Granulocyte-macrophage Colony-stimulating Factor Expression Research Articles

Oncolytic immunotherapeutic virus in HCC: Can it compete with molecular therapies?

Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a random- ized phase 2 dose-finding trial (n = 30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15, and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose- related survival in individuals with HCC.

Mycobacterium tuberculosis-induced expression of granulocyte-macrophage colony stimulating factor is mediated by PI3-K/MEK1/p38 MAPK signaling pathway

Members of the colony stimulating factor cytokine family play important roles in macrophage activation and recruitment to inflammatory lesions. Among them, granulocyte-macrophage colony stimulating factor (GM-CSF) is known to be associated with immune response to mycobacterial infection. However, the mechanism through which Mycobacterium tuberculosis (MTB) affects the expression of GM-CSF is poorly understood. Using PMA-differentiated THP-1 cells, we found that MTB infection increased GM-CSF mRNA expression in a dosedependent manner. Induction of GM-CSF mRNA expression peaked 6 h after infection, declining gradually thereafter and returning to its basal levels at 72 h. Secretion of GM-CSF protein was also elevated by MTB infection. The increase in mRNA expression and protein secretion of GM-CSF caused by MTB was inhibited in cells treated with inhibitors of p38 MAPK, mitogen-activated protein kinase kinase (MEK-1), and PI3-K. These results suggest that up-regulation of GM-CSF by MTB is mediated via the PI3-K/MEK1/p38 MAPK-associated signaling pathway. [BMB Reports 2013; 46(4): 213-218]

Suppressed expression of granulocyte macrophage colony-stimulating factor in oviduct ampullae of obese cows

Obese heifers have been found to produce fewer excellent-grade embryos than lean and normal heifers due to unknown mechanisms. Oviducts synthesize granulocyte macrophage colony-stimulating factor (GMCSF) to promote embryogenesis, and GMCSF expression may be down-regulated in the oviducts of obese cows. The present study evaluated the relationship between the degree of obesity and GMCSF expression in the ampullary or isthmic section of oviducts in lean [n=5; body condition score (BCS) on a 5-point scale, 2.5], normal (n=6; BCS, 3.0), and obese (n=5; BCS, 4.0) Japanese Black cows. GMCSF mRNA and protein expression in the ampulla, measured by real-time PCR and western blotting, respectively, were less (P<0.05) in the obese group than in the normal group. mRNA and GMCSF protein did not differ significantly in the isthmus among the three groups. The obese group had less GMCSF immuno-reactivity in the tunica mucosa, the primary site of GMCSF gene expression, of the ampulla than the normal and lean groups. In conclusion, unlike normal and lean cows, obese cows had suppressed GMCSF gene expression in the ampulla.

Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer

Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n=30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P=0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC.

Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12

BackgroundAn adenovirus that expresses both interleukin (IL)-12 and granulocyte-macrophage colony-stimulating-factor (GM-CSF) has been proven to be very effective in treating several tumors, but causes serious normal tissue toxicities.MethodsIn this study, a novel adenoviral vector was constructed by placing the human GM-CSF gene under the control of the CMV-IE promoter and human IL-12 gene under the control of heat shock protein 70B gene promoter. Both hGM-CSF and hIL-12 expressions in virus-infected tumor cells were analyzed in vitro and in vivo when underlying single or multiple rounds of hyperthermia.ResultsWe observed constitutive high expression of human GM-CSF and heat-induced expression of human IL-12 after a single round of hyperthermia post viral infection. The heat-induced hIL-12 expression exhibited a pulse-like pattern with a peak at 24 hrs followed by a decline 48 hrs post heat stress. Repeated heat treatment was more effective in inducing hIL-12 expression than a one-time heat treatment. Interestedly, we also observed that constitutive expression of hGM-CSF could be stimulated by heat stress in tested tumor cells.ConclusionOur study provided a novel strategy for combined gene therapy that allows constitutive expression of a non-toxic gene such as GM-CSF and heat-induced expression of a toxic gene such as IL-12. In addition, our study also showed that hyperthermia can be used to trigger gene expression in temporal and special manner.

