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.

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.

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.

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.

Application of pegylated recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF) for the prevention of neutropenia in triple negative breast cancer patients older than 65 years during adjuvant chemotherapy

Objective The aim of this study was to compare the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF)and recombinant human granulocyte colonystimulating factor(rhG-CSF)for the prevention of neutropenia in elderly breast cancer patients during adjuvant chemotherapy.Methods A total of 45 oncology inpatients with breast cancer,who received adjuvant chemotherapy and were older than 65 years from May 2017 to October 2018 in the General Hospital of the Northern Theater of the Chinese people’s Liberation Army,were included.Epirubivin Cyclophoshamide-Docetaxel(EC-T)sequential adjuvant chemotherapy was chosen.Forty-five patients were randomly divided into two groups;25 patients in the treatment group were treated with PEG-rhG-CSF and 20 patients in the control group were not treated with PEG-rhG-CSF,but only rhG-CSF.The experimental group was treated with the PEG-rhG-CSF at the end of chemotherapy for 24–48 h,with a 6 mg subcutaneous injection once per chemotherapy cycle.In the control group,rhG-CSF was administered after 48 h of chemotherapy,with a 100μg subcutaneous injection,1/d,d 1–7.The dosage could be increased step by step with the exacerbation of neutropenia.The primary aims of this study was to discover the incidence of leukopenia,neutropenia,neutrophilic fever,and adverse reactions in the two groups.Results The incidence of neutropenia,neutrophilic fever and adverse reactions decreased in the treatment group compared to the control group,but no significant difference existed between two groups(P>0.05).Patients in treatment group had a lower,but not statistically significant,incidence of adverse reactions(P>0.05).Conclusion Applying PEG-rhG-CSF could be effective in preventing neutropenia in elderly patients with postoperative adjuvant chemotherapy to treat breast cancer.It may effectively control the occurrence of neutropenia after chemotherapy and reduce the chance of infection.The incidence of side effects,such as fever and bone pain,was low.The adverse drug reactions were well tolerated by patients,which could ensure the smooth progress of chemotherapy.

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 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.

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.

Mobilization Efficiency of Granulocyte Colony-stimulating Factor and Stem Cell Factor to Bone Marrow Mononuclear Cells and Mechanisms

The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed, and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells. Kunming mice were randomly divided into two groups. The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 μg/kg and SCF at a dose of 25 μg/kg every day for 5 days, and those in control group were given isodose physiological saline. The MNCs were separated, counted and cultured, and the colony-forming unit-fibroblast (CFU-F) was evaluated. CD34+CXCR4+ MNCs were sorted by flow cytometry. The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA, and that of CXCL12 mRNA in bone marrow was measured by RT-PCR. The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P<0.01). The factors had a dramatic effect on the expansion capability of CFU-F (P<0.05). Flow cytometric of bone marrow MNCs surface markers revealed that CD34+CXCR4+ cells accounted for 44.6%±8.7% of the total CD34+ MNCs. Moreover, G-CSF/SCF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. In this study, it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.

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.

A New Protocol for Solubilization, Refolding and Purification of Recombinant Human Granulocyte Colony-stimulating Factor in Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.

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.

Effects of granulocyte colony-stimulating factor and stem cell factor, alone and in combination, on the biological behaviours of bone marrow mesenchymal stem cells

Aim: The effects of granulocyte colony- stimu-lating factor (G-CSF) and stem cell factor (SCF) on the proliferation and osteogenic differentia-tion capacity of bone marrow mesenchymal stem cells (MSCs) were studied in the experi-ment. Methods: Bone marrow MSCs were col-lected from rabbits successfully, and treated with various concentrations of G-CSF, SCF or a combination of the two. Flow cytometric ana-lyse, MTT test, CFU-F assay, and alkaline phosphatase (ALP) activity measurement were employed. Results: The results of flow cytome-try showed that immunophenotype of the cells were CD29+/CD45-, CD105+/ CD34–, CD90+/ HLADR–. MSCs were shown to constitutively express low levels of c-kit which could be en-hanced by SCF. G-CSF and SCF had an obvious facilitative effect on the proliferation of MSCs in a dose-dependent fashion. In addition, G-CSF and SCF would be effective in reversibly pre-venting their differentiation, as showed by the decrease of ALP activity, leading to self-renewal rather than differentiative cell divisions. The effects of G-CSF were superior to SCF. And cells in the group treated with combination of G-CSF and SCF showed more powerful effects than the groups treated with G-CS, SCF, or none of the two. Conclusion: On the whole, these studies demonstrated that MSCs responsed to G-CSF, SCF, and to G-CSF plus SCF in a manner that suppressed differentiation, and promotes proliferation and self-renewal, and support the view that these factors could act synergistically.

Granulocyte Colony-Stimulating Factor Enhances the Anticancer Effects of Cisplatin against Lung Cancer by Promoting Angiogenesis

The G-CSF is used as a therapeutic drug of the febrile neutropenia in lung cancer chemotherapy, however, there were few reports that showed the effects of combination effects of G-CSF and anticancer drugs against lung cancer. In the present study, we investigated the effects of G-CSF and the combination effects of G-CSF and cisplatin on lung cancer growth. We investigated the effect of G-CSF against the LL-2 and KLN-205 cells by MTT assay and tried to detect the G-CSF receptor by RT-PCR. Next, to analyze the G-CSF effects in vivo, we transplanted the LL-2 into C57BL/6 mice, intraperitoneally administered G-CSF (30 micro/kg/day) with or without cisplatin (5 mg/kg), measured the tumor size and analyzed pathologically by HE and immunostaining. In vitro analyses, G-CSF showed no effects in LL-2 and KLN-205 cells, and RT-PCR revealed no G-CSF receptor mRNA. In vivo analyses, G-CSF alone did not significantly suppress tumor growth. However, concurrent G-CSF administration with cisplatin significantly enhanced the tumor suppressing effect of cisplatin in early stage of tumor growth. The analysis data of vWF immunostaining indicated that the neovascularization in the peripheral region of the tumors was more enhanced in G-CSF treatment mice. ELISA assay revealed that G-CSF did not influence the serum concentration of TNF-alpha and IL-12 in tumor-bearing mice. This study suggests that concurrent (combination) administration of cisplatin with G-CSF is a safe and effective method for enhancing anticancer effects and reducing chemotherapeutic agent-induced myelosuppression.

Granulocyte-colony stimulating factor therapy improves survival in patients with hepatitis B virus-associated acuteon-chronic liver failure

AIM:To evaluate the safety and efficacy of granulocyte-colony stimulating factor(G-CSF) therapy in patients with hepatitis B virus(HBV)-associated acuteon-chronic liver failure(ACLF).METHODS:Fifty-five patients with HBV-associated ACLF were randomized into two groups:the treatment group and the control group.Twenty-seven patients in the treatment group received G-CSF(5 μg/kg per day,six doses) treatment plus standard therapy,and 28 patients in the control group received standard therapy only.The peripheral CD34 + cell count was measured consecutively by flow cytometry.Circulating white blood cell count,biochemical parameters,and other clinical data of these patients were recorded and analyzed.All patients were followed up for a period of 3 mo to evaluate the changes in liver function and survival rate.RESULTS:The peripheral neutrophil and CD34 + cell counts in the G-CSF group increased on day 3 from the onset of therapy,continued to rise on day 7,and remained elevated on day 15 compared to those of the control group.Child-Turcotte-Pugh score of patients in the treatment group was improved on day 30 from the onset of G-CSF therapy,compared to that in the controls(P = 0.041).Model for End-Stage of Liver Disease score of patients in the treatment group was improved on day 7(P = 0.004) and remained high on day 30 from the onset of G-CSF therapy(P < 0.001) compared to that in controls.After 3 mo of follow-up observation,the survival rate in the treatment group(48.1%) was significantly higher than that in the control group(21.4%)(P = 0.0181).CONCLUSION:G-CSF therapy promoted CD34 + cell mobilization in patients with HBV-associated ACLF,and improved the liver function and the survival rate of these patients.

Capillary Zone Electrophoresis Investigation of Interactions between Granulocyte-colony Stimulating Factor and Dextran Sulfate/Carrageenan Oligosaccharide

The interactions between granulocyte-colony stimulating factor (G-CSF) and dextran sulfate / κ-carrageenan oligosaccharide were studied by capillary zone electrophoresis. Dextran sulfate could strongly interact with G-CSF and the complex was detected. The binding constant and stoichiometry were determined to be 1.2×106 (mol/L)-1 and 3:1, respectively. However, the interaction between κ-carrageenan oligosaccharide and G-CSF was not found.

Administration of granulocyte colony stimulating factor after liver transplantation leads to an increased incidence and severity of ischemic biliary lesions in the rat model

AIM： Recently it has been reported that granulocyte colony stimulating factor （G-CSF） can induce hypercoagulability in healthy bone marrow donors. It is conceivable that the induction of a prothrombotic state in a recipient of an organ graft with already impaired perfusion might cause further deterioration in the transplanted organ. This study evaluated whether G-CSF treatment worsens liver perfusion following liver transplantation in the rat model. METHODS： A non-arterialized rat liver transplantation model was employed to evaluate the effect of G-CSF treatment on the liver in a syngeneic and allogeneic strain combination. Study outcomes included survival time and liver damage as investigated by liver enzymes and liver histology. Observation times were 1 d, 1 wk and 12 wk. RESULTS： Rats treated with G-CSF had increased incidence and severity of biliary damage following liver transplantation. In these animals, hepatocellular necrosis was accentuated in the centrilobular region. These lesions are indicative of impaired perfusion in G-CSF treated animals. CONCLUSION： G-CSF should be used with caution in recipients of liver transplantation, as treatment might enhance preexisting, undetected perfusion problems and ultimately lead to ischemia induced biliary complications .

The Role of C-Reactive Protein, Granulocyte Colony Stimulating Factor and Total Antioxidant Capacity in Diagnosis of Acute Appendicitis

Background and Aim: Despite the fact that acute appendicitis is the most common surgical emergency all around the world, its diagnosis is still based on clinical evaluation and accuracy of the diagnosis depending on experience. The aim of this study is to evaluate the role of inflammatory markers in diagnosis of acute appendicitis. Material and Method: The study includes 77 cases with histopathologically proven acute appendicitis and 17 control cases. Blood samples were obtained from all cases and C-reactive protein (CRP), Granulocyte Colony Stimulating Factor (G-CSF) and Total Antioxidant Capacity (TAC) were measured. Findings: In cases with acute appendicitis, CRP and G-CSF levels were found to be related to acute appendicitis;however, TAC was not affected by the disease process. Moreover, CRP and G-CSF levels were correlated with the disease severity. Conclusion: Both CRP and G-CSF can be used in diagnosis of acute appendicitis. Furthermore, increased CRP level can be a marker to show advanced cases. However, G-CSF is not an effective marker to show disease severity.