Plasma Levels Of Granulocyte Colony-stimulating Factor Research Articles

MST1 controls murine neutrophil homeostasis via the G-CSFR/STAT3 axis.

The release of neutrophils from the bone marrow into the blood circulation is essential for neutrophil homeostasis and the protection of the organism from invading microorganisms. Granulocyte colony-stimulating factor (G-CSF) plays a pivotal role in this process and guides granulopoiesis as well as the release of bone marrow neutrophils into the blood stream both during homeostasis and in case of infection through activation of the G-CSF receptor/signal transduction and activation of transcription 3 (STAT3) signaling pathway. Here, we investigated the role of the mammalian sterile 20-like kinase 1 (MST1) for neutrophil homeostasis and neutrophil mobilization. We found increased plasma levels of G-CSF in Mst1 -/- mice compared to wild type mice both under homeostatic conditions as well as after stimulation with the proinflammatory cytokine TNF-α. In addition, G-CSF-induced mobilization of neutrophils from the bone marrow into the blood circulation in vivo was markedly reduced in the absence of MST1. Interestingly, this was not accompanied by differences in the number of blood neutrophils. Addressing the underlying molecular mechanism of MST1-regulated neutrophil mobilization, we found reduced STAT3 phosphorylation and impaired upregulation of CXCR2 in Mst1 -/- bone marrow neutrophils compared to wild type cells, while JAK2 phosphorylation was not altered. Taken together, we identify MST1 as a critical modulator of neutrophil homeostasis and neutrophil mobilization from the bone marrow, which adds another important aspect to the complex role of MST1 in regulating innate immunity.

In vitro characterization of granulocyte-colony stimulating factor (G-CSF) production by dendritic cells and macrophages during Streptococcus suis infection

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and emerging zoonotic agent. Infections induce an exacerbated inflammation that can result in sudden death (septic shock) and meningitis. Though neutrophilic leukocytosis characterizes S. suis infection, the mediators involved are poorly understood. Among them, granulocyte-colony stimulating factor (G-CSF), a pro-inflammatory cytokine, triggers proliferation of neutrophil progenitors and neutrophil mobilization. However, the systemic production of G-CSF induced during S. suis infection, the cell types involved, and the underlying mechanisms remain unknown. In a S. suis serotype 2 mouse model of systemic infection, plasma levels of G-CSF rapidly increased after infection. S. suis activation of DCs and macrophages resulted in high (> 1000 pg/mL) and comparable production levels of G-CSF, as measured by ELISA. By using mutant strains deficient in capsular polysaccharide (CPS) or lipoprotein maturation in combination with purified lipoteichoic acid (LTA) from the latter mutant strain, it was showed that G-CSF production is mainly mediated by S. suis lipoproteins. The Toll-like receptor (TLR) pathway via myeloid differentiation primary response 88 (MyD88) is required for G-CSF production by DCs and macrophages following S. suis activation, with a partial involvement of TLR2. On the other hand, TLR2-independant G-CSF production induced by S. suis requires internalization and bacterial DNA might play a role in this pathway. Finally, these signals activated nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways leading to G-CSF production. In conclusion, this study demonstrated for the first time that S. suis induces G-CSF production in vivo and DCs and macrophages are key cellular sources of this cytokine mediator, mainly via the binding of lipoproteins to TLR2. The CPS significantly reduced this activation, confirming the powerful role of this component in S. suis virulence. As such, this study contributes to better understand how DCs and macrophages produce G-CSF in response to S. suis, and potentially to other streptococci.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear.Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry.Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells.Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

Abstract B13: Plasma G-CSF levels are predictive of lack of response to zoledronic acid treatment in reducing breast cancer recurrence

