stimulated gene 54 (ISG54), was inhibited. The expression of IFN regulatory factor-1 (IRF-1) and MHC class I antigens was downregulated at protein levels in hematopoietic cell lines (U937, THP1). In contrast to GM-CSF, granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3) did not influence the IFN-induced Stat1 activation. To explore the molecular mechanism of suppression of Stat1 tyrosine phosphorylation, we investigated the induction and activation of cytokine-inducible SH2-containing protein/suppressor of cytokine signaling (CIS/SOCS) molecules and phosphatases on GM-CSF treatment. In contrast to G-CSF and IL-3, GM-CSF strongly induced the expression of CIS1 and SOCS2 at mRNA levels, but overexpression of CIS1 or SOCS2 in HEK293 cells did not show inhibition of Stat1 tyrosine phosphorylation upon IFN treatment. In PTP assays, on GM-CSF incubation, no enhanced src homology 2 domain tyrosine phosphatase 1 and 2 (SHP1 and SHP2) activity was detectable. However, GM-CSF-induced downregulation of Tyk2 and Jak1 tyrosine phosphorylation as well as Tyk2 protein levels likely contributed to the reduced Stat1 tyrosine phosphorylation. In hematopoietic cells, GM-CSF antagonizes IFN-induced signals by a block in Stat1 activation.

Phase II Study of Ketoconazole Plus Granulocyte-Macrophage Colony-Stimulating Factor for Prostate Cancer: Effect of Extent of Disease on Outcome

The efficacy of ketoconazole plus the immunostimulatory cytokine granulocyte-macrophage colony-stimulating factor was prospectively evaluated in patients with castration resistant prostate cancer with and without metastases. Eligible patients had progressive castration resistant prostate cancer by consensus criteria and had received no prior immunotherapy, chemotherapy or ketoconazole. Patients received 400 mg ketoconazole orally 3 times daily, and 20 mg hydrocortisone orally each morning and 10 mg hydrocortisone orally each evening. Granulocyte-macrophage colony-stimulating factor (250 microg/m2) was administered subcutaneously on days 15 to 28 of each 28-day cycle. Progression was defined as disease progression or toxicity. A total of 49 patients were enrolled, including 37 with radiographically evident metastases and 12 with prostate specific antigen only disease. Median patient age was 68 years (range 52 to 84) and median prostate specific antigen was 23.1 ng/ml (range 5.4 to 306.5). Time to progression, which was the primary study end point, was 9.7 months for all patients. Ten of the 30 treatment failures showed radiographic progression and 6 were due to toxicity, while treatment failure in 14 of 30 patients (47%) consisted only of increasing prostate specific antigen. Median time to progression was 6.9 and 15.4 months in patients with and without metastases, respectively (p = 0.01). Of 48 patients 36 (75%, 95% CI 60-86) experienced a 50% or greater decrease in prostate specific antigen. Four grade 4 events occurred that were unrelated to the study drug. Grade 3 events related to study therapy in more than 1 patient consisted of fatigue in 7 (14%). Combined ketoconazole and granulocyte-macrophage colony-stimulating factor yields a high response rate and it is an option for patients with castration resistant prostate cancer. Time to progression in patients without metastases is significantly longer than in those without metastases.

Use of granulocyte-macrophage colony-stimulating factor to reverse anergy in otherwise immunologically healthy children

