Combination Of Granulocyte-macrophage Colony-stimulating Factor Research Articles

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and β-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and β-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3β-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

Phase II trial of granulocyte-macrophage colony-stimulating factor plus thalidomide in older patients with castration-resistant prostate cancer.

The objective of this study was to assess the efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with thalidomide for prostate-specific antigen (PSA) reduction in older patients (aged ≥70 years, life expectancy of >1 year) with castration-resistant prostate cancer (CRPC). A total of 11 CRPC patients were treated with 300 µg GM-CSF administered subcutaneously on days 1, 3 and 6 of weeks 1 and 2 of each cycle. Thalidomide was gradually increased to reach the study dose of 100 mg/day. The patients were assessed every 4 weeks with therapy continuing to 4 months. All 11 patients exhibited a decrease in PSA levels and 3 patients (27.2%) exhibited a PSA decrease of >50% in cycle 1. In cycle 2, 8 patients exhibited decreasing PSA levels. A total of 3 patients (27.2%) had a PSA rebound, with 1 patient exhibiting a PSA rebound of >50%. In cycle 3, 10 patients exhibited continuously decreasing PSA levels, with 2 patients (18.2%) exhibiting a PSA decrease of >50%; 1 patient (27.2%) had a PSA rebound of <50%. In cycle 4, 9 patients exhibited continuously decreasing PSA levels and 2 patients (18.2%) had a PSA rebound of >50%. All 11 patients in this study exhibited a decrease in PSA levels, with a median decrease of 92.2%. Therapy was well tolerated, with the majority of the patients experiencing only one adverse event. In conclusion, the combination of GM-CSF with thalidomide was found to be clinically effective and well tolerated by elderly CRPC patients. Therefore, GM-CSF plus thalidomide may be considered a viable treatment option for such patients.

Granulocyte macrophage colony-stimulating factor is required for aortic dissection/intramural haematoma.

Aortic dissection and intramural haematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear. Here we show that granulocyte macrophage colony-stimulating factor (GM-CSF) is a triggering molecule for this condition. Transcription factor Krüppel-like factor 6 (KLF6)-myeloid-specific conditional deficient mice exhibit this aortic phenotype when subjected to aortic inflammation. Mechanistically, KLF6 downregulates expression and secretion of GM-CSF. Administration of neutralizing antibody against GM-CSF prevents the condition in these mice. Conversely, administration of GM-CSF in combination with aortic inflammation to wild-type mice is sufficient to induce the phenotype, suggesting the general nature of effects. Moreover, patients with this condition show highly increased circulating levels of GM-CSF, which is also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory molecule causative of this aortopathy, and modulation of this cytokine might be an exploitable treatment strategy for the condition.

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ.

Current status of granulocyte-macrophage colony-stimulating factor in the immunotherapy of melanoma.

In 2012, it was estimated that 9180 people in the United States would die from melanoma and that more than 76,000 new cases would be diagnosed. Surgical resection is effective for early-stage melanoma, but outcomes are poor for patients with advanced disease. Expression of tumor-associated antigens by melanoma cells makes the disease a promising candidate for immunotherapy. The hematopoietic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) has a variety of effects on the immune system including activation of T cells and maturation of dendritic cells, as well as an ability to promote humoral and cell-mediated responses. Given its immunobiology, there has been interest in strategies incorporating GM-CSF in the treatment of melanoma. Preclinical studies with GM-CSF have suggested that it has antitumor activity against melanoma and can enhance the activity of anti-melanoma vaccines. Numerous clinical studies have evaluated recombinant GM-CSF as a monotherapy, as adjuvant with or without cancer vaccines, or in combination with chemotherapy. Although there have been suggestions of clinical benefit in some studies, results have been inconsistent. More recently, novel approaches incorporating GM-CSF in the treatment of melanoma have been evaluated. These have included oncolytic immunotherapy with the GM-CSF–expressing engineered herpes simplex virus talimogene laherparepvec and administration of GM-CSF in combination with ipilimumab, both of which have improved patient outcomes in phase 3 studies. This review describes the diverse body of preclinical and clinical evidence regarding use of GM-CSF in the treatment of melanoma.