Abstract Some breast cancer patients have no evidence of metastatic disease at the time of original diagnosis, yet ~30-40% of patients will experience recurrent breast cancer. Over 90% of breast cancer deaths occur due to distant spread of the disease, and bone is the most common site of breast cancer metastasis. Bisphosphonates, such as zoledronic acid (ZA), are used to treat patients with osteolytic disease, including metastatic breast cancer. ZA has been demonstrated to reduce disseminated tumor cells (DTCs) and breast cancer recurrence, but not all patients see this benefit and it is not yet clear what predicts benefit from adjuvant ZA treatment; the mechanism of action for this effect of ZA in reducing breast cancer remains undetermined. Here, we establish that ZA renders osteoclast progenitor cells (OCPs) tumor suppressive, primarily by directing their lineage potential. In instances where the OCP lineage potential was modulated via systemic or tumor-derived granulocyte-colony stimulating factor (G-CSF), OCPs did not differentiate into tumor-suppressive populations but instead into mature osteoclasts. High G-CSF was sufficient to abrogate the tumor-suppressive effect of ZA. Furthermore, we determined that in a subset of patient plasma samples from the AZURE clinical trial where stage II/III breast cancer patients were randomized to receive standard systemic treatment alone with adjuvant ZA treatment, patients who had baseline plasma G-CSF levels >23 pg/mL had reduced disease-free survival as assessed over a 10-year period post treatment initiation compared to patients with baseline plasma G-CSF levels <23 pg/mL (p=0.02). These findings indicate that patients with higher baseline plasma G-CSF levels will not likely observe reduction in breast cancer recurrence with adjuvant ZA treatment, and in fact may have worse prognosis with adjuvant ZA treatment as compared to control treatment. Our data are the first to demonstrate that ZA mediates its tumor-suppressive function via the OCP population and implicate capitalizing on the differentiation potential of the OCPs to maximize patient response to adjuvant ZA treatment in breast cancer risk of recurrence. Citation Format: Jessalyn M. Ubellacker, Ninib Baryawno, Nicolas Severe, Molly J. DeCristo, Jaclyn Sceneay, Marie-Therese Haider, Catherine S. Rhee, Yuanbo Qin, Ingunn Holen, Walter M. Gregory, Janet E. Brown, Robert E. Coleman, David T. Scadden, Sandra S. McAllister. Plasma G-CSF levels are predictive of lack of response to zoledronic acid treatment in reducing breast cancer recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B13.

Association between leukocyte telomere length and angiogenic cytokines in knee osteoarthritis.

The aims of this study were to compare leukocyte relative telomere length (RTL) in knee osteoarthritis (OA) patients and healthy controls and to investigate associations between plasma angiogenic cytokine concentrations and leukocyte RTL. Eighty knee OA patients and 60 age-matched controls were enrolled. Leukocyte RTL was assessed using real-time quantitative polymerase chain reaction (qPCR). Angiogenic cytokines were measured by a multiplex immunoassay. Leukocyte RTL in knee OA patients was significantly lower than that in healthy controls (1.1 ± 0.4 vs. 1.3 ± 0.6, P = 0.039). Plasma angiopoietin-2, follistatin, granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial growth factor (VEGF) levels in knee OA patients were higher than those in controls (P < 0.01). Correlation analysis revealed significant negative correlations between leukocyte RTL and plasma levels of HGF (r = -0.377, P = 0.017), VEGF (r = -0.405, P = 0.009) and G-CSF (r = -0.347, P = 0.026). In contrast, plasma angiopoietin-2, follistatin, IL-8, leptin, platelet-derived growth factor-BB and PECAM-1 were not correlated with leukocyte RTL. Telomere length was shortened in knee OA patients compared to healthy controls. Plasma HGF, VEGF and G-CSF were negatively correlated with leukocyte RTL, suggesting involvement of telomere shortening and these cytokines in knee OA.

G-CSF Predicts Cardiovascular Events in Patients with Stable Coronary Artery Disease.

