Effect of Human Macrophage Colony-stimulating Factor on Granulopoiesis and Survival in Bone-marrow-transplanted Mice

Human macrophage colony-stimulating factor (hM-CSF) has been isolated from normal human urine and purified to a homogenous protein. The effect of hM-CSF on granulopoiesis was investigated in BALB/c mice transplanted with a suboptimal number of bone marrow cells. Lethally irradiated (7.8 Gy) mice were transplanted with 1 × 10⁶ syngeneic mouse bone marrow cells and treated with a daily intraperitoneal dose of 64 μg/kg of hM-CSF for 5 days following the transplant. The hM-CSF injection resulted in stimulation of the recovery of blood neutrophils as well as an increase in the number of granulocyte-macrophage progenitor cells (CFU-GM) in the femur and spleen. The survival of lethally irradiated mice was dependent on the cell number transplanted; most mice transplanted with 2 × 10⁴ cells died within 2 weeks. The recovery of hematopoiesis in mice transplanted with 2 × 10⁴ cells was modestly but significantly stimulated by hM-CSF administration initiated from 5 days before or 1 day after transplantation for a 5-day period. Furthermore, the hM-CSF administrations markedly reduced the mortality in these mice during the early period after the transplantation. Since anaerobic bacteria were frequently detected in arterial blood immediately before the deaths but were not found in the surviving mice, it is speculated that early deaths occurring within 2 weeks after the transplant may be caused by opportunistic infections, and hM-CSF injection may prevent these mortal infections through its stimulating effect on monocyte-macrophage functions that are responsible for the production of hematopoietic regulators.     

Induction of Tumor Necrosis Factor in Mice by Recombinant Human Macrophage Colony-stimulating Factor

The effect of recombinant human macrophage colony-stimulating factor (rhM-CSF) on endogenous production of tumor necrosis factor (TNF) was investigated in mice. The intravenous injection of lipopolysaccharide (LPS) after the administration of rhM-CSF via the same route induced the production of endogenous cytotoxic activity in serum as assessed by using TNF-sensitive miirine L929 cells. The intravenous injection of LPS alone or rhM-CSF alone did not induce cytotoxic activity. The priming effect of rhM-CSF was transient and the optimal duration between injections of primer and trigger was 3 h, while the optimal duration between trigger injection and serum sampling was 1 h after LPS injection. Moreover, preinjection of rhM-CSF enhanced the priming effect of recombinant mouse interferon-γ. No triggering effect of rhM-CSF was observed. The cytotoxic activity in the serum was completely neutralized by anti-mouse TNF-α polyclonal antibody. These results indicate that rhM-CSF can be used as a priming agent for endogenous production of TNF in vivo , and raise the possibility of using rhM-CSF in cancer immunotherapy.     

Subcutaneous Administration of Recombinant Human Granulocyte Colony-stimulating Factor (KRN8601) in Intensive Chemotherapy for Patients with Advanced Lung Cancer

The efficacy and toxicity of recombinant human granulocyte colony-stimulating factor (rh G-CSF, KRN8601) given subcutaneously was evaluated in patients with advanced lung cancer undergoing intensive chemotherapy. Twenty-nine and 30 patients with or without prior therapy were enrolled in this study. At dose levels of 50, 90 and 130 μg/m² of rh G-CSF for 14 consecutive days after chemotherapy, the mean neutrophil nadir counts, the mean neutrophil nadir ratios and the duration of neutropenia (days of < 1000/mm³) were significantly improved. No significant differences were seen in frequency and duration of febrile episodes (>38°C). When rh G-CSF is given subcutaneously, the dose required for an equal effect in alleviating neutropenia is 50% of that required when it is given intravenously. The monocyte counts in the peripheral blood were also significantly increased after chemotherapy cycles with rh G-CSF. The cumulative plasma concentration of rh G-CSF showed a decrement after 7–9 days despite maintenance of the same dose of rh G-CSF for the entire 14 days. In conclusion, 50–130 μg/m² of sc rh G-CSF increased the neutrophil nadir count and shortened the duration of neutropenia in patients undergoing intensive chemotherapy for lung cancer without intolerable side effects.     

