CO-EXPRESSION OF MACROPHAGE COLONY-STIMULATING FACTOR WITH ITS RECEPTOR IN HUMAN HEPATOMA CELLS AND ITS POTENTIAL ROLES

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P<0.001). The M-CSF/M-CSF-R expression levels of PBMC from hepatoma patients were higher than those of LMT patients (P<0.01,P<0.05) and the normal people (P<0.001). The hepatoma cell lines showed strong positive for M-CSF and M-CSF-R production. Both B5 and RE2 displayed a dose-dependent inhibitory effect on the growth and proliferation of hepatoma cells. Conclusion: The study indicates a co-expression model for M-CSF-R in hepatoma cells, suggesting an involvement of M-CSF/M-CSF-R in growth signaling of those malignant cells. The M-CSF/M-CSF-R seems to function through an autonomy mechanism in human hepatoma.

Granulocyte-macrophage colony-stimulating factor （GM-CSF） and T-cell responses： what we do and don＇t know

Granulocyte-macrophage colony-stimulating factor （GM-CSF） is an important hematopoietic growth factor and immune modulator. GM-CSF also has profound effects on the functional activities of various circulating leukocytes. It is produced by a variety of cell types including T cells, macrophages, endothelial cells and fibroblasts upon receiving immune stimuli. Although GM-CSF is produced locally, it can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense. Recent intensive investigations are centered on the application of GM-CSF as an immune adjuvant for its ability to increase dendritic cell （DC） maturation and function as well as macrophage activity. It is used clinically to treat neutropenia in cancer patients undergoing chemotherapy, in AIDS patients during therapy, and in patients after bone marrow transplantation. Interestingly, the hematopoietic system of GM-CSF-deficient mice appears to be normal; the most significant changes are in some specific T cell responses. Although molecular cloning of GM-CSF was carried out using cDNA library oft cells and it is well known that the T cells produce GM-CSF after activation, there is a lack of systematic investigation of this cytokine in production by T cells and its effect on T cell function. In this article, we will focus mainly on the immunobiology of GM-CSF in T cells.

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor(HSVGM-CSF) in pancreatic carcinoma.METHODS: Tumor blocks were homogenized in a sterile grinder in saline.The homogenate was injected into the right armpit of each mouse.After vaccination,the mice were randomly assigned into four groups: a control group,a high dose HSVGM-CSFgroup [1 × 107plaque forming units(pfu)/tumor],a medium dose HSVGM-CSF group(5 × 106pfu/tumor) and a low dose HSVGM-CSF group(5 × 105pfu/tumor).After initiation of drug administration,body weights and tumor diameters were measured every 3 d.Fifteen days later,after decapitation of the animal by cervical dislocation,each tumor was isolated,weighed and stored in 10% formaldehyde solution.The drug effectiveness was evaluated according to the weight,volume and relative volume change of each tumor.Furthermore,GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1,2,3 and 4 d after injection of HSVGM-CSF.RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group,and dosedependent effects were observed: the relative tumor proliferation rates of the low dose,medium dose and high dose groups on 15 d after injection were 45.5%,55.2% and 65.5%,respectively.The inhibition rates of the tumor weights of the low,middle,and high dose groups were 41.4%,46.7% and 50.5%,respectively.Furthermore,the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF.The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.CONCLUSION: Our study provides the first evidence that HSVGM-CSFcould inhibit the growth of pancreatic cancer.The enhanced GM-CSF expression might be responsible for the phenomenon.

Neuroprotective Effect of Granulocyte Colony-stimulating Factor in a Focal Cerebral Ischemic Rat Model with Hyperlipidemia

Granulocyte colony-stimulating factor (G-CSF) has been demonstrated to have neuroprotective effects in rat model with focal cerebral ischemia through anti-apoptotic pathways and by promoting proliferation of neural stem cells. In the present study, we examined the neuroprotective effect of G-CSF in an acute focal cerebral ischemia rat model with lipid metabolism disorder. Eighty male SD rats were randomly divided into normal diet control group (NC group) and high-fat diet group (HFD group) (n = 40 in each). In HFD group, rats were fed on high fat diet to induce atherosclerosis. After 29 days, 4 rats from each group were sacrificed to evaluate the effects of different diets, and the middle cerebral artery occlusion (MCAO) was performed in the rest of the rats. MCAO rats received either G-CSF (50 μg·kg–1·mL–1) or phosphate buffered saline (PBS) injection through the external jugular vein for 5 days, which was followed by 5-bromo-deoxy uridine (BrdU, i.p., 50 mg/kg) injection for another 7 days. To evaluate the effects of G-CSF treatment on neurological function, the modified neurological severity score (mNSS) was calculated. The vascular distribution, ischemic cells proliferation, cell apoptosis and the expression of vascular endothelial growth factor (VEGF) were measured to determine the effects of G-CSF treatment. Our results showed that G-CSF-treated rats had a lower mNSS than PBS-treated rats in both NC group and HFD group. G-CSF injection promoted endothelial cell proliferation and vascular regeneration, and inhibited cell apoptosis. The serum and tissue levels of VEGF were significantly increased after G-CSF treatment. It is concluded that G-CSF exerts its neuroprotective effect in focal cerebral ischemia rats with hyperlipidemia by enhancing angiogenesis, promoting cells proliferation, decreasing cell apoptosis, and increasing local VEGF expression.