Molecular cloning, recombinant expression and characterization of GMCSF from the rhesus monkey, Macaca mulatta

Recent studies have found that, in addition to hematopoiesis, granulocyte–macrophage colony-stimulating factor (GMCSF) plays pivotal roles in multiple immune disorders. The gene encoding Macaca mulatta GMCSF (mmGMCSF) was cloned from stimulated peripheral blood mononuclear cells (PBMCs). Concanavalin A (Con A) and mismatched allogeneic antigen-stimulation significantly increased the production of mmGMCSF by monkey PBMCs. The gene encoding mature mmGMCSF was first expressed as a soluble fusion protein in Escherichia coli, and native mmGMCSF was further prepared by protease cleavage. The recombinant mmGMCSF induced antigen-presenting dendritic cells from monkey PBMCs, suggesting a central role of mmGMCSF in the immune system of the rhesus monkey. Although the predicted mature mmGMCSF protein differs from human GMCSF (hGMCSF) at six amino acid residues, mmGMCSF showed a strong ability to support human TF-1 cell survival. Additional co-immunoprecipitation experiments revealed that mmGMCSF cross reacts with the hGMCSF receptor (hGMCSFR). In addition, the hGMCSF-neutralizing agents hGMCSFR-Fc and anti-hGMCSF antibody reduced the biological effects of mmGMCSF on TF-1 cells. These results suggest that recombinant mmGMCSF might be used for the in vitro evaluation of novel hGMCSF-neutralizing agents prior to the in vivo preclinical evaluation of these agents in the rhesus monkey model.

Potential toxicological effects of amines used for carbon capture and storage and their degradation products

With the increasing use of amine solvents for carbon capture, there is a critical need to characterize the potential health effects of these materials and their degradation products formed within the capture system or downwind of release. This study evaluated the acute inhalation health risk of exposure to degraded mixtures of monoethanolamine (MEA), methyldiethanolamine (MDEA), and piperazine (PIP). Degradation was achieved in a heated (150C) and pressurized (approximately 15 psi) stainless steel vessel by adding NO2 and purified air over approximately 75 days. Laboratory mice were exposed to the degraded mixture (as well as the neat amines in separate experiments) 6hrs/day for 7 consecutive days in a whole-body inhalation chamber. Bronchoalveolar lavage (BAL) was carried out 18 to 24hours post-exposure and inflammatory cells were counted in lavage fluid. Cytokine expression and oxidative stress were measured in lung tissue. We found that exposure to degraded MEA resulted in significant increases in total cells, neutrophils, and lymphocytes, and the strongest cytokine response compared to control mice and compared to MEA-exposed mice. Neither MDEA nor PIP, or their degradant mixtures, caused increased inflammatory cells in BAL fluid. The degraded MEA atmosphere also caused a statistically significant decrease in oxidative stress in mouse lung. The degraded MDEA atmosphere caused the strongest cytokine response of the amines tested, producing statistically significant increases in cytokine growth-related oncogene (GRO-KC), monocyte chemotactic protein -1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GMCSF) expression compared to the control and to MDEA alone. The degraded PIP atmosphere showed a statistically significant increase in GMCSF expression. This investigation represents a hazard evaluation and not a dose-response assessment; however, the results suggest that our approach can be used successfully to screen potential solvents for carbon capture for acute human health impacts.

Patients undergoing long-term treatment with antihypertensive eye drops responded positively with respect to their ocular surface disorder to oral supplementation with antioxidants and essential fatty acids