T-cell anergy, as measured by delayed hypersensitivity skin testing, is associated with increased susceptibility to infection. Because the repertoire of effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) includes enhancement of antigen processing and presentation by antigen-presenting cells, GM-CSF has been used to augment immune function in human immunodeficiency virus-induced and other viral illness-induced immune dysfunction and to affect positively immune function in a wide variety of disorders. To attempt reversal of T-cell anergy using GM-CSF in 3 otherwise immunologically healthy children with severe recurrent and persistent viral respiratory tract infections and in one child with recurrent bacterial sepsis. After written informed consent and baseline data were obtained, the study participants were administered 3 two-week cycles of GM-CSF. Delayed hypersensitivity skin testing and laboratory tests were repeated 2 weeks after the third cycle and subsequently as clinically indicated. All 4 children developed delayed hypersensitivity by skin testing, and all demonstrated markedly decreased number and severity of infection. Improvement persisted in all patients for at least 1 year. A single cycle of additional treatment in 2 patients reestablished delayed hypersensitivity and decreased infection, both of which persisted throughout the follow-up period of 4 or more years. GM-CSF treatment reversed T-cell anergy in 4 children. Reestablishment of delayed hypersensitivity was associated with a significant decrease in infection. Although further studies will be needed, use of GM-CSF should be considered as an immune modulator in patients with T-cell anergy and recurrent infections.

Differential immunostimulating effect of Granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF) and Interferon γ (IFNγ) after severe trauma

Severe trauma leads to an increased vulnerability to bacterial sepsis. In the present study, we compared the immunostimulating potential of granulocyte-colony stimulating-factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma). Prospective clinical experimental study. University hospital intensive care unit and research facility. 6 patients with an Injury Severity Score (ISS) of more than 25 points. Heparinized blood samples of severely injured patients and 12 healthy volunteers were incubated in vitro with 10 ng/ml GM-CSF, 10 ng/ml G-CSF or 10 ng/ml IFN-gamma for 6 h. Flow cytometry: HLA-DR expression on monocytes, B- and T-lymphocytes. ELISA: LPS-induced TNFalpha and IL-10 production. In all patients reduced cytokine production and HLA-DR expression on monocytes was established. After administration of GM-CSF and IFN-gamma it in vitro, the level of HLA-DR expression on monocytes and the it ex vivo TNFalpha-synthesis increased while only GM-CSF increased significantly IL-10-liberation after LPS-stimulation. However, only IFN-gamma had the capacity to enhance HLA-DR on B- and T-lymphocytes. G-CSF it in vitro had no significant effect on the measured parameter. These data suggest that GM-CSF and IFN-gamma may serve to support immune functions in severely injured patients.

HIV and Other Lentiviral Infections Cause Defects in Neutrophil Chemotaxis, Recruitment, and Cell Structure: Immunorestorative Effects of Granulocyte-Macrophage Colony-Stimulating Factor

Patients with HIV infection exhibit deficits in bacterial and fungal clearance, and possibly depressed innate immunity. In this study, we observed that neutrophils from HIV-infected patients have a profound defect in chemotaxis in response to endogenous (IL-8) and bacterial (fMLP) chemoattractants, which was directly correlated with peripheral CD4(+) lymphocyte levels but not plasma viral load. A similar chemotactic defect was observed in the feline immunodeficiency virus (FIV) model of HIV infection. Intravital microscopy of FIV-infected animals revealed marked impairment in the in vivo recruitment of leukocytes; specifically integrin-dependent neutrophil adhesion and emigration induced by bacterial products. Treatment of FIV-infected animals with GM-CSF re-established both neutrophil recruitment (rolling, adhesion, and emigration) and in vitro chemotaxis to the levels seen in uninfected animals. This restoration of neutrophil responses was not due to GM-CSF-mediated priming. Rather, HIV and FIV infections resulted in defective neutrophil development, with an ensuing reduction in neutrophil granularity and chemotactic receptor expression. GM-CSF therapy restored neutrophil granularity, implying restoration of normal neutrophil development. Together, our findings underscore the fundamental defects in innate immunity caused by lentivirus infections, while also indicating that GM-CSF may be a potential immunorestorative therapy for HIV-infected patients.

