Human Macrophage Colony-stimulating Factor Research Articles

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Replication and expression of oncolytic herpes simplex virus type Ⅱ in tumors

This study aimed to measure the duration and replication level of oncolytic herpes simplex virus type 2 (oHSV2) at the tumor injection site in BALB/c mice. Additionally, the expression level of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HSV-2 antibody in the serum was also measured. High and low doses of oHSV2-Fluc (firefly luciferin, Fluc) were injected into the mice's tumors to track the change and duration of fluorescence expression. The copy number of oHSV2 gene in tumor tissues was determined using quantitative real-time polymerase chain reaction (qPCR). Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of hGM-CSF and HSV-2 antibody in the serum. The tumor volume in the high-dose group was significantly lower than that in the control group (P < 0.01). Intratumor injection of oHSV2-Fluc showed that the carried Fluc could continue to express in the tumor, with fluorescence still detectable at day 11 and declining to undetectable level by day 18. The mRNA expression of oHSV2 was detected in tumor tissues of both high and low dose groups on day 9 using qPCR. ELISA results showed that the levels of HSV2 antibody and hGM-CSF in both high and low dose groups were significantly increased compared to the control group (P < 0.05) after collecting orbital blood. These findings suggest that oHSV2 can replicate in the tumor and sustainably express exogenous factors, thus effectively targeting and killing the tumor. Furthermore, intratumoral injection of oHSV2 resulted in higher levels of hGM-CSF and HSV-2 antibodies found in the mice's serum.

Low Serum Vitamin D Associated With Increased Tumor Size and Higher Grade in Premenopausal Canadian Women With Breast Cancer

IntroductionThis study investigated the association of Vitamin D with tumor characteristics in pre and postmenopausal women. Patients and MethodsA prospective cohort of 476 women with incident stage I-III breast cancer (BC) in Alberta, Canada comprised the study population. Vitamin D was measured as 25(OH)D concentration in serum samples collected at diagnosis (presurgery and prior to treatment initiation). Tumor characteristics including size, grade, receptor status, stage and nodal status were evaluated in regression models for association with Vitamin D and measured cytokines in models adjusted for menopausal status. ResultsMore than half of the women were diagnosed as stage I and Luminal A/B, most were postmenopausal, had sufficient Vitamin D levels, and were 56.6 years of age on average. Higher vitamin D levels were associated with decreased tumor size for all women with larger effect seen in premenopausal status. Insufficient vitamin D levels were significantly associated with increased risk of higher grade, but only in premenopausal women. Elevated human granulocyte macrophage colony-stimulating factor was an independent risk factor associated with increased risk of higher-grade tumors. ConclusionWomen with sufficient Vitamin D levels at BC diagnosis had smaller and lower grade tumors compared to women with insufficient vitamin D, especially among premenopausal women. Maintaining adequate vitamin D levels in premenopausal women could improve prognostically important BC characteristics at diagnosis.

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

Efficacy of Low-Dose rhGM-CSF Treatment in a Patient With Severe Congenital Neutropenia Due to CSF3R Deficiency: Case Report of a Novel Biallelic CSF3R Mutation and Literature Review.

This study reports the clinical manifestations, genetics, and efficacy of treatment with the efficacy of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-GSF) of a 2-year-old female patient with severe congenital neutropenia (SCN) type 7 (SCN7) caused by novel biallelic mutations in the colony-stimulating factor 3 receptor (CSF3R) gene. Genetic diagnosis of the patient was performed by whole-exome and Sanger sequencing. Expression of the CSF3R gene in the peripheral neutrophils of the patient was detected by real-time PCR and Western blotting. The patient presented with recurrent suppurative tonsillitis and decreased absolute neutrophil count <0.5 × 109/L. Novel heterozygous mutations were found to be inherited from each parent (maternal c.690delC [p.met231Cysfs*32] and paternal c.64+5G>A). The patient's neutrophils had lower CSF3R mRNA and protein levels than those of the parents. Low-dose rhGM-CSF (3 μg/kg/day once a week) prevented recurrent infection in the patient. These results demonstrate that the clinical manifestations of SCN7 with biallelic CSF3R mutations and downregulated CSF3R can be effectively treated with rhGM-CSF.

Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.

LncRNA TUG1 positively regulates osteoclast differentiation by targeting v-maf musculoaponeurotic fibrosarcoma oncogene homolog B

Osteoclast differentiation-mediates bone resorption is the key biological basis of orthodontic treatment while the specific mechanism of osteoclastogenesis remains unclear. This study aims to explore the underlying mechanism of the osteoclast differentiation from the perspective of long non-coding RNA (LncRNA). In the present study, the osteoclast differentiation of CD14+ peripheral blood mononuclear cells (PBMCs) was induced by recombinant human macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), and LncRNA TUG1 expression was dramatically elevated during this process. Functionally, the silence of TUG1 in CD14+ PBMCs decreased tartrate-resistant acid phosphatase (TRAP)-positive cell numbers and the protein levels of TRAP, nuclear factor of activated T cell c1 (NFATc1), and osteoclast-associated receptor (OSCAR), whereas increased V‐maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB) protein level. The subsequent experiments confirmed that TUG1 lessened the MafB protein level via accelerating its degradation. Then, the interference of MafB reversed the inhibitory effect of si-TUG1 on osteoclastogenesis, including increased the TRAP-positive cell numbers and up-regulated the protein levels of osteoclast markers. Finally, the in vivo experiments displayed that the increased TUG1 levels could promote tooth movement and bone resorption via facilitating osteoclast differentiation in the rat model of orthodontic tooth movement. In summary, TUG1 overexpressed during the process of osteoclast differentiation and positively regulated osteoclast differentiation by targeting MafB.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E.coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

Effects of recombinant human granulocyte macrophage colony stimulating factor gel on treatment of full-thickness frostbite wounds on foot and hand

Objective: To explore the effects of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) gel on treatment of thefull-thickness frostbite wounds on foot and hand. Methods: From November 2013 to April 2017, a total of 45 patients of 71 full-thickness frostbite wounds on foot and hand meeting the inclusion criteria were admitted to the First Hospital of Jilin University and the prospective randomized controlled study was done. The patients were divided into rhGM-CSF group of 24 patients with 35 wounds and control group of 21 patients with 36 wounds according to the random number table. There were 20 males and 4 females, aged (38±13) years among patients in rhGM-CSF group, and there were 19 males and 2 females, aged (36±14) years among patients in control group. Patients in 2 groups were performed with the same systemic treatment of rewarming, anti-inflammation, pain relief, anti-infection, anti-coagulation, and thrombolysis. Wounds of patients in rhGM-CSF group and control group were respectively treated with rhGM-CSF gel and aloe vera gel for external usage with 10 mg for every square centimeter and dressing change once every 24 hours, until wounds healed completely. The wound inflammatory response was scored on treatment day (TD) 1, 3, 7, 14, wound secretion was collected for bacteria culture and positive bacteria detection rate was calculated before treatment and on TD 6 and 12, adverse drug reaction after drug use was observed, and the complete wound healing time was recorded. Data were processed with Fisher's exact probability test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results: The scores of wound inflammatory response of patients in 2 groups on TD 1 and 3 were close (t=0.37, 2.93, P>0.05). The scores of wound inflammatory response of patients on TD 7 and 14 in rhGM-CSF group were significantly higher than those in control group (t=5.77, 5.83, P<0.01). The results of bacteria culture of wound secretion of patients in 2 groups before treatment were negative. The positive bacteria detection rates of wound secretion of patients in rhGM-CSF group on TD 6 and 12 were 5.71% (2/35) and 22.86% (8/35), which were slightly lower than 13.89% (5/36) and 30.56%(11/36) in control group respectively, but there was no significantly statistical difference (P>0.05). No adverse drug response occurred in patients in rhGM-CSF group, while 1 patient in control group had adverse drug response, with symptoms of redness and swelling of wounds and patchy erythema on skin around wounds, which were alleviated by irrigating with normal saline. The complete wound healing time of patients in rhGM-CSF was (12.3±0.5) d, which was significantly shorter than (16.5±0.8) d in control group (t=24.89, P<0.05). Conclusions: The topical rhGM-CSF gel has effects of shortening time of wound healing and reducing inflammatory response of wound on treatment of full-thickness frostbite wounds on foot and hand, which is safe in clinical application.