Impaired granulocyte-macrophage colony-stimulating factor bioactivity accelerates surgical recurrence in ileal crohn's disease

AIM To examine the relationship between elevated granulocyte-macrophage colony-stimulating factor(GMCSF) auto-antibodies(Ab) level and time to surgical recurrence after initial surgery for Crohn's disease(CD). METHODS We reviewed 412 charts from a clinical database at tertiary academic hospital. Patients included in the study had ileal or ileocolonic CD and surgical resection of small bowel or ileocecal region for management of disease. Serum samples were analyzed for serological assays including GM-CSF cytokine, GM-CSF Ab, ASCA Ig G and Ig A, and genetic markers including SNPs rs2066843, rs2066844, rs2066845, rs2076756 and rs2066847 in NOD2, rs2241880 in ATG16 L1, and rs13361189 in IRGM. Cox proportional-hazards models were used to assess the predictors of surgical recurrence.RESULTS Ninety six percent of patients underwent initial ileocecal resection(ICR) or ileal resection(IR) and subsequently 40% of patients required a second ICR/IR for CD. GMCSF Ab level was elevated at a median of 3.81 mcg/mL. Factors predicting faster time to a second surgery included elevated GM-CSF Ab [hazard ratio(HR) 3.52, 95%CI: 1.45-8.53, P = 0.005] and elevated GM-CSF cytokine(HR = 2.48, 95%CI: 1.31-4.70, P = 0.005). Factors predicting longer duration between first and second surgery included use of Immunomodulators(HR = 0.49, 95%CI: 0.31-0.77, P = 0.002), the interaction effect of low GM-CSF Ab levels and smoking(HR = 0.60, 95%CI: 0.45-0.81, P = 0.001) and the interaction effect of low GM-CSF cytokine levels and ATG16 L1(HR = 0.65, 95%CI: 0.49-0.88, P = 0.006).CONCLUSION GM-CSF bioavailability plays a critical role in maintaining intestinal homeostasis. Decreased bioavailability coupled with the genetic risk markers and/or smoking results in aggressive CD behavior.

Pharmacokinetic Study of a Novel Recombinant Human Granulocyte Colony-stimulating Factor in Rats

Objective To study the pharmacokinetics of a novel recombinant human granulocyte colonystimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. Methods The pharmacokinetics of rhG-CSFa and conventional (wild type,WT) granulocyte colonystimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20,50,or 100 μg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro,the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat’s whole blood or serum. Results Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that,at each dose tested,for either route of drug administration,the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF,and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa,respectively. Conclusion rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.

Mobilization of Peripheral Blood Stem Cells Using Regimen Combining Docetaxel with Granulocyte Colony-stimulating Factor in Breast Cancer Patients

Objective：To evaluate the effectiveness and safety of the mobilization of peripheral blood hematopoietic stem cells by combining docetaxel with granulocyte colony-stimulating factor（G-CSF） in breast cancer patients.Methods：A total of 57 breast cancer patients were treated with docetaxel 120 mg/m2.When the white blood cell（WBC） count decreased to 1.0×109/L,patients were given G-CSF 5-g/kg daily by subcutaneous injection until the end of apheresis.Peripheral blood mononuclear cells（MNC） were isolated by Cobe Spectra Apheresis System.The percentage of CD34＋ cell was assayed by flow cytometry.Results：At a median 6 of days（range 3-8） after the administration of docetaxel,the median WBC count decreased to 1.08×109/L（range 0.20-2.31）.The median duration of G-CSF mobilization was 3 days（range 2-7）.The MNC collection was conducted 8-12 days（median 10 days） after docetaxel treatment.The median MNC was 5.35×108/kg（range 0.59-14.07）,the median CD34＋ cell count was 2.43×106/kg（range 0.16-16.69）.The CD34＋ cell count was higher than 1.00×106/kg in 47 of 57 cases（82.46%） and higher than 2.00×106/kg in 36 cases（63.16%）.The CD34＋ cell count was higher than 2.00×106/kg in 27 collections（23.68%）.The MNC count and the CD34＋ cell count were correlated with the bottom of WBC after docetaxel chemotherapy（r=0.364,0.502,P=0.005,0.000）.The CD34＋ cell count was correlated with the MNC count（r=0.597,P=0.000）.The mobilization and apheresis were well tolerated in all patients.Mild perioral numbness and numbness of hand or feet were observed in 3 cases.No serious adverse events were reported.Conclusion：Mobilization of peripheral blood hematopoietic stem cell by combining docetaxel with G-CSF was effective and safety in breast cancer patients.

