Granulocyte Colony-stimulating Factor Stimulation Research Articles

Upregulation of microRNA-125b by G-CSF promotes metastasis in colorectal cancer.

Although there are reports of miR-125b being dysregulated in colorectal cancer (CRC) and associated with CRC progression, little is known about its intrinsic regulatory mechanisms. Here we detected the expression of miR-125b in CRC tissues, subsequently investigated the effect of miR-125b on the proliferation, apoptosis, cell cycle and metastasis on CRC cells. Our results showed that the expression of miR-125b was significantly decreased in CRC tissues comparing to adjacent tissues. However, with the stimulation of Granulocyte colony-stimulating factor (G-CSF), which was highly expressed in CRC tissues, the expression of miR-125b could be improved. Analysis of patient samples revealed that miR-125b presented a clear association with poor differentiation, positive lymph node metastasis, and advanced TNM stage. Overexpression of miR-125b inhibited cell proliferation, triggered G2/M cell cycle arrest, induced subsequent apoptosis, and promoted cell migration and invasion. Moreover, luciferase reporter assays and western blot clarified that the myeloid cell leukemia 1 (MCL1) was a direct target of miR-125b. Thus overexpression of MCL1 attenuated the pro-metastasis function of miR-125b in CRC cell lines. In addition, the protein expression level of MCL1 was decreased in CRC tissues from patients with positive lymph node metastasis, which had high miR-125b expression. Collectively, our study suggested that miR-125b induced by G-CSF plays a promoting role in the metastasis of CRC by targeting MCL1, which may serve as a novel therapeutic target for CRC metastasis.

Influence of GCSF stimulation on sCD40L release kinetic

It has been demonstrated by in vitro experiments using inhibitors that CD40L is cleaved from the surface of activated platelets by matrix-metalloproteinases (MMP). Additionally, after granulocyte colony stimulating factor (GCSF) activation of peripheral stem cell donors a MMP elevation is induced, and stem cell products contain a considerable amount of platelets. Therefore stem cell donations (peripheral stem cell apheresis (PBSC) as well as bone marrow (BM) donation) were used as an in vivo model to observe alterations of the sCD40L release kinetic and its effects on the respective stem cell products. AND METHODS: The study included twelve healthy stem cell donors (PBSC n =6 , BMn = 6). sCD40L concentrations, relative MMP activities and blood cell compositions were determined in peripheral blood as well as in the respective stem cell products before, during and after donation and/or during cell product storage. RESULTS: In stem cell products, sCD40L concentrations were manifold elevated (range from 1121 to 4123 pg/mL) in comparison to concentrations of peripheral blood samples (range from 39 to 334 pg/mL). Dependent on storage duration a sCD40L accumulation in the products could be observed. MMP concentrations were elevated by GCSF stimulation and resulted in an about 2-fold increase of sCD40L release kinetic. In parallel, MMP concentrations were increased in the stem cell products (PBSC as well as BM products). In the peripheral blood of PBSC donors, the sCD40L concentration decreased after apheresis in correlation to a decrease in platelet count. CONCLUSION: As known from platelet concentrates, an accumulation of sCD40L could also be observed in stem cell products pointing out the importance of sCD40L release by platelets. Stem cell products showed elevated MMP concentrations compared to peripheral blood and additionally during stem cell apheresis, sCD40L release kinetic is accelerated by MMP elevation.

RUNX1 haploinsufficiency results in granulocyte colony-stimulating factor hypersensitivity.

RUNX1/AML1 is among the most commonly mutated genes in human leukemia. Haploinsufficiency of RUNX1 causes familial platelet disorder with predisposition to myeloid malignancies (FPD/MM). However, the molecular mechanism of FPD/MM remains unknown. Here we show that murine Runx1+/− hematopoietic cells are hypersensitive to granulocyte colony-stimulating factor (G-CSF), leading to enhanced expansion and mobilization of stem/progenitor cells and myeloid differentiation block. Upon G-CSF stimulation, Runx1+/− cells exhibited a more pronounced phosphorylation of STAT3 as compared with Runx1+/+ cells, which may be due to reduced expression of Pias3, a key negative regulator of STAT3 signaling, and reduced physical sequestration of STAT3 by RUNX1. Most importantly, blood cells from a FPD patient with RUNX1 mutation exhibited similar G-CSF hypersensitivity. Taken together, Runx1 haploinsufficiency appears to predispose FPD patients to MM by expanding the pool of stem/progenitor cells and blocking myeloid differentiation in response to G-CSF.

Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for granulocyte colony-stimulating factor (G-CSF) as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been elucidated in humans. Here, we investigate the changes in mRNA and miRNA expression in successive stages of neutrophil development following in vivo administration of G-CSF in humans, mimicking emergency granulopoiesis. Blood samples were collected from healthy individuals after five days of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry and extracted RNA was subjected to microarray analysis. mRNA levels were compared to previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. miRNA expression was investigated in the most mature cell population to determine G-CSF-induced changes in circulating neutrophils. G-CSF substantially affected mRNA and miRNA expression patterns, demonstrating significant impact on neutrophil development and function. 1110 mRNAs were differentially expressed more than 2-fold with G-CSF while the treatment induced changes in the levels of 73 miRNAs in the mature population. In addition, G-CSF treatment reduced the levels of four out of five measured granule proteins in mature neutrophils including hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. Cell cycle analysis pointed towards an induced proliferative capacity of myelocytes. These results indicate that multiple biological processes are altered in order to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Sensory neuropathy hampers nociception-mediated bone marrow stem cell release in mice and patients with diabetes.

Aims/hypothesisUpon tissue injury, peripheral sensory neurons release nociceptive factors (e.g. substance P [SP]), which exert local and systemic actions including the recruitment of bone marrow (BM)-derived haematopoietic stem and progenitor cells (HSPCs) endowed with paracrine pro-angiogenic properties. We herein explore whether diabetic neuropathy interferes with these phenomena.MethodsWe first investigated the presence of sensory neuropathy in the BM of patients with type 2 diabetes by immunohistochemistry and morphometry analyses of nerve size and density and assessment of SP release by ELISA. We next analysed the association of sensory neuropathy with altered HSPC release under ischaemia or following direct stimulation with granulocyte colony-stimulating factor (G-CSF). BM and circulating HSPCs expressing the neurokinin 1 receptor (NK1R), which is the main SP receptor, were measured by flow cytometry. We finally assessed whether an altered modulation of SP secretion interferes with the mobilisation and homing of NK1R-HSPCs in a mouse model of type 2 diabetes after limb ischaemia (LI).ResultsNociceptive fibres were reduced in the BM of patients and mice with type 2 diabetes. Patients with neuropathy showed a remarkable reduction in NK1R-HSPC mobilisation under ischaemia or upon G-CSF stimulation. Following LI, diabetic mice manifested an altered SP gradient between BM, peripheral blood and limb muscles, accompanied by a depressed recruitment of NK1R-HSPCs to the ischaemic site.Conclusions/interpretationSensory neuropathy translates into defective liberation and homing of reparative HSPCs. Nociceptors may represent a new target for treatment of diabetic complications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3735-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

ETV6/ARG Oncoprotein Induces Autonomous Cell Growth By Enhancing c-Myc Expression through STAT5 Activation in the Acute Promyelocytic Leukemia Cell Line HT93A