Efficacy, Prognosis and Safety of Decitabine Combined with Low-Dose Cytarabine in the Treatment of Elderly Patients with Relapsed/Refractory Acute Myeloid Leukemia

To investigate the efficacy, prognosis and safety of decitabine combined with low-dose CAG regimen in the treatment of elderly patients with acute myeloid leukemia （AML）. The clinical data of 40 elderly patients with relapsed/refractory AML （69-85 years old） admitted to our hospital from January 2014 to August 2016 were analyzed retrospectively. 40 patients were divided into combination therapy group and CAG group according to different treatment methods. 20 patients of the combination therepy group were treated with decitabine combined with low-dose CAG （decitabine, 15 mg/m2, d 1; aclarithromycin, 10 mg/m2, d 3-6; Cytidine, 10 mg/m2, d 1-14; recombinant human granulocyte macrophage colony-stimulating factor （G-CSF） for injection, 200 μg/（m2·d）, d 1-14）. 20 patients of CAG group were treated by the standard CAG protocol （acralmycin 20 mg/m2, d 1-4; cytarabine for injection, 15 mg/m2, d 1-14; G-CSF 400 μg/（m2·d）, d 1-14）. One course of treatment lasted for 2 weeks, after 2 courses of continuous medication, the complete remission rate （CR）, overall remission rate （ORR）, overall survival （OS）, 1-year survival rate, hemoglobin, white blood cells, platelets improvement, and incidence of adverse reactions were compared. In combination therapy group the CR was 55.00% （11/20）, OR was 85.00% （17/20）, but in the CAG group CR was 30.00% （6/20）, and OR was 50.00% （10/20）. Till to February 2018, out of 40 patients 17 survived, 20 died, and 3 failed to be followed-up. The median follow-up time was 12 （2 to 35） months; the median survival time in the comtination therapy group was 13 （2-35） months, and the 1-year OS rate was 70.00%, and the median survival time of the CAG group was 10 （2-31） months, and the 1-year OS rate was 50.00%, without staistical significance between the 2 groups （P>0.05）. After treatment, the WBC and Plt counts in the combination therapy group were higher than those in the CAG group, but the Hb level was lower than that in the CAG group with statistically significant difference （P<0.05）. In the combination therapy group, the incidence of lung infection, nausea and vomiting was higher than that of the CAG group （65.00% vs 25.00%, 50.00% vs 20.00%）, with statistically significant difference （P<0.05）. Decitabine combined with low-dose CAG regimen is effective for the treatment of relapsed/refractory AML in the elderly. Compared with the standard CAG regimen, the long-term efficacy of this regimen is not different significantly, but its adverse reactions are increase, thus the preventive treatment should be given in time.

Boosting Abscopal Response to Radiotherapy with Sargramostim: A Review of Data and Ongoing Studies.

Drug development in oncology today routinely focuses on approaches that utilize the patients’ immune system to destroy the malignancy. Combinatorial approaches of antineoplastic agents, both new and old, are being incorporated in the armamentarium of cancer treatments. The overarching goal of therapy remains the achievement of a complete and durable response with long term remission or cure. One approach in advancing treatment is aimed at strategies that improve immunological memory to induce long lasting immunity against the tumor. Although radiation therapy has not traditionally been thought to elicit an immunological effect, an increasing number of reports document the induction of an immune response against a tumor that kills cancer cells distant to the original site of treatment after local irradiation to a tumor. This phenomenon is called an abscopal effect. Since radiation alone is rarely associated with such a response, it is being combined with immuno-oncology drugs in an attempt to enhance response. One such strategy combines sargramostim, a recombinant human granulocyte macrophage colony stimulating factor (rhu GM-CSF), with radiotherapy. GM-CSF is a cytokine secreted by multiple cells types that promotes maturation of dendritic cells and enables the presentation of tumor-associated antigens to generate a T-cell response. This review article discusses the outcomes of clinical trials and case reports examining the efficacy and safety of combining radiation therapy with this immunomodulatory agent. We will also examine future studies and challenges facing the translation of this therapeutic approach.

Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants.

As cutaneous melanoma (CM) currently remains with a bleak prognosis, thorough investigation of new treatment options are of utmost relevance. In the phase II/III randomized clinical trial (CASVAC-0401), the repeated immunization of stages IIB-III CM patients with the irradiated, allogeneic cellular CSF-470 vaccine plus the adjuvants bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) demonstrated a significant benefit over IFN-alpha2B treatment in distant metastasis-free survival. Here we present on the short and long term immune monitoring results after completing the 2-year protocol; a continuation of the previous report by Mordoh et al. (1). We demonstrate that the repeated CSF-470 vaccinations stimulated a long term cellular and humoral immunity response directed against the vaccine antigens. In the case of 2 patients, we are able to show that a similar immune response was generated against autologous antigens. Evaluation of inhibitory receptor co-expression on patient's T cells indicates that the vaccination protocol did not stimulate T cell exhaustion. In order to better understand the basis for the efficacious vaccine responses observed, we investigated the short term immune events following vaccine injection. A significant increase in C-reactive protein (CRP) and IL-6 was observed 24 h after vaccination, with in vitro studies suggesting IL-6 production occurs in the vaccine site. We demonstrate that CRP enhances the cytotoxicity of peripheral blood mononuclear cells (PBMC) against melanoma cells in an in vitro model. Additionally, CRP stimulates the release of pro and anti-inflammatory cytokines from PBMC. As our results demonstrate that successive vaccinations with CSF-470 plus adjuvants promoted an increase in both anti-tumor innate and adaptive immunity, we propose a subsequent model of action.

An Efficient and Rapid Two-step Purification Method for Active Human Macrophage Colony-stimulating Factor from Escherichia coli

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of mononuclear phagocytes. To be biologically active, M-CSF must form a homodimer linked by disulfide bridges. However, the refolding step of recombinant M-CSF is not only extremely laborious but also rate-limiting. Here, we describe an efficient and simplified method for refolding and purifying M-CSF from Escherichia coli. A truncated M-CSF (amino acids 36–181) was tagged with histidine, expressed, and then purified using nickel affinity columns under denaturing conditions. Our redox refolding buffer containing a mixture of reduced and oxidized glutathione and arginine correctly refolded M-CSF. Chromatography on Q-sepharose columns selectively purified the M-CSF dimer from the monomeric form. The dimeric nature of the purified M-CSF was confirmed using multi angle light scattering combined with high performance liquid chromatography. Moreover, cell proliferation and osteoclast differentiation assays using bone marrow-derived macrophages demonstrated that the recombinant M-CSF was biologically active ex vivo.

Recombinant human granulocyte macrophage colony-stimulating factor for the prevention and treatment of oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma

Objective To observe the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on the prevention and treatment of oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma. Methods A total of 64 patients with locally advanced nasopharyngeal carcinoma from April 2015 to April 2017 in the Second Affiliated Hospital of Xi'an Jiaotong University were enrolled. The patients were randomly divided into the observation group (32 cases) and the control group (32 cases) according to the random number table method. Both groups were treated with intensity modulated radiotherapy (IMRT) and synchronized with TP regimen (docetaxel + cisplatin) chemotherapy for 2 cycles. Both groups were treated with mouthwash at the beginning of radiotherapy to prevent oral mucositis. The observation group was given rhGM-CSF mouthwash, and the control group was given compound borax mouthwash. The oral mucositis and the oral pain during the treatment and the end of the treatment were evaluated by using American Radiation Oncology Group (RTOG)grading criteria and visual analogue scoring method (VAS) grading criteria. The time of onset of oral mucositis and the total time of radiotherapy in both groups was also recorded. Results All the patients were treated with concurrent chemoradiotherapy. The total radiotherapy time in the observation group was less than that in the control group [(46.4±1.6) vs. (48.2±3.2) d, t=-2.720, P=0.009]. The clinical total effective rate was 93.8% (30/32) in the observation group and 96.9% (31/32) in the control group respectively (χ 2=0.35, P = 0.554). The occurrence of grade 1, 2 and 3 oral mucositis in the observation group was (20.9±2.5), (29.3±2.4), and (34.5±1.8) d respectively, which was latter than that in the control group [(16.3±2.0), (24.2±2.2) and (31.0±2.2) d] respectively (t = 8.125, P = 0.000; t = 8.840, P = 0.000; t = 6.944, P = 0.001). During concurrent chemoradiotherapy, the incidence of grade 3 oral mucositis in the observation group was lower than that in the control group [31.2% (10/32) vs. 56.2% (18/32); Z = -2.197, P = 0.028]. At the end of concurrent chemoradiotherapy, the total incidence of grade 2 and grade 3 oral mucositis in the observation group was lower than that in the control group [53.1% (17/32) vs. 81.2% (26/32); Z = -2.708, P = 0.007]. The incidence of moderate and severe oral pain caused by oral mucositis in the observation group was lower than that in the control group [46.9% (15/32) and 6.2% (2/32) vs. 59.4% (19/32) and 15.6% (5/32), respectively; Z = -2.009, P = 0.045]. Conclusion rhGM-CSF mouthwash can delay the occurrence of oral mucositis, reduce the incidence of oral mucositis and oral pain to effectively prevent and treat oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma, which is worthy of clinical application. Key words: Nasopharyngeal neoplasms; Radiotherapy; Antineoplastic combined chemotherapy protocols; Stomatitis; Granulocyte macrophage colony-stimulating factors, recombinant

Single Platform for Gene and Protein Expression Analyses Using Luminescent Gold Nanoclusters.

A single platform for gene and protein expression studies is proposed to pursue rapid diagnostics. A common method to synthesize gold (Au) nanoclusters on both DNA and protein template was developed using a benchtop device. The method of synthesis is rapid and versatile and can be applied to different classes of DNA/protein. Employing luminescent Au nanoclusters as the signal-generating agents, the device enables carrying out reverse transcriptase polymerase chain reaction and array-based analyses of multiple genes/proteins simultaneously using switchable holders and custom-designed software. The device and methods were applied to evaluate gene profiling related to apoptosis in HeLa cancer cells and further to analyze the protein expressions of glutathione-S-transferase (GST) and GST-tagged human granulocyte macrophage colony-stimulating factor (GST-hGMCSF) recombinant proteins purified from bacterial strains of BL21(DE3) Escherichia coli (E. coli). The device with user-friendly methods for diagnosis using the luminescence of Au nanoclusters offers potential use in disease diagnostics with a vision to extend health care facilities especially to remote geographical locations.

Effects of intratumoral injection of immunoactivator after microwave ablation on antitumor immunity in a mouse model of hepatocellular carcinoma.

This study investigated the effects of intratumoral injection of immunoactivator after microwave ablation on antitumor immunity in a mouse model of hepatocellular carcinoma. Hepatocellular carcinoma cell line Hepa1-6 was subcutaneously injected into C57/B6 mice to establish a mouse model of hepatocellular carcinoma. When tumor diameter reached 8 mm, microwave ablation was performed for 3 min with temperature controlled at 55°C. Cytokine sustained-release microspheres (CytoMPS) containing human interleukin-2 (hIL-2) and mouse granulocyte macrophage colony-stimulating factor (mGM-CSF) were injected into the tumor of mice in the experimental group (n=5) at 3, 7 and 14 days after ablation, while sustained-release microspheres containing no cytokine were used in the control group (n=5). Mice were sacrificed on the 17th day after ablation, and CD4+ and CD8+ T cells in peripheral blood were counted by flow cytometry. Spleen was collected from the mice to isolate lymphocytes. Lactate dehydrogenase (LDH) release assay was used to determine the cytotoxicity of spleen cells to Hepal-6 cells. Injection of CytoMPS after ablation increased the percentage of CD4+ and CD8+ T cells in peripheral blood. Cytotoxicity of CD8+ CTL to Hepal-6 is significantly higher in experimental group than in control group (P<0.01). The results showed that intratumoral injection of CytoMPS containing hIL-2 and mGM-CSF can significantly increase the proportion of CD4+ and CD8+ T cells in blood and increase the cytotoxicity of CTL cells to tumor cells in mice with hepatocellular carcinoma.