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.

Granulocyte colony-stimulating factor-producing squamous cell carcinoma of the tongue exhibiting characteristic fluorine-18 deoxyglucose accumulation on positron emission tomography–computed tomography: A case report

BACKGROUND Granulocyte colony-stimulating factor(G-CSF)is a cytokine produced in inflammatory environments that induces differentiation and proliferation of neutrophils in bone marrow.We report a rare case of aggressive G-CSFproducing squamous cell carcinoma of the tongue exhibiting fluorine-18 deoxyglucose(FDG)accumulation in primary lesion,metastatic lymph nodes,spleen,and bone marrow on positron emission tomography–computed tomography(PET/CT).CASE SUMMARY We report a 58-year-old female with a rapid enlarged lingual mass with partial necrosis.Blood test results from the initial examination revealed a leukocyte count of 21380/μL.On PET/CT,extensive FDG accumulation was observed in the tongue and bilateral cervical lymph nodes,with elevated FDG accumulation in the spleen and bone marrow although no distant metastases were observed.We performed partial glossectomy and bilateral neck dissection.Immunohistochemical staining with G-CSF antibodies on biopsy specimen and resected samples revealed that both specimens were G-CSF positive.This is a rare case of G-CSF producing tongue carcinoma with elevated FDG accumulation in the spleen and bone marrow.CONCLUSION In patients with the tongue cancer and hyperleukocytosis,where FDG accumulations in the spleen and bone marrow are observed using PET/CT and when these accumulations are not caused by metastasis,G-CSF-producing tumors,with associated poor prognosis,should be considered.

Granulocyte colony-stimulating factor-producing hepatocellular carcinoma with abrupt changes

Granulocyte colony-stimulating factor(G-CSF)-producing tumor is one of the rare types of cancer clinically characterized by an elevated fever and white blood cell(WBC) increment. Although G-CSF producing tumors have been reported in several types of cancer including those of the lungs, cervix and bladder, G-CSF producing hepatocellular carcinoma is extremely rare. Here, we report the case of a rapidly growing and poorly differentiated hepatocellular carcinoma producing G-CSF. The patient showed symptoms of continuous high fever, stomach pain and cough, and high serum WBC counts, C-reactive protein(CRP) and G-CSF levels were found in laboratory tests. After a radical hepatectomy, the patient completely recovered from the above symptoms and inflammatory state. The serum levels of G-CSF were reduced to normal levels after radical surgery. An immunohistochemical analysis revealed the overexpression of G-CSF in the cytoplasm of certain hepatocellular carcinoma(HCC) cell. The patient's serum WBC, CRP and G-CSF levels remained within normal levels in the six months after surgery without recurrence. This is the 9^(th) case report of G-CSF producing hepatocellular carcinoma in English literature. We review the clinical characteristics of the G-CSF producing HCC and discuss a possible treatment strategy.

DETERMINATION OF SERUM SOLUBLE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR LEVELS IN PATIENTS WITH HEMATOLOGICAL DISEASES

Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ～ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.

Granulocyte colony-stimulating factor increases extracellular glutamic acid uptake and suppresses free radicals in an experimental model of amyotrophic lateral sclerosis

Excitatory amino acid toxicity and free radical damage play important roles in amyotrophic lateral sclerosis. Granulocyte colony-stimulating factor （G-CSF） protects nerve cells exposed to high-concentrations of glutamic acid, suggesting positive effects in the treatment of amyotrophic lateral sclerosis. The present study induced in vitro motor neuron injury using glutamic acid excitotoxicity, and the biochemical effects of G-CSF on glutamic acid concentration were determined. In addition, the effects of G-CSF on superoxide dismutase, glutathione peroxidase activity in motor neurons, and malondialdehyde and nitric oxide contents were analyzed. Immunohistochemistry was performed to measure neuronal survival. Results revealed that G-CSF significantly suppressed free radical activity, inhibited excitotoxicity, and reduced apoptosis and loss of motor neurons in the anterior horn of the spinal cord.