Enhanced Antitumor Effect of Recombinant Human Tumor Necrosis Factor in Combination with Recombinant Human Granulocyte Colony-stimulating Factor in BALB/c Mice

The synergistic antitumor effect of tumor necrosis factor (TNF) and granulocyte colony-stimulating factor (G-CSF) was investigated. G-CSF was administered subcutaneously to BALB/c mice inoculated with Meth-A cells at a dose of 2.5 μ g/day for 5 consecutive days. When TNF (1 × 10³ U) was administered intravenously to mice which had been pretreated with G-CSF, tumor growth showed a 74.1% inhibition 17 days after the tumor cell inoculation, compared to that of untreated mice. In this experiment, G-CSF significantly ( P <0.025) enhanced the antitumor effect of TNF. The in vitro cytotoxicity of TNF (10 U/ml) towards Meth-A cells was increased about 5.2-fold in the presence of neutrophils (E/T=50) as compared to the cytotoxicity obtained with TNF alone. A combination of TNF and G-CSF (50 ng/ml) in the presence of neutrophils, resulted in a 2.1 times greater cytotoxicity against Meth-A cells as compared to that obtained without G-CSF. Significant augmenting effects of G-CSF on superoxide (O₂⁻) production by TNF-stimulated neutrophils were observed. These observation suggest that the neutrophil plays an important role in the antitumor action of TNF on Meth-A cells, and that the antitumor effect of TNF is enhanced by combination with G-CSF.     

Enhancement of Cytosine Arabinoside Cytotoxicity by Granulocyte/Macrophage Colony-stimulating Factor and Granulocyte Colony-stimulating Factor in a Human Myeloblastic Leukemia Cell Line

Enhancement of the cytotoxicity of cytosine arabinoside (ara-C) by granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), and the mechanisms involved, were studied in the AML-193 human leukemia cell line. AML-193 cells require GM-CSF and G-CSF(CSFs) for optimum growth, and 24 h deprivation of CSFs decreased DNA synthesis measured in terms of ³H-thymidine incorporation. The DNA synthesis gradually recovered upon addition of CSFs. To examine the sensitivity to ara-C under different growth conditions, two groups of cell suspensions, one pretreated with CSFs after 24 h deprivation (CSFs(+) cells), and the other held continuously under CSFs-free conditions (CSFs(-) cells), were exposed to 1.0 μg/ml of ara-C for 16 h. In clonogenic assays, CSFs(+) cells showed higher sensitivity to ara-C than CSFs(-) cells. These cell groups showed no significant difference in ara-C triphosphate accumulation or retention, though the amount of ara-C incorporated into the acid-insoluble fraction was two times greater in CSFs(+) cells than CSFs(-) cells, and that difference became even clearer in the retention pools. These data suggest that the enhancement of cytotoxicity by CSFs was due to the promotion of ara-C incorporation into DNA as a result of an increase of the cell fraction in the S phase.     

Effects of the in vivo Administration of Recombinant Human Granulocyte Colony-stimulating Factor Following Cytotoxic Chemotherapy on Granulocytic Precursors in Patients with Malignant Lymphoma1

