心力衰竭患者外周血粒细胞-巨噬细胞集落刺激因子与白细胞介素8的变化及其意义

目的检测心力衰竭患者外周血粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素8(IL-8)及白细胞的水平,并探讨他们在心力衰竭中的关系.方法59例心力衰竭患者,根据NYHA分级分成心功能Ⅱ、Ⅲ、Ⅳ级3组,测定血清GM-CSF及IL-8水平采用放射免疫法,并镜检血WBC数.并与年龄、性别相匹配的正常对照组(19例)进行比较.结果心力衰竭组外周血GM-CSF、IL-8及WBC水平与正常对照组比较有显著性差异(P＜0.01),且随心力衰竭程度的加重而更明显;不同病因的心力衰竭患者之间无显著差异(P＞0.05);心力衰竭患者外周血GM-CSF与IL-8及WBC呈显著正相关(r值分别为0.602,0.541;P＜0.01).结论心力衰竭患者外周血GM-CSF、IL-8及WBC水平增高,并随心衰程度的加重而加重;提示炎症反应可能参与了心力衰竭的发生和发展,而GM-CSF在其中可能起重要作用.     

Granulocyte-macrophage colony-stimulating factor as an arteriogenic factor in the treatment of ischaemic stroke

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces arteriogenic growth of collateral vessels after occlusion of cardiac or peripheral arteries. Recently, evidence has been provided that arteriogenesis also occurs in the brain under conditions of reduced arterial blood supply. Hemispheric hypoperfusion induced by unilateral carotid and bilateral vertebral artery occlusion (three-vessel occlusion, [3-VO]) led to the growth of the anterior and posterior segments of the circle of Willis, which is the main collateral pathway between the origins of the anterior, middle and posterior cerebral arteries. GM-CSF applied subcutaneously at daily doses of 40 μg·kg^-1 resulted in the marked acceleration of this process. Within one week after the onset of treatment, the diameter of the posterior segment of the circle of Willis enlarged to 170% of control, blood flow and the haemodynamic reserve capacity of the brain returned to normal, and haemodynamic stroke, induced after 3-VO by systemic hypotension, was greatly alleviated. GM-CSF-induced stimulation of arteriogenesis in the hypoperfused brain thus provides powerful protection against ischaemic stroke.     

Granulocyte-macrophage colony-stimulating factor as an arteriogenic factor in the treatment of ischaemic stroke

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces arteriogenic growth of collateral vessels after occlusion of cardiac or peripheral arteries. Recently, evidence has been provided that arteriogenesis also occurs in the brain under conditions of reduced arterial blood supply. Hemispheric hypoperfusion induced by unilateral carotid and bilateral vertebral artery occlusion (three-vessel occlusion, [3-VO]) led to the growth of the anterior and posterior segments of the circle of Willis, which is the main collateral pathway between the origins of the anterior, middle and posterior cerebral arteries. GM-CSF applied subcutaneously at daily doses of 40 microg.kg(-1) resulted in the marked acceleration of this process. Within one week after the onset of treatment, the diameter of the posterior segment of the circle of Willis enlarged to 170% of control, blood flow and the haemodynamic reserve capacity of the brain returned to normal, and haemodynamic stroke, induced after 3-VO by systemic hypotension, was greatly alleviated. GM-CSF-induced stimulation of arteriogenesis in the hypoperfused brain thus provides powerful protection against ischaemic stroke.     

Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro.

Interest in human dendritic cells (DC) has been heightened recently by the discovery that this cell type is a primary target of the human immunodeficiency virus, the causative agent of AIDS. DC are bone marrow-derived cells with an extraordinarily potent ability to promote the immunological activity of T lymphocytes. Unfortunately, since DC constitute less than 0.5% of peripheral blood mononuclear cells and die within a few days of their isolation, they are not readily accessible to study. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine with well-recognized effects on granulocyte and macrophage maturation, profoundly affects the morphology and viability of DC isolated from peripheral blood. GM-CSF not only promotes DC survival but also induces DC differentiation to mobile, reversibly adherent cells with long-branched projections. DC cultured in GM-CSF survive for up to 6 wk and retain their ability to stimulate the proliferation of T cells in allogeneic and autologous mixed leukocyte reactions.     