Differential Effects of Granulocyte-Macrophage Colony-Stimulating Factor upon Neutrophil Activation as Evaluated by Flow Cytometric and Morphological Assays

Differential Effects of Granulocyte-Macrophage Colony-Stimulating Factor upon Neutrophil Activation as Evaluated by Flow Cytometric and Morphological Assays

Granulocyte-macrophage colony-stimulating factor inhibits tumour growth.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine antitumour responses was examined. Sixty mice received Lewis lung carcinoma implants and were then randomized to receive GM-CSF 1 microgram/day, GM-CSF 0.5 microgram/day or saline for 10 days and studied with regard to tumour volume, carcass weight and food intake. Macrophage antitumour mechanisms including oxygen free radical production and nitric oxide release were studied in peritoneal macrophages after co-culture with GM-CSF in vitro and in vivo. GM-CSF 1 microgram/day decreased tumour growth after 5 days (mean(s.e.m.) 0.62(0.14) versus 1.24(0.19) cm3, P = 0.017). GM-CSF upregulated macrophage antitumour mechanisms by enhancing the in vivo production of superoxide radicals (mean(s.e.m.) 0.69(0.06) versus 0.45(0.10) nmol, P < 0.05) and nitric oxide (mean(s.e.m.) 48(3) versus 24(4) mumol, P < 0.01). GM-CSF functions through the enhancement of macrophage tumoricidal activity, suggesting a therapeutic potential for this cytokine in the tumour-bearing host.

Preliminary Experience with Low Concentration of Granulocyte-Macrophage Colony-Stimulating Factor: A Potential Regulator in Preimplantation Mouse Embryo Development and Apoptosis

To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development of preimplantation mouse embryos. Mouse 2-cell embryos were collected and cultured in P-1 medium supplemented with GM-CSF at different concentrations. Using reverse transcription-polymerase chain reaction, expression Bcl-2 and Bax mRNA in blastocyst were evaluated in the GM-CSF group and control group. Apoptosis detection was performed using the in situ apoptosis detection kit in mouse blastocysts. The statistical significance of the data was analyzed using t-test and chi-square test. The development of blastocyst increased to 89% in the addition of GM-CSF (0.125 ng/mL), compared to controlled group (80%). The number of cells staining for apoptosis was lower in GM-CSF group than that in the control group. Bcl-2 expression was found to be upregulated in blastocysts in the GM-CSF supplemented group compared to the control group. These results suggest that GM-CSF might be an important regulator in embryo development.

GM-CSF activates RhoA, integrin and MMP expression in human monocytic cells.

Monocyte migration is one of the key events occurring in the early stage of atherosclerosis. This process includes monocytic adhesion to and penetration through the arterial intima. In such an environment, many factors stimulate the monocytes to enhance integrin activation and extracellular matrix degradation. To investigate the coordinative operation of these two events in relation to monocyte migration, we paid particular attention to the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on monocytes in terms of RhoA activation and matrix metalloproteinase (MMP) expression. RhoA and integrin clustering were activated by GM-CSF, monocyte chemoattractant protein-1 (MCP-1) and platelet-derived growth factor-BB (PDGF-BB) in human monocytic cell lines. Furthermore, enhancement of migration was observed with stimulation by MCP-1 and PDGF-BB. Granulocyte-macrophage colony-stimulating factor did not enhance the migration, even though it activated RhoA and integrin. However, GM-CSF is known to stimulate monocytes to express MCP-1, suggesting the presence of an indirect mechanism for GM-CSF-mediated migratory activity. In contrast, only GM-CSF enhanced the expression of MMP-1 and MMP-9. These results provide evidence that GM-CSF has multiple functions enhancing monocytic migration via RhoA and integrin activation, and via MMP expression.

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

Erythropoietin overcomes imatinib-induced apoptosis and induces erythroid differentiation in TF-1/bcr-abl cells.

Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following "suicide" gene therapy in a murine colon carcinoma model.

In prodrug-activated ("suicide") gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected "bystander" cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving "suicide" gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to "bystander" killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.