BackgroundGlaucoma and dry eye disorders (DEDs) are frequent comorbidities. The antioxidant and anti-inflammatory properties of essential polyunsaturated fatty acids have been extensively studied in relation to eye diseases.ObjectiveOur objective was to determine the effects of oral supplementation with a combined formulation of antioxidants and essential polyunsaturated fatty acids on expression of cytokines and chemokines in tears from patients with DEDs or primary open-angle glaucoma (POAG).MethodsParticipants (n = 97) were distributed into three groups: (1) individuals with nonsevere DEDs (DEDG), (2) individuals with nonadvanced POAG (POAGG), and (3) healthy controls. These groups were randomized into two subgroups: one received a daily antioxidant and essential polyunsaturated fatty acid supplement (two pills) for 3 months (+S), and the other did not (−NS). Participants were interviewed and ophthalmologically examined. Concentrations of specific cytokines and chemokines in reflex tears were determined by multiplexed particle-based flow cytometry. The data were analyzed statistically (SPSS version 15.0).ResultsComparison of the results from the DEDG and POAGG patients showed significant differences in tear expression of granulocyte-macrophage colony-stimulating factor (P = 0.008), tumor necrosis factor α (P = 0.005), vascular endothelial growth factor (P = 0.038), interleukin-4 (P = 0.030), and interleukin-6 (P = 0.044). The main signs and symptoms of dry eyes such as dryness, burning, photophobia, eye heaviness, and blurred vision, as well as positive changes in eyelashes, hair, nails and skin, were significantly improved in DEDG +S and POAGG +S patients relative to unsupplemented patients.ConclusionInflammation biomarkers were differentially expressed in glaucomatous tears, but the differences changed upon antioxidant/essential polyunsaturated fatty acid supplementation. Chronic instillation of antihypertensive eye drops must be considered for integrating protocols to glaucoma standards of care.

Abstract A59: Block/homo polyplex micelle-based GM-CSF gene therapy via intraperitoneal administration elicits antitumor immunity against peritoneal dissemination and exhibits safety potentials in mice and monkeys.

Abstract A block/homo-mixed polyplex micelle, comprising of cationic homo polymer: {N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} P[Asp(DET)] and block copolymer: polyethylene glycol (PEG) -P[Asp(DET)], has been reported to exhibit the efficient transgene expression in vivo by intratracheal and systemic administration. In the present study, we investigated the potential of immunogene therapy by intraperitoneal (i.p.) administration of block/homo polyplex micelles for peritoneal dissemination. For evaluation of transgene expression in vivo, block/homo polyplex micelles showed 12-fold higher level in luciferase expression evaluated by bioluminescence imaging system at 24 h after the i.p. administration compared with block polyplex micelles in nude mice bearing peritoneal dissemination. The distribution of polyplex micelles in tumor nodules was similarly present for both polyplex micelles, where the intracellular uptake of plasmid DNA was detected for block/home polyplex micelles but little for block polyplex micelles. The i.p. administration of block/homo polyplex micelles encapsulating granulocyte macrophage colony stimulating factor (GM-CSF) gene elicited GM-CSF expression dominantly in tumors and less in normal organs (liver and spleen), inhibited the tumor growth and prolonged survival rate for the mice harboring disseminated pancreatic cancer more significantly compared with the mock. The antitumor effect of GM-CSF gene therapy was mediated via the activation of natural killer cells. For safety evaluation, block/homo polyplex micelles indicated almost no adverse events for patho-physical findings and blood examinations in mice and cynomolgus monkeys, although slight increases in serum fibrinogen were observed in the monkey model. In conclusion, block/homo polyplex micelle-based immunogene therapy via i.p. administration may be a safe and effective approach for suppressing intractable peritoneal dissemination. Citation Format: Masahiro Ohgidani, Koichi Furugaki, Shinkai Kentaro, Yumi Kunisawa, Keiji Itaka, Kazunori Kataoka, Kenji Nakano. Block/homo polyplex micelle-based GM-CSF gene therapy via intraperitoneal administration elicits antitumor immunity against peritoneal dissemination and exhibits safety potentials in mice and monkeys. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A59.