Background: A fusion gene comprising ETS-variant gene 6 (ETV6) and ABL- related gene (ARG) has been identified in some patients with acute leukemia. Although the ETV6/ARG fusion gene is considered to contribute to the leukemogenesis, the detailed underlying mechanism is still unknown. Here, we investigated the role of ETV6/ARG fusion genes by exposing the HT93A cell line, which was established from a patient with relapsed acute promyelocytic leukemia carrying ETV6/ARG fusion gene, to the tyrosine kinase inhibitor, nilotinib.Material and Methods: HT93A cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum with or without nilotinib ± 50 ng/ml of granulocyte colony-stimulating factor (G-CSF). The cells were intracellularly stained anti-phosphorylated ARG (Y429), anti-phosphorylated STAT3 (Y705), anti-phosphorylated STAT5 (Y694), anti-p21/Waf1, anti-p27/Kip1, and anti-c-Myc polyclonal antibodies. Thereafter, protein expression and the degree of phosphorylation were evaluated with a flow cytometer. Cell growth was evaluated by trypan blue exclusion test, cell cycle analysis, and by intracellular staining with propidium iodide followed by flow cytometric analysis. Experiments were independently repeated at least thrice. Results shown are the mean ± standard deviation (SD) values. Data were analyzed using Student’s t-test, and p < 0.05 was considered statistically significant.Results: ARG was predominantly activated in HT93A cells and nilotinib treatment inhibited ARG phosphorylation. HT93A cells were sensitive to nilotinib, which inhibited cell growth in a dose-dependent manner for 96 h of incubation. Nilotinib treatment for 48 h significantly increased the probability of cells in the G0/G1 phase and decreased that in the S+G2/M phase. Further, nilotinib alone inhibited the expression of both phosphorylated STAT3 and STAT5. Moreover, nilotinib downregulated the expression levels of p21/Waf1 and c-Myc. However, p27/Kip expression level did not alter. Because the activation of STAT3 and/or STAT5 was assumed as the major pathway induced by ETV6/ARG oncoprotein, HT93A cells were coincubated with 50 ng/mL G-CSF in the presence or absence of nilotinib. After stimulation with G-CSF for 60 min, STAT5 was significantly phosphorylated in both nilotinib-treated and untreated cells; the expression level of phosphorylated STAT5 returned to the control level when treated with nilotinib and G-CSF together. In contrast, STAT3 phosphorylation was unaltered by G-CSF stimulation in nilotinib-treated and untreated cells. G-CSF alone had an insignificant effect on cell growth. The downregulation of the expression levels of p21/Waf1 and c-Myc by nilotinib was abolished by the combination of G-CSF. G-CSF had slight effect on HT93A cell cycle distribution; results of the combination therapy with nilotinib and G-CSF returned the cell cycle distribution to the control level.Conclusions: We demonstrated that ETV6/ARG inhibition by nilotinib abrogates autonomous cell growth via the downregulation of c-Myc through the inactivation of STAT5 but not of STAT3. Our data suggest that ETV6/ARG oncoprotein has a contributory effect for the autonomous cell growth and is a potential target in the treatment of leukemia. DisclosuresIriyama:Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria. Hatta:Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria. Takei:Novartis: Research Funding; Kyowa Hakko Kirin: Research Funding.

Clozapine promotes the proliferation of granulocyte progenitors in the bone marrow leading to increased granulopoiesis and neutrophilia in rats.

Clozapine is an atypical antipsychotic that is limited in its use due to the risk of idiosyncratic agranulocytosis. The bone marrow is suspected to be the site of the reaction, and indirect measurements in patients suggest that neutrophil production and maturation are altered in the marrow by clozapine. Specifically, the majority of patients have elevated neutrophil counts at the start of treatment, often paired with increased serum granulocyte-colony stimulating factor (G-CSF). Employing a rat model of clozapine treatment, we set out to determine if the neutrophilia observed at the start of treatment is characteristic of G-CSF-associated bone marrow stimulation. Female Sprague-Dawley rats were treated with 30 mg/kg/day of clozapine for 10 days, and sustained neutrophilia was evident after 1 week of treatment paired with spikes in G-CSF. Within the bone marrow, clozapine was found to induce proliferation of the granulocyte progenitor colonies as measured by a methylcellulose assay. This led to elevated granulopoiesis observed by H&E and myeloperoxidase staining of bone marrow slices. Increased release of neutrophils from the marrow to the circulation was measured through 5-bromo-2'-deoxyuridine labeling in vivo, and these neutrophils appeared to be less mature based on (a) a decrease in the nuclear lobe count and (b) increased expression of surface CD62L. Furthermore, faster transit of the neutrophils through the marrow was suggested by a shift toward elevated numbers of neutrophils in the bone marrow maturation pool and increased CD11b and CD18 staining on the less mature neutrophils residing in the marrow. Taken together, these data indicate that clozapine stimulates the bone marrow to produce more neutrophils in a manner that is characteristic of endogenous G-CSF stimulation, and it is consistent with the inflammatory response observed in patients treated with clozapine.

Efficacy and safety of early G-CSF administration in patients with head and neck cancer treated by docetaxel-cisplatin and 5-fluorouracil (DCF protocol): a retrospective study.

Induction chemotherapy with docetaxel-cisplatin and 5-fluorouracil (DCF) for locally advanced head and neck cancers (HNC) is associated with a high risk of severe neutropenia or febrile neutropenia (FN). We conducted a retrospective study to evaluate the efficacy and safety of administering granulocyte colony-stimulating factor (G-CSF) on day 3 (D3) during chemotherapy (early G-CSF stimulation) versus after the end of chemotherapy, as per current guidelines (i.e., after the end of 5-FU perfusion; D7), and its impact on patient outcomes. Patients ≥19 years old, with advanced HNC who received DCF induction chemotherapy (D and P 75 mg per meter squared (mg/m(2)) on day 1 and 5-FU 750 mg/m(2)/day from D1 to D5), were included in the analysis. Data of 70 patients were analyzed from 01 January 2003 to 01 December 2010. Mean age was 56 years (range 45 to 77 years). Thirty-six patients (51.4 %) received pegfilgrastim on D7, and 28 (40 %) started G-CSF prophylaxis during chemotherapy; 12 (17.1 %) had daily filgrastim and 16 (22.9 %) pegfilgrastim on D3. Overall response rate (ORR) was 89.6 % (three early deaths due to infectious complications; 4.3 %). The 3-year overall survival (OS) rate was 72.8 %. FN rate was 14.3 % and chemotherapy delay was 12.9 %. In the D7 G-CSF arm, incidence of grade 3-4 neutropenia (p = 0.023), FN (p = 0.029), and cycle delays (p = 0.006) was statistically higher than the "early" G-CSF arm. A decrease of OS was observed at 2 years (from 85.1 to 63.5 %) of chemotherapy discontinuation or FN (p = 0.0348). Early administration of G-CSF is safe and seems to be more effective than D7. Future prospective trials are required to confirm our results.

Safety and feasibility of long term administration of recombinant human granulocyte-colony stimulating factor in patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuronal disease resulting in a loss of the upper and lower motor neurons and subsequent death within three to four years after diagnosis. Mouse models and preliminary human exposure data suggest that the treatment with granulocyte-colony stimulating factor (G-CSF) has neuro-protective effects and may delay ALS progression. As data on long-term administration of G-CSF in patients with normal bone marrow (BM) function are scarce, we initiated a compassionate use program including 6 ALS patients with monthly G-CSF treatment cycles. Here we demonstrate that G-CSF injection was safe and feasible throughout our observation period up to three years. Significant decrease of mobilization efficiency occurred in one patient and a loss of immature erythroid progenitors was observed in all six patients. These data imply that follow-up studies analyzing BM function during long-term G-CSF stimulation are required.

MiR-155 is associated with the leukemogenic potential of the class IV granulocyte colony-stimulating factor receptor in CD34⁺ progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV-overexpressing (G-CSFRIV(+)) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV(+) cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV(+) HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference-mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV(+) HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV(+) HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV(+) HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.

Loss of GADD34 induces early age‐dependent deviation to the myeloid lineage

Hematopoietic stem cells (HSCs) generate all known hematopoietic lineages and are capable of self-renewal. Upon aging, myeloid-biased HSCs are maintained, whereas lymphoid-biased HSCs are lost. GADD34 protein is expressed in myeloid-lineage cells and has been cloned from them. However, the function of GADD34 in the myeloid lineage has not yet been elucidated. Here, we show that early age-dependent deviation to the myeloid lineage occurs in GADD34-deficient mice. Early increases of GR-1(int)CD11b(+) and GR-1(high)CD11b(+) neutrophils were observed in the spleen, bone marrow (BM) and blood of GADD34-deficient mice. We found that BM Lin(-) c-Kit(+) Sca1(+) and Lin(-) c-Kit(+) Sca1(-)cells expressed GADD34 protein without stimulation and increased GADD34 expression following intravenous injection of Staphylococcus aureus (S.aureus). These cell populations were high in GADD34-deficient BM and were increased by the injection of S. aureus. Because of the increase in granulocyte colony-stimulating factor (G-CSF) induced by S. aureus injection, we examined the signaling pathway from the G-CSF receptor (G-CSFR). We found that phosphorylation of signal transducer and activator of transcription factor 3 was highly increased in GADD34-deficient Lin(-) BM cells by the stimulation of G-CSF. These results indicate that GADD34 binds to Lyn and inhibit G-CSFR signaling. We show here that GADD34 works to inhibit the proliferation and differentiation of HSCs or myeloid precursor cells and maintains homeostatic differentiation of neutrophil-lineage cells to avoid early immunological senescence.

Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF–triggered granulopoiesis

We found that hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF-triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.

Distinct Signal Transduction Abnormalities and Erythropoietin Response in Bone Marrow Hematopoietic Cell Subpopulations of Myelodysplastic Syndrome Patients

Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34(+), CD45(+), or CD71(+)CD45(-) bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. In CD71(+)CD45(-) cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71(+)CD45(-) cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34(+) and CD45(+) MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34(+) cells. Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71(+)CD45(-) cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations.

Clinical Outcome of 50 Progressive Multiple Sclerosis Patients Treated with Cellular Therapy in Iraq

Multiple Sclerosis is a disease characterized by multifocal areas of demyelination in the brain and spinal cord, with associated inflammatory cell infiltrates, reactive gliosis, and axonal degeneration. It typically presents in young adults with episodic neurologic dysfunction, our aim is to find new simple method to treat multiple sclerosis by hematopoietic stem cells derived from peripheral blood. 50 patients suffering from multiple sclerosis worsening despite pharmacological treatment were treated by means of several intrathecal injections of peripheral blood cells harvested by aphaeresis after G-CSF(granulocyte colony stimulating factor) treatment. 24 patients (48% ) had a reduction of EDSS score. 8 patients had a relapse, but it was milder than usual and more easily controlled by cortisone. Since mesenchymal cells increase in the peripheral blood after G-CSF stimulation, a peripheral blood harvest seems easier and cheaper than mesenchymal cells cultivation prior to the injection. It seems a reasonable treatment for progressive multiple sclerosis.

IL-17A stimulates granulocyte colony-stimulating factor production via ERK1/2 but not p38 or JNK in human renal proximal tubular epithelial cells

We investigated the potential role of IL-17A in the induction of granulocyte colony-stimulating factor (G-CSF), a critical granulopoietic growth factor, in human renal proximal tubular epithelial cells. Human renal proximal tubular cells (HK-2, ATCC) were used to characterize the effects of IL-17A or IL-17F on G-CSF production, using ELISA, real-time RT-PCR, and immunoblotting. The cell surface expression of IL-17 receptors (IL-17Rs) was analyzed by flow cytometry. IL-17A stimulation of proximal tubular cells led to a dose- and time-dependent increase in secreted G-CSF. This effect was dependent on mRNA transcription and protein translation. Real-time RT-PCR demonstrated that G-CSF mRNA expression reached a maximum level at 6 h following IL-17A stimulation and that this increase was dose dependent. Both IL-17RA and IL-17RC were expressed on proximal tubular cells. IL-17A also enhanced TNF-α- or IL-1β-mediated G-CSF secretion from cells. Additionally, IL-17A induced MAPK (ERK1/2 but not p38 MAPK or JNK) activation, and pharmacological inhibitors of MEK1/2 (U0126) but not of p38 MAPK (SB203580) or JNK (SP600125), significantly blocked the IL-17A-mediated G-CSF release. We demonstrated the potential ability of IL-17A to induce G-CSF in renal proximal tubular cells. It is proposed that IL-17A may play an important role in neutrophil transmigration and activation via stimulation of G-CSF in tubular injury.

Hematologic Recovery in Patients Who Are Treated With Autologous Stem Cells Transplantation Taken From Bone Marrow After Granulocyte–Colony-Stimulating Factor Stimulation

Background We sought to compare hematologic recovery between patients who did or did not receive granulocyte–colony-stimulating factor (G-CSF)-stimulated bone marrow (rich bone marrow [RBM]). Materials and Methods The study subjects were 20 patients whose bone marrow was taken without prior stimulation with G-CSF and 15 patients in whom bone marrow was taken after previous G-CSF mobilization. The bone marrow harvest took place on the fifth day after G-CSF initiation. The bone marrow aliquot was 20 mL/kg. Results The median value of nucleated cells obtained from patients without G-CSF preparation was 3.65 × 10 8/kg. The median value of nucleated cells from RBM patients was 4.83 × 10 8/kg. The median value of stem cells obtained from patients without G-CSF preparation was 0.96 × 10 6/kg versus 1.9 × 10 6/kg from RBM patients. The median time to recovery of the hematopoietic system based on an increase in PLT value >20 g/L was 12.6 days for RBM versus 18.8 days without G-CSF preparation. The median time to recovery of the hematopoietic system based on assessment of growth ANC>0.5 g/L was 13.0 days for RBM versus 17.8 days without G-CSF stimulation. Significantly higher values of nucleated cells and increased stem cells were observed among RBM patients compared with those whose bone marrow was harvested without any stimulation ( P = .01). There was faster recovery of the hematopoietic system in cases where bone marrow was collected after G-CSF: PLT >20 g/L ( P = .015) and ANC >0.5 g/L ( P = .01). We also observed that the use of stimulated bone marrow shortened hospital stay after the administration of hematopoietic cells to 17.3 days compared with 23.1 days among patients receiving hematopoietic cells from nonstimulated bone marrow. The number of complications during transplantation was comparable in both cases, the most frequent ones being febrile neutropenia and grade III and IV mucositis. Conclusion RBM is a better method to obtain stem cells from bone marrow. Stimulated bone marrow shows faster engraftment compared with nonstimulated bone marrow helping patients who fail to generate are adequate number of stem cells from peripheral blood.

G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase

AbstractSrc activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF–stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

Venous vessel pathology — chronic cerebrospinal venous insufficiency in multiple sclerosis

Background: Now it is not known which cell type is best for inducing therapeutic angiogenesis in end-stage critical limb ischemia (CLI). We hypothesized that mononuclear cells mobilized with granulocyte colony stimulating factor (G-CSF) into peripheral blood are as effective as highly selective and purified CD133+ and CD34+ endothelia progenitor cells (EPCs). This would have significant clinical implications with regard to study designs and costs of stem cell treatment regimens. Methods: Patients with critical ischemia of lower extremity donated peripheral blood stem cells after 5 days of stimulation with G-CSF. Five patients in each group were treated with CD 34, CD 133 or mononuclear cells after G-CSF stimulation. We analyzed expression of cells surface markers CD34, VEGFR-2, CD133, CD31 and CD144. Results: There were no differences in pain, ABI, ulcer healing or amputation rate between the three groups. Overall limb salvage rate was 12/15. Only in 4/15 patients, after a mean follow-up of 11 months, did control MRA examinations show stem-cell-induced neoangiogenesis: in two patients after CD34 cell injections, in one case after CD 133 treatment and in one case after treatment with mononuclear cells. In the remaining cases, MRA analysis did not show any traces of neoangiogenesis. Injection of purified CD133+ or CD34+ EPCs did not yield better results than unselected mononuclear cells after G-CSF stimulation. Paracrine factors produced by the mononuclear fraction of mobilized peripheral blood may contribute more to the amelioration of critical limb ischemia than one particular cell type. Conclusion: Stem cell therapy will play an increasingly important role in the near future. Most patients can be treated with unselected cells, which further reduce costs of cell-based therapy.

Serious Adverse Drug Reactions (sADRS) Associated with Hematopoietic Growth Factors: A Systematic Review From the Southern Network on Adverse Reactions (SONAR) Program