MP34-06 GMCSF GENE THERAPY AND SIRNA TARGETING COX-2 IN A MURINE MODEL OF BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2014MP34-06 GMCSF GENE THERAPY AND SIRNA TARGETING COX-2 IN A MURINE MODEL OF BLADDER CANCER Sin Mun Tham, Kesavan Esuvaranathan, and Ratha Mahendran Sin Mun ThamSin Mun Tham More articles by this author , Kesavan EsuvaranathanKesavan Esuvaranathan More articles by this author , and Ratha MahendranRatha Mahendran More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is a good model for gene therapy as candidate genes can be transferred directly to the tumor without surgical intervention. Different cytokine genes successfully induced tumor regression in murine models of bladder cancer. In our studies, intravesical gene therapy with either Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF) or siRNA targeting Cox-2 (Cyclooxygenase-2) (siCox2) successfully cured some mice. This study investigates the efficacy of combined therapy in an orthotopic bladder cancer model. METHODS C57BL/6 mice with bladder tumors received liposome-mediated GMCSF (2.5μg) and siCox2 (1μM/mouse) twice a week for 3 weeks for a total of 6 treatments. As controls, mice received each of these therapies singly as well as saline and non-targeting siRNA (siCon). After 28 days, bladders were harvested, weighed and gene expression changes were analyzed by polymerase chase reaction (PCR). RESULTS 30-33% of mice were cured of bladder tumors with intravesical GMCSF or siCox2 therapy. Local therapy with siCox2 alone resulted in significantly smaller tumors and better survival rates. Mice that were cured after receiving siCox2 therapy showed significant increase in activin receptor-like kinase 7 (Alk7), CD4 and angiotensin II receptor 2 (Agtr2). However, GMCSF therapy did not have an additive effect on the therapeutic effects of siCox2. Few gene expression changes were observed between combined GMCSF and siCox2 against GMCSF and siCon, in which significant increase was observed only in CD4. CONCLUSIONS While local therapy with GMCSF gene therapy or silencing of Cox2 with siRNA have anti-tumor activity individually against bladder cancer in mice, combined therapy did not enhance the beneficial effects. More candidate genes have to be studied to find synergistic anti-tumour effects against bladder cancer. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e364 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Sin Mun Tham More articles by this author Kesavan Esuvaranathan More articles by this author Ratha Mahendran More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identification of Immune Factors Regulating Antitumor Immunity Using Polymeric Vaccines with Multiple Adjuvants

The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study, we used polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, to identify dendritic cell (DC) subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of granulocyte macrophage colony-stimulating factor (GM-CSF) and various Toll-like receptor (TLR) agonists affected 70% to 90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40 mm(2)), resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs, plasmacytoid DCs (pDC), along with local interleukin (IL)-12, and granulocyte colony-stimulating factor (G-CSF) concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Furthermore, vaccine studies in Batf3(-/-) mice revealed that CD8(+) DCs are required to affect tumor protection, as vaccines in these mice were deficient in cytotoxic T lymphocytes priming and IL-12 induction in comparison with wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, pDCs, IL-12, and G-CSF play important roles in priming effective antitumor responses with these vaccines.

