Acceleration of the hemopoietic reconstitution in mice undergoing bone marrow transplantation by recombinant human granulocyte colony-stimulating factor.

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.     

重组人粒细胞集落刺激因子促进骨髓间充质干细胞增殖

目的探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠骨髓间充质干细胞(MSCs)增殖的影响。方法将昆明小鼠随机分为2组(n=15),G-CSF组采髓前皮下注射重组人粒细胞集落刺激因子(rhG-CSF)80μg·kg-1·d-1,连用5d,对照组皮下注射等量生理盐水。每组分别于最后一次注射后6h、12h及7d(168h)取小鼠骨髓,分离、培养MSCs,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪检测单个核细胞(MNCs)细胞周期及CFU-F细胞表面抗原特征。结果应用rhG-CSF后,培养MNCs所获得的CFU-F数目增加(P<0.01),CFU-F数与取材时间(6h、12h、7d)呈负相关(P<0.05),但12h取材者与7d取材者比较,CFU-F数目的差异无统计学意义(P>0.05)。MNCs培养形成的CFU-F,其细胞表面抗原呈CD34-、CD133-、CD90+、CD105+,分别占2.5%、3.1%、67.0%和78.0%。G-CSF组各时间点获得的MNCs,其G0/G1期细胞百分率较对照组低(P<0.05),而S+G2/M期增高(P<0.05),且6h取材者S+G2/M期细胞百分率明显高于12h和7d取材者(P<0.05)。结论rhG-CSF可促进骨髓MNCs进入细胞增殖周期,增殖的高峰在应用G-CSF后6h左右。     

Ovariectomy fails to augment bone resorption and marrow B lymphopoiesis in granulocyte colony-stimulating factor transgenic mice

The mechanism of pathological bone loss induced by estrogen deficiency has not been fully elucidated. It has been shown in recent animal studies that increased B lymphopoiesis induced by estrogen deficiency is involved in the mechanism of stimulated bone resorption. Mice transgenic for granulocyte colony-stimulating factor (G-CSF) (G-Tg) exhibit generalized osteopenia with an increase in osteoclast number and enhancement of bone resorption, which coexists with enhanced hematopoiesis. When ