Granulocyte-macrophage colony-stimulating factor protects mice against hepatocellular carcinoma by ameliorating intestinal dysbiosis and attenuating inflammation

BACKGROUND Hepatocellular carcinoma(HCC)is the third leading cause of cancer mortality worldwide.The gut microbiota can help maintain healthy metabolism and immunity.Granulocyte-macrophage colony-stimulating factor(GM-CSF)is a critical factor in promoting health and homeostasis;it promotes intestinal immunity,stimulates bone marrow precursors to generate macrophage colonies,and enhances the antibacterial and antitumor activity of circulating monocytes.As such,GM-CSF may protect against HCC development by regulating immunity as well as intestinal microecology.AIM To investigate the impact of GM-CSF on the gut microbiome and metabolic characteristics of HCC.METHODS Thirty-six male BALB/c nude mice were divided into three groups:Control(n=10),HCC(n=13),and HCC+GM-CSF(GM-CSF overexpression,n=13).We utilized HCC cells to establish orthotopic transplantation tumor models of HCC with normal and over-expressing GM-CSF.Liver injury,immune inflammatory function and intestinal barrier function were evaluated.The fecal microbiome and metabolome were studied using 16S rRNA absolute quantification sequencing and gas chromatography-mass spectrometry.RESULTS GM-CSF overexpression significantly affected the gut microbiome of mice with HCC and resulted in a high abundance of organisms of the genera Roseburia,Blautia and Butyricimonass,along with a significant reduction in Prevotella,Parabacteroides,Anaerotruncus,Streptococcus,Clostridium,and Mucispirillum.Likewise,GM-CSF overexpression resulted in a substantial increase in fecal biotin and oleic acid levels,along with a prominent decrease in the fecal succinic acid,adenosine,fumaric acid,lipoic acid,and maleic acid levels.Correlation analysis revealed that the intestinal microbiota and fecal metabolites induced by GM-CSF were primarily involved in pathways related to reducing the inflammatory response,biotin metabolism,and intestinal barrier dysfunction.CONCLUSION GM-CSF can protect against HCC development by regulating immunity and modulating the abundance of specific intestinal microorganisms and their metabolites.This study provides new insights into the therapeutic approaches for HCC.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke

Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of middle cerebral artery occlusion. Results showed reduced infarct volume, decreased number of apoptotic cells, improved neurological functions, increased angiogenic factor expression, and increased vascular density in the ischemic boundary zone in rats that underwent GM-CSF-BMSCs transplantation compared with the BMSCs group. Experimental findings suggested that GM-CSF-BMSCs could serve as a potential therapeutic strategy for ischemic stroke and are superior to BMSCs alone.

EFFECTS OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR GENE ENCODED VACCINIA VIRUS VECTOR ON MURINE PULMONARY METASTATIC MELANOMA

A recombinant vaccinia virus expressing murine granulocyte-macrophage colony-stimulating factor (VVGM-CSF) was tested for its antitumor activity.Murine pulmonary metastasis was established by injecting 20×10~5 B16F10 melanoma cells into the tail vein of C57BL/6 mice. Three days after B16F10 inoculation,WGM-CSF or VVTK, a thymidine kinase gene deficient control vaccinia virus, were injected intraperitoneally twice weekly for 2 weeks. Two weeks later, the mice were sacrificed and pulmonary metastasis fool counted.The results demonstrated that VVGM-CSF treatment significantly decreased the number of pulmonary metastasis and prolonged the survival time of tumorbearing mice. Cytotoxic and phagocytic activities of the peritoncal macrophages were found to be markedly elevated in mice treated with WGM-CSF. Nitric oxide released from the macrophages was also found to be increased. These data, together with our other results,strongly demonstrated that continuous secretion of GMCSF and activation of macrophages might pal-tially explain the therapeutic effects of VVGM-CSF on murine pulmonary metastasis.

Granulocyte-macrophage colony-stimulating factor and interleukin 4 induce the malignant transformation of the bone marrow- derived human adult mesenchymal stem cells

Background The purpose of the study was to examine the effects of granulocyte-macrophage colony-stimulating factor （GM-CSF） and interleukin 4 （IL-4） on the bone-marrow-derived human adult mesenchymal stem cells （hMSCs）. Methods The hMSCs were isolated and cultured with GM-CSF and IL-4 for a period of one month. A single colony of transformed cells was then isoloated and their phenotype was characterized by morphology, surface marker expression, and in vivo tumorigenesis.Results After one month culture, the transformed mesenchymal cells exhibited the morphology and phenotype similar to those of tumor cells, and also caused multiple fast growing lung deposits when it was injected into immunodeficient mice.Conclusion Cytokines-driven malignant transformation of hMSCs may be a useful model for studying signaling pathways initiating malignant transformation of hMSC.

Effects of granulocyte-macrophage colony stimulating factor on the repair of vessel intima damaged by balloon