Changes of Circulating Platelet-Derived Growth Factor and Granulocyte Colony-Stimulating Factor in Patients with Condyloma Acuminatum

Objective: To study the expression levels of platelet-derived growth factor (PDGF) and granulocyte colony-stimulatingfactor (G-CSF) in peripheral blood and their role in thepathogenesis of Condyloma acuminatum (CA). Methods: Sera were taken from 70 patients with Condylomaacuminatum and compared with 35 healthy controls. PDGFand G-CSF in serum were quantitated using a dual antibodysandwich enzyme-linked immunoabsorbent assay (ELISA). Results: Serum concentrations of PDGF and G-CSF weresignificantly increased in patients with Condylomaacuminatum (CA) compared to controls (P<0.001 and P<0.005respectively). Serum levels of PDGF and G-CSF correlatedwith clinical severity of CA, but no significant difference wasobserved between different duration of disease groups. Asignificant positive correlation was noticed between neutrophilcount and G-CSF levels (γ=0.38, P<0.001), and the neutrophilcount showed no significant correlation with PDGE Conclusion: The results indicated that increased expressionof PDGF an -CSF in peripheral blood might be involved in pathogenesis of CA.

Granulocyte colony-stimulating factor promotes growth of processes,growth associated protein 43 and microtubule-associated protein 2 expression in cultured rat retinal ganglion cells in vitro

Following granulocyte colony-stimulating factor （G-CSF） treatment,the growth of processes in cul-tured rat retinal ganglion cells （RGCs） in vitro,expression of growth associated protein 43,and expression of microtubule-associated protein 2 mRNA expression were significantly increased.In contrast,RhoA/Rock protein content was significantly reduced by G-CSF treatment.These results indicate that G-CSF promotes the growth of processes in RGCs and increases the expression of growth-associated protein 43 and microtubule-associated protein 2 mRNA by inhibiting the RhoA/Rock pathway,thereby benefiting axonal repair in RGCs exposed to hypoxia.

Granulocyte colony-stimulating factor as a novel adjunct to improve hepatitis B vaccination

Hepatitis B vaccination is successful in 95% of individuals. In the remainder, despite repeated attempts, immunization often remains unsuccessful. 'Non-response' leaves the individual susceptible to infection. Various strategies have been employed to overcome this. These include the use of adjuncts alongside conventional vaccines which activate immune responses. In this case report we demonstrate the successful use of the hematopoietic growth factor Granulocyte colonystimulating factor (G-CSF) as a vaccine adjunct in an individual who had previously failed conventional vaccination three times. The patient tolerated the regimen without any side effects and achieved a hepatitis B surface antibody titer greater than 100 IU/L. Use of G-CSF as a vaccine adjunct for hepatitis B has not previously been reported and the outcome in this case suggests that the use of G-CSF in this context warrants further exploration.

An autopsy case of granulocyte-colony-stimulating-factor- producing extrahepatic bile duct carcinoma

A 79-year-old man was referred to this department due to the presence of extrahepatic bile duct carcinoma with a tumor at the left chest wall. The lesion was suspected to be a metastasis of bile duct carcinoma to the left wall, however, computed tomography (CT) revealed no regional lymph node or liver metastases. In addition, cytological and pathological examinations did not show malignancy. At the time of admission, the white blood cell count was 21 460 cells/μL (neutrophils, 18 240 cells/μL) and this elevated to 106 040 before death. In addition, serum granulocyte colony-stimulating factor (G-CSF) was elevated. At 28 d after admission, the patient died. An autopsy showed a poorly differentiated adenocarcinoma with sarcomatous change, which had slightly invaded into the pancreas around the bile duct, and was found in the distal bile duct with multiple metastases to the chest wall, lung, kidney, adrenal body, liver, mesentery, vertebra and mediastinal and para-aortic lymph nodes, without locoregional lymph node and liver metastasis. The cancer cells showed positive immunohistochemical staining for anti-G-CSF antibody. This is believed to be the first report of an extrahepatic bile duct carcinoma that produces G-CSF. Since G-CSF-producing carcinoma and sarcomatous change of the biliary tract leads to poor prognosis, early diagnosis and treatment are needed. When infection is ruled out, the G-CSF in serum should be examined. In addition, examinations such as bonescintigraphy and chest CT should also be considered for distant metastasis.

Anti-apoptotic effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemic rats

The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor （50 pg/kg） over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA （a key protease for apoptosis in the cerebral ischemia zone）, lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.