Immunological Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Autologous Tumor Vaccine in Patients With Renal Cell Carcinoma

Biological therapy for renal cell carcinoma (RCC) uses agents that mobilize immune effector cells which are able to recognize and destroy cancer. We evaluated the effects of weekly then monthly autologous tumor vaccine combined with daily granulocyte macrophage-colony stimulating factor (GM-CSF) in patients with RCC as a method of stimulating antigen presenting cells. Eligible patients with pathological stage II to IV RCC were entered into this pilot study. Autologous tumor vaccine (0.5 to 1 x 107 irradiated tumor cells) admixed with 250 microg GM-CSF per vaccine was given subcutaneously weekly for 4 weeks and then monthly for 4 months. GM-CSF (125 microg/m2) was given subcutaneously for 13 days after vaccine injection 1 and injections 4 to 8. Treatment related tumor specific CD4 and CD8 positive T cell precursors were assessed. A total of 22 patients were entered into this study. Patients were stratified by bulk of disease (group 1, 9 patients with micrometastatic disease, and group 2, 13 patients with macrometastatic disease). In general treatment was well tolerated. Of 9 patients in group 1 7 remained disease-free after nephrectomy. In group 2, 6 patients had stable (46.2%) and 7 patients had progressive disease (53.8%). Statistically significant treatment related increases in CD4 (p = 0.028) and CD8 (p = 0.018) positive tumor specific T cell precursors were observed for the entire group of patients. Changes in CD4 and CD8 positive precursors correlated significantly with each other (p = 0.0001). This correlation was seen in the 2 patient subpopulations as well (group 1 p = 0.003, group 2 p = 0.013). Patients with minimal disease, and with changes in CD4 and CD8 positive tumor specific T cell precursors greater than the median appeared to have an improved time to progression as well as a survival benefit. GM-CSF and autologous vaccine can be given safely in combination to patients with renal cell cancer. We observed treatment related changes in tumor specific circulating lymphocyte populations.

Effect of granulocyte-macrophage colony-stimulating factor on the immune response of circulating monocytes after severe trauma.

Severe injury compromises functions of the antigen-presenting immune cells, resulting in an increased vulnerability toward bacterial sepsis. Support of the immune capabilities contributes a desirable therapeutic option in high-risk patients. Factors possessing immunostimulating properties such as granulocyte-macrophage colony-stimulating factor (GM-CSF) may serve as potential tools to compensate immunosuppression caused by severe trauma. In the present study, therefore, GM-CSF was examined with regard to its capacity to overcome trauma-induced down-regulation of immune functions. Prospective clinical experimental study. University hospital intensive care unit and research facility. Severely injured patients with >25 points on the Injury Severity Score. Blood samples of severely injured patients were incubated in vitro with 10 ng/mL GM-CSF for 6 hrs. Human leukocyte antigen (HLA)-DR expression on monocytes was analyzed by flow cytometry, lipopolysaccharide-induced tumor necrosis factor (TNF)alpha and interleukin-10 production of blood samples was measured by means of enzyme-linked immunoabsorbent assay. Compared with blood specimens of healthy donors, ex vivo endotoxin-induced TNF alpha production and HLA-DR expression on monocytes were significantly reduced in blood of trauma patients. Ex vivo treatment of blood specimens with GM-CSF increased HLA-DR expression and TNF alpha production stimulated by lipopolysaccharides in both healthy volunteers and patients on day 1 after trauma. Blood samples of patients with an uneventful recovery showed nearly normal TNF alpha synthesis and HLA-DR expression after 2-3 wks, whereas TNF alpha production and HLA-DR expression of patients with sepsis and multiple organ failure remained at low levels. In the sepsis/multiple organ failure group, GM-CSF also enhanced HLA-DR expression and TNF alpha production, although the levels of the volunteers' blood were not reached. The presented data show that trauma- and sepsis-induced depression of monocyte functions can be counteracted by GM-CSF in vitro, suggesting that this substance may serve as support of immune functions in severely injured patients.