Granulocyte-colony-stimulating-factor (G-CSF) induces mobilization of progenitor cells but may also exert pro-inflammatory and pro-thrombotic effects. Treatment with recombinant G-CSF after acute myocardial infarction is currently under examination and has been associated with in-stent restenosis. However, it is not known whether plasma levels of endogenous G-CSF are also associated with an increased cardiovascular risk. Therefore we included 280 patients with angiographically proven stable coronary artery disease. G-CSF was measured by specific ELISA and patients were followed for a median of 30 months for the occurrence of major adverse cardiovascular events (MACE: death, myocardial infarction, re-hospitalization). Those with cardiac events during follow-up showed significant higher G-CSF levels (32.3 pg/mL IQR 21.4–40.5 pg/mL vs. 24.6 pg/mL IQR 16.4–34.9 pg/mL; p<0.05) at baseline. Patients with G-CSF plasma levels above the median had a 2-fold increased risk for MACE (p<0.05). This was independent from established cardiovascular risk factors. In addition, G-CSF above the median was a predictor of clinical in-stent restenosis after implantation of bare-metal stents (6.6% vs. 19.4%; p<0.05) but not of drug-eluting stents (7.7% vs. 7.6%; p = 0.98). This data suggests that endogenous plasma levels of G-CSF predict cardiovascular events independently from established cardiac risk factors and are associated with increased in-stent restenosis rates after implantation of bare metal stents.

Therapeutic exercise in improving acute lung injury: a long distance to be covered.

Skeletal muscle weakness is a common finding among critically ill patients, and many factors have been recognized to trigger its initiation early in the patients’ course in intensive care unit (ICU) (1). In acute respiratory distress syndrome (ARDS), a clinical condition which is characterized by acute respiratory failure, skeletal muscle weakness acquired in ICU is present in 25-50% of patients, and is independently associated with mortality and long term morbidity in survivors (2). Several clinical research studies suggest that early mobilization of critically ill patients improves clinically meaningful outcomes for the patient (3), however, without having clarified the mechanisms underlying this implementation. We read with interest the study by Files et al. (4) who through their experiments suggest a unifying mechanism underlying the beneficial effects of therapeutic exercise in mice with lung injury. In their study authors exercised ALI mice for 2 days after lipopolysaccharides (LPS) instillation. They found that a short duration of moderate intensity exercise attenuated muscle ring finger 1 (MuRF 1)-mediated atrophy of the limb and diaphragm and improved limb muscle force generation. Importantly, exercise also limited neutrophilic influx into the alveolar space through modulation of a co-ordinated systemic neutrophil chemokine response. Furthermore, in ALI mice exercise reduced granulocyte colony-stimulating factor (G-CSF), and in vivo blockade of the G-CSF receptor lead to recapitulation of the lung. Similarly, in humans with acute respiratory failure, early mobility therapy led to greater decrements over time in G-CSF plasma levels compared to control patients, confirming the relevance of G-CSF as a mediator of the improved outcomes of mobilized/exercised critically ill patients.

Daily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress

Propranolol has been shown previously to decrease the mobilization of hematopoietic progenitor cells (HPCs) after acute injury in rodent models; however, this acute injury model does not reflect the prolonged period of critical illness after severe trauma. Using our novel lung contusion/hemorrhagic shock/chronic restraint stress model, we hypothesize that daily administration of propranolol will decrease prolonged mobilization of HPCs without worsening lung healing. Male Sprague-Dawley rats underwent 6days of restraint stress after undergoing lung contusion or lung contusion/hemorrhagic shock. Restraint stress consisted of a daily 2-hour period of restraint interrupted every 30minutes by alarms and repositioning. Each day after the period of restraint stress, the rats received intraperitoneal propranolol (10mg/kg). On day 7, peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) and stromal cell-derived factor 1 via enzyme-linked immunosorbent assay and for mobilization of HPCs using c-kit and CD71 flow cytometry. The lungs were examined histologically to grade injury. Seven days after lung contusion and lung contusion/hemorrhagic shock, the addition of chronic restraint stress significantly increased the mobilization of HPC, which was associated with persistently increased levels of G-CSF and increased lung injury scores. The addition of propranolol to lung contusion/chronic restraint stress and lung contusion/hemorrhagic shock/chronic restraint stress models greatly decreased HPC mobilization and restored G-CSF levels to that of naïve animals without worsening lung injury scores. The daily administration of propranolol after both lung contusion and lung contusion/hemorrhagic shock subjected to chronic restraint stress decreased the prolonged mobilization of HPC from the bone marrow and decreased plasma G-CSF levels. Despite the decrease in mobilization of HPC, lung healing did not worsen. Alleviating chronic stress with propranolol may be a future therapeutic target to improve healing after severe injury.

