Expression Of Granulocyte Colony-stimulating Factor Receptor Research Articles

Evaluation of the Effect of TNF-α on the Expression of G-CSF and M-CSF in the Monocytes of Multiple Sclerosis Patients

Background: Multiple sclerosis (MS) is an inflammatory autoimmune disease in which the myelin sheaths of nerve cells in the brain and spinal cord are damaged. This damage can disrupt the abilities of parts of the nervous system that are responsible for communication and cause many physical signs and symptoms. Granulocyte colony-stimulating factor (G-CSF) is a major extracellular regulator of hematopoiesis and the innate immune system. Macrophage colony-stimulating factor (M-CSF) is a glycoprotein that is effective in hematopoiesis and stimulates the production of numerous cytokines. It also plays a key role in stimulating the clearance of myelin by the brain’s innate immune cells, which is a prerequisite for proper remyelination and myelin repair. The aim of the study was to investigate the effect of overlapping and non-overlapping cytokines in MS. Methods: Overall, 20 MS patients referred to the MS clinic of Imam Reza hospital with mild, moderate, and severe stages were selected for investigation. The blood samples were taken from the patients, and magnetic-activated cell-sorting monocyte cells were separated from peripheral blood mononuclear cells (PBMCs). The purity of the monocyte cells was determined using flow cytometry and the CD14 antibody. The monocyte cells were then cultured and stimulated with tumor necrosis factor (TNF) over a time course of 0, 6, 12, 24, and 48 hours. After the end of stimulation, the cells were collected, and the expression level of G-CSF and M-CSF was evaluated by real-time polymerase chain reaction (RT-PCR). Results: The expression of G-CSF and M-CSF receptors decreased significantly over time as a time course of stimulation, with the lowest value 12 hours after stimulation. Comparing G-CSF and M-CSF, the decreasing ratio has been the same. The level of TNF-α function was evaluated as a time course. Conclusion: The results of RT-PCR showed that TNF-α significantly decreases the expression of innate immune system receptors such as G-CSF and M-CSF. This reduction was in surface receptors such as G-CSF and M-CSF.

Exploring the Effects of G-CSF Mobilization on the Immune Function of Mucosal-Associated Invariant T Cell Subsets By Single-Cell RNA-Seq