Cytokine-enhanced intravenous oncolytic virotherapy

BackgroundOncolytic viruses are emerging as promising cancer therapies; a key challenge in their use is delivery of virus to tumour targets in the face of host immunity. In a recent translational clinical study we found that intravenously administered reovirus successfully reaches liver tumours, despite patients all having high concentrations of neutralising anti-reovirus antibodies. Functional virus was recoverable from several white-cell subsets, suggesting that systemically administered virus evades neutralisation by close cell-association (hitch-hiking). We hypothesised that this occurrence could be manipulated to enhance virotherapy by expanding the numbers of carrier cells in the circulation before viral administration. MethodsWe used immune competent mice (C57BL/6) that had been vaccinated with reovirus, or phosphate buffered saline (PBS) control, before establishing syngeneic melanoma (B16-Ova) tumours on their flank. Mice were then treated with one of three cytokines—interleukin 2, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF)—or PBS as control, for 3 days, either before or after virotherapy (reovirus) or PBS as control therapy. Unvaccinated mice were also tested with this regimen. Tumours were then measured daily or harvested for correlative analyses. Mice were humanely killed when tumours reached 1 cm in any dimension. Eight mice were used in each treatment group and experiments were repeated at least twice to ensure consistency of the findings. FindingsTreatment with GM-CSF enhanced the number of circulating granulocytes, and enhanced macrophage infiltration into tumours. When reovirus-immune mice were treated with GM-CSF before virotherapy with reovirus, tumours regressed in all mice and were undetectable 60 days later; but reovirus-immune mice treated with either interleukin 2 or G-CSF before virotherapy, or treated with GM-CSF alone, all developed tumours that necessitated euthanasia. Moreover the combination of GM-CSF and reovirus was only effective if GM-CSF was administered before reovirus. The efficacy of GM-CSF plus reovirus was considerably diminished in unvaccinated reovirus-naive mice, but was restored if three cycles of GM-CSF plus reovirus were administered, suggesting that in this regimen anti-Rep antibodies contributed to the efficacy of therapy. InterpretationWe found that oncolytic virotherapy with reovirus is enhanced by vaccinating mice against the virus and pretreatment with GM-CSF. Further in-vivo experiments, and associated in-vitro assays, are underway to characterise the mechanism involved. Pragmatically these data suggest a novel method for enhancing oncolytic virotherapy that can be readily implemented because both reovirus and GM-CSF are used clinically; accordingly a translational clinical trial protocol is being developed to explore this possibility further. FundingUK Medical Research Council, Royal Society of Medicine.

Abstract 3336: The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF.

Abstract The Inhibitor of Apoptosis (IAP) protein family is able to inhibit cell death and promote survival signaling triggered by a wide variety of stimuli. Two members of this family in particular, cIAP1 and cIAP2, play key roles in enabling tumor necrosis factor alpha (TNFα)-induced nuclear factor-kappa beta (NF-kB) activation and inflammatory responses. Birinapant (TL32711) is a bivalent Smac Mimetic that antagonizes multiple IAP family members resulting in the conversion of TNFα-induced survival signaling into an apoptotic response, an effect which is more prominent in tumor cells than their normal counterparts. Combining birinapant with agents capable of increasing the levels of TNFα in the tumor microenvironment represents a novel approach to cancer treatment. We have tested the ability of interferon-alpha/-beta (IFN-α, IFN-β) and granulocyte macrophage colony stimulating factor (GM-CSF), both known to be immunomodulatory agents, for their ability to stimulate TNFα production by peripheral blood mononuclear cells (PBMC). Treatment with each cytokine induced the production of TNFα by cultured PBMC as measured by ELISA. Supernatants of IFN-α/-β and GM-CSF-treated PBMC cultures were able to sensitize resistant tumor cells to Smac Mimetics in a TNFα-dependent manner. Furthermore, the combination of birinapant and either IFN-β or GM-CSF synergistically inhibited the development of RENCA tumors in a syngeneic mouse prevention model. Due to the highly aggressive nature of the syngeneic tumors, this model is performed by simultaneous implantation of tumor cells and initiation of dosing of test agents. Activity in this model is measured by prevention of tumor outgrowth. Birinapant in combination with IFN-β resulted in only 5 of 18 measurable tumors, versus 19 of 20 tumors in vehicle-treated mice, 16 of 20 tumors in IFN-β alone-treated mice and 13 of 20 tumors in birinapant alone-treated mice. In addition, when tumor growth occurred it was delayed in mice treated with the combination. Similarly, the birinapant and GM-CSF combination treatment resulted in 9 of 20 tumors, versus 20 of 20 tumors in the vehicle-treated mice, 20 of 20 tumors in the GM-CSF alone-treated mice, and 16 of 20 tumors in the birinapant alone-treated mice. In addition when tumors did develop in the combination-treated mice, their growth was substantially delayed: 4 of 10 mice treated with the combination of birinapant and GM-CSF were still on study on day 85 post initiation - approximately 40 days post dosing - with no measureable tumor. The combination of birinapant with immunomodulatory agents that induce TNF represents a potential novel therapeutic strategy. Citation Format: Christopher A. Benetatos, Jennifer M. Burns, Ernest C. Borden, Daniel Lindner, Yasuhiro Mitsuuchi, Mark A. Mckinlay, Gurpreet Singh Kapoor, Eric M. Neiman, Martin E. Seipel, Guangyao Yu, Martin Graham, David Weng, Stephen M. Condon, C. Glenn Begley, Srinivas Chunduru. The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2013-3336

Granulocyte—Macrophage Colony-Stimulating Factor Enhances the Humoral Immune Responses of Mouse Zona Pellucida 3 Vaccine Strategy Based on DNA and Protein Coadministration in BALB/c Mice

We recently demonstrated that co-administration of mouse zona pellucida 3 (mZP3) DNA and protein vaccine enhanced the contraception of mice by increasing humoral immune responses. In this study, we try to use granulocyte-macrophage colony-stimulating factor (GM-CSF) to further improve the humoral immune responses induced by mZP3 DNA and protein co-administration. BALB/c mice were intranasally pre-injected with GM-CSF 4 days before co-administration. Compared to DNA and protein coadministration without GM-CSF, the combination of GM-CSF and coadministration significantly enhances humoral immune responses, especially the level of secretory immunoglobulin A (sIgA) in vaginal washes. The enhanced antibody responses are correlated with the upregulated level of interleukin 4 (IL-4) and enhanced maturation of dendritic cells (DCs). Thus, GM-CSF is a potential candidate adjuvant to be used for the development of a safe and effective contraceptive vaccine.

Mobilization of Hematopoietic Progenitors from Normal Donors Using the Combination of Granulocyte-Macrophage Colony-Stimulating Factor and Granulocyte Colony-Stimulating Factor Results in Fewer Plasmacytoid Dendritic Cells in the Graft and Enhanced Donor T Cell Engraftment with Th1 Polarization: Results from a Randomized Clinical Trial

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) both mobilize CD34+ stem cells into the blood when administered before apheresis but have distinct effects on dendritic cell (DC) differentiation. We previously demonstrated that the combination of GM+G-CSF results in fewer plasmacytoid DCs (pDCs) when used to mobilize peripheral blood stem cells for autologous transplantation. To test the hypothesis that the content of pDCs in an allograft can be modulated with the cytokines used for mobilization, we randomized the human leukocyte antigen–matched sibling donors of 50 patients with hematological malignancies to a mobilization regimen of either GM+G-CSF (n = 25) or G-CSF alone (n = 25). Primary and secondary endpoints included the cellular constituents of the mobilized grafts, the kinetics of posttransplantation immune reconstitution, and clinical outcomes of the transplantation recipients. Grafts from donors receiving GM+G-CSF contained equivalent numbers of CD34+ cells with fewer pDCs and T cells, with a higher fraction of Th1-polarized donor T cells than G-CSF mobilized grafts. Immune recovery was enhanced among recipients of GM+G-CSF. Survival was not significantly different between transplantation recipients in the two arms. The use of GM+G-CSF modulates immune function and recovery after allogeneic transplantation and should be explored in larger studies powered to evaluate clinical outcomes.

Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models

BackgroundThe tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Incubating bone marrow (BM) precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg) metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression.ResultsExposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln) and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK) was activated during MDSC maturation in GM-CSF and IL-6–treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was decreased in MSC-1 cells when L-Arg metabolizing enzymes were inhibited. Finally, inhibition of AMPK activity by the specific inhibitor Compound C (Comp-C) resulted in the inhibition of L-Arg metabolizing enzyme activity and abolished MDSCs immunosuppressive activity.ConclusionsWe anticipate that the inhibition of AMPK and the control of metabolic fluxes may be considered as a novel therapeutic target for the recovery of the immunosurveillance process in cancer-bearing hosts.