Mesenchymal stem cells reverse trauma and hemorrhagic shock-induced bone marrow dysfunction

BackgroundLung contusion (LC) followed by hemorrhagic shock (HS) causes persistent bone marrow (BM) dysfunction lasting up to 7 d after injury. Mesenchymal stem cells (MSCs) are multipotent cells that can hasten healing and exert protective immunomodulatory effects. We hypothesize that MSCs can attenuate BM dysfunction after combined LCHS. Materials and methodsMale Sprague–Dawley rats (n = 5–6 per group) underwent LC plus 45 min of HS (mean arterial pressure of 30–35). Allogeneic MSCs (5 × 106 cells) were injected intravenously after resuscitation. At 7 d, BM was analyzed for cellularity and growth of hematopoietic progenitor cell (HPC) colonies (colony-forming unit–erythroid; burst-forming unit–erythroid; and colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte). Flow cytometry measured %HPCs in peripheral blood; plasma granulocyte colony-stimulating factor (G-CSF) levels were measured via enzyme-linked immunosorbent assay. Data were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. ResultsAs previously shown, at 7 d, LCHS resulted in 22%, 30%, and 24% decreases in colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte, burst-forming unit–erythroid, and colony-forming unit–erythroid colony growth, respectively, versus naive. Treatment with MSCs returned all BM parameters to naive levels. There was no difference in %HPCs in peripheral blood between groups; however, G-CSF remained increased up to 7 d after LCHS. MSCs returned G-CSF to naive levels. Plasma from animals receiving MSCs was not suppressive to the BM. ConclusionsOne week after injury, the persistent BM dysfunction observed in animals undergoing LCHS is reversed by treatment with MSCs with an associated return of plasma G-CSF levels to normal. Plasma from animals undergoing LCHS plus MSCs was not suppressive to BM cells in vitro. Treatment with MSCs after injury and shock reverses BM suppression and returns plasma G-CSF levels to normal.

Low-dose cyclophosphamide improves survival in a murine treatment model of sepsis.

Sepsis is a complex medical condition characterized by a systemic inflammatory response in the setting of infection. We hypothesized that combining antibiotics plus an immunosuppressant would protect against the morbidity and mortality of polymicrobial sepsis in mice better than would antibiotics alone. We used a murine cecal-ligation-and-puncture model in which mice were treated either with imipenem plus cyclophosphamide or imipenem alone. Titration to a low cyclophosphamide dose revealed that combination therapy increased survival by 20% compared with imipenem alone (56% vs. 36%, P < 0.001). To investigate the mechanism by which combination therapy did this, we reviewed quantitative and qualitative markers of the systemic immune response, end-organ damage, and the local immune response at the site of injury. Cyclophosphamide treatment was not associated with depletion of peripheral leukocytes or differences in pulmonary damage. However, mice that received combination therapy had higher plasma granulocyte colony-stimulating factor levels than did those treated with antibiotics alone. In addition, mice treated with cyclophosphamide had higher levels of bacterial colonization in intestinal Peyer's patch lymph nodes at 72 h after the septic insult. Intraperitoneal macrophage phenotypes and phagocytosis activity did not differ between groups. We conclude that the inflammatory response plays a significant role in the mortality of polymicrobial sepsis and that the regulation of this element is both feasible and beneficial in this disease model.

The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice.