Macrophage colony stimulating factor enhances non-small cell lung cancer invasion and metastasis by promoting macrophage M2 polarization

Objective: To investigate the key cytokine which polarizes M2 macrophages and promotes invasion and metastasis in non-small cell lung cancer (NSCLC). Methods: After co-culture with A549 cells in vitro, the proportion of CD14(+) CD163(+) M2 macrophages in monocytes and macrophage colony stimulating factor (M-CSF) levels in culture supernatant were detected by flow cytometry, ELISA assay and real-time qPCR, respectively. The effects of CD14(+) CD163(+) M2 macrophages on invasion of A549 cells and angiogenesis of HUVEC cells were measured by transwell assay and tubule formation assay, respectively. The clinical and prognostic significance of M-CSF expression in NSCLC was further analyzed. Results: The percentage of CD14(+) CD163(+) M2 macrophages in monocytes and the concentration of M-CSF in the supernatant followed by co-culture was (12.03±0.46)% and (299.80±73.76)pg/ml, respectively, which were significantly higher than those in control group [(2.80±1.04)% and (43.07±11.22)pg/ml, respectively, P< 0.05]. Human recombinant M-CSF promoted M2 polarization of macrophages in vitro. M2 macrophages enhanced the invasion of A549 cells (66 cells/field vs. 26 cells/field) and the angiogenesis of HUVEC cells (22 tubes/field vs. 8 tubes/field). The mRNA expression of M-CSF in stage Ⅰ-Ⅱ patients (16.23±4.83) was significantly lower than that in stage Ⅲ-Ⅳ (53.84±16.08; P<0.05). M-CSF levels were associated with poorer overall survival and disease-free survival in NSCLC patients (P<0.05). Conclusions: Tumor-derived M-CSF can induce CD14(+) CD163(+) M2 polarization of macrophages, which can further promote the metastasis and angiogenesis of NSCLC. M-CSF could be used as a potential therapeutic target of NSCLC.

Recombinant human granulocyte macrophage colony stimulating factor in deep second-degree burn wound healing.

Background:The aim of this study was to explore the effects of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) on deep second-degree burn wound healing.Methods:In this study, 95 patients with a total of 190 burn wounds were treated with either rhGM-CSF or placebo, separated into 2 groups by treatment type. Wound healing rate, wound healing time, histopathological condition, and scar scale were all compared between the 2 groups.Results:The healing rates in the rhGM-CSF group were remarkably higher than those in the placebo group (P < .01). The wound healing time in the rhGM-CSF group (18.8 ± 7.6 days) was significantly shorter than that in the placebo group (25.5 ± 4.6 days, P < .01). On the 14th day and 28th day, the average optical density of vascular endothelial factor (VEGF) in the rhGM-CSF group was larger than that in the placebo group. Meanwhile, the average optical density of fibroblast growth factor (FGF) in the rhGM-CSF group was also larger than that in the placebo group. Furthermore, the Vancouver scar scale scores of pigmentation, pliability, height, and vascularity were notable lower in the rhGM-CSF group than those in the placebo group (P < .01).Conclusion:The results suggest that rhGM-CSF can significantly accelerate deep second-degree burn wound healing.