The dysfunction of vascular endothelial cells plays a key role in startingand facilitating restenosis. The acceleration of intima repair and the recovery of endothelialfunction would reduce the restenosis rate. This study was undertaken to assess the effect ofgranulocyte-macrophage colony stimulating factor ( GM-CSF) on the repair of damaged iliac arteries.Twenty-four male New Zealand white rabbits undergoing primary iliac artery deendothelialization wererandomly divided into two groups ( GM-CSF group and control group) . The GM-CSF group received asubcutaneous injection of GM-CSF (10 μg ? kg^(-1) ? d^(-1) ) , and the control groupwas given a subcutaneous injection of equivalent saline. The iliac arteries of all animals weredamaged by balloon after 7 days. The levels of nitric oxide ( NO) were detected before, 1 week, 2weeks and 4 weeks after angioplasty. The repair and hyperplasia of the intima were observedmicroscopically and the indices of stenosis were evaluated by computerized planimetry after 4 weeksof angioplasty. The NO levels of the GM-CSF group were higher than those of the control group 2weeks and 4 weeks after angioplasty [91.92 +-11.57) μmol/L vs. (81. 67 +- 12. 18) μmol/L; (97. 67+- 10. 13 ) ( μmol/L vs. (83. 16 +-12. 64) μmol/L]. Four weeks after balloon damage,histological examination showed that neointima formation, vascular smooth muscle cells and fibroustissue of the GM-CSF group were less than those of the control group. The endothelium of the GM-CSFgroup was more integrated, and stenosis of lumen was slighter than that of the control group.Morphometry showed the lumen area of the GM-CSF group was larger than that of the control group[(1.27 +-0. 31) mm^2 vs. (0. 92 +- 0. 24) mm^2 ] , the neointimal area and percent of intimahyperplasia were significantly smaller than those of the control group [ (0. 85 +-0. 34) mm vs. (1.18 +-0. 38) mm^2; (40 +- 7)% vs. (55 +- 6)%]. GM-CSF could facilitate the repair of the intima,reduce neointima formation, better the function of the endothelium, and decrease the rate ofrestenosis.

Clinical Study of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor on Chemotherapy-Induced Leukopenia.

We have studied the efficacy and safery of recombinant human granulocyte-macrophate colony-stimulating factor

Serum profiles of circulating granulocyte-macrophage colony-stimulating factor in acute myocardial infarction and relation with post-infarction left ventricular function

Accumulating evidence indicates that inflammation plays an important role in cardiac repairing and remodeling after acute myocardial infarction （AMI）, process of which is mediated by a cytokine reaction cascade. 1 Granulocytemacrophage colony-stimulating factor （GM-CSF） is a cytokine, which belongs to the family of haemopoietic cell colony-stimulating factor and regulates the proliferation and differentiation of myeloid progenitor cells. In addition to its growthpromoting effects, this pro-inflammation cytokine stimulates the function of mature neutrophils, monocytes and eosinophils, including regulation of leukocyte adhesion, augmentation of surface antigen expression, superoxide anion generation, enhancement or induction of other cytokine production.

EXPRESSION OF RHGM-CSF GENE IN EUKARYOCYTE BY LIPOFECTION

Objective: To recombinant the nearly natural human granulocyte-macrophage-colony stimulating factor (GM-CSF) for supplying more safe and steady expressed cytokine in clinic. Method: The eukaryotic recombinant pcDNA3.1-GM-CSF plasmid which was controlled by the CMV promoter was transferred into CHO cell by lipofectamine, selected by G418 and the positive clones was got. The recombinant vector which was rejoined into the groups of DNA of CHO was identified by PCR. Results: The results showed that the protein of rhGM-CSF was about 28 KD by using ELISA, SDS-PAGE and Western blot. Conclusion: rhGM-CSF was expressed steadily and highly. The rhGM-CSF will be of more use value.

Cytokine levels in patients with chikungunya virus infection

Objective: To investigate cytokine profile in patients with chikungunya virus (CHIKV) infection. Methods: Twenty eight pairs of serum samples collected from CHIKV infected patients during the outbreak of chikungunya fever in South Thailand in 2008 were obtained. A multiple cytokine assay for detection of 17 cytokines was performed. Results: In the acute stage of CHIKV infection, the patients had significantly higher levels of interleukin-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and tumor necrosis factor alpha than the control ( P<0.001, P=0.023, P=0.015, P <0.001 and P=0.024, respectively). When the disease developed to the recovery stage, the patients had significantly lower levels of interleukin-6, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and macrophage inflammatory protein beta than in the acute stage ( P<0.001). Conclusions: This study provides additional information that these cytokines could play roles in pathogenesis of CHIKV infection and could be used as disease biomarkers or drug targets.

Stem cells as an option for the treatment of COVID-19

The application of stem cells is among the many strategies currently available for the treatment of multiple diseases.Stem cells are characterized as undifferentiated cells that have the ability to differentiate towards multiple lineages and selfrenewal,among other attributes.Since the first umbilical cord stem cell transplant for the treatment of Fanconi anemia,the use of stem cells for the treatment of multiple diseases,including coronavirus disease 2019,has increased,showing promising results that require evaluation through research studies that include a longer follow-up time.Therefore,the main objective of this Letter is to provide an update on the use of stem cells in the treatment of severe acute respiratory syndrome coronavirus 2,as well as identify the main challenges and limitations presented by this type of therapy.

Effect of cigarette smoke extract on lipopolysaccharide-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background Lipopolysaccharide （LPS） forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase （MAPK） signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract （CSE） and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation. Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used： blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase （ERK1/2）, p38 MAPK and c-Jun N-terminal kinase （JNK）. The expression of cytokines of MAPK transduction pathway （granulocyte-macrophage colony stimulating factor （GM-CSF） and mRNA of IL-8） in the primary epithelial cells of respiratory tract was also determined. Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group （P〈0.05）; no significant difference was found between CSE-stimulation group and blank control group （P〉0.05）; there was a significant difference between CSE ＋ LPS group and blank control group and between CSE ＋ LPS group and LPS group （P〈0.05）. The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased （P〈0.05） and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation （P〈0.05） and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise （P〈0.05）, and the level was the highest 8 hours after the stimulation （P〈0.01）. Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA （P〉0.05）, but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower. Conclusions LPS stimulation can significantly increase the phosphorylation of ERK1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.