Acquisition of microbes by the neonate, which begins immediately during birth, is influenced by gestational age and mother’s microbiota and modified by exposure to antibiotics1. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of sepsis after 4 days of life, known as late-onset sepsis (LOS)2, a disorder critically controlled by neutrophils3, but a role for the microbiota in regulating neutrophil behavior in the neonate has not been described. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number of microbes in the intestine, altered the structure of intestinal microbiota and changed the pattern of microbial colonization. These changes were associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage restricted progenitor cells in the bone marrow. Antibiotic-exposure of dams attenuated the postnatal granulocytosis by reducing the number of interleukin (IL) 17-producing cells in intestine and consequent production of granulocyte colony stimulating factor (G-CSF). Relative granulocytopenia contributed to increased susceptibility of antibiotic-exposed neonatal mice to Escherichia coli K1 and Klebsiella pneumoniae sepsis, which could be partially reversed by administration of G-CSF. Restoration of normal microbiota, through TLR4- and MYD88-dependent mechanism, induced accumulation of IL17-producing type 3 innate lymphoid cells (ILC) in the intestine, promoted granulocytosis, and restored the IL17-dependent resistance to sepsis. Specific depletion of ILCs prevented the IL17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis and host resistance to sepsis in the neonates.

Do all β-blockers attenuate the excess hematopoietic progenitor cell mobilization from the bone marrow following trauma/hemorrhagic shock?

Severe injury results in increased mobilization of hematopoietic progenitor cells (HPC) from the bone marrow (BM) to sites of injury, which may contribute to persistent BM dysfunction after trauma. Norepinephrine is a known inducer of HPC mobilization, and nonselective β-blockade with propranolol has been shown to decrease mobilization after trauma and hemorrhagic shock (HS). This study will determine the role of selective β-adrenergic receptor blockade in HPC mobilization in a combined model of lung contusion (LC) and HS. Male Sprague-Dawley rats were subjected to LC, followed by 45 minutes of HS. Animals were then randomized to receive atenolol (LCHS + β1B), butoxamine (LCHS + β2B), or SR59230A (LCHS + β3B) immediately after resuscitation and daily for 6 days. Control groups were composed of naive animals. BM cellularity, %HPCs in peripheral blood, and plasma granulocyte-colony stimulating factor levels were assessed at 3 hours and 7 days. Systemic plasma-mediated effects were evaluated in vitro by assessment of BM HPC growth. Injured lung tissue was graded histologically by a blinded reader. The use of β2B or β3B following LCHS restored BM cellularity and significantly decreased HPC mobilization. In contrast, β1B had no effect on HPC mobilization. Only β3B significantly reduced plasma G-CSF levels. When evaluating the plasma systemic effects, both β2B and β3B significantly improved BM HPC growth as compared with LCHS alone. The use of β2 and β3 blockade did not affect lung injury scores. Both β2 and β3 blockade can prevent excess HPC mobilization and BM dysfunction when given after trauma and HS, and the effects seem to be mediated systemically, without adverse effects on subsequent healing. Only treatment with β3 blockade reduced plasma G-CSF levels, suggesting different mechanisms for adrenergic-induced G-CSF release and mobilization of HPCs. This study adds to the evidence that therapeutic strategies that reduce the exaggerated sympathetic stimulation after severe injury are beneficial and reduce BM dysfunction.

The Role of Plasma Granulocyte Colony Stimulating Factor and Bone Marrow Dysfunction after Severe Trauma