We examined the effects of the in vivo administration of recombinant granulocytc colony-stimulating factor (rhG-CSF) on granulocytic precursors in the bone marrow of 4 patients with malignant lymphoma who received chemotherapy. Patients were treated with rhG-CSF at doses of 100–800 μg/ m²/day intravenously for 14 days only in the first course of chemotherapy (G-CSF course) followed by the second course of chemotherapy without rhG-CSF which was used as a control course. In the G-CSF course, white blood cell counts (WBCs) demonstrated a biphasic response consisting of a first peak observed within a few days after the initiation of rhG-CSF administration, and a second peak observed on the last day of rhG-CSF injection or the day after. In the second peak, the incidence of granulocyte-macrophage colony-forming units (CFU-GM) in mononucleated bone marrow cells did not change significantly after treatment with rhG-CSF as compared with a control. However, since the number of nucleated cells in the bone marrow increased, the absolute number of CFU-GM in the bone marrow increased. The number of mature and immature granulocytes in the bone marrow increased. These findings suggest that G-CSF stimulates the proliferation and differentiation of granulocytic precursors in the bone marrow in granulocytopenic patients who received cytotoxic drugs and causes mature granulocytes to be released from the bone marrow.     

Proliferative Effect of Human Granulocyte Colony-stimulating Factor on Blast Cells of Acute Promyelocytic Leukemia

The effect of human granulocyte colony-stimulating factor (G-CSF) on leukemic cells of acute promyelocytic leukemia (APL) was examined. Mononuclear cells obtained from bone marrow cells containing more than 90% blasts from seven APL patients were incubated in the presence of G-CSF using semisolid and liquid culture systems. On day 7, the cells from all the patients produced many clusters consisting of 8–40 cells. These cells appeared to be promyelocyte-like blast cells in four patients and had differentiated to more mature neutrophils in three patients. On day 14, the number of clusters decreased except for two patiens. Blast cells from the two patients showing the increase of blast clusters could proliferate in a liquid culture containing G-CSF. Blast cells cultured for 14 days formed many secondary cultures after replating on a methylcellulose medium. Moreover, chromosomal analyses of blasts cultivated in the presence of G-CSF for 7 days showed t(15;17) in all metaphases in one patient. It appears that the leukemic cells from APL patients could proliferate in the presence of G-CSF.     

Macrophage Colony-stimulating Factor (M-CSF) Prevents Infectious Death Induced by Chemotherapy in Mice, While Granulocyte-CSF Does Not

To clarify the effect of granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF/CSF-1) on chemotherapy-induced infection, we estimated the effect of those CSFs on a mouse model under severe myelosuppression. First, we established an animal model in which 48.9% (22/45) of C3H/Hej mice died of sepsis related to severe myelosuppression after intraperitoneal administration of a single dose (9 mg/kg) of mitomycin C (MMC). G-CSF or M-CSF was administered to this model on various administration schedules after chemotherapy, and the effect of those CSFs on survival rates, peripheral blood granulocyte counts, expression of adhesion molecules (CD11a, CD11b, CD18) on granulocytes and granulocyte function (phagocytosis and superoxide anion production) were examined. In all G-CSF administration groups, peripheral blood granulocyte counts were increased, but improvements in expression of adhesion molecules such as CD11a and CD18, and granulocyte function were less marked and survival rates were not unproved. Meanwhile, when M-CSF was administered from 1 to 7 days after chemotherapy, granulocyte and platelet counts were increased, and moreover, expression of adhesion molecules and granulocyte function were markedly improved. Furthermore, the survival rate was significantly improved to 77.8% (28/36) compared with the MMC group ( P <0.05). Positive rate of blood culture examination at 7 days after chemotherapy in the M group was 0%, and was significantly lower than that in the G group (40%) and the MMC group (40%) ( P <0.05). These results demonstrated that it is important not only to increase the granulocyte counts, but also to improve granulocyte functions for preventing infection under myelosuppression after chemotherapy.     

聚乙二醇化重组人粒细胞集落刺激因子预防化疗后中性粒细胞减少症临床疗效观察中性粒细胞减少症临床疗效观察

比较聚乙二醇化重组人粒细胞集落刺激因子（PEG-rhG-CSF）和重组人粒细胞集落刺激因子（rhG-CSF）预防化疗后中性粒细胞减少症的有效性和安全性。方法：采用随机自身交叉对照,选择初治恶性肿瘤患者接受2个周期相同方案的化疗,其中试验周期给予PEG-rhG-CSF 100 μg/kg皮下注射一次,对照周期每日一次皮下注射rhG-CSF 5 μg/kg,直至外周血中性粒细胞绝对值（ANC）在低谷后连续两次检查≥5.0×109/L。结果：入组78例患者,在76个试验周期和74个对照周期中,ANC<1.5×109/L的发生率分别为30.26%和21.16%,持续时间分别为2.34 d和2.31 d;ANC<0.5×109/L的发生率分别为3.8%和3.0%;抗生素使用率分别为11.59%和9.60%（P均<0.05）。试验药和对照药的不良反应均为骨骼肌肉疼痛、乏力、发热、头晕等,发生率与严重程度相似。结论：PEG-rhG-CSF一次给药的疗效和不良反应与rhG-CSF多次给药相似。     

Leukemic Cell Lysis by Activated Human Macrophages: Significance of Membrane-associated Tumor Necrosis Factor

In this study, we analyzed the mechanism(s) of leukemic cell lysis by human macrophages. Peripheral blood monocyte-derived macrophages were activated with recombinant interferon-γ and lipopoly-saccharide and their lytic activity against two leukemic cell lines (K562 and HL-60 cells) was assessed by an ¹¹¹In releasing assay. Activated macrophages lysed these leukemic cells, and the lytic activity against leukemic cells was almost completely inhibited by anti-tumor necrosis factor (TNF) antibody. The macrophage-lysate prepared from activated macrophages also exhibited significant lytic activity against leukemic cells; this lytic activity was inhibited by anti-TNF antibody. The leukemic cells that we used for the cytotoxicity assays were resistant to recombinant TNF. The culture supernatant of activated macrophages did not show any lytic activity. These findings suggest that cell-associated TNF plays a role in macrophage-mediated cytotoxicity against leukemic cells.     

基因重组人粒细胞集落刺激因子防治CHOP和CAF方案化疗所致白细胞减少症的临床研究

作者采用随机分组、自身交叉对比的方法，观察了基因重组人粒细胞集落刺激因子（ＲｅｃｏｍｂｉｎａｎｔＨｕｍａｎＧｒａｎｕｌｏｃｙｔｅＣｏｌｏｎｙ－ＳｔｉｍｕｌａｔｉｎｇＦａｃｔｏｒ，ｒｈＧ－ＣＳＦ，以下简称ｒｈＧ－ＣＳＦ）对２３例肿瘤患者ＣＨＯＰ和ＣＡＦ方案化疗所致白细胞和中性粒细胞减少的防治作用及毒副反应。结果表明，ｒｈＧ－ＣＳＦ可以减轻化疗过程中白细胞和中性粒细胞下降的程度，缩短白细胞和中性粒细胞降至正常值以下的持续时间，促进其早日恢复，使化疗可以如期进行。ｒｈＧ－ＣＳＦ副反应轻微，安全可靠。     

儿童神经母细胞瘤化疗后严重感染的影响因素及重组人粒细胞刺激因子的预防效果

目的 探讨儿童神经母细胞瘤化疗后严重感染的影响因素及重组人粒细胞刺激因子(rhG-CSF)的预防效果.方法 回顾性分析儿童神经母细胞瘤114例临床资料,包括患儿性别、年龄、临床分期、原发部位、远处转移、血清神经元特异性烯醇化酶(NSE)水平、手术切除、化疗药物、化疗维持时间,统计所有患儿化疗后严重感染发生率并分析判断以...     

Comparison of Peripheral Blood Stem Cells Mobilized with Granulocyte Colony-stimulating Factor with or without Prior Standard-dose Chemotherapy in Patients with Malignancy

We studied the effect of granulocyte colony-stimulating factoron the magnitude of peripheral blood stem cell mobilizationin patients with malignancy. The leukapheresis products mobilizedwith granulocyte colony-stimulating factor alone at a steadystate (a period of full hematopoietic recovery) (group 1) werecompared with those obtained after cytotoxic chemotherapy usinggranulocyte colony-stimulating factor (group 2). In group 1,six patients underwent six courses of stem cell collection witha median of 20 l leukapheresis. In group 2, 10 patients underwent12 courses of stem cell collection with a median of 10 l leukapheresis.Median yields of group 1 vs. group 2 were mononuclear cells(x10⁹), 21.9 vs. 11.6; CD34⁺ cells (x10⁶/l), 14.5 vs. 17.1;colony-forming unit for granulocyte-macrophage (/ml), 223 vs.1193; burst-forming unit for erythroid (/ml), 29 vs. 71; colony-formingunit for erythroid (/ml), 42 vs. 29; colony-forming unit formegakaryocyte (/ml), 26 vs. 59. While there were no statisticallysignificant differences in the number of CD34⁺ cells betweenthe two groups, granulocyte-macrophage-committed progenitorcells were more enriched in the apheresis products of group2. The correlation between CD34⁺ cells and colony-forming unitfor granulocyte-macrophage was poor. Our results demonstratethat granulocyte colony-stimulating factor can mobilize a sufficientnumber of progenitor cells into the peripheral blood for stemcell transplantation with or without prior chemotherapy.     

Transduction of the Macrophage Colony-stimulating Factor Gene into Human Multidrug Resistant Cancer Cells: Enhanced Therapeutic Efficacy of Monoclonal Anti-P-glycoprotein Antibody in Nude Mice

To develop a therapeutic modality for overcoming multidrug-resistant (MDR) cancer with anti-MDR1 antibody, we examined the effect of macrophage colony-stimulating factor (M-CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti-MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M-CSF gene inserted into an expression vector to establish gene-modified cells capable of producing low (ML-AD10), intermediate (MM-AD10) and high (MH-AD10) amounts of M-CSF. Systemic administration of MRK17 resulted in significant dose-dependent inhibition of subcutaneous growth of ML-AD10 tumors. In contrast, systemic administration of recombinant M-CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M-CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH-AD10 cells producing a large amount of M-CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti-MDR1 mAb and M-CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.     

低剂量格拉诺赛特（rhG－CSF）防治肺癌CE方案化疗后中性粒细胞减少症的临床研究

作者采用随机分组、自身交叉对比的方法，观察了低剂量格拉诺赛特（Ｇｒａｎｏｃｙｔｅ，ｒｈＧ－ＣＳＦ）对２２例肺癌ＣＥ方案（卡铂＋足叶乙甙）化疗后白细胞和中性粒细胞减少症的防治作用及毒副反应。患者随机分成Ａ、Ｂ两组（各１１例）。Ａ组第一周期化疗后加用格拉诺赛特，第二周期单用化疗；Ｂ组第一周期单用化疗，第二周期化疗后加用格拉诺赛特。格拉诺赛特在化疗药物未次给药后４８ｈ起，５０μｇ１次／日皮下注射。结果表明，该剂量的格拉诺赛特可以明显减轻化疗过程中白细胞和中性粒细胞下降的程度，降低中性粒细胞严重降低的发生率，缩短其在正常值以下的持续时间，促进早日恢复，缩短化疗周期的间隔时间，使化疗可以如期进行。低剂量的格拉诺赛特疗效确切，副反应轻微。     

乳腺癌荷瘤小鼠白细胞介素-1活性水平体外动态研究

应用ＭＡ７３－２３４乳腺癌津白Ⅱ号小鼠动物模型，探讨了随病程进展而不断增大的肿瘤负荷对Ｍφ抗瘤效应如Ｍφ分泌的ＩＬ－１活性水平、Ｍφ吞噬功能ＭＭＡＤＣＣ效应的动态影响。结果发现，Ｍφ分泌的ＩＬ－１活性水平在病程早期即明显下降，提示该功能更易受到肿瘤负荷的影响，从而可能会中枢性地抑制宿主免疫系统抗肿瘤效应的发挥，成为逃避宿主免疫工攻击的分子机制之一；而Ｍφ吞噬功能、ＭＭＡＤＣＣ效应在病程早期或中期当肿瘤负荷较小时有所加强，而晚期在肿瘤负荷较大时则减弱。     

惠尔血治疗肺癌化疗所致的白细胞减少的疗效观察

目的：观察麒麟啤酒株式会社生产的基因重组人粒细胞集落刺激因子—惠尔血治疗化疗所致白细胞减少的疗效。方法：用惠尔血治疗３０ 例肺癌化疗所致的白细胞减少与用一般升白细胞中药治疗３４ 例肺癌化疗所致的白细胞减少进行了对比观察。结果：１）一般升白细胞中药和惠尔血均能使化疗后白细胞下降的患者恢复至正常范围（４×１０９／Ｌ） ,但惠尔血组白细胞回升至正常范围的时间平均４．３ 天,而一般升白细胞中药则需１０．３ 天。两组相比,差异有显著性。结论：惠尔血可明显缩短白细胞降至正常值以下的持续时间,有利于化疗的顺利进行。２） 白细胞降至Ⅲ度～Ⅳ度的患者,用惠尔血治疗比一般升白细胞中药恢复正常所用的时间短,故为争取化疗时间,在Ⅲ度～Ⅳ度白细胞下降的患者最好用粒细胞集落刺激因子—惠尔血治疗。     

Augmentation and Suppression of Release of Tumor Necrosis Factor from Macrophages by Negatively Charged Phospholipids

We recently reported that some lipid species of cell membranes and lipoproteins induced the growth of peripheral macrophages. In this study, the effects of phospholipids on tumor necrosis factor (TNF)-releasing activity of macrophages were examined. Ten to 20 μg/ml of cardiolipin, which is a suboptimal concentration for macrophage growth-stimulation, augmented macrophage TNF release triggered by lipopolysaccharide (LPS) in vitro. This priming effect appeared with 1 day of preincubation and was still potent on day 3, whereas the priming effect of interferon-γ (IFN-γ) peaked at 3 h and then gradually decreased. In contrast, a high concentration of cardiolipin (40 μg/ml) which is optimal for the induction of macrophage growth, completely suppressed LPS-triggered TNF release from not only untreated macrophages but also IFN-γ-primed macrophages. The suppressive effect was potent even with 3 h preincubation, was still potent on day 3, and was not abolished by indomethacin. Cardiolipin had scarcely any effect on the triggering activity of LPS. Similar augmentative and suppressive activities were observed in peroxidized phosphatidylserine, which is also highly active in inducing macrophage growth, but was not found in native phosphatidylserine, which is less active, nor in phosphatidylcholine, which is an inactive species toward macrophage growth. These results suggest that lipids may be important endogenous factors in regulating both activation and growth states of peripheral macrophages.     

Ribonucleotide reductase activity and growth of glutathione-depleted mouse leukemia L1210 cells in vitro

L1210 cells treated with -buthionine-(S/R)-sulfoximine (BSO) had glutathione (GSH) and non-protein thiol levels only 15% that of control. These GSH-depleted cells grew as well as the control L1210 cells and there was no decrease in ribonucleotide reductase activity in situ as measured by the conversion of [¹⁴C]cytidine to deoxytidine nucleotides and incorporation into DNA. Further, when these BSO-stressed cells were treated with hydroxyurea or IMPY, there was no potentiation of the inhibition caused by hydroxyurea or IMPY alone. These data indicate that the glutathione/glutaredoxin system of ribonucleotide reductase is not the sole carrier of reducing equivalents from NADPH for the reduction of the 2′-position of the corresponding ribonucleoside 5′-diphosphate; and that glutathione is not critical in regenerating the tyrosyl free-radical on the M2 subunit which is destroyed by the hydroxyurea or 2,3-dihydro-1H-pyrazolo-[2,3-a]imidazole (IMPY) treatment.