A DNA vaccination regime including protein boost and electroporation protects cattle against foot-and-mouth disease

Protection against foot-and-mouth disease (FMD) using DNA technology has been documented for sheep and pigs but not for the highly susceptible species of cattle.Twenty-five Holstein Friesian cross-bred cattle were vaccinated twice, 21days apart, with a DNA vaccine containing the capsid coding region (P1) along with the non-structural proteins 2A, 3C and 3D (pcDNA3.1/P1-2A3C3D) of O1 Kaufbeuren alone or coated onto PLG (d,l-lactide-co-glycolide) microparticles. In some pcDNA3.1/P1-2A3C3D was also combined with an adjuvant plasmid expressing bovine granulocyte macrophage colony stimulating factor (GM-CSF). DNA vaccinations were administered intramuscularly with, or without, the use of electroporation and at 42days post primary vaccination cattle received a protein boost of 146S FMD virus (FMDV) antigen and non-structural protein 3D. For comparison, four cattle were vaccinated with a conventional FMD vaccine and two more included as unvaccinated controls. Apart from those immunised with PLG microparticles all cattle were challenged with 105 TCID50 cattle adapted O1 Lausanne FMDV virus at day 93 post primary vaccination.All DNA vaccinated cattle regardless of regime developed good humoral and cell mediated responses prior to challenge. The best overall virus neutralising antibody, IFN-γ and clinical protection (75%) were seen in the cattle whereby the DNA was delivered by electroporation. In contrast, only 25% of cattle vaccinated with the DNA vaccine without electroporation were clinically protected. The addition of GM-CSF in combination with electroporation further improved the efficacy of the vaccine, as demonstrated from the reduction of clinical disease and virus excretions in nasal swabs.We thus demonstrate for the first time that cattle can be clinically protected against FMDV challenge following a DNA prime-protein boost strategy, and particularly when DNA vaccine is combined with GM-CSF and delivered by electroporation.

The value of rituximab for the treatment of fludarabine-refractory chronic lymphocytic leukemia: a systematic review and qualitative analysis of the literature

The increase of fludarabine-resistant chronic lymphocytic leukemia (CLL) presents a new treatment challenge. The aim of this review is to evaluate the efficacy and safety of rituximab for patients with fludarabine-refractory CLL. Medline, Embase, The Cochrane Library and selected conference proceedings were searched. Seventeen relevant publications reporting stratified data were identified. Treatments included: rituximab in combination with etanercept, alemtuzumab, bendamustine or methylprednisolone alone, with fludarabine and cyclophosphamide (FCR), with oxaliplatin as well as fludarabine and cytarabine, with cyclophosphamide as well as fludarabine and alemtuzumab (CFAR), and with cytarabine, cisplatinum and dexamethasone (DHAP). One study evaluated rituximab with granulocyte-macrophage colony-stimulating factor in combination with alternating cyclophosphamide, liposomal daunorubicin, vincristine, dexamethasone and methotrexate plus Ara-C. One study evaluated rituximab as monotherapy. Of the nine studies considering overall response, eight reported rates above 50% (four reported rates above 75%). Median overall survival was 37 months for FCR, 11 months for CFAR, 20 months for rituximab with methylprednisolone, 30 months for rituximab with alemtuzumab and 44 months for an FCR/CFAR mixed treatment. The identified studies indicate that regimens containing rituximab may be highly efficacious in the fludarabine-refractory CLL setting. Nevertheless, further research is needed to facilitate the choice of treatment for the clinician.

GM-CSF contributes to prompt healing of ecthyma gangrenosum lesions in kidney transplant recipient

Ecthyma gangrenosum (EG) is an unusual, potentially fatal cutaneous disease, commonly associated with Pseudomonas aeruginosa septicemia. We report the case of a 61-year-old man admitted to the Nephrology Department for fever, leukopenia and inguinal and scrotal painful lesions. Physical examination revealed inguinal and scrotal macules, nodules, blisters and ulcers with central necrosis. P. aeruginosa was isolated from an ulcer. EG was diagnosed. Because of the severe leukopenia, granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered until the white blood cell count significantly increased. Based on antibiogram, intravenous ceftazidime and teicoplanin were given for 11 days. Cutaneous manifestations were completely healed in about 2 months. We suggest that the combination of GM-CSF with appropriate antibiotics can resolve EG and avoid or minimize the risk of septicemia in immunosuppressed patients.