Bone marrow dysfunction is common in severely injured trauma patients, with release of hematopoietic progenitor cells (HPC) into the peripheral blood. Granulocyte colony stimulating factor (G-CSF) is a potent stimulator of HPC mobilization. We hypothesized that plasma G-CSF levels are elevated after trauma and correlate with postinjury anemia and infection. Blood from 83 severely injured patients was collected at several time points for determination of G-CSF levels and HPC mobilization and compared with that from healthy volunteers. Data were categorized by age, sex, Injury Severity Score (ISS), and whether the patient was in shock. Hemoglobin and transfusion requirements and hospital-acquired infection data were recorded. Data are expressed as mean ± SEM. After trauma, there is a 50-fold increase in plasma levels of G-CSF in trauma patients compared with controls (1,640.4 ± 304.3 pg/mL vs 33.0 ± 6.8 pg/mL, p < 0.001). Patients who presented in shock had 5-times higher G-CSF levels than nonshock trauma patients and a 75-fold increase compared with controls (2,528.7 ± 536.4 pg/mL vs 728.0 ± 191.0 pg/mL vs 33.0 ± 6.8 pg/mL, p < 0.001). Age, sex, and ISS had no effect on G-CSF levels. Mobilization of HPC was sustained for up to 10 days after injury and involved multiple cells types. Higher G-CSF levels were also associated with lower hemoglobin levels and greater transfusion requirements 3 weeks after injury and a higher incidence of hospital-acquired pneumonia and bacteremia. Plasma G-CSF is markedly elevated after injury and is greater in patients who present in shock. The rise in G-CSF was also associated with prolonged mobilization of HPC. Elevation of G-CSF in humans after severe trauma may play a significant role in the development of post-traumatic bone marrow dysfunction, anemia, and infection.

Prolonged circulation and in vivo efficacy of recombinant human granulocyte colony-stimulating factor encapsulated in polymeric micelles

To improve the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) and decrease dosing frequency, polyethylene glycol polyglutamate block copolymers were used as delivery carriers. The block copolymers are partially substituted with hydrophobic octyl or benzyl groups to form a micellar structure in aqueous media and encapsulate the protein. G-CSF is encapsulated in the polymeric micelles with a diameter of 60–70 nm. The present study was designed to evaluate the plasma pharmacokinetics, G-CSF release and in vivo efficacy of G-CSF-encapsulating micelles. Pharmacokinetic studies in rats showed highly enhanced plasma retention of the micelles compared with native G-CSF. The AUC (area under the curve) of the octyl-based polymer formulation showed a 5-fold increase, compared with native G-CSF. Size-exclusion chromatography of the blood from rats injected with the micelles demonstrated the release of G-CSF from the micelles in the blood circulation. The pharmacokinetic behavior supports the in vivo studies showing that the micelles display a comparable efficacy to PEGylated G-CSF. Simultaneous pharmacokinetic analysis of released and encapsulated G-CSF plasma levels showed that the G-CSF release occurs with the first-order kinetics and the half-life is 4.8 h. In conclusion, G-CSF is endowed by the polymeric micelles with prolonged half-life and increased efficacy without any chemical modification.

Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma

To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function. Peripheral blood mononuclear cells (PBMCs) from healthy control subjects and from patients with newly diagnosed glioma were stimulated with anti-CD3/anti-CD28 and T cells assessed for intracellular expression of interferon (IFN)-γ. Antibody staining of PBMCs from glioma patients and healthy donors (CD33, HLADR, CD15, and CD14) followed by 4-color flow cytometry analysis-defined MDSC levels in the peripheral blood. To assess the role of MDSCs in suppressing T cell IFNγ production, PBMCs were depleted of MDSCs using anti-CD33 and anti-CD15 antibody-coated beads prior to T cell stimulation. Enzyme-linked immunosorbent assays were used to assess plasma arginase activity and the level of granulocyte colony-stimulating factor (G-CSF). Patients with glioblastoma have increased MDSC counts (CD33+HLADR-) in their blood that are composed of neutrophilic (CD15(+); >60%), lineage-negative (CD15(-)CD14(-); 31%), and monocytic (CD14(+); 6%) subsets. After stimulation, T cells from patients with glioblastoma had suppressed IFN-γ production when compared with healthy, age-matched donor T cells. Removal of MDSCs from the PBMCs with anti-CD33/CD15-coated beads significantly restored T cell function. Significant increases in arginase activity and G-CSF levels were observed in plasma specimens obtained from patients with glioblastoma. The accumulation of MDSCs in peripheral blood in patients with glioma likely promotes T cell immune suppression that is observed in this patient population. Increased plasma levels of arginase and G-CSF may relate to MDSC suppressor function and MDSC expansion, respectively, in patients with glioma.

β-Blockade Protection of Bone Marrow Following Trauma: The Role of G-CSF

Following severe trauma, there is a profound elevation of catecholamine that is associated with a persistent anemic state. We have previously shown that β-blockade (βB) prevents erythroid growth suppression and decreases hematopoietic progenitor cell (HPC) mobilization following injury. Under normal conditions, granulocyte colony stimulating factor (G-CSF) triggers the activation of matrix metalloprotease-9 (MMP-9), leading to the egress of progenitor cells from the bone marrow (BM). When sustained, this depletion of BM cellularity may contribute to BM failure. This study seeks to determine if G-CSF plays a role in the βB protection of BM following trauma. Male Sprague-Dawley rats were subjected to either unilateral lung contusion (LC) ± βB, hemorrhagic shock (HS) ± βB, or both LC/HS ± βB. Propranolol (βB) was given immediately following resuscitation. Animals were sacrificed at 3and 24h and HPC mobilization was assessed by evaluating BM cellularity and flow cytometric analysis of peripheral blood for HPCs. The concentration of G-CSF and MMP-9was measured in plasma by ELISA. BM cellularity is decreased at 3h following LC, HS, and LC/HS. HS and LC/HS resulted in significant HPC mobilization in the peripheral blood. The addition of βB restored BM cellularity and reduced HPC mobilization. Three h following HS and LC/HS, plasma G-CSF levels more than double, however LC alone showed no change in G-CSF. βB significantly decreased G-CSF in both HS and LC/HS. Similarly, MMP-9is elevated following LC/HS, and βB prevents this elevation (390 ± 100pg/mL versus 275 ± 80pg/mL). βB protection of the BM following shock and injury may be due to reduced HPC mobilization and maintenance of BM cellularity. Following shock, there is an increase in plasma G-CSF and MMP-9, which is abrogated by βB and suggests apossible mechanism how βB decreases HPC mobilization thus preserving BM cellularity. In contrast, βBprotection of BM following LC is not mediated by G-CSF. Therefore, the mechanism of progenitor cell mobilization from the BM is dependent on the type of injury.

Granulocyte colony-stimulating factor expression as a prognostic biomarker in non-small cell lung cancer

Hematologic paraneoplastic alternations may not be very common, but they have been observed in a small number of patients. Granulocytosis has been described in several malignancies and its common mechanism may be associated with granulocyte colony-stimulating factor (G-CSF) production by the tumor. High neutrophilia (150,000-240,000 white blood cell count) observed in two patients with non-small cell lung cancer led us to run the present trial. The purpose of this study was to compare the white blood cell counts and the G-CSF plasma levels of the patients and classify the results into groups, as well as to determine the survival rates of the patients in each of the groups. The histological specimens and plasma of two patients as well as the plasma of another 87 patients with several malignancies, were examined. The plasma G-CSF levels were determined using a quantitative sandwich immunoassay technique (Quantikine; RySD systems) according to the manufacturer's instructions and all samples were tested in triplicate. It was found that 12 patients had a white blood cell count increased beyond normal as well as a high G-CSF plasma level and the survival of these patients was shorter as compared to the rest of the patients. We assume that these findings may indicate that increased G-CSF levels may function as a biomarker of survival.

Stem Cell-Like Human Endothelial Progenitors Show Enhanced Colony-Forming Capacity After Brief Sevoflurane Exposure: Preconditioning of Angiogenic Cells by Volatile Anesthetics

Endothelial progenitor cells play a pivotal role in tissue repair, and thus are used for cell replacement therapies in "regenerative medicine." We tested whether the anesthetic sevoflurane would modulate growth or mobilization of these angiogenic cells. In an in vitro model, mononuclear cells isolated from peripheral blood of healthy donors were preconditioned with sevoflurane (3 times 30 min at 2 vol% interspersed by 30 min of air). Colony-forming units were determined after 9 days in culture and compared with time-matched untreated control. Using magnetic cell sorting, CD133+/CD34+ endothelial progenitors were enriched from human umbilical cord blood, and vascular endothelial growth factor (VEGF), VEGFR2 (KDR), granulocyte colony-stimulating factor (G-CSF), STAT3, c-kit, and CXCR4 expressions were determined in sevoflurane-treated and untreated cells by real-time reverse transcriptase polymerase chain reaction. In a volunteer study with crossover design, we tested whether sevoflurane inhalation (<1 vol% end-tidal concentration) would mobilize endothelial progenitor cells from the bone marrow niche into the circulation using flow cytometry of peripheral blood samples. VEGF and G-CSF plasma levels were also measured. In vitro sevoflurane exposure of mononuclear cells enhanced colony-forming capacity and increased VEGF mRNA levels in CD133+/CD34+ cord blood cells (P = 0.017). Sevoflurane inhalation in healthy volunteers did not alter the number of CD133+/CD34+ or KDR+/CD34+ endothelial progenitors in the circulation, but increased the number of colony-forming units (P = 0.034), whereas VEGF and G-CSF plasma levels remained unchanged. Sevoflurane preconditioning promotes growth and proliferation of stem cell-like human endothelial progenitors. Hence, it may be used to promote perioperative vascular healing and to support cell replacement therapies.

Decreased Plasma Levels of Granulocyte-Colony Stimulating Factor (G-CSF) in Patients with Early Alzheimer's Disease

Alzheimer's disease (AD) is characterized by massive neuronal cell loss in the brain. Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor that promotes neuroprotective effects and supports neurogenesis in the brain. In the present study, we found significantly lower G-CSF plasma levels in 50 early AD patients in comparison with 50 age-matched healthy controls. In AD patients, G-CSF levels showed a significant inverse correlation with amyloid-beta (Abeta1-42) levels in cerebrospinal fluid, but not with levels of tau protein in cerebrospinal fluid or Mini-Mental Status Examination scores. In addition, G-CSF plasma levels were significantly inversely correlated with age in AD patients and healthy controls. In conclusion, decreased G-CSF plasma levels in early AD patients may contribute to a deficient hematopoietic brain support with putative pathogenic relevance. Further studies are needed to examine whether a modulation of hematopoietic growth factors such as G-CSF could be a promising new therapeutic strategy for AD.

Plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury.

To evaluate the association between plasma granulocyte colony-stimulating factor (G-CSF) levels and clinical outcomes including mortality in patients with acute lung injury (ALI), and to determine whether lower tidal volume ventilation was associated with a more rapid decrease in plasma G-CSF over time in patients with ALI. Retrospective measurement of G-CSF levels in plasma samples that were collected prospectively as part of a large multicenter clinical trial. Intensive care units in ten university centers. The study included 645 patients enrolled in the National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome Clinical Network trial of lower tidal volumes compared with traditional tidal volumes for ALI. Baseline plasma levels of G-CSF were associated with an increased risk of death and a decrease in ventilator-free days and organ failure-free days in multivariate analyses controlling for ventilation strategy, age, and sex (Odds ratio death 1.2/log10 increment G-CSF, 95% confidence interval 1.01 to 1.4). Stratification of G-CSF levels into quartiles revealed a strong association between the highest levels of G-CSF and an increased risk of death and decreased ventilator-free days and organ failure-free days in multivariate analyses controlling for ventilation strategy, Acute Physiology and Chronic Health Evaluation III score, Pao2/Fio2 ratio, creatinine, and platelet count (p < 0.05). Subgroup multivariate analysis of patients with sepsis as their risk factor for ALI revealed a U-shaped association between mortality and G-CSF levels such that risk increased linearly from the second through fourth (highest) quartiles, yet also increased in the first (lowest) quartile. G-CSF levels decreased over time in both tidal volume groups, and there was no statistical difference in the extent of decrease between ventilator strategies. In patients with ALI, plasma G-CSF levels are associated with morbidity and mortality, but these levels are not influenced by tidal volume strategy. In patients with sepsis-related ALI, a bimodal association between baseline plasma G-CSF levels and subsequent morbidity and mortality from this disease was found.