Granulocyte Colony-stimulating Factor Pretreatment Research Articles

Prospective, randomized, double-blind phase 2B trial of the TLPO and TLPLDC vaccines to prevent recurrence of resected stage III/IV melanoma: a prespecified 36-month analysis

BackgroundThe tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is made by ex vivo priming matured autologous dendritic cells (DCs) with yeast cell wall particles (YCWPs) loaded with autologous tumor lysate...

Augmented anti-tumour effect of lipopolysaccharide with G-CSF without enhancing body weight loss in mice bearing MH134 hepatoma

BackgroundAlthough lipopolysaccharide (LPS) is reported effective for cancer, developmental application as anti-tumour therapeutic agents have been prevented due to its severe toxicity. Since antitumour action of LPS was suggested to be partly related to the function of neutrophil, we investigated the effects of granulocyte colony stimulating factor (G-CSF), a cytokine enhancing neutrophil function, on antitumour activity of bacterial LPS against a murine syngeneic hepatoma MH134. MethodsEffect of G-CSF (30 μg/kg i.v. pretreatment, 4 days) and LPS (20μg/mouse) on tumor growth and survival days of mice bearing MH134 hepatoma were monitored. Results4 day treatment of G-CSF 30 μg/kg increased the neutrophil level with statistical significance. On the MH134 hepatoma bearing mice, LPS significantly inhibited the tumour growth. Although G-CSF pretreatment alone did not inhibit the growth, once combined with LPS, significant inhibition was observed compared with LPS group. Tumour regression was demonstrated in combination group (6/12 mice), and survival of the mice in the combination group exceeded those of the LPS monotherapy group without enhancing the body weight loss. ConclusionsCombination of G-CSF and LPS prominently inhibit the tumour growth and elongated the survival days without enhancing a toxic response to LPS treatment.

Granulocyte colony-stimulating factor significantly decreases density of hippocampal caspase 3-positive nuclei, thus ameliorating apoptosis-mediated damage, in a model of ischaemic neonatal brain injury.

Ischaemic brain injury is a major complication in patients undergoing surgery for congenital heart disease, with the hippocampus being a particularly vulnerable region. We hypothesized that neuronal injury resulting from cardiopulmonary bypass and associated circulatory arrest is ameliorated by pretreatment with granulocyte colony-stimulating factor (G-CSF), a cytokine and an anti-apoptotic neurotrophic factor. In a model of ischaemic brain injury, 4 male newborn piglets were anaesthetized and subjected to deep hypothermic circulatory arrest (DHCA) (cooled to 18°C, DHCA maintained for 60 min, rewarmed and recovered for 8-9 h), while 4 animals received G-CSF (34 µg/kg, intravenously) 2 h prior to the DHCA procedure. At the end of each experiment, the animals were perfused with a fixative, the hippocampus was extracted, cryoprotected, cut and the brain sections were immunoprocessed for activated caspase 3, a pro-apoptotic factor. Immunopositive neuronal nuclei were counted in multiple counting boxes (440 × 330 µm) centred on the CA1 or CA3 hippocampal regions and their mean numbers compared between the different treatment groups and regions. G-CSF pretreatment resulted in significantly lower counts of caspase 3-positive nuclei per counting box in both the CA1 [52.2 ± 9.3 (SD) vs 61.6 ± 8.4, P < 0.001] and CA3 (41.2 ± 6.9 vs 60.4 ± 16.4, P < 0.00002) regions of the hippocampus as compared to DHCA groups. The effects of G-CSF were significant for pyramidal cells of both regions and for interneurons in the CA3 region. In an animal model of ischaemic brain injury, G-CSF reduces neuronal injury in the hippocampus, thus potentially having beneficial effect on neurologic outcomes.

Lipopolysaccharide-binding protein (LBP) blockade augments the protective effect of granulocyte colony-stimulating factor (G-CSF) in a rat sepsis model.

The effect of granulocyte colony-stimulating factor (G-CSF) on sepsis is discussed controversially in clinical studies. We previously demonstrated that G-CSF treatment induced lipopolysaccharide (LPS) sensitization via up-regulation of LPS-binding protein (LBP). We hypothesized that the futile effect of G-CSF-treatment in sepsis might be due to its ability to up-regulate LBP. Therefore, blockade of LBP may attenuate the G-CSF-induced LPS sensitization and protect animals from polymicrobial sepsis. Endogenous LBP levels were up-regulated by pretreatment with G-CSF, and the LBP protein was blocked by administration of a specific blocking peptide-LBPK95A. Polymicrobial sepsis was induced by intraperitoneal injection of feces slurry. Rats were monitored every 3 up to 72 h to observe the survival rate. Tissue injury, bacterial infiltration, local inflammatory response, and neutrophil infiltration at 0, 2, and 12 h after the septic insult were analyzed. The survival benefit of G-CSF pretreatment was improved when combined with LBPK95A treatment (control vs. G-CSF vs. combi: 36% vs. 56% vs. 93%; P < 0.05). Combined treatment of G-CSF and LBPK95A was associated with the minimal tissue damage. Treatment with LBPK95A significantly inhibited the neutrophil infiltration without interfering with the bacterial clearance. The G-CSF-induced inflammatory sensitization effect was inhibited by LBPK95A, indicated by the decrease of cytokines expression, and the activation of nuclear factor kappa B and signal transducer and activator of transcription 3 signaling pathway. In conclusion, these results suggested that the effect of prophylactic augmentation of the host's response via G-CSF pretreatment was further enhanced by inhibition of the up-regulation of LBP.

G-CSF pretreatment aggravates LPS-associated microcirculatory dysfunction and acute liver injury after partial hepatectomy in rats.

Liver dysfunction is a serious complication in the early phase following major liver resection or liver transplantation and might be aggravated by the translocation of bacteria and lipopolysaccharide (LPS). As a preventive strategy, granulocyte colony-stimulating factor (G-CSF) is prophylactically applied in patients who are subjected to major surgery. However, we previously demonstrated that G-CSF can induce LPS sensitization. In this study, we aimed to evaluate the effects of G-CSF pretreatment on hepatic microcirculatory disturbances and postoperative liver dysfunction after 70 % partial hepatectomy (PH) in rats. PH alone was well tolerated by all animals (100 % survival rate, slight liver damage and inflammation). LPS application after 70 % PH caused moderate inflammation, microcirculatory disturbances and hepatic damage and led to a 24-h survival rate of 30 % after the operations. In the G-CSF-LPS-PH group, all of the rats died within 4 h with severe inflammatory responses and liver damage (i.e., pronounced erythrocyte congestion and neutrophil infiltration). Portal hypertension and microcirculatory disorders (i.e., inhomogeneous perfusion, sinusoidal dilatation and reductions on functional capillary density) were more pronounced in the G-CSF-LPS-PH group. In conclusion, increased circulating LPS levels were associated with an imbalanced inflammatory response and microcirculatory dysfunction that preceded liver damage and subsequent dysfunction following surgery. G-CSF-pretreatment aggravated microcirculatory disturbances and liver damage, which might have been related to G-CSF-induced LPS sensitization.

Heme oxygenase-1 induction contributes to renoprotection by G-CSF during rhabdomyolysis-associated acute kidney injury

Granulocyte colony-stimulating factor (G-CSF) is renoprotective during acute kidney injury (AKI) induced by ischemia and cisplatin nephrotoxicity; however, the underlying mechanism is not entirely clear. Rhabdomyolysis is another important clinical cause of AKI, due to the release of nephrotoxins (e.g., heme) from disrupted muscles. The current study has determined the effects of G-CSF on rhabdomyolysis-associated AKI using in vivo and in vitro models. In C57BL/6 mice, intramuscular injection of glycerol induced AKI, which was partially prevented by G-CSF pretreatment. Consistently, glycerol-induced renal tissue damage was ameliorated by G-CSF. In addition, animal survival following the glycerol injection was improved from ∼30 to ∼70% by G-CSF. In cultured renal tubular cells, hemin-induced apoptosis was also suppressed by G-CSF. Interestingly, G-CSF induced heme oxygenase-1 (HO-1, a critical enzyme for heme/hemin degradation and detoxification) in both cultured tubular cells and mouse kidneys. Blockade of HO-1 with protoporphyrin IX zinc(II) (ZnPP) could largely diminish the protective effects of G-CSF. Together, these results demonstrated the renoprotective effects of G-CSF in rhabdomyolysis-associated AKI. Notably, G-CSF may directly protect against tubular cell injury under the disease condition by inducing HO-1.

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats.

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.

The Antiapoptotic Effect of Granulocyte Colony-stimulating Factor Reduces Infarct Size and Prevents Heart Failure Development in Rats

Background/Aim. Granulocyte colony-stimulating factor (G-CSF) reduces myocardial injury and improves cardiac function after myocardial infarction (MI). We investigated the early alterations provided by G-CSF and the chronic repercussions in infarcted rats. Methods. Male Wistar rats (200-250g) received vehicle (MI) or G-CSF (MI-GCSF) (50 µg/kg, sc) at 7, 3 and 1 days before MI surgery. Afterwards MI was produced and infarct size was measured 1 and 15 days after surgery. Expression of anti- and proapoptotic proteins was evaluated immediately before surgery. 24 hours after surgery, apoptotic nuclei were evaluated. Two weeks after MI, left ventricular (LV) function was evaluated, followed by in situ LV diastolic pressure-volume evaluation. Results. Infarct size was decreased by 1 day pre-treatment before occlusion (36±2.8 vs. 44±2.1% in MI; P<0.05) and remained reduced at 15 days after infarction (28±2.2 vs. 36±1.4% in MI; P<0.05). G-CSF pretreatment increased Bcl-2 and Bcl-xL protein expression, but did not alter Bax in LV. Apoptotic nuclei were reduced by treatment (Sham: 0.46±0.42, MI: 15.5±2.43, MI-GCSF: 5.34±3.34%; P<0.05). Fifteen days after MI, cardiac function remained preserved in G-CSF pretreated rats. The LV dilation was reduced in MI-G-CSF group as compared to MI rats, being closely associated with infarct size. Conclusion. The early beneficial effects of G-CSF were essentials to preserve cardiac function at a chronic stage of myocardial infarction.

Effects of granulocyte-colony stimulating factor on bone marrow-derived progenitor cells in murine cardiac transplantation

Granulocyte-colony stimulating factor (G-CSF) mobilizes progenitors from the bone marrow (BM) and into the circulation. In cardiac transplantation, G-CSF pretreatment of both donors and recipients has been found to improve cardiac function. The aim of this study was to examine whether the observed benefit of G-CSF pretreatment in cardiac transplantation involves vascular repopulation by host progenitor cells. Progenitor cells were exposed to immunosuppressive agents±G-CSF. The effect of drug treatment on total cell counts, proliferation, angiogenesis, apoptosis, and tubule formation was assessed. C57BL/6BM-GFP chimeric recipients underwent cardiac transplantation. Host progenitor cell seeding was evaluated on hearts 14 and 30 days post-transplant. G-CSF treatment of BM-derived progenitor cells in vitro improved survival, proliferation, and angiogenesis of the cells despite treatment with immunosuppressive agents. G-CSF pretreatment of BM-GFP transgenic recipient mice prior to heart transplantation resulted in increased re-endothelialization at 30 days post-transplant in G-CSF pretreated allografts (9.3±2.2%) relative to nonpretreated allografts (3.4±1.6%). G-CSF pretreated allografts also demonstrated a reduction of intimal narrowing in vessels of the transplanted heart. These findings suggest that G-CSF pretreatment leads to elevated numbers of host progenitor cells which may contribute to reconstitution of damaged allograft blood vessels.

Granulocyte Colony-stimulating Factor Reduces Mortality by Suppressing Ventricular Arrhythmias in Acute Phase of Myocardial Infarction in Rats

Our aim was to evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on early cardiac arrhythmias after myocardial infarction (MI) and the impact on survival. Male Wistar rats received repeated doses of 50 mug/kg G-CSF (MI-GCSF group) or vehicle (MI group) at 7, 3, and 1 days before surgery. MI was induced by permanent occlusion of left coronary artery. The electrocardiogram was obtained before occlusion and then for 30 minutes after surgery. Events and duration of ventricular arrhythmias were analyzed. The levels of connexin43 (Cx43) were measured by Western blot immediately before MI production. Survival was significantly increased in MI-GCSF pretreated group (74% versus 52.9% MI, P < 0.05). G-CSF pretreatment also significantly reduced the ventricular premature beats when compared with the untreated-MI group (201 +/- 47 versus 679 +/- 117, P < 0.05). The number and the duration of ventricular tachycardia were smaller in the MI-G-CSF group, as well as the number of ventricular fibrillation episodes (10% versus 69% in MI, P < 0.05). Cx43 levels were significantly increased by G-CSF treatment (1.27 +/- 0.13 versus 0.86 +/- 0.11; P < 0.05). The MI size 24 hours after occlusion was reduced by G-CSF pretreatment (36 +/- 3% versus 44 +/- 2% of left ventricle in MI group; P < 0.05). The increase of Cx43 expression in the heart may explain the reduced incidence in ventricular arrhythmias in the early phases after coronary artery occlusion in rats, thus increasing survival after MI.

Pretreatment with granulocyte colony‐stimulating factor attenuated renal ischaemia and reperfusion injury via activation of PI3/Akt signal pathway

Granulocyte colony-stimulating factor (G-CSF) has been shown to exert protective effects in various tissues and experimental models of ischaemia-induced injury. However, the mechanism of renoprotective action in ischaemia/reperfusion (I/R) renal injury of G-CSF was unknown. Male C57BL/6J mice, subjected to renal ischaemia for 45 min, 48 h and 7 days reperfusion, were administered either saline, wortmannin, G-CSF, and G-CSF plus wortmannin 3 days prior to I/R. Saline-treated group served as the control. At 48 h and 7 days of reperfusion, the mice were killed. Significantly, renal dysfunction and morphological injury were identified at 48 h and 7 days after I/R. Wortmannin pretreatment worsened the renal injury significantly. However, G-CSF pretreatment significantly attenuated renal injury, reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive ratio of renal tubular epithelial cells and inflammation cytokine expression in the kidney. Moreover, G-CSF pretreatment inhibited the expression of Bax and increased the expression of bcl-2 and p-Akt in the kidney. Wortmannin blunted the beneficial effects of G-CSF. The cytoprotective action of G-CSF against I/R injury seems to be associated with its anti-apoptotic action mediated by upregulation of p-Akt signal pathway.

Granulocyte-colony stimulating factor upregulates ErbB2 expression on breast cancer cell lines and converts primary resistance to trastuzumab

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.

Administration of granulocyte colony-stimulating factor to recipients followed by intra-bone marrow–bone marrow transplantation accelerates acceptance of allogeneic bone marrow cells in mice

We have recently established a novel method for bone marrow transplantation: intra-bone marrow–bone marrow transplantation (IBM–BMT), by which the rapid recovery of donor-derived hematopoiesis can be expected even when reduced radiation doses are used. In this paper, we examine, using mice, whether the combination of pretreatment of recipients with granulocyte-colony-stimulating factor (G-CSF) and IBM–BMT can induce a more rapid recovery of donor-derived hematopoiesis than IBM–BMT alone. We first pretreated recipients with recombinant human (rh) G-CSF (250 μg/kg/day) for 5 consecutive days (days −6 to −2). On day −1, the recipients were irradiated, and IBM–BMT was carried out on day 0. On day 12, we performed colony-forming units of spleen (CFU-S) assays. The combination of G-CSF pretreatment and IBM–BMT augmented the CFU-S counts, the weight of spleens, and the numbers of donor-derived hematopoietic cells. We next analyzed the mechanisms underlying these effects of G-CSF and found that (i) G-CSF induces Th2 polarization, which can prevent graft rejection, and (ii) G-CSF augments natural suppressor activity, which suppresses graft rejection. The combination of G-CSF pretreatment and IBM–BMT can produce the rapid recovery of donor-derived hematopoiesis and suppress graft rejection. This method would lighten the burden on patients in allogeneic BMT.

Granulocyte Colony-Stimulating Factor Facilitates the Angiogenesis Induced by Ultrasonic Microbubble Destruction

Ultrasonic destruction of microbubbles (US/MB) in the microcirculation causes local inflammatory cell infiltration, which has been shown to induce angiogenesis. Granulocyte colony-stimulating factor (G-CSF), which mobilizes myelomonocytic cells from the bone marrow and enhances vascular endothelial growth factor (VEGF) release from these cells, has also been applied to therapeutic angiogenesis induction. In the present study, we sought to examine the potential of G-CSF pretreatment to enhance the angiogenic effect of US/MB. Ischemic hindlimbs in mice were treated with either a predetermined minimal effective dose (300 μg/kg) of G-CSF, US/MB alone or G-CSF pretreatment followed by US/MB at seven days after removal of the femoral artery. Ultrasonic destruction of microbubbles was performed as intermittent pulsed local insonation using a diagnostic ultrasound scanner at a peak negative pressure of 1.4 MPa after intravenous injection of perfluorocarbon microbubbles. At 21 days after the treatment, we quantified the surface vascularity using a grid method and the capillary density using an alkaline phosphatase stain. Relative to the capillary density in normal muscle, the capillary density in the treated limbs was restored to 74 ± 13% by G-CSF alone and 90 ± 20% by US/MB alone ( p < 0.05 vs. both untreated and G-CSF alone), and further increased to 101 ± 21% by G-CSF pretreatment. The collateral growth induced by the combination of G-CSF pretreatment and US/MB was 2.8- and 1.4-fold greater than the growth induced by G-CSF alone and US/MB alone, respectively ( p < 0.05 for both). Thus, pretreatment with a single minimal effective dose of G-CSF can augment the angiogenic effect of US/MB. (E-mail: komori@med.kagawa-u.ac.jp)

Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice

Neointimal formation following percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since reendothelialization is one of the determinant factors for the development of neointimal formation, we examined the effects of granulocyte colony-stimulating factor (G-CSF) on reendothelialization and neointimal formation after vascular injury in mice. Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. G-CSF pretreatment significantly accelerated reendothelialization and decreased neointimal formation following vascular injury; however, this inhibitory effect of G-CSF was diminished when G-CSF was started following the injury. Flow cytometry analysis revealed that G-CSF treatment increased the number of endothelial progenitor cells (EPCs: CD34+/Flk-1+) in the peripheral circulation. Vascular injury was also produced in 2 types of mice whose bone marrow was replaced with that of enhanced green fluorescent protein- and Tie2/LacZ-transgenic mice. In the reendothelialized artery of these mice, few bone marrow-derived EPCs were detected. Furthermore, G-CSF treatment reduced the serum level of interleukin (IL)-6. G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI.

Increased FDG bone marrow uptake after intracoronary progenitor cell therapy

Patients with coronary artery disease who undergo FDG, PET for therapy monitoring after intracoronary progenitor cell infusion (PCT) show an increased bone marrow uptake in some cases. AIM of the study was to evaluate the systemic bone marrow glucose metabolism in this patient group after PCT. FDG bone marrow uptake (BMU), measured as standardized uptake value (SUVmax) in the thoracic spine, was retrospectively evaluated in 23 control patients who did not receive PCT and in 75 patients who received PCT 3 +/- 2.2 days before PET scanning. Five out of them were pretreated with granulocyte colony-stimulating factor (G-CSF) 5 days prior to PCT and 10 +/- 1.2 days before PET scanning. In 39 patients who received only PCT without G-CSF and underwent PET therapy monitoring 4 months later, baseline and follow up bone marrow uptake were measured. Leucocytes, C-reactive protein (CRP) levels and the influence of nicotine consumption were compared with the BMU. In patients (n = 70) who received PCT without G-CSF, BMU median (1.3) was slightly, but significantly higher than in the controls (1.0) (p = 0.02) regardless nicotine consumption. BMU did not change significantly 4 months later (1.2) (p = 0.41, n.s.). After G-CSF pretreatment, patients showed a significantly higher bone marrow uptake (3.7) compared to patients only treated with PCT (1.3) (p = 0.023). Leucocyte blood levels were significantly higher in patients with a BMU > or =2.5 compared to patients with a bone marrow SUVmax <2.5 (p<0.001). CRP values did not correlate with the BMU (rho -0.02, p = 0.38). Monitoring PCT patients, a slightly increased FDG BMU may be observed which remains unchanged for several months. Unspecific bone marrow reactions after PCT may be associated with increased leucocyte blood levels and play a role in the changed systemic glucose BMU. In addition, pretreatment with G-CSF shows an intense amplification of BMU.

Granulocyte Colony-Stimulating Factor Prior to Low-Dose Total Body Irradiation as Conditioning Regimen in Hematopoietic Stem Cell Transplantation for Gene Therapy.

Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. Nonmyeloablative conditioning regimens such as low-dose total body irradiation (LDTBI) can enhance competitive engraftment in the absence of a selective advantage for HSC. Granulocyte colony-stimulating factor (G-CSF) treatment of recipients prior to LDTBI has been shown to enhance engraftment of fresh bone marrow (BM) cells, but the underlying mechanisms and whether this combined approach can enhance long-term engraftment of genetically transduced donor cells have not been defined. We examined whether pre-treatment of recipient mice with G-CSF prior to LDTBI improves long-term donor engraftment of retroviral vector-transduced HSC in murine X-linked chronic granulomatous disease (X-CGD). In initial studies, we verified that pre-treatment of wild type mice with 4 ug G-CSF SC BID for 5 days prior to irradiation with either 160 cGy or 300 cGy significantly increased long-term engraftment of congenic fresh BM cells (see Table).Donor → HostCell doseLDTBI (cGy)Donor chimerism + vs - G-CSF (Mean ±SD)C57BL/6J → PtrcaPep3b/Boy J20 x 106 fresh BM16065.2±0.9% vs 43.4±3.5%, n=4 and 5 mice each; p=0.0002 (4 months after transplant)C57BL/6J-EGFP → C57BL/6J2.5 x 106 fresh BM30055.25±4.5% vs 28.7±5.3%, n=4mice each; p=0.0002 (6 months after transplant)C57BL/6J male X-CGD → C57BL/6J female X-CGD5 x 106 transduced BM30051.0±13.1% vs 30.4±7.8%, n=5mice each; p=0.02 (3 months after transplant)We next investigated 2 potential mechanisms by which G-CSF prior to LDTBI might enhance long-term engraftment of HSC. First, analysis of host BM 20 hours after transplantation showed a significantly lower frequency of homed donor HSC in G-CSF+160cGy treated mice compared to 160 cGy-conditioned mice (1 ± 0.13% vs 3.5 ± 0.7% in one experiment, and 3.4 ± 0.8% vs 9.5 ± 2.3% in another experiment, n= 20 mice). Second, in a competitive repopulation assay, we observed a substantial decrease in marrow long-term repopulating ability (LTRA) in mice treated with G-CSF + 160 cGy compared to 160 cGy alone (10.5% vs 48.6%, n= 5 mice in each group; p= 0.0002). These results suggest that administration of G-CSF prior to LDTBI does not increase initial homing of donor cells but further impairs LTRA of recipient BM compared to LDTBI alone. In the context of gene therapy, we transduced male X-CGD BM with MSCV-gp91Neo vector and infused these transduced cells into female X-CGD mice treated ± G-CSF + 300 cGy. Donor cell engraftment, measured by FISH for Y chromosome, at 3 months post-transplant was significantly increased in recipients pretreated with G-CSF (see Table). NADPH oxidase activity (by 123-dihydrorhodamine assay) was reconstituted in 9.75 ± 5.5% of neutrophils compared to 6.6 ± 2.5% without G-CSF pretreatment. The calculated fraction of donor cells with oxidase activity was similar in both groups (18.8 ± 6.6% vs 21.4 ± 11%, respectively) (p= 0.67). These results suggest that the use of G-CSF prior LDTBI provides a competitive advantage for long-term engraftment by decreasing LTRA in the host and may be a useful strategy to increase engraftment of fresh and retroviral transduced donor BMC.

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-γ production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.

Granulocyte colony-stimulating factor directly affects human monocytes and modulates cytokine secretion.

Recent reports have indicated that monocytes express receptors for the granulocyte colony-stimulating factor (G-CSF). The direct effects of G-CSF on cytokine secretion in monocytes were examined. A monocytic cell line NOMO-1 that secretes multiple cytokines upon stimulation with lipopolysaccharide (LPS) was used. Normal human monocytes were purified by negative selection using magnetic beads. Cells pretreated with or without G-CSF were stimulated with LPS, and the subsequent concentrations of cytokines and chemokines in supernatants were determined by sandwich enzyme-linked immunosorbent assay. NOMO-1 cells were found to express receptors for G-CSF. Although G-CSF stimulation did not induce cytokine secretion, pretreatment with G-CSF significantly attenuated LPS-stimulated secretion of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin (IL)-12 in NOMO-1 cells. Simultaneously, however, G-CSF pretreatment apparently enhanced LPS-induced secretion of IL-10 and monocyte chemoattractant protein-1, whereas secretions of IL-1beta, IL-6, and IL-8 were unaffected. When normal human monocytes from healthy volunteers were similarly examined, marked individual variations in LPS-induced secretion of cytokines were observed. Although some exceptions exist, a similar tendency as to the effects of G-CSF treatment on cytokine secretions as that in NOMO-1 cells was observed in human monocytes. Our data suggest that G-CSF directly affects monocytes and modulates their cytokine secretion. NOMO-1 cells can provide an alternate model for in vitro culture of monocytes to investigate the effects of G-CSF on cytokine secretion by these cells.

Granulocyte colony-stimulating factor enhances host defenses against bacterial pneumonia following peritonitis in nonneutropenic rats.

Polymorphonuclear cell functions frequently are impaired in critically ill patients, and restoration of normal functions could help to prevent nosocomial infections. The aim of this study was to evaluate the effects of pretreatment with granulocyte colony-stimulating factor (G-CSF) on bacterial pneumonia induced 48 hrs after peritonitis (cecal ligation and puncture [CLP]) in rats. Controlled animal study. Research laboratory of an academic institution. Male Sprague-Dawley rats. First, the CLP model was characterized. Second, alveolar endotoxin instillation allowed us to evaluate the ability of neutrophils to migrate to airspaces after CLP was assessed. In the last set of experiments, CLP was followed by G-CSF treatment as a preventive therapy for subsequent bacterial superinfection induced by alveolar instillation. CLP induced a brief increase in proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta) at the 6th hr followed by a longer-lived anti-inflammatory response (interleukin-10 increase from days 1 to 3) in plasma, compared with healthy rats. Impaired neutrophil migration to alveolar spaces denoting immunoparalysis was evidenced after endotracheal endotoxin instillation following CLP, compared with non-CLP rats challenged with endotoxin. No such impairment was found when G-CSF (100 microg/kg: glycosylated recombinant human G-CSF, Lenograstim) was given before endotoxin. G-CSF (100 microg/kg 24 and 48 hrs after CLP) given before endotracheal instillation increased bacterial clearance, as shown by counts in both bronchoalveolar lavage (8.9 x 10 +/- 2.8 x 10 colony-forming units/mL vs. 3.3 x 10 +/- 1.5 x 10 colony-forming units/mL with saline) and lung tissue (4.2 x 10 +/- 1.0 x 10 colony-forming units/g vs. 1.5 x 10 +/- 0.6 x 10 colony-forming units/g with saline). Furthermore, G-CSF pretreatment kept clearance in CLP rats similar to that in non-CLP rats challenged with. These results suggest that G-CSF (Lenograstim) may enhance host defenses in rats with peritonitis and immunoparalysis.