Optimizing DC Vaccination by Combination With Oncolytic Adenovirus Coexpressing IL-12 and GM-CSF

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21+) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4+CD25+ T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.

Combined GM-CSF treatment and M-CSF inhibition of tumor-associated macrophages induces dendritic cell-like signaling in vitro

Macrophages demonstrate plasticity, and tumor-associated macrophages (TAM) can function as immunosuppressive cells in the tumor microenvironment. Therefore, in this study, we aimed to reprogram TAM in vitro with cytokine signal alteration. Granulocyte macrophage colony stimulating factor (GM-CSF) treatment alone did not lead to changes in the expression of M1 (including IL-1β, TNFα and CXCL-10) or M2 (including CD36, CD206 and CCL17) molecules by TAM in vitro, although they adopted a round morphology and were less adhesive to the culture dish. When macrophage colony stimulating factor (M-CSF) signals were suppressed by siRNA against the M-CSF receptor (M-CSFR) in conjunction with GM-CSF treatment, the signal transduction pathway of TAM was altered, and the expression of STAT1, STAT5 and STAT6, which are usually expressed by dendritic cells, was increased. However, the same treatment did not alter the TAM expression pattern of M1/M2 marker molecules. With respect to the NF-κB pathway, GM-CSF and M-CSFR siRNA combination treatment significantly induced the expression of p65, which is usually not expressed by TAM, while p50 and p105 expression by TAM was not affected by the treatment. These findings indicate that our model could not redirect TAM to a monocyte-derived dendritic cell-like phenotype based on the analysis of M1/M2 marker expression, but it was able to modify cell signaling pathways toward a dendritic cell-like pattern. Therefore, the present data suggest that TAM demonstrate plasticity toward dendritic cell-like signal transduction patterns, and that the alteration of the tumor microenvironment has the potential to reverse the immunosuppressive properties of TAM.

Subcutaneous injection containing IL-3 and GM-CSF ameliorates stab wound-induced brain injury in rats

Macrophage-like cells densely accumulate in stab wound-induced brain lesions in rats. Many of these cells express the macrophage marker Iba1 and the oligodendrocyte progenitor cell marker NG2 chondroitin sulfate proteoglycan (NG2), and have been termed BINCs (brain Iba1 +/NG2 + cells). Results from our previous study showed that BINCs elicit neuroprotective action, and agents inducing BINC activation or proliferation are expected to ameliorate traumatic brain injuries (TBIs). In the present study, TBI was established by inserting a needle into the cerebrum and moving the needle in a longitudinal, fan-like movement. Isolated BINCs from these stab lesions expressed mRNAs encoding receptors for interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). When this mixture of cytokines was added to the cultured BINCs, expression of mRNAs encoding insulin-like growth factor-1, hepatocyte growth factor, and proliferating cell nuclear antigen increased. The cytokine mixture induced enhanced wound healing in BINCs-brain cell co-cultures in vitro. Stab wounds in the rats resulted in significant brain tissue loss at 2 months post-lesion. However, tissue loss was reduced by 40% when the combination of IL-3 and GM-CSF was subcutaneously injected 7 times (once per day) beginning at 2 or 3 days post-lesion (dpl). BINCs are highly proliferative and an intraperitoneal injection of 5-fluorouracil (5FU) at 2 dpl eliminated the BINCs, resulting in death of the rats. The cytokine mixture injection significantly reduced mortality of the 5FU-treated rats. These results suggest that the combination of IL-3 and GM-CSF serves as a promising agent to ameliorate TBI via action on BINCs.