Background: Mucosal-associated invariant T cells (MAITs) are a group of mucosal-enriched unconventional T cells that can exert a variety of immune functions including pro-inflammatory, cytotoxic and immunomodulatory effects, whereas with the characteristics of innate immune cells. A few studies including our previous work have confirmed the relationship between MAITs and gut acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). In addition, we further proved that specific gut microbiota can promote MAIT17 subset activation, and MAITs has a direct immunosuppressive effect on T cells in vitro, which may potentially explain our results that higher number of MAITs in granulocyte colony-stimulating factor (G-CSF) mobilized grafts is associated with lower incidence of gut aGVHD. G-CSF has been widely used to mobilize hematopoietic stem/progenitor cells before HSCT. It also plays an important immunomodulatory role in inducing immune tolerance by regulating both innate and adaptive immune cells, especially T cells. Therefore, we used single-cell RNA sequencing to further explore the different effects of G-CSF mobilization on the immune function of each subset of MAITs. Methods & Cases: We isolated MAITs from the peripheral blood (PB) of 4 healthy donors pre- and post-G-CSF mobilization by flow cytometry pre-transplantation. 8 samples were performed by single-cell RNA-seq (Figure 1A). One of these recipients who received these 4 healthy donor grafts developed gut aGVHD after transplantation. Through single-cell bioinformatics analysis, we compared the sequencing data from pre- and post-G-CSF-mobilized MAITs of doners, and from G-CSF-mobilized donors in the group of recipients with or without gut aGVHD after transplantation. Results: By performing UMAP cluster analysis on these 8 samples, according to the expression of key cell type-associated marker genes of each cluster, MAITs were annotated into 14 subsets (Figure 1A and 1B). Among these subsets, Cluster 1(CD8_effector_EOMES), 4(CD4_effector_CCR6), 5(CD4_immature_CCR7), 6(CD8_effector_RORC), 12(CD4_immunomodulatory) increased significantly after G-CSF mobilization (Figure 1C). By comparing the bioinformatical data of MAITs of pre- and post-G-CSF mobilization, the results showed that molecular markers responsible for MAIT cell hyporesponsiveness were upregulated, including potential direct or indirect modulation mediated by the IL-4/IL-13 signaling pathway (FOS, JUNB, DDIT4, NFKBIA, MT2A, VIM, etc.) (Figure 1D) or ligand receptor (G-CSF-CSF3R) interactions (Figure 1E). Besides, a specific subset of MAITs defined as Cluster 12-CD4_immunomodulatory MAITs (Figure 1A) was found that played the most important role in immune regulation after G-CSF stimulation, which specifically expressed G-CSF receptor CSF3R. Furthermore, IL-17 signaling pathway was up-regulated and GVHD pathway was down-regulated in Cluster 12 post-G-CSF mobilization (Figure 1F), which suggested the Cluster 12 is an important subset of MAITs anti-GVHD. Compared to the sequencing data of donors in the group of recipients with gut aGVHD after G-CSF mobilization (post-G-CSF-gut aGVHD), we also observed the higher expression of immunosuppressive genes (JUN, DUSP2, JUNB, VIM, etc.) of doner MAITs in group without gut aGVHD after mobilization (post-G-CSF-no gut aGVHD). In addition, in the post-G-CSF-no gut aGVHD group, the expression of SOCS3, STAT3, CCR6 (Figure 1G) and other key markers that may potentially explain the anti-GVHD effect of MAITs was upregulated, as well as the ability of tissue repair and gut-directed migration of MAITs was upregulated (Figure 1H), while its cytotoxic effector functions were not significantly affected. Conclusions: Our results indicated that G-CSF mobilization can enhance the immunosuppressive effect of MAITs. Cluster 12 (CD4_immunomodulatory) is an important immunoregulatory and anti-GVHD subset of MAITs, contributing to the differentiation of MAITs towards MAIT17. The upregulation of markers such as SOCS3, CCR6, STAT3 that involved in CSF3R-JAK-STAT pathway, as well as markers related to mucosal tissue repair may partially explain the protective effect from gut aGVHD of G-CSF mobilized MAITs. These results also enlighten us that gut aGVHD might be prevented by effectively expanding the specific immunosuppressive subset of MAIT cells in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

A review of granulocyte colony-stimulating factor receptor signaling and regulation with implications for cancer.

Granulocyte colony-stimulating factor receptor (GCSFR) is a critical regulator of granulopoiesis. Studies have shown significant upregulation of GCSFR in a variety of cancers and cell types and have recognized GCSFR as a cytokine receptor capable of influencing both myeloid and non-myeloid immune cells, supporting pro-tumoral actions. This systematic review aims to summarize the available literature examining the mechanisms that control GCSFR signaling, regulation, and surface expression with emphasis on how these mechanisms may be dysregulated in cancer. Experiments with different cancer cell lines from breast cancer, bladder cancer, glioma, and neuroblastoma are used to review the biological function and underlying mechanisms of increased GCSFR expression with emphasis on actions related to tumor proliferation, migration, and metastasis, primarily acting through the JAK/STAT pathway. Evidence is also presented that demonstrates a differential physiological response to aberrant GCSFR signal transduction in different organs. The lifecycle of the receptor is also reviewed to support future work defining how this signaling axis becomes dysregulated in malignancies.

Granulocyte Colony-Stimulating Factor Restored Impaired Spermatogenesis and Fertility in an AML-Chemotherapy Mice Model.

Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

The development of a new muscle atrophy treatment method based on the elucidation of the sarcomere synthesis mechanism

Muscle mass is controlled via sarcomere levels and conditions brought on by long-term rest can lead to muscle wasting and significant decreases in sarcomere levels. Assistant Professor Nozomi Hayashiji, Center for Genomic and Regenerative Medicine, Juntendo University, Japan, is investigating the mechanisms of skeletal muscle homeostasis in order to develop new treatments for muscle atrophy. There is little knowledge about how sarcomeres are synthesised within the human body due to a lack of suitable animal models but in 2015, Hayashiji made an important discovery surrounding the existence of granulocyte colony-stimulating factor (G-CSF) receptor-deficient mice that demonstrated a resistance to adult sarcomere synthesis . This could pave the way to uncovering the mechanisms that regulate sarcomere production. Currently, Hayashiji is working to shed light on the factors involved in the sarcomere synthesis pathway. To do this, she is using a synthesis resistance model and hopes the work will lead to new treatments to help patients affected by diseases that lead to muscle atrophy. Little is known about the expression of G-CSF receptors (G-CSFR) in muscle fibres and Hayashiji is seeking to uncover the pathways relating to sarcomere production downstream of the G-CSFR and elucidate the role of G-CSFR in sarcomere synthesis using a G-CSFR-deficient mouse model that she developed in previous work. Ultimately, Hayashiji hopes that her research will have an important impact worldwide, benefiting patients with muscle atrophy and also potentially proving beneficial within sports.