AbstractAbstract 2555Erythrpoiesis stimulating agents (ESAs) and the granulocyte hematopoietic growth factors granulocyte colony stimulating factor (G-CSF) and pegylated G-CSF revolutionized supportive care approaches to anemia and neutropenia, respectively. Thrombopoietin-receptor agonists improve antibody-mediated thrombocytopenia management. For these drugs, as with all agents, a comprehensive understanding of sADRs is important, however, often difficult to do. We reviewed sADRS described in meta-analyses and web-site materials maintained by regulatory agencies and manufacturers reports (1989 to July 2010; n=2,094). Overall, 28 systematic reviews, six package inserts, three medication guides, four FDA Risk Evaluation and Mitigation Systems (REMS) materials, 16 Summaries of Product Characteristics/Product Assessment Reports were included. For ESAs, common sADRs associated with increased relative risks (RRs) included: mortality among cancer patients with chemotherapy-associated anemia or cancer patients not receiving active treatment (RRs: 1.1–1.2); cerebrovascular and cardiovascular events among chronic kidney disease patients ((RRs: 1.3, 1.9), and venous thromboembolism in the cancer setting (RRs;16-1.7). For G-CSF/pegylated-GCSF, rare instances of myelodysplasia/acute myeloid leukemia (MDS/AML) associated with statistically increased RRs were reported among breast cancer patients receiving chemotherapy in trials reported from one NCI-sponsored clinical trials group, one meta-analysis, and one review of Medicare/SEER databases (RR: 1.9–2.1), although the absolute risks were very small (< 2 per 100,000 treated patients). Other rarely reported sADRs (total N < 20) included splenic rupture following G-CSF stimulation of healthy peripheral blood stem donors. Bone marrow fibrosis and collagen deposition were reported with thrombopoietin receptor agonists, although the implications of this finding is uncertain. Also, instances of VTEs were reported among persons who developed high platelet counts following administration of these thrombopoeitin receptor agonists. SADRs are commonly associated with ESA administration in a range of settings, are uncommon occurrences with G-CSF/pegylated G-CSF, and are not well characterized for thrombopoieitin receptor agonists. Formal pharmacovigilance initiatives are ongoing, including FDA mandated REMS with restricted distribution requirement for ESAs in the cancer setting (APPRISE) and the NEXUS and the PROMACTA CARES programs for thrombopoeitin receptor agonists.ESAs (Cancer)ESAs (CKD)G-CSF/Pegylated G-CSFVTEMortalityCerebrovascular and CVDAML/MDSRR1.57 and 1.671.10 to 1.171.34 and 1.922.14 and 1.9295% CI1.31–1.87 and 1.35–2.061.02–1.20 and 1.06–1.301.03–1.74 and 1.38–2.261.12–4.08 and 1.19–3.07Baseline RiskAbsolute RiskCommonCommonCommonRare (2/100,000) Disclosures:Bennett:Pfizer: Consultancy.

Comparison of Outcomes after Transplantation of G-CSF–Stimulated Bone Marrow Grafts versus Bone Marrow or Peripheral Blood Grafts from HLA-Matched Sibling Donors for Patients with Severe Aplastic Anemia

We compared outcomes of patients with severe aplastic anemia (SAA) who received granulocyte-colony stimulating factor (G-CSF)-stimulated bone marrow (G-BM) (n = 78), unstimulated bone marrow (BM) (n= 547), or peripheral blood progenitor cells (PBPC) (n = 134) from an HLA-matched sibling. Transplantations occurred in 1997 to 2003. Rates of neutrophil and platelet recovery were not different among the 3 treatment groups. Grade 2-4 acute graft-versus-host disease (aGVHD) (relative risk [RR] = 0.82, P = .539), grade 3-4 aGVHD (RR = 0.74, P = .535), and chronic GVHD (cGVHD) (RR = 1.56, P = .229) were similar after G-BM and BM transplants. Grade 2-4 aGVHD (RR = 2.37, P = .012) but not grade 3-4 aGVHD (RR = 1.66, P = .323) and cGVHD (RR = 5.09, P < .001) were higher after PBPC transplants compared to G-BM. Grade 2-4 (RR = 2.90, P < .001), grade 3-4 (RR = 2.24, P = .009) aGVHD and cGVHD (RR = 3.26, P < .001) were higher after PBPC transplants compared to BM. Mortality risks were lower after transplantation of BM compared to G-BM (RR = 0.63, P = .05). These data suggest no advantage to using G-BM and the observed higher rates of aGVHD and cGVHD in PBPC recipients warrants cautious use of this graft source for SAA. Taken together, BM is the preferred graft for HLA-matched sibling transplants for SAA.