Rituximab and CHOP Chemotherapy Plus GM-CSF for Previously Untreated Diffuse Large B-Cell Lymphoma in the Elderly: A Wisconsin Oncology Network Study

Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) may potentiate rituximab activity by upregulating CD20 expression and activating effector cells necessary for antibody-dependent cellular cytotoxicity. GM-CSF was combined with standard rituximab + CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone) chemotherapy (R-CHOP) in the treatment of elderly patients with de novo diffuse large B-cell lymphoma (DLBCL). Thirty-eight patients over the age of 60 years with newly diagnosed DLBCL were treated with R-CHOP every 21 days for 6-8 cycles and GM-CSF 250 µg/m2 per day on days 3-10. Patients were evaluated for response after cycles 4, 6, and 8. The primary endpoint was the rate of complete response, and secondary endpoints were progression-free survival (PFS), event-free survival, and overall survival (OS). Thirty-eight patients were enrolled, with a median age of 72 years, and 29% of patients having high-risk disease (International Prognostic Index [IPI] score ≥ 4). A complete or unconfirmed complete response (CR) was achieved in 53% of patients. After a median follow-up of 51.1 months, the 3-year PFS and OS were 78% and 84%. Twenty-one percent of patients discontinued protocol treatment because of chemotherapy-related toxicity and 16% because of GM-CSF toxicity. Dose intensity for planned chemotherapy cycles was 81.1%. These data suggest that survival outcomes may be modestly improved when GM-CSF is combined with R-CHOP in the treatment of elderly DLBCL. GM-CSF had toxicity precluding planned administration in 16% of patients, which may limit usefulness of this agent. Further investigation of GM-CSF in combination with rituximab-containing chemotherapy is warranted.

Abstract 5623: Synergy of the immunomodulators GM-CSF and imiquimod for peptide-based therapeutic vaccines

Abstract All approved immunomodulators currently in use with therapeutic peptide-based cancer vaccines in are of only moderate efficacy in terms of direct activation of antigen-presenting cells which is a key prerequisite for efficient priming of cytotoxic T cells (CTLs). Moreover, induction of immune responses against tumor-associated antigens (TAAs) is further hampered by an in most cases immunosuppressive environment in cancer patients and by effective tolerization of the immune system against self-antigens, as the majority of TAAs is not exclusively expressed in tumors but also to some extent present on normal tissues. Because the reliable induction of tumor-directed immune responses is crucial for the efficacy of therapeutic cancer vaccines, novel immunomodulator regimens have to be introduced into clinical testing to achieve a better clinical outcome. Therefore, we analyzed safety and efficacy of several approved immunomodulatory substances and combinations thereof in a dedicated pre-clinical screening program. The combination of subcutaneously (s.c.) applied granulocyte-macrophage colony-stimulating factor (GM-CSF) with topical application of the TLR7 ligand imiquimod resulted in synergistic enhancement of peptide-vaccine induced CTL responses without any signs of toxicity or cumulated adverse effects. GM-CSF applied s.c. combined with imiquimod cream applied topically to the shaved skin of C57BL/6 mice at the vaccination site significantly increased the number of vaccine-specific CTLs induced by s.c. vaccinations with model MHC class I-binding peptides compared with either substance alone. The induced immune response was comparable to the effect of CpG deoxyoligonucleotides or poly-IC, two potent immunomodulators currently not approved and therefore not widely available for clinical trials in humans. However, the timing of imiquimod application in relation to the vaccination schedule is crucial, as the synergistic effect of imiquimod and GM-CSF was only observed, if imiquimod was applied at the day of vaccination and/or 24 h later, but not if imiquimod was applied the day before peptide vaccination. In total, 45 mice were immunized with the combination of GM-CSF and imiquimod without any signs of local or systemic toxicities. Further results from in vitro activation assays with human PBMCs and isolated antigen-presenting cells and the distinct molecular mechanisms of immunostimulation employed by both substances provide further support for the anticipated safety of this regimen in humans. GM-CSF and imiquimod is therefore considered as a promising and safe alternative immunomodulator regimen for future clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5623.