Transplanted hepatocytes rescue mice in acetaminophen-induced acute liver failure through paracrine signals for hepatic ATM and STAT3 pathways.

Acute liver failure constitutes a devastating condition that needs novel cell and molecular therapies. To elicit synergisms in cell types of therapeutic interest, we studied hepatocytes and liver sinusoidal endothelial in mice with acetaminophen-induced acute liver failure. The context of regenerative signals was examined by transplants in peritoneal cavity because it possesses considerable capacity and allows soluble signals to enter the systemic circulation. Whereas transplanted hepatocytes and liver sinusoidal endothelial cells engrafted in peritoneal cavity, only the former could rescue mice in liver failure by improving injury outcomes, activating hepatic DNA damage repair, and inducing liver regeneration. The cytokines secreted by donor hepatocytes or liver sinusoidal endothelial cells differed and in hepatocytes from mice undergoing acetaminophen toxicity major cytokines were even rendered deficient (eg, G-CSF, VEGF, and others). Significantly, recapitulating hepatotoxicity-related DNA damage response in cultured cells identified impairments in ATM and JAK/STAT3 intersections since replacing cytokines produced less from injured hepatocytes restored these pathways to avoid acetaminophen hepatotoxicity. Similarly, hepatocyte transplantation in acute liver failure restored ATM and JAK/STAT3 pathways to advance DNA damage/repair and liver regeneration. The unexpected identification of novel hepatic G-CSF receptor expression following injury allowed paradigmatic studies of G-CSF supplementation to confirm the centrality of this paracrine ATM and STAT3 intersection. Remarkably, DNA damage/repair and hepatic regeneration directed by G-CSF concerned rebalancing of regulatory gene networks overseeing inflammation, metabolism, and cell viability. We conclude that healthy donor hepatocytes offer templates for generating specialized cell types to replace metabolic functions and regenerative factors in liver failure.

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Neuroprotection through G-CSF: recent advances and future viewpoints.

Granulocyte-colony stimulating factor (G-CSF), a member of the cytokine family of hematopoietic growth factors, is 19.6kDa glycoprotein which is responsible for the proliferation, maturation, differentiation, and survival of neutrophilic granulocyte lineage. Apart from its proven clinical application to treat chemotherapy-associated neutropenia, recent pre-clinical studies have highlighted the neuroprotective roles of G-CSF i.e., mobilization of haemopoietic stem cells, anti-apoptotic, neuronal differentiation, angiogenesis and anti-inflammatory in animal models of neurological disorders. G-CSF is expressed by numerous cell types including neuronal, immune and endothelial cells. G-CSF is released in autocrine manner and binds to its receptor G-CSF-R which further activates numerous signaling transduction pathways including PI3K/AKT, JAK/STAT and MAP kinase, and thereby promote neuronal survival, proliferation, differentiation, mobilization of hematopoietic stem and progenitor cells. The expression of G-CSF receptors (G-CSF-R) in the different brain regions and their upregulation in response to neuronal insult indicates the autocrine protective signaling mechanism of G-CSF by inhibition of apoptosis, inflammation, and stimulation of neurogenesis. These observed neuroprotective effects of G-CSF makes it an attractive target to mitigate neurodegeneration associated with neurological disorders. The objective of the review is to highlight and summarize recent updates on G-CSF as a therapeutically versatile neuroprotective agent along with mechanisms of action as well as possible clinical applications in neurodegenerative disorders including AD, PD and HD.

Characterization of G-CSF receptor expression in medulloblastoma.