粒-巨噬细胞集落刺激因子治疗血液肿瘤化疗所致口腔黏膜炎的疗效观察

[目的]研究粒-巨噬细胞集落刺激因子(GM-CSF)悬液对治疗血液肿瘤化疗所致口腔黏膜炎(OM)的疗效。[方法]选取136例血液肿瘤化疗后出现口腔黏膜炎病人,随机将136例病人分为观察组和对照组,对照组用替硝唑漱口液漱口,观察组予以GM-CSF混悬液涂布溃疡处,比较两组不同部位和程度OM的愈合情况。[结果]观察组与对照组OM愈合时间比较差异有统计学意义(P<0.01);唇部OM愈合最早,硬腭部OM愈合最晚。[结论]对血液肿瘤化疗所致的OM,应用GM-CSF混悬液涂布溃疡处明显优于常规替硝唑漱口液治疗。     

Sunflower seed oil-enriched product can inhibit Ehrlich solid tumor growth in mice

BACKGROUND: Sunflower seed oil (SSO) effect on solid and ascitic forms of Ehrlich tumor was evaluated. METHODS: Solid or ascitic Ehrlich tumor-bearing Swiss mice were treated daily, by subcutaneous route, with 200 microl of SSO. The solid tumor-bearing footpad was measured every 3 days and ascitic tumor-bearing mice had their ascites collected and quantified. At the end of the SSO treatment, the total cell number in lymphoid organs was quantified. RESULTS: Subcutaneous treatment with SSO inhibits the solid tumor growth and increases lymph node cell number in animals with solid tumor, but has no effect on animals with ascitic tumor. CONCLUSIONS: SSO can delay the solid tumor growth, possibly due to better absorption of this treatment by draining lymph nodes.     

Neutralizing anti-insulin-like growth factor receptor 1 antibodies inhibit receptor function and induce receptor degradation in tumor cells

Insulin-like growth factor receptor 1 (IGFR1) plays a crucial role in oncogenic transformation [C. Sell et al., Mol. Cell. Biol., 14: 3604-3612,1994]. Compared with the normal human mammary epithelial cell line MCF12A, MCF7 human mammary carcinoma cells overexpress IGFR1 on the cell surface. To measure the effects of IGFR1 inhibition on tumor cells, we tested two mouse neutralizing antibodies against human IGFR1 in cell-based assays. Both MAB391 and anti-IR3 antibodies inhibit IGFR1 autophosphorylation upon IGF-I ligand stimulation with IC50s of 0.58 and 0.80 nM, respectively. When cells were treated with neutralizing anti-IGFR1 antibodies for > or = 4 h, the total receptor level was dramatically decreased. IGF-I-stimulated activation of AKT was also inhibited by anti-IGFR1 antibodies. Furthermore, MAB391 and anti-IR3 inhibited the growth of MCF7 cells in soft agar. In addition to MCF7 cells, MAB391 also inhibited IGFR1 autophosphorylation and induced IGFR1 down-modulation in HT29 colorectal and Du145 prostate cancer cells. Therefore, neutralizing antibodies against IGFR1 represent a valid approach to inhibit growth of tumor cells.     

Phagocytosis of tumor cells by human monocytes cultured in recombinant macrophage colony-stimulating factor

Macrophages and cultured human monocytes can mediate efficient antibody-dependent cytotoxicity (ADCC) against human tumor cells using monoclonal antibodies (mAbs). The mechanism of this killing is usually assumed to involve secreted factors (reactive oxygen intermediates, tumor necrosis factor, or other cytotoxic factors) leading to target cell lysis. In this study, we present evidence that phagocytosis of intact target cells is the principal mechanism of antitumor cytotoxicity in our in vitro model of ADCC by cultured monocytes. Human monocytes cultured in recombinant human macrophage colony-stimulating factor ingested up to 100% of fluorochrome-labeled melanoma and neuroblastoma target cells, in the presence of an appropriate antitumor mAb. Electron microscopy demonstrated phagocytosis of intact tumor cells by cultured monocytes during ADCC. All of the radionuclide in radiolabeled target cells was taken up by monocytes during phagocytosis. By preventing the release of radioisotope tracers, phagocytosis thus prevents the detection of this very efficient form of cytotoxicity by most conventional assays.     

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.     

短程高剂量重组人粒细胞-集落刺激因子对小鼠外周血造血干细胞的动员作用

背景:外周血造血干细胞用于损伤组织的修复及再生需要快速有效的动员方法.常规的动员方案多采用重组人粒细胞-集落刺激因子或大剂量化疗联合重组人粒细胞-集落刺激因子,该方案耗时长、程序复杂.目的:对常规的外周血造血干细胞动员方案进行改进,观察短程高剂量重组人粒细胞-集落刺激因子动员KM小鼠外周血造血干细胞的效果.设计、时间及地点:于2006-09/2007-03在大连医科大学附属第二医院血液科实验室开展的随机对照动物实验.材料:雄性8周龄KM小鼠24只,以给药方式分为3组;常规给药组、短程高剂量给药组、生理盐水对照组.方法:常规给药组,皮下注射重组人粒细胞-集落刺激因子.0.2μg次,2次/d,共6d;短程高剂量给药组,前4d注射等量生理盐水,后2d皮下注射重组人粒细胞-集落刺激因子.2.5μg次,2次/d;生理盐水对照组,每日注射等量生理盐水,共6d.主要观察指标:各组小鼠外周血白细胞计数、粒-巨核细胞集落数.结果:常规剂量组及短程大剂量组小鼠外周血白细胞迅速增高,对照组无明显变化.粒-巨核细胞集落培养7d后,常规剂量组和短程高剂量组的粒-巨核细胞集落计数较对照组显著升高达50倍以上(P<0.01),常规剂量组和短程高剂量组两组间比较差异无显著性意义(P0.05).结论:短程高剂鼠重组人粒细胞-集落刺激因子动员小鼠外周血造血干细胞,有着良好的可行性及有效性,是对小鼠外周血造血干细胞动员模型的成功改进     

Granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-12 synergize with calcium ionophore to enhance dendritic cell function

The authors previously showed that monocytes treated with calcium ionophore (CI) acquire characteristics of mature dendritic cells (DC) in part through a calcineurin-dependent pathway. In this study, the authors evaluated the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), and interleukin-12 (IL-12) alone or in combination with CI to induce DC characteristics in peripheral blood monocytes. Monocytes obtained by leukapheresis and countercurrent centrifugal elutriation were cultured with calcium, cytokines, or both, profiled by flow cytometry, and assessed for antigen uptake and sensitization of autologous CD8+ T cells to antigen. Monocytes treated with the combination of GM-CSF, IL-2, and IL-12 resulted in immunophenotypic and antigen uptake profiles typical of immature DC, including loss of surface CD14 expression, de novo low-level expression of B7.1, negligible CD83 expression, marked enhancement of CD40 and ICAM-1, and high major histocompatibility complex class I and II levels. A high level of antigen uptake by macro-pinocytosis was observed. In contrast, CI treatment significantly up-regulates B7.1, B7.2, CD40, CD54, and CD83 and substantially down-regulates CD14 and macro-pinocytosis, a profile consistent with mature DC. Many CI-induced modulations, but none resulting from cytokine treatment alone, were inhibited by the calcineurin phosphatase inhibitor cyclosporin A. Compared with monocytes treated with CI alone, combined treatment of monocytes with GM-CSF, IL-2, IL-12, and CI augmented B7.1 and CD83 expression and enhanced sensitization of autologous CD8+ T cells to melanoma-antigen-derived peptides. These results suggest that several independent pathways of DC activation can cooperatively enhance the function of monocyte-derived DC.     

Differential effects of granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor on tumor necrosis factor and interleukin-1 production in human monocytes

Human peripheral blood monocytes (PBM) cultured in the presence of 100-5,000 u/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) for 24 hr secreted small quantities of tumor necrosis factor (TNF), but not interleukin-1 (IL-1). The activation of PBM to produce TNF was weak and could be blocked by polyclonal anti-GM-CSF anti-serum. Neither LPS nor IL-2 were synergistic with GM-CSF in the production of TNF or IL-1. IFN gamma alone did not induce either cytokine, but in the presence of GM-CSF it caused a synergistic (100-fold) increase in TNF but not IL-1 production. Macrophage colony-stimulating factor (M-CSF) alone or in combination with LPS, IFN gamma or IL-2 did not stimulate PBM to produce TNF or IL-1 in 24 hr culture.     

Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies

Introduction

The insulin-like growth factor type 1 (IGF-1) receptor contributes importantly to transformation and survival of tumor cells both in vitro and in vivo, and selective antagonists of the IGF-1 receptor (IGF-1R) activity represent an attractive experimental approach for human cancer therapy.

Methods

Using a phage display library, we identified several high-affinity fully human monoclonal antibodies with inhibitory activity against both human and rodent IGF.1Rs.

Results

These candidate therapeutic antibodies recognized several distinct epitopes and effectively blocked ligand-mediated receptor signal transduction and cellular proliferation in vitro. They also induced IGF-1R downregulation and catabolism following antibody-mediated endocytosis. These antibodies exhibited activity against human, primate, and rodent IGF-1Rs, and dose-dependently inhibited the growth of established human tumors in nude mice.

Conclusion

These fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.     

Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony-stimulating factor

We performed a clinical study of five patients with melanoma to evaluate the immunobiological effects of retrovirally transduced autologous tumor cells given as a vaccine to prime draining lymph nodes. Patients were inoculated with both wild-type (WT) and GM-CSF gene-transduced tumor cells in different extremities. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were removed. There was an increased infiltration of dendritic cells (DCs) in the GM-CSF-secreting vaccine sites compared with the WT vaccine sites. This resulted in a greater number of cells harvested from the GM-CSF-VPLNs compared with the WT-VPLNs at a time when serum levels of GM-CSF were not detectable. Four of five patients proceeded to have the adoptive transfer of GM-CSF-VPLN cells secondarily activated and expanded ex vivo with anti-CD3 MAb and IL-2. One patient had a durable complete remission of metastatic tumor. Utilizing cytokine (IFN-gamma, GM-CSF, IL-10) release assays, GM-CSF-VPLN T cells manifested diverse responses when exposed to tumor antigen in vitro. In two of two patients, GM-CSF-VPLN T cell responses were different from those of matched WT-VPLN cells. This study documents measurable immunobiologic differences of GM-CSF-transduced tumor cells given as a vaccine compared with WT tumor cells. The complete tumor remission in one patient provides a rationale to pursue this approach further.     

An uncleavable form of pro-scatter factor suppresses tumor growth and dissemination in mice

Scatter factor (SF), also known as hepatocyte growth factor, is ubiquitously present in the extracellular matrix of tissues in the form of an inactive precursor (pro-SF). In order to acquire biological activity, pro-SF must be cleaved by specific proteases present on the cell surface. The mature form of SF controls invasive cues in both physiological and pathological processes through activation of its receptor, the Met tyrosine kinase. By substituting a single amino acid in the proteolytic site, we engineered an unprocessable form of pro-SF (uncleavable SF). Using lentivirus vector technology, we achieved local or systemic delivery of uncleavable SF in mice. We provide evidence that (a) uncleavable SF inhibits both protease-mediated pro-SF conversion and active SF-induced Met activation; (b) local expression of uncleavable SF in tumors suppresses tumor growth, impairs tumor angiogenesis, and prevents metastatic dissemination; and (c) systemic expression of uncleavable SF dramatically inhibits the growth of transplanted tumors and abolishes the formation of spontaneous metastases without perturbing vital physiological functions. These data show that proteolytic activation of pro-SF is a limiting step in tumor progression, thus suggesting a new strategy for the treatment or prevention of the malignant conversion of neoplastic lesions.     

肝细胞生长因子与成纤维生长因子联合诱导入脐带间充质干细胞向肝样细胞的分化

背景:成纤维生长因子可促进间充质干细胞增殖、贴壁生长,但对其诱导间充质干细胞向肝细胞分化的实验报道为数不多.当肝细胞生长因子质量浓度达1 μg/L时,可促进肝细胞有丝分裂,它是正常肝细胞最强的促有丝分裂剂.目的:体外分离培养人脐带间充质干细胞,拟揭示其生物学特性及在细胞因子联合诱导下向肝样细胞分化的能力.设计、时间及地点:细胞学体外观察,于2008-08/2009-04在暨南大学血研所完成.材料:脐带取自健康足月胎儿,产妇对实验知情同意,由广州华侨医院提供.肝细胞生长因子、成纤维生长因子为美国Peprotech产品.方法:Ⅳ型胶原酶消化+差速贴壁法分离培养人脐带间充质干细胞,取传至第3代细胞,进行细胞表面抗原分析、细胞周期测定,检测其成脂、成骨能力.取第5代细胞.调整细胞密度为5×10~9 L~(-1),分为2组:对照组用含体积分数为5%胎牛血清的DMEM/F12培养液培养;诱导组在其基础上,添加20 μg/L肝细胞生长因子、10 μg/L成纤维生长因子联合诱导其向肝样细胞分化.主要观察指标:人脐带间充质干细胞的生物学特性,人脐带间充质干细胞体外向肝样细胞的分化情况.结果:成功从人脐带中分离并纯化得到间充质干细胞,第3代细胞92.2%处在G_0/G_1期;表达CD29,CD44,CD105,不表达造血细胞标志CD34,CD45;油红O染色后胞浆中呈现红色颗粒,碱性磷酸酶染色后细胞质呈黑色,具有成脂、成骨能力.经肝细胞生长因子、成纤维生长因子联合诱导10 d后,RT-PCR及Western blot检测结果显示细胞表达肝细胞特异性抗原甲胎蛋白、白蛋白,对照组均呈阴性表达.结论:人脐带中含有丰富的间充质干细胞,其具有较强的多向分化潜能,经肝细胞生长因子与成纤维生长因子联合诱导后,易向肝样细胞分化.     

Differentially expressed genes in radioresistant nasopharyngeal cancer cells: gp96 and GDF15

Radiotherapy is the major treatment modality for nasopharyngeal cancer (NPC), but in some cases, the disease is radioresistant. We designed this study to identify genes that may be involved in this resistance. We first established two radioresistant subclone cell lines derived from NPC parental cell lines (NPC-076 and NPC-BM1) by treating the cells with four rounds of sublethal ionizing radiation. cDNA microarray analysis was then done, comparing the two resistant cell lines with their corresponding parental cell lines. Seven genes were found to be up-regulated in radioresistant subclones, including gp96 and GDF15, which had shown highest overexpressions. We constructed small interfering RNA plasmids (gp96si and GDF15si) and transfected them into NPC cells to knock down these genes and examine whether this changed their response to radiation. Both gp96si and GDF15si transfectants had radiation-induced growth delay and reduction in colonogenic survival compared with control cells. Knockdown of either gp96 or GDF15 increased the proportion of the cells in G(2)-M phase, the most radiosensitive phase of the cell cycle. We have therefore identified at least two genes, gp96 and GDF15, involved in radioresistance of NPC cell lines and showed that knockdown of the genes enhances radiosensitivity.     

Tumor necrosis factor alpha stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/macrophage colony-stimulating factor

TNF-alpha has been shown to antagonize the proliferative effects of growth factors present in crude conditioned media from PHA-stimulated leukocytes or cell lines on the clonogenic cells of acute myeloblastic leukemia (AML) (19,21). In the present study, we investigated the responses of AML blasts to TNF-alpha in the presence of defined growth factors (recombinant granulocyte/macrophage-CSF [rGM-CSF], recombinant granulocyte-CSF [rG-CSF], rIL-3, and rIL-1) and under conditions described for autocrine stimulation (32). While TNF-alpha antagonized the stimulatory effects of G-CSF and IL-3 on blast progenitors, TNF-alpha did not affect blast colony formation in the presence of IL-1. Unexpectedly, TNF-alpha significantly enhanced blast proliferation in the presence of GM-CSF. Further, TNF-alpha also acted synergistically with an endogenous source of growth stimulatory signal to promote proliferation of blast clonogenic cells. Thus, on human leukemic cells, TNF-alpha appears to be a molecule that is at least bifunctional, having the ability to either support or inhibit cell proliferation, depending on the other growth factors present. It is postulated that the proliferative response of blast progenitors to TNF-alpha under conditions that favor autocrine stimulation may represent one property that allows the cells to escape from negative regulation and proliferate in AML.