Comparative phenotypic and functional analyses of the effects of autologous plasma and recombinant human macrophage-colony stimulating factor (M-CSF) on porcine monocyte to macrophage differentiation

Porcine monocyte-derived macrophages (moMΦ) have been employed as a model cell in numerous studies of the porcine immune system. However, the lack of a standardized method for moMΦ differentiation hampers the comparison of results coming from the use of different laboratory protocols. In this study we compared the use of varying concentrations of autologous plasma (10, 20 and 30% v/v) or recombinant human macrophage-colony stimulating factor (hM-CSF; 50, 100, and 200ng/ml) to differentiate porcine monocytes into macrophages. Changes in cell morphology and surface marker expression were assessed by confocal microscopy and flow cytometry. Macrophage differentiation was evaluated by analysing TNF-α response to LPS stimulation and determining cytokine secretion patterns under both basal conditions and after classical and alternative activation. The effects of the differentiation methods on metabolic activity and susceptibility to infection with the myelotropic African swine fever virus (ASFV) were also evaluated. Monocytes cultured using the different culture conditions tested augmented in dimension and cellular complexity, but increasing porcine plasma concentrations resulted in a dose dependent enhancement in granularity and a marked pleomorphism. As expected, CD163, MHC class II DR and CD203a expression were up-regulated in both hM-CSF (M-CSF-moMΦ) and autologous plasma cultured macrophages (AP-moMΦ), although a lower percentage of CD163+ cells were found following differentiation with high percentages of porcine plasma. We observed enhanced number of viable cells using high concentration of hM-CSF compared to porcine plasma, suggesting a proliferative effect. Irrespective of differentiation conditions, monocyte differentiation into macrophages resulted in an increased susceptibility to ASFV and yielded larger amounts of LPS-induced TNF-α. AP-moMΦ showed a higher basal release of IL-1RA compared to those cultured with hM-CSF and displayed a reduced ability to respond to classical activation, suggesting that the use of high percentages of porcine plasma led to the acquisition of a M2-like phenotype. We conclude that all the protocols tested in this study can be considered as suitable to produce porcine moMΦ, although the use of hM-CSF provides high responsiveness to M1 polarization. Since a higher phenotypic and functional inter-animal variability was observed in AP-moMΦ, we propose that the use of low concentration of hM-CSF should be adopted as the method of choice to provide a better reproducibility between experiments.

Molecular mechanism and functional role of macrophage colony‑stimulating factor in follicular granulosa cells.

Our previous demonstrated that macrophage colony‑stimulating factor (M‑CSF) stimulated the production of estradiol (E2) and progesterone (P) in luteinized granulosa cells (GCs), and that its secretion may be regulated by follicle‑stimulating hormone (FSH). The present study aimed to examine the effect of M‑CSF alone or with Letrozole treatment on the function of non‑luteinizing granulosa cells, using the COV434cell line, and its interaction with FSH. Human luteinized granulosa cells (LGC) were isolated from the follicular fluid of superovulated infertile patients (average age, 30.8±2.1years) undergoing an intracytoplasmic sperm injection. The LGC were cultured with various concentrations of recombinant human macrophage colony stimulating factor (rhM‑CSF; 0, 10, 25, 50 or 100ng/ml), rhM‑CSF+Letrozole (10‑6mol/l), rhFSH (0, 10, 25, 50 or 100IU/ml), or rhFSH+Letrozole (10‑6 mol/l). E2 concentrations in the media were measured using ELISA. The expression levels of the FSH receptor and the M‑CSF receptor were detected via reverse transcription‑quantitative polymerase chain reaction. Following COV434cell treatment with M‑CSF, cell proliferation was quantified using the MTS assay and protein expression was detected by western blotting. It was demonstrated that M‑CSF and FSH stimulated the production of E2. The production of FSH receptors was enhanced by rhM‑CSF or rhM‑CSF+Letrozole invitro in a dose‑dependent manner. It was observed that rhFSH promoted the expression of the M‑CSF receptor, at a certain concentration. Proliferation of COV434cells increased in a dose‑dependent manner following treatment with RhM‑CSF. Furthermore, M‑CSF induced the phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38; however, the level of E2 in the medium was not altered when the cells were pretreated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580. The present study suggested that M‑CSF may be important in regulating the response of GCs to gonadotropin and may have a promotive effect in the early phase of follicular development. The biological effects of M‑CSF may partially be attributed to activation of the JNK and p38 signaling pathways. M‑CSF may represent a novel follicular development regulator agent in the future.