BackgroundIdentifying mechanisms of medulloblastoma recurrence is a key to improving patient survival, and targeting treatment-resistant subpopulations within tumors could reduce disease recurrence. Expression of the granulocyte colony-stimulating factor receptor (G-CSF-R, CD114) is a potential marker of cancer stem cells, and therefore we hypothesized that a subpopulation of medulloblastoma cells would also express CD114 and would demonstrate chemoresistance and responsiveness to G-CSF.MethodsPrevalence of CD114-positive (CD114+) cells in medulloblastoma cell lines, patient-derived xenograft (PDX) tumors, and primary patient tumor samples were assessed by flow cytometry. Growth rates, chemoresistance, and responses to G-CSF of CD114+ and CD114-negative (CD114−) cells were characterized in vitro using continuous live cell imaging and flow cytometry. Gene expression profiles were compared between CD114+ and CD114− medulloblastoma cells using quantitative RT-PCR.ResultsCD114+ cells were identifiable in medulloblastoma cell lines, PDX tumors, and primary patient tumors and have slower growth rates than CD114− or mixed populations. G-CSF accelerates the growth of CD114+ cells, and CD114+ cells are more chemoresistant. The CD114+ population is enriched when G-CSF treatment follows chemotherapy. The CD114+ population also has higher expression of the CSF3R, NRP-1, TWIST1, and MYCN genes.ConclusionsOur data demonstrate that a subpopulation of CD114+ medulloblastoma cells exists in cell lines and tumors, which may evade traditional chemotherapy and respond to exogenous G-CSF. These properties invite further investigation into the role of G-CSF in medulloblastoma therapy and methods to specifically target these cells.

Impairment of G-CSF receptor on granulocytic progenitor cells causes neutropenia in protein malnutrition.

It is well known that protein malnutrition (PM) states can affect hematopoiesis, leading to severe leukopenia and reduced number of granulocytes, which act as the first line of defense, and are important to the innate immune response. The aim of this study was to elucidate some of the mechanisms involved in the impairment of granulopoiesis in PM. Male C57BL/6 mice were submitted to PM with a low-protein diet containing 2% protein. Control mice were fed a 12% protein-containing diet. Bone marrow histology and the percentage of granulocytic progenitors were evaluated after in vivo granulocyte-colony stimulating factor (G-CSF) stimulus. Cell proliferation, STAT3 signaling, and the expression of G-CSF receptor were evaluated in hematopoietic progenitor cells. Malnourished animals presented with leukopenia associated with reduced number of granulocytes and reduced percentage of granulocytic progenitors; however, no differences were observed in the regulatory granulopoietic cytokine G-CSF. Additionally, the malnourished group presented with impaired response to in vivo G-CSF stimulus compared with control animals. PM was implicated in decreased ability of c-Kit+ cells to differentiate into myeloid progenitor cells and downregulated STAT3 signaling. Furthermore, the malnourished group exhibited reduced expression of G-CSF receptor on granule-monocytic progenitors. This reduced expression was not completely reversible with G-CSF treatment. This study implies that PM promotes intrinsic alterations to hematopoietic precursors, which result in hematologic changes, mainly neutropenia, observed in peripheral blood in PM states.

Antiapoptotic Effect of Granulocyte-Colony Stimulating Factor After Peripheral Nerve Trauma

Granulocyte-colony stimulating factor (G-CSF) has been observed to have direct protective effects on neurons after stroke in experimental models and in humans. In the present study, the antiapoptotic effects of G-CSF on spinal α-motoneurons after inducement of peripheral sciatic nerve lesions were evaluated in a rat model. Of 48 rats, 24 were treated with G-CSF and 24 were treated with glucose 5% solution (control group). The spinal cord of 6 rats in each group were removed at days 1, 4, 7, and 14. The α-motoneurons of spinal cord section L4-L6 were counted and investigated for the expression of choline acetyltransferase (ChAT), G-CSF receptor (G-CSFR), and Bcl-2 and Bax proteins. Additionally, α-motoneuron fluorescence double staining was performed for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. Without G-CSF treatment, the number of ChAT-positive α-motoneurons on the lesion side was significantly decreased (P < 0.001). The number of α-motoneurons with Bcl-2 and G-CSFR positivity on the lesion side was significantly decreased (P < 0.05). In contrast, the number of α-motoneurons with Bax positivity was significantly greater (P < 0.05). After G-CSF treatment, the differences in the number of α-motoneurons on the 2 sides were not statistically significant. Fluorescence double staining of α-motoneurons was positive for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. The results indicated that G-CSF has neuroprotective properties in spinal α-motoneurons and contributes to antiapoptotic effects after peripheral nerve lesions. The relevance of G-CSF, its precise mode of action, and the effect of these findings in clinical situations remains to be elucidated and require examination in further studies.

Clostridium perfringens \u03b1-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock

During bacterial infection, granulocyte colony-stimulating factor (G-CSF) is produced and accelerates neutrophil production from their progenitors. This process, termed granulopoiesis, strengthens host defense, but Clostridium perfringens α-toxin impairs granulopoiesis via an unknown mechanism. Here, we tested whether G-CSF accounts for the α-toxin-mediated impairment of granulopoiesis. We find that α-toxin dramatically accelerates G-CSF production from endothelial cells in response to Toll-like receptor 2 (TLR2) agonists through activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Meanwhile, α-toxin inhibits G-CSF-mediated cell proliferation of Ly-6G+ neutrophils by inducing degradation of G-CSF receptor (G-CSFR). During sepsis, administration of α-toxin promotes lethality and tissue injury accompanied by accelerated production of inflammatory cytokines in a TLR4-dependent manner. Together, our results illustrate that α-toxin disturbs G-CSF-mediated granulopoiesis by reducing the expression of G-CSFR on neutrophils while augmenting septic shock due to excess inflammatory cytokine release, which provides a new mechanism to explain how pathogenic bacteria modulate the host immune system.

G-CSF administration favours SDF-1 release and activation of neutrophils and monocytes in recipients of autologous peripheral blood progenitor cells

G-CSF is a growth factor widely used to mobilise CD34+ progenitor cells for clinical applications. The present study aimed to assess expression of G-CSF receptor (CSF3R) on neutrophils and monocytes, as well as SDF-1 and G-CSF serum levels in relation to efficacy of G-CSF-induced mobilisation for autologous transplantation. For this purpose, 105 patients with haematological disorders and 46 healthy controls were investigated.Before mobilisation patients were characterised with significantly higher percentage of CSF3R expressing neutrophils (p < 0.001) and monocytes (p = 0.002), than controls. G-CSF administration resulted in a decrease of CSF3R+ neutrophils (p < 0.001) and monocytes (p < 0.001), while presence of G-CSF receptor on neutrophils tended to negatively affect mobilisation yield (p = 0.075).G-CSF concentration increased during mobilisation (p < 0.001). On the 5th day of mobilisation a positive correlation was observed between G-CSF and SDF-1 serum levels (p < 0.001) and the number of CD34+ cells released from bone marrow seemed to be related to both G-CSF (p = 0.036) and SDF-1 levels (p = 0.084).As compared to Hodgkin’s lymphoma patients, those with multiple myeloma had lower basal percentage of CSF3R+ neutrophils (p = 0.014) while Non-Hodgkin’s lymphoma cases exhibited higher G-CSF (p = 0.026) and SDF-1 (p = 0.006) concentration on mobilisation day 5. Hodgkin’s lymphoma patients were also characterised with worse mobilisation efficacy than multiple myeloma (p = 0.022) and Non-Hodgkin’s lymphoma (p = 0.013) patients.These results suggest that both SDF-1 and G-CSF play a role in HSC release into peripheral blood and show that G-CSF administration affects expression of CSF3R on monocytes and neutrophils, implying potential role of these cell subpopulations in mobilisation process.

VITAMIN E ACTIVATES EXPRESSION OF C/EBP ALPHA TRANSCRIPTION FACTOR AND G-CSF RECEPTOR IN LEUKEMIC K562 CELLS

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562cells. RNA extracted from K562cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). We have not found detectable expression of C/EBP alpha in K562cells. Upon 48-h culture with vitamin E at a dose of 100µM, K562cells expressed both C/EBP alpha and G-CSFR. VitaminE restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562cells. In this setting, G-CSFR expression in vitaminE treated K562cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

Leukocytosis, but Not Thrombocytosis, May Constitute a Prothrombotic Pathway in Women with Ovarian Cancer: A Cohort Study

Venous thromboembolism (VTE) occurs at a higher rate in cancer patients and is frequently associated with well-known prothrombotic risk factors such as prolonged bed rest, central venous catheters, diverse oncological treatments and invasive procedures. Patients may share one or more risk factors. In addition, there seem to be tumor-driven prothrombotic pathways. Thus different tumors may promote activation of coagulation either directly through the expression of procoagulant molecules such as tissue factor or indirectly through the secretion of factors that in turn induce thrombocytosis or leukocytosis. Certain pathways may be more characteristic of a particular cancer cell type. Figuring out what these pathways are in different tumors may help better understand how these risk factors may be targeted.Regarding ovarian cancer, it has been suggested that tumor-driven elevated platelet counts may be the pathway promoting thrombosis (Hisada Y and Mackman N. Blood 2017 :blood-2017-03-743211). Indeed platelet counts above 450,000/ul are reported in close to a third of women with ovarian cancer at diagnosis and murine models show aberrant IL-6/IL-6R expression by malignant cells which induce hepatic thrombopoietin secretion driving thrombocytosis (Stone RL et al N Engl J Med. 2012, 16;366(7):610-8) In fact, high IL-6 levels have been documented in woman with ovarian cancer. Another pathway is tumor-induced leucocytosis through expression of granulocyte colony stimulating factor (G-CSF) by ovarian cancer cells. Broad G-CSF and GCSF receptor expression has been reported across all ovarian cancer primary cell lines. Despite these observations, to our knowledge there are no reports showing that high platelet and/or leukocyte counts indeed correlate with VTE.We sought to evaluate the incidence and prognostic impact of VTE in women with an initial diagnosis of ovarian cancer as well as the prevalence and impact of thrombocytosis or leukocytosis on the incidence of VTE and on survival. We retrospectively looked at a cohort of women with epithelial ovarian cancer admitted at the NCI in Mexico between Jan-Nov 2017. Baseline pretreatment complete blood counts were obtained in 118 consecutive women (mean age 53; 19 - 82 years). We obtained pathological staging data, doppler US and CT images were reviewed for corroboration of extent of VTE. Standard statistical analysis was carried out using SPSS software version 20 (SPSS Inc., Chicago, IL).With a median follow up of 6.5 months (range: 0.5 - 13.7) the incidence of VTE was 13.4% (n=16). Consistent with other reports, patients often developed VTE at diagnosis 44% (n=7/16) associated with tumor activity before having been subjected to treatment. Six women developed VTE in association with surgery (mean of 24.6 days post-operatively; range 4- 58). The other 3 developed VTE in association with neoadjuvant chemotherapy (without surgery). As compared to patients with serous carcinoma, those with endometrioid carcinoma had a higher risk of VTE (8/91: 8.8%; 4/9: 44.4%, respectively, p<0.001) yet the number of patients with endometriod carcinoma is small. Women with VTE showed a trend to having a more advanced TNM stage at diagnosis, stage IV (44.7% vs 75%; p=0.083).Despite a high prevalence of thrombocytosis 37.8% (n=45) using cutoff values of >350x 103/□l or 450 x 103/□l. this was not a risk factor for symptomatic thrombosis in our patients. The median platelet counts for all patients were 379 x 103/□l (range 31 - 914 x 103/□l), with no differences in patients with or without VTE: 407x 103/□l (107 - 914 x103/□l) and 378 103/□l (31 - 792 x103/□l) p=0.779, respectively. Notably, pretreatment leukocytosis, neutrophilia and monocytosis were all found to be associated with VTE. Women with VTE had higher median leukocyte counts (8.9 [4.5 - 27.3] vs 7.3 [3.1 - 29.3] 103/□l, p=0.051). Median neutrophyl counts were higher in VTE patients (6.5[3.2 - 23.6] vs. 5.1[1.8 vs.27.4] 103/mm3, p=0.036). Likewise, median monocyte count was higher among VTE patients (0.7 [0.3 - 1.7] vs. 0.5[0.3 - 1.2], p=0.042). Although mortality data is still premature, VTE was associated with a higher mortality rate at both six (27% vs 7%), and at 12 months (40% vs 13.4%; p:0.003) Fig 1. Thrombocytosis did not correlate with survival Fig 2. Thus leukocytosis, but not thrombocytosis, may constitute a prothrombotic pathway in women with ovarian cancer. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of granulocyte colony-stimulating factor receptor in colitis-associated colonic carcinogenesis

Objective: To investigate the expression of granulocyte-colony stimulating factor receptor (G-CSFR) in a mouse model of colitis-associated cancer (CAC), and the roles of G-CSFR positive immune cells in the development of CAC. Methods: The C57BL/6 mouse model of CAC was established by azoxymethane and dextran sulphate sodium. Three different stages in the development of CAC, including inflammation (AD1), mild dysplasia (AD2) and adenocarcinoma (AD3) were simulated. Colon tissue was digested into single cell suspension and the expressions of G-CSF and G-CSFR were analyzed by real-time PCR and fluorescence activated cell sorter (FACS). The expressions of G-CSFR on T cell, macrophage and neutrophil were analyzed by FACS. Results: The establishment of mouse model can effectively simulate the disease progression of CAC. The results of real-time PCR detection showed that the expression level of G-CSF mRNA in AD1, AD2 and AD3 groups were 1.2, 7.3 and 18.0-fold changes of the control group, respectively. The differences between AD2, AD3 and control groups were statistically significant (P<0.05). G-CSFR mRNA levels in AD1, AD2 and AD3 groups were 1.5, 2.2 and 4.5-fold changes of the control group, respectively. The difference between AD3 and control groups was statistically significant (P<0.05). FACS showed that the percentages of CD45(+) G-CSFR(+) cells in colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.84±1.77)%, (41.48±4.15)%, (44.84±8.54)% and (57.76±1.95)%, respectively.The percentages of CD45(+) G-CSFR(+) cells in AD2 and AD3 groups were significantly higher than that of control group (P<0.05). The percentages of CD45(+) G-CSFR(+) macrophage in the colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.54±5.88)%, (47.14±5.25)%, (42.49±7.80)% and (29.25±8.24)%, respectively. The percentages of CD45(+) G-CSFR(+) T cells in these groups were (30.04±6.87)%, (29.65±8.08)%, (33.75±7.37)% and (33.32±9.85)%, respectively. The percentages of CD45(+) G-CSFR(+) granulocyte were (2.39±2.10)%, (4.05±1.56)%, (3.62±2.67)% and (2.26±0.85)%, respectively (P<0.05). The percentages of G-CSFR(+) macrophage and G-CSFR(+) T cells were significantly higher than that of G-CSFR(+) granulocyte (P<0.05). The differences between AD1 and control group, AD2 and control group, AD1 and AD2 group, AD2 and AD3 group were statistically significant (P<0.05). Conclusions: The expression of G-CSFR is significantly up-regulated in the development of CAC. The enrichment of G-CSFR(+) macrophages in the colon tissue suggests G-CSFR(+) macrophages participate in the development of CAC.

Highly Expressed Granulocyte Colony-Stimulating Factor (G-CSF) and Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) in Human Gastric Cancer Leads to Poor Survival.

BackgroundChemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear.Material/MethodsSeventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined.ResultsThe levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation.ConclusionsThese data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.

Effect of G-CSF in vitro Stimulation on Distribution of Peripheral Lymphocyte Subsets in the Healthy Persons

To investigate the effect of granulocyte-colony stimulating factor (G-CSF) in vitro stimulation on the distribution of lymphocyte subset in healthy human. Peripheral blood mononuclear cells (PBMNCs) were collected from 8 healthy volunteers by density gradient centrifugation on Ficoll-PaqueTM. In vitro 200 ng/ml G-CSF or 200 ng/ml G-CSF plus 10 µg/ml ConA directly act on PBMNCs, then the colleted cells were cultivated for 3 days. Lymphocyte subsets were stained with the corresponding fluoresce labeled antibodies and detected by flow cytometry. The levels of T cells in G-CSF group and G-CSF+ConA group were both higher than that in the control group (P<0.001, P<0.05). However, there were not significantly different in B cells and NK cells levels among the 3 groups. Furthermore, analysis of the effect of G-CSF on T cell subsets indicated that the levels of CD4+T cells and CD8+T cells in G-CSF group were both significantly higher than those in control group (P<0.01, P<0.05), Treg cells was not different between G-CSF and control group. Compared with the control group, the level of CD4+T cells, CD8+T cells and Treg cells in G-CSF+ConA group significantly increased (P<0.05, P<0.01, P<0.01). Analysis of G-CSF receptor (G-CSFR) expression showed that G-CSFR expression on T cells in G-CSF+ConA group dramatically increased, as compared with control group (P<0.01). The levels of CD4+T cells and CD8+T cells in healthy human peripheral blood can be increased by G-CSF stimulation. ConA can enhance the level of T cells and induce G-CSFR expression on T cells.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia

ABSTRACTObjective: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients.Method: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle.Results: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib.Conclusion: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.