Protection Against Cyclophosphamide-Induced Myelosuppression by ZPDC Glycoprotein (24 kDa)

Immunomodulatory agents are often used to reduce myelosuppression and enhance immune response for cancer treatment. Cyclophosphamide (CTX) can induce oxidative stress in bone marrow resulting in suppression of anti-oxdiantive enzymes and causes myelosuppression. We isolated glycoprotein from Zanthoxylum piperitum DC fruit (ZPDC), and it consists of a carbohydrate (18%) and a protein (82%). The objective of this study was to investigate its protective activity against CTX-induced myelosuppression in Balb/c (n=6/group). The mice were orally administrated by ZPDC glycoprotein (10 and 20 mg/kg, BW) for 1 week in the presence or absence of CTX. Intracellular reactive oxygen species (ROS), anti-oxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)], cyclin kinase inhibitors (CKIs: p53, p21 and p27), cyclin D1/ cyclin dependent kinase (CDK) 4, PCNA and cytokines [interleukin (IL)-3, and granulocyte⁄ macrophage-colony-stimulating factor (GM-CSF)] were evaluated using biochemical activity, Western blot analysis, and ELISA. The results obtained from this study showed that CTX decreased spleen and thymic indices, bone marrow cellularity and expression of cyclin D1/CDK4 and PCNA, but it increased CKIs, whereas ZPDC glycoprotein (20 mg/kg, BW) resulted in vice versa in CTX-induced Balb/c. Expression of IL-3 and GM-CSF were normalized by ZPDC glycoprotein. Thus, this study suggested that ZPDC glycoprotein prevents oxidative stress and myelosuppression in CTX-induced mice and might be a potential immunomodulatory agent.

Expression of Human Granulocyte-Macrophage Colony-Stimulating Factor in Stably-Transformed BmN and Sf-9 Cells and Silkworms by a Non-Transposon Vector

Expression of Human Granulocyte-Macrophage Colony-Stimulating Factor in Stably-Transformed BmN and Sf-9 Cells and Silkworms by a Non-Transposon Vector

GM‐CSF enhances tumor invasion by elevated MMP‐2, ‐9, and ‐26 expression

Granulocyte–macrophage colony-stimulating factor (GM-CSF) promotes tumor progression in different tumor models in an autocrine and paracrine manner. However, at the same time GM-CSF is used in cancer therapies to ameliorate neutropenia. We have previously shown in GM-CSF and G-CSF expressing or negative skin or head and neck squamous cell carcinoma that GM-CSF expression is associated with a highly angiogenic and invasive tumor phenotype. To determine the functional contribution of GM-CSF to tumor invasion, we stably transfected a GM-CSF negative colon adenocarcinoma cell line HT-29 with GM-CSF or treated the same cell line with exogenous GM-CSF. While GM-CSF overexpression and treatment reduced tumor cell proliferation and tumor growth in vitro and in vivo, respectively, it contributed to tumor progression. Together with an enhanced migratory capacity in vitro, we observed a striking increase in tumor cell invasion into the surrounding tissue concomitant with the induction of an activated tumor stroma in GM-CSF overexpressing or GM-CSF treated tumors. In a complex 3D in vitro model, enhanced GM-CSF expression was associated with a discontinued basement membrane deposition that might be mediated by the increased expression and activation of MMP-2, -9, and -26. Treatment with GM-CSF blocking antibodies reversed this effect. The increased presence and activity of these tumor cell derived proteases was confirmed in vivo. Here, expression of MMP-26 protein was predominantly located in pre- and early-invasive areas suggesting MMP-26 expression as an early event in promoting GM-CSF dependent tumor invasion.

Lung microenvironment contributes to the resistance of alveolar macrophages to develop tolerance to endotoxin*

Endotoxin tolerance corresponds to reprogramming of mononuclear phagocytes after iterative encounters with toll-like receptor agonists aimed to dampen the inflammatory response. We investigated why this phenomenon cannot be observed with murine alveolar macrophages. Animal study. Research institution laboratory. rag2-/-, rag2γc-/-, cd3ε-/-, µ-/-, il-15-/-, Jα18-/-, ifnγr-/-, il-18r-/-, and wild-type mice. Alveolar macrophages were harvested from untreated mice or after injection of endotoxin. Alveolar macrophages were activated in vitro with endotoxin (lipopolysaccharide), and tumor necrosis factor production was monitored. In contrast to monocytes or peritoneal macrophages, alveolar macrophages did not display endotoxin tolerance in an ex vivo model after injection of endotoxin. An in vivo systemic inhibition of granulocyte-macrophage colony-stimulating factor or interferon-γ allowed the induction of alveolar macrophage endotoxin tolerance, which was also observed in interferon-γ receptor-deficient mice. Using mice missing different leukocyte subsets and adoptive cell transfers, we demonstrated the involvement of B lymphocytes in interferon-γ production within the lung microenvironment and in the prevention of alveolar macrophage endotoxin tolerance. Furthermore, we demonstrated the importance of interleukin-18 in preventing alveolar macrophage endotoxin tolerance through studies of interleukin-18 messenger RNA expression in il-18r-/- mice and injection of interleukin-18 in rag2-/- and µ-/- mice. Our results support the conclusion that at homeostasis in the lungs, constitutive expression of granulocyte-macrophage colony-stimulating factor, interleukin-18, interferon-γ and possibly interleukin-15, and a cross-talk between B lymphocytes and alveolar macrophages create a microenvironment specific to the lungs that prevents alveolar macrophages from becoming tolerant to endotoxin.

Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system

The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.

Proteasome Inhibition Augments Cigarette Smoke-Induced GM-CSF Expression in Trophoblast Cells via the Epidermal Growth Factor Receptor

Maternal cigarette smoking has adverse effects on pregnancy outcomes. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential cytokine for a normal pregnancy. We investigated the impact of cigarette smoke extract (CSE) on GM-CSF expression in human cytotrophoblast cells and suggested a cellular mechanism underlying the CSE-induced GM-CSF expression. An immortalized normal human trophoblast cell line (B6Tert-1) was treated with CSE. The viability and proliferation of the CSE-treated B6Tert-1 cells were evaluated, and the expression of GM-CSF in these cells was quantified at the mRNA and the protein levels by means of reverse-transcription and quantitative polymerase chain reaction (RT-qPCR); and enzyme-linked immunosorbent assay (ELISA), respectively. Human trophoblast cells treated with CSE had an increased expression of GM-CSF at both the mRNA and the protein levels. The CSE-induced GM-CSF expression was synergistically enhanced by the addition of the proteasome inhibitor MG-132, but inhibited by AG-1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase. Furthermore, CSE treatment increased the phosphorylation of the extracellular-signal regulated kinases (ERK1/2) in the trophoblast cells. The expression of other growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular endothelial growth factor (VEGF) was also evaluated. Our data suggested that cigarette smoking and proteasome inhibition synergistically up-regulate GM-CSF cytokine expression by activating the EGFR signaling pathway.

Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4+ T cells and γδ T cells (171.24)

Abstract Recent findings have demonstrated an indispensable role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). However the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1 signaling centrally regulates GM-CSF production during EAE. IL-1R1 is required to induce GM-CSF production following polyclonal T cell stimulation under both TH17 (i.e. IL-23) and TH1 (i.e. IL-12) stimulatory conditions. Furthermore, we demonstrate that γδ T cells are also potent producers of GM-CSF during the inflammatory response and that their secretion of GM-CSF is similarly dependent on IL-1 signaling. Interestingly, we find that the expression of GM-CSF by myelin-reactive T cells during EAE is not restricted to discrete TH1 and TH17 cell lineages. Rather GM-CSF can be co-expressed with both IFNγ and IL-17. Inflammasome-derived IL-1β was identified to be an upstream amplifier of GM-CSF producing T cells during the priming phase of EAE. Finally, we show a central role for MYD88 in the control of GM-CSF production by both CD4+ T cells and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R1/MYD88 signaling axis in the regulation of GM-CSF production.

Effect of alveolar epithelial cell plasticity on the regulation of GM-CSF expression

Local pulmonary expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically important for defense of the pulmonary alveolar space. It is required for surfactant homeostasis and pulmonary innate immune responses and is protective against lung injury and aberrant repair. Alveolar epithelial cells (AEC) are a major source of GM-CSF; however, the control of homeostatic expression of GM-CSF is incompletely characterized. Increasing evidence suggests considerable plasticity of expression of AEC phenotypic characteristics. We tested the hypothesis that this plasticity extends to regulation of expression of GM-CSF using 1) MLE-12 cells (a commonly used murine cell line expressing some features of normal type II AEC, 2) primary murine AEC incubated under standard conditions [resulting in rapid spreading and loss of surfactant protein C (SP-C) expression with induction of the putative type I cell marker (T1α)], or 3) primary murine AEC on a hyaluronic acid/collagen matrix in defined medium, resulting in preservation of SP-C expression. AEC in standard cultures constitutively express abundant GM-CSF, with further induction in response to IL-1β but little response to TNF-α. In contrast, primary cells cultured to preserve SP-C expression and MLE-12 cells both express little GM-CSF constitutively, with significant induction in response to TNF-α and limited response to IL-1β. We conclude that constitutive and cytokine-induced expression of GM-CSF by AEC varies in concert with other cellular phenotypic characteristics. These changes may have important implications both for the maintenance of normal pulmonary homeostasis and for the process of repair following lung injury.

Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor Expression, a Biomarker of Memory CD8+ T Cell Immunity and Vaccine Efficacy

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) exhibit overlapping activities and are produced by activated T cells. In this study, the role of IL-3 and GM-CSF in CD8+ T cells were assessed following HIV-1 prime-boost immunization. Data indicate that the expression of IL-3/GM-CSF by HIV-specific effector CD8+ T cells is vaccine delivery route and time dependent, where purely systemic, intramuscular i.m./i.m. vaccination induce elevated levels of IL-3 in HIV-specific CD8+ T cells compared to purely mucosal, intranasal i.n./i.n. immunization. Interestingly, the GM-CSF expression was optimal only following i.n./i.m. delivery. Data also revealed that peak IL-3 mRNA and protein expression in CD8+ T cells were detected 16-20 h of KdGag197-205 peptide stimulation, whereas the expression kinetics of GM-CSF was similar to IFN-γ. Next the IL-3 and GM-CSF expression in HIV-specific CD8+ T cells were assessed at acute, effector and memory stages of immunity using i.n./i.m. delivery of FPV-HIV/VVHIV control vaccine compared to a novel IL-13Rα2 adjuvanted HIV-vaccine (FPV-HIV IL-13Rα2/VV-HIV IL-13Rα) that has shown to induce excellent high avidity CD8+ T cells with greater protective immunity. The IL-13Rα2 adjuvanted vaccine induced greatly elevated HIV-specific memory CD8+IL-3+ and also CD8+IL-3+IFN-γ+ T cells compared to the control vaccine, where the expression in memory phase was greater than effector T cells. Both the control and IL- 13Rα2 adjuvanted vaccines, elicited elevated but similar numbers of antigen-specific GM-CSF+ memory CD8+ T cells. Data suggest that induction of both IL-3 and GM-CSF play a role in maintenance of antigen-specific memory CD8+ T cells which is linked to better protective immunity. These results also demonstrate that route of delivery, time post vaccination, expression kinetics/ length of antigen exposure, should not be neglected when evaluating the vaccine efficacy. Also IL-3/GM-CSF expression by memory CD8+ T cells could be a biomarker of protective immunity.

Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced Hematological Toxicity in Non-Tumor-Bearing Mice

Active hexose correlated compound (AHCC) is known as a dietary supplement derived from an extract of a basidiomycete mushroom. The present study was conducted to evaluate the role of AHCC in alleviating the side effects, particularly hematological toxicity, in non-tumor-bearing mice receiving monotherapy with gemcitabine (GEM). The results from the GEM treatment groups with and without AHCC administration were compared to control group that received vehicle alone. The GEM alone treatment reduced peripheral leukocytes and hemoglobin, and bone marrow cell viability in spite of no influence on body weight, food consumption, and renal and hepatic parameters. Supplementation with AHCC significantly alleviated these side effects. The colony forming assay of bone marrow cells revealed that AHCC improved reduction of colony forming unit-granulocyte macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) related to GEM administration. However, when mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) was examined using a quantitative reverse transcription polymerase chain reaction (RT-PCR), AHCC showed no effect for the mRNA levels of their hematopoietic growth factors. These results support the concept that AHCC can be beneficial for cancer patients with GEM treatment through alleviating the hematotoxicity.