Granulocyte-macrophage Colony-stimulating Factor Gene Research Articles

Dietary Selenium Nanoparticles Improved Growth and Health Indices in Asian Seabass (Lates calcarifer) Juveniles Reared in High Saline Water.

A 60-day study was carried out to determine the effect of dietary selenium nanoparticles (SeNP) on growth, digestive enzymes, and health status of Asian seabass (Lates calcarifer, 46.5 ± 0.2 g) juveniles reared in high saline water (48 ppt). Five levels of SeNP were added to a basal diet (45% protein, 15% lipid), including control (0), 0.5 (SeNP0.5), 1.0 (SeNP1), 2 (SeNP2), and 4 (SeNP4) mg SeNP kg-1 diet. Fish were stocked into fifteen 2,000 L tanks (50 fish tank-1) filled with 1,800 L sand-filtered seawater (26.5 ± 1.5°C, 48.0 ± 0.2 ppt) in a flow-through system. Each dietary treatment was performed in three replicates. The growth rate positively increased in both linear and quadratic trends with increasing dietary SeNP level (P < 0.05). The liver Se concentration increased with increasing SeNP in diet (P < 0.05). Gut total protease, trypsin, chymotrypsin, alkaline phosphatase (ALP), lipase, and α-amylase activities were significantly enhanced in the SeNP4 group compared to the other treatments (P < 0.05). Antioxidant capacity improved in fish-fed SeNP2 and SeNP4 diets regarding catalase and superoxide dismutase activities and the liver glutathione content. Serum lysozyme and hemolytic activities and white blood cells' respiratory burst activity in the control were lower than in fish-fed SeNP-supplemented diets. Serum total protein, globulin, and globulin/albumin ratio in fish-fed SeNP1, SeNP2, and SeNP4 diets were higher than the other groups (P < 0.05). The interleukin-10 and granulocyte-macrophage colony-stimulating factor genes' relative transcription levels in the gut of fish-fed SeNP4 were higher than the other groups. Serum cholesterol, triglycerides, ALP, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly decreased in fish with increasing SeNP content in the diet. In conclusion, supplementing the diet with 4 mg kg-1, SeNP was recommended to improve growth and health indices in L. calcarifer juveniles reared in high saline water (48 ppt).

Bound polyphenols in insoluble dietary fiber of navel orange peel modulate LPS-induced intestinal-like co-culture inflammation through CSF2-mediated NF-κB/JAK-STAT pathway.

Our laboratory previously extracted bound polyphenols (BPP) in insoluble dietary fiber from navel orange peel (NOP-IDF), and the aim of this study was to investigate the anti-inflammatory activity and potential molecular mechanisms of BPP by establishing an LPS-induced intestinal-like Caco-2/RAW264.7 co-culture inflammation model. The results demonstrated that BPP reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the production of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxidative species (ROS) during the inflammatory damage process. Furthermore, BPP alleviated the lipopolysaccharides (LPS)-induced intestinal barrier damage by attenuating the decrease in trans-epithelial electrical resistance (TEER), diamine oxidase (DAO) activity, and intestinal alkaline phosphatase (IAP) activity, as well as the downregulation of ZO-1, Occludin, and Claudin-1 protein expression levels. RNA-seq results on RAW264.7 cells in the co-culture model showed that the NF-κB and JAK-STAT pathways belonged to the most significantly affected signaling pathways in the KEGG analysis, and western blot confirmed that they are essential for the role of BPP in intestinal inflammation. Additionally, overexpression of the granulocyte-macrophage colony-stimulating factor (CSF2) gene triggered abnormal activation of the NF-κB and JAK-STAT pathways and high-level expression of inflammatory factors, while BPP effectively improved this phenomenon. The above results suggested that BPP could inhibit intestinal inflammatory injury and protect intestinal barrier integrity through CSF2-mediated NF-κB and JAK-STAT pathways.

The GM-CSF/CCL17 pathway in obesity-associated osteoarthritic pain and disease in mice

We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r=-0.458and p=0.0096), with elevated circulating CCL17 levels (r=0.2108and p=0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

In vitro Characterization of Enhanced Human Immune Responses by GM-CSF Encoding HSV-1-Induced Melanoma Cells.

PurposeWe studied the innate and adaptive immune response against melanoma cells after JS-1 (wild-type herpes simplex virus 1, wt HSV-1) or Talimogene laherparepvec (T-VEC) infection and evaluated the antitumoral efficacy in human melanoma cells. We analyzed the putative synergistic biological and immunological effects of JS-1 or T-VEC combined with cytostatic drugs in human tumor and immune cells. T-VEC is a genetically modified strain of HSV-1. Genetic modifications (insertion of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene) were made to attenuate the virus and increase selectivity for cancer cells. In addition to the direct oncolytic effect, we investigated the immune stimulatory effects of T-VEC by comparing it with JS-1. JS-1 is identical T-VEC except for the inserted GM-CSF gene.Materials and MethodsWe analyzed the effects of T-VEC and JS-1 with cytostatic drugs in human tumor-immune cell coculture experiments. After coculture, the surface markers CD80, CD83 and CD86 were measured by fluorescence-activated cell sorting and the cytokines, interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α and GM-CSF, by enzyme-linked immunosorbent assays. Furthermore, we analyzed the potential of the viruses to induce T cell activation, measured on the basis of CD4, CD8 and CD69. Analysis of these markers and cytokines allows for conclusions to be drawn concerning the maturation of dendritic cells (DCs) and the immunostimulatory effects of the treatment.ResultsWe documented increased activation of human cytotoxic T lymphocytes after infection by both HSV-1 strains and treatment with cytostatic drugs without significant differences between T-VEC and JS-1.ConclusionWe demonstrated an immune response as a result of infection with both viruses, but T-VEC was in vitro not stronger than JS-1. The immunostimulatory effects of the viruses could be partially increased by chemotherapy, providing a rationale for future preclinical studies designed to explore T-VEC in combined regimens.

Effect of Niacin on Growth Performance, Intestinal Morphology, Mucosal Immunity and Microbiota Composition in Weaned Piglets.

Simple SummaryThe protective effect of niacin on growth performance and gut health of weaned piglets and the underlying mechanism remains unclear despite it being a common additive in pig diets. The present study aimed to investigate the effect of niacin on growth performance, intestinal morphology, intestinal mucosal immunity, and colonic microbiota in weaned piglets. Our results show that niacin supplementation significantly improved the growth performance in piglets as compared with those given a niacin receptor antagonist. Niacin also significantly improved the relative abundance of beneficial bacteria in the colon and alleviate the inflammatory response in the intestinal mucosa as compared with control piglets and those given a niacin receptor antagonist. These results provide new insight into the beneficial effects of niacin on growth performance and gut health in weaned piglets.This study aimed to investigate the effects of niacin on growth performance, intestinal morphology, intestinal mucosal immunity, and colonic microbiota in weaned piglets. A total of 96 weaned piglets (Duroc × (Landrace × Yorkshire), 21-d old, 6.65 ± 0.02 kg body weight (BW)) were randomly allocated into 3 treatment groups (8 replicate pens per treatment, each pen containing 4 males; n = 32/treatment) for 14 d. Piglets were fed a control diet (CON) or the CON diet supplemented with 20.4 mg/kg niacin (NA) or an antagonist for the niacin receptor GPR109A (MPN). The results showed that NA or MPN had no effect on ADG, ADFI, G/F or diarrhea incidence compared with the CON diet. However, compared with piglets in the NA group, piglets in the MPN group had lower ADG (p = 0.042) and G/F (p = 0.055). In comparison with the control and MPN group, niacin supplementation increased the villus height and the ratio of villus height to crypt depth (p < 0.05), while decreasing the crypt depth in the duodenum (p < 0.05). Proteomics analysis of cytokines showed that niacin supplementation increased the expression of duodenal transforming growth factor-β (TGF-β), jejunal interleukin-10 (IL-10) and ileal interleukin-6 (IL-6) (p < 0.05), and reduced the expression of ileal interleukin-8 (IL-8) (p < 0.05) compared with the control diet. Piglets in the MPN group had significantly increased expression of ileal IL-6, and jejunal IL-8 and interleukin-1β (IL-1β) (p < 0.05) compared with those in the control group. Piglets in the MPN group had lower jejunal IL-10 level and higher jejunal IL-8 level than those in the NA group (p < 0.05). The mRNA abundance of duodenal IL-8 and ileal granulocyte-macrophage colony-stimulating factor (GM-CSF) genes were increased (p < 0.05), and that of ileal IL-10 transcript was decreased (p < 0.05) in the MPN group compared with both the control and NA groups. Additionally, niacin increased the relative abundance of Dorea in the colon as compared with the control and MPN group (p < 0.05), while decreasing that of Peptococcus compared with the control group (p < 0.05) and increasing that of Lactobacillus compared with MPN supplementation (p < 0.05). Collectively, the results indicated that niacin supplementation efficiently ensured intestinal morphology and attenuated intestinal inflammation of weaned piglets. The protective effects of niacin on gut health may be associated with increased Lactobacillus and Dorea abundance and butyrate content and decreased abundances of Peptococcus.

Enhanced immunogenicity of foot and mouth disease DNA vaccine delivered by PLGA nanoparticles combined with cytokine adjuvants

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P < 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.

Impaired Immune Function in Patients With Chronic Postsurgical Hypoparathyroidism: Results of the EMPATHY Study.

Despite the pivotal role of calcium signaling in immune response, little is known about immune function in patients affected by hypoparathyroidism. This work aimed to evaluate immune function in hypoparathyroidism. The Evaluation of iMmune function in Postsurgical and AuToimmune HYpoparathyroidism (NCT04059380) is a case-control, cross-sectional study set in an Italian referral center. Participants included 20 patients with postsurgical hypoparathyroidism (12 females) and 20 age- and sex-matched controls. Main outcome measures included calcium metabolism assessment, peripheral blood mononuclear cells (PBMC) profiling via flow cytometry, parathyroid hormone receptor 1 (PTHr1) expression analysis using immunofluorescence and PrimeFlow RNA assay, gene expression analysis via real-time polymerase chain reaction, cytokine measurement, and evaluation of infectious disease frequency and severity. Immune cell profiling revealed decreased monocytes, regulatory, naive, and total CD4+ T lymphocytes, which correlated with total calcium, ionized calcium, and PTH levels, in patients with hypoparathyroidism. Patients with hypoparathyroidism had a higher CD3-CD56+ natural killer (NK) cell count, which inversely correlated with calcium, PTH, and vitamin D levels. Furthermore, they exhibited decreased tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor gene expression and decreased circulating TNF levels. Gene expression and immunofluorescence analysis confirmed PTHr1 expression in all PBMC lineages; however, the percentage of cells expressing PTHr1 was lower, whereas the intensity of PTHr1 expression in monocytes, total T lymphocytes, CD8+CD4+ and CD4+ T lymphocytes, and total NK cells was higher in patients with hypoparathyroidism. This study describes for the first time the immune alterations in patients with hypoparathyroidism receiving conventional therapies, supporting the immunoregulatory role of PTH and proposing an explanation for the increased susceptibility to infections observed in epidemiological studies.

Regulation of Cytokine and Growth Factor Gene Expression in Human Bone Marrow Stromal Cells Transformed With Simian Virus 40

Marrow stromal cells are thought to regulate hematopoiesis by producing colony-stimulating factors (CSFs) and other cytokines, either constitutively or in response to mediators such as interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF alpha). The mechanisms by which these inflammatory cytokines induce CSF expression in stromal cells are not fully defined. In this study, we used human marrow stromal cells transformed by simian virus 40 (SV-MSCs) to study growth factor and cytokine gene regulation in response to IL-1 alpha and TNF alpha. IL-1 alpha induced significant and prolonged increases in steady-state mRNA levels for interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), granulocyte-macrophage CSF (GM-CSF), and, to a lesser extent, granulocyte-CSF (G-CSF); this induction was not dependent on new protein synthesis. Nuclear run-on analyses showed that IL-1 alpha transcriptionally activated the genes for IL-6, GM-CSF, and IL-1 beta, while TNF alpha transcriptionally induced expression of IL-6 and IL-1 beta. Furthermore, mRNA for IL-6 and IL-1 beta was dramatically superinduced by the combination of cycloheximide and TNF alpha. When SV-MSCs were cultured in semisolid medium, they formed colonies of blast-like cells that, when replated on plastic, resumed adherent growth. These "colony-derived" cell lines, unlike the parental SV-MSCs from which they were derived, constitutively expressed colony-stimulating activity and mRNA for GM-CSF, G-CSF, IL-6, and IL-1 beta. In this report, we show that the expression of IL-6 and IL-1 beta mRNA in the colony-derived cell lines was due, at least in part, to constitutive transcriptional activation of these genes (similar to the findings in IL-1 alpha- and/or TNF alpha-stimulated parental SV-MSCs). However, in contrast to the transcriptional activation of the GM-CSF gene seen in cytokine-induced parental SV-MSCs, GM-CSF transcripts accumulated in the colony-derived cell lines by a posttranscriptional mechanism.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1.

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.

Inhibition of melanoma by survivin-specific lymphocytes combined with CCL17 and granulocyte-macrophage colony-stimulating factor in a mouse syngeneic model.

As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better.

Regulation of cytokine production by treating with Chinese tonic herbs in human peripheral blood mononuclear and human acute monocytic leukemia cells

Chinese tonic herbs are often used as remedies for the treatment of cancer or immunity related diseases. Cytokine regulation is one of the pathways that modulates the immune system. In the present study, we investigated the effects of commonly used Chinese herbs on cytokine regulation in human peripheral blood mononuclear cells (PBMC) and human acute monocytic leukemia cell line, THP-1. Among the 15 selected Chinese herbs, Paeonia lactiflora exhibited inhibitory effect on phytohemagglutinin (PHA)-activated PBMC proliferation and induced IFN-γ production. We also used a multiplexed flow cytometric bead-based assay to analyze the release of 17 cytokines and reverse transcription polymerase chain reaction (RT-PCR)/quantitative real-time PCR (qRT-PCR) to evaluate gene expression. Cuscuta chinensis induced both interleukin-8 and MIP-1β release in THP-1 cells and influenced mRNA transcription. Dioscorea opposita, Cinnamomum osmophloeum, and Glycyrrhiza uralensis enhanced secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenced GM-CSF gene expression. As such, different Chinese tonic herbs may regulate immunomodulatory functions in human cells via diverse processes such as the modulation of different cytokines and may be used as adjuvant immunity-boosting drugs. The present study is the first to use high-throughput multiplexed flow cytometric method to analyze cytokine regulatory effects of herbal extracts.

The Clinicopathological Impact of Granulocyte-Macrophage Colony-Stimulating Factor Gene Expression and Different Molecular Prognostic Biomarkers in Egyptian Acute Myeloid Leukemia Patients.

Background:Acute myeloid leukemia (AML) is characterized by clonal expansion of myeloid precursors with diminished capacity for differentiation. It develops as the consequence of a series of genetic changes in a hematopoietic precursor cell. Purpose This study aimed to investigate the correlation between GM-CSF gene expression and different molecular prognostic markers such as FLT3-ITD, NPM1 mutation A and CEBPA gene expression in 100 Egyptian AML patients. As well as, correlation with the response to induction therapy, DFS andOS in these patients. Methodology:Quantitative assessment of GM-CSF gene expression was performed by qRT-PCR. Additional prognostic molecular markers were determined as FLT3-ITD and NPM1 mutation A together with quantitative assessment of CEBPA gene expression by qRT-PCR. Results:Patients with high GM-CSF expression levels had better OS and DFS with p value 0.004 and 0.02, respectively. However, no statistically significant difference between low andhigh GM-CSF gene expression was found regarding the response to therapy (p value= 0.08). Most patients with low CEBPA expression had resistant disease together with poor OS and DFS (P value = <0.001 for each). Our results showed that patients with high CEBPA gene expression whether GM-CSF gene expression was high or low had significant higher complete remission rates (p value = 0.1 for each). However, low GM-CSF gene expression andlow CEBPA gene expression showed poor response to treatment. Conclusion:Our findings suggest that molecular diagnostic biomarkers for AML are an essential tool that improves prognostication andhence better patients’ management.

An Oncolytic Vaccinia Virus Armed with GM-CSF and IL-24 Double Genes for Cancer Targeted Therapy.

PurposeTargeted oncolytic vaccinia virus is an attractive candidate for cancer therapy due to its replication causing lysis of infected tumor cells as well as a delivery vector to overexpress therapeutic transgenes. This study constructed a novel oncolytic vaccinia virus carrying granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-24 (IL-24) double genes to improve efficacy for cancer therapy.MethodsVaccinia virus co-expressing GM-CSF and IL-24 based on Chinese Guang9 strain (VG9-GMCSF-IL24) was constructed with disruption of the viral thymidine kinase (TK) gene. The cytotoxicity of VG9-GMCSF-IL24 in various cell lines was assessed by MTT. The synergistic antitumor effect of VG9-GMCSF-IL24 in vivo was assessed on multiple tumor models.ResultsIn vitro cytotoxicity assay showed that VG9-GMCSF-IL24 exerted a strongly cytotoxic effect on cancer cells, but with no significant cytotoxicity to normal cells. Significant tumor growth inhibition and prolonged survival were observed in different tumor models treated with VG9-GMCSF-IL24. Additionally, systemic and specific antitumoral immunity was investigated in vivo, and enhanced antitumor immunity was observed in VG9-GMCSF-IL24-treated mice.ConclusionOur results indicated that VG9-mediated GM-CSF and IL-24 co-expression performed cooperative and overlapping antitumor effect. As a novel and effective therapeutic strategy for cancer, the combination of oncolysis and immunotherapy with vaccinia virus carrying one or more immunostimulatory genes may have a satisfactory clinical application prospect.

Cloning, Optimization of Periplasmic Expression and Purification of Recombinant Granulocyte Macrophage-Stimulating Factor in Escherichia coli BL21 (DE3).

Background:Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by Escherichia coli. However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of E. coli BL21 (DE3).Materials and Methods:The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the pelB secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 104 IU/μg.Conclusion:The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by E. coli.

Abstract 5911: A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer

Abstract Background: Combination of cytotoxic treatments with immunotherapeutic agents seem more efficacious than using either strategy alone. Oncolytic viral vectors carrying immunostimulatory genes like GM-CSF have emerged as treatment options in various tumors. Previously, we have shown that combination of suicide gene therapy either with GM-CSF or vector-induced dendritic cells has significant anti-tumor efficacy. In the current study, we aimed to combine the cytotoxic effect of the suicide gene cytosine deaminase and the immunostimulatory effect of granulocyte macrophage colony stimulating factor in a single vector construct. Methods: We have constructed a bicistronic adenoviral vector carrying cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) genes combined with an IRES element and driven by CMV promoter (Ad-CMV-CDiresGMCSF). We tested the in vitro efficacy of the vector in various tumor cell lines and in a mouse model of colon cancer. Results: Our bicistronic vector Ad-CMV-CDiresGMCSF showed significant in-vitro cytotoxic effects on tumor cell lines similar to the vector carrying CD gene alone when 5-FC added. Likewise, the GM-CSF producing efficacy of the bicistronic vector was comparable to the vector carrying GM-CSF gene alone. In the syngeneic colon cancer model, the bicistronic vector carrying CD and GM-CSF genes yielded significant tumor shrinkage and overall survival when compared to the control suicide vector carrying CD gene alone. Accordingly, anti-tumor immune response parameters including CTL assay and immune cell infiltration of tumor tissue were significantly improved in the Ad-CMV-CDiresGMCSF vector treated group (p&amp;lt;0.01). Conclusions: The Ad-CMV-CDiresGMCSF vector construct suggests a potential for the treatment of established tumors by inducing significant tumor killing and tumor-specific immune response. Citation Format: Hakan Akbulut, Arzu Coleri, Gunce Sahin, Yucheng Tang, Albert Deisseroth, Fikri Icli. A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5911.

Suppressed calcineurin-dependent gene expression identifies lung allograft recipients at increased risk of infection.

Lung transplant immunosuppression regimens generally include the calcineurin inhibitor tacrolimus. We hypothesized that mean residual expression (MRE) of calcineurin-dependent genes assesses rejection and infection risk better than does tacrolimus trough. We prospectively followed 44 lung allograft recipients at 2 to 18months posttransplant and measured changes in whole blood interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor gene expression following a tacrolimus dose. Posttransplant duration, immunosuppressive medication levels, and bronchoscopic rejection and infection assessments were compared with MRE by using generalized-estimating equation-adjusted models. Prednisolone effect on MRE was assessed ex vivo in blood samples from nontransplanted controls. Tacrolimus concentration inhibiting 50% of cytokine production (IC50 ) was measured in a pretransplant subset. Results showed that MRE did not change with diagnosis of rejection but that airway infection was associated with a 20% absolute decrease (95% confidence interval 11%-29%). MRE increased with time after transplant but was not associated with tacrolimus trough. Interestingly, MRE correlated inversely with corticosteroid dose in the study cohort and ex vivo. Pretransplant tacrolimus IC50 depended on the cytokine measured and varied between individuals, suggesting a range in baseline responses to tacrolimus. We conclude that MRE identifies infection risk in lung allograft recipients, potentially integrating calcineurin inhibitor and steroid effects on lymphocyte effector function.

Endoplasmic reticulum stress in perivascular adipose tissue promotes destabilization of atherosclerotic plaque by regulating GM-CSF paracrine

BackgroundPerivascular adipose tissue (PVAT) accelerates plaque progression and increases cardiovascular risk. We tested the hypothesis that PVAT contributed to plaque vulnerability and investigated whether endoplasmic reticulum stress (ER stress) in PVAT played an important role in vulnerable plaque.MethodsWe transplanted thoracic aortic PVAT or subcutaneous adipose tissue as a control, from donor mice to carotid arteries of recipient apolipoprotein E deficient (apoE−/−) mice after removing carotid artery collar placed for 6 weeks. Two weeks after transplantation, ER stress inhibitor 4-phenyl butyric acid (4-PBA) was locally administrated to the transplanted PVAT and then animals were euthanized after 4 weeks. Immunohistochemistry was performed to quantify plaque composition and neovascularization. Mouse angiogenesis antibody array kit was used to test the angiogenic factors produced by transplanted adipose tissue. In vitro tube formation assay, scratch wound migration assay and mouse aortic ring assay were used to assess the angiogenic capacity of supernatant of transplanted PVAT.ResultsUltrastructural detection by transmission electron microscopy showed transplanted PVAT was a mixed population of white and brown adipocytes with abundant mitochondria. Transplanted PVAT increased the intraplaque macrophage infiltration, lipid core, intimal and vasa vasorum neovascularization and MMP2/9 expression in plaque while decreased smooth muscle cells and collagen in atherosclerotic plaque, which were restored by local 4-PBA-treatment. Antibody array analysis showed that 4-PBA reduced several angiogenic factors [Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), MCP-1, IL-6] secreted by PVAT. Besides, conditioned medium from 4-PBA treated-PVAT inhibited tube formation and migration capacity of endothelial cells and ex vivo mouse aortic ring angiogenesis compared to conditioned medium from transplanted PVAT. mRNA expression and protein levels of GM-CSF were markedly elevated in adipocytes under ER stress which would be suppressed by 4-PBA. In addition, ER stress enhanced NF-κB binding to the promoter of the mouse GM-CSF gene in adipocytes confirmed by Chromatin immunoprecipitation analyses.ConclusionsOur findings demonstrate that ER stress in PVAT destabilizes atherosclerotic plaque, in part through increasing GM-CSF paracrine via transcription factor NF-κB.

Combined Interleukin 12 and Granulocyte-macrophage Colony-stimulating Factor Gene Therapy Synergistically Suppresses Tumor Growth in the Murine Fibrosarcoma

Background: Interleukin (IL-12) and the granulocyte-monocyte colony-stimulating factor gene (GM-CSF) have been used as immunotherapeutic agents in cancer gene therapy as they activate dendritic cells and boost anti-tumor immune responses. IL-12 and GM-CSF have different roles in anti-tumor immune response. Objectives: The aim of present study was to investigate the anti-tumor effects of combined gene therapy with GM-CSF and IL-12 in a fibrosarcoma mouse model. Methods: To investigate the combined therapeutic effect of GM-CSF and IL-12, WEHI 164 tumor cells were transfected with Murine GM-CSF (m-GM-CSF) and Murine IL-12 (m-IL-12) genes, using Lipofectamine. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/c mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of GM-CSF, IL-12 and IFN-γ was studied by real-time PCR and immunoblotting. The expression of Ki-67 (a proliferation marker) in tumor masses was studied by immunohistochemistry staining. Results: The tumor size was reduced in IL-12 + GM-CSF group (P < 0.0001); the results of western blotting and real-time PCR demonstrated that IL-12, GM-CSF and IFN-γ expression increased in IL-12+GM-CSF group (with a relative expression of: 2.86, 1.98, and 2.560). Immunohistochemistry staining indicated that Ki-67 expression was reduced in IL-12 + GM-CSF group. Conclusions: Combination therapy with GM-CSF and IL-12 displayed significant therapeutic effects and represented a promising gene therapy strategy for cancer.

Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice – role of regulatory T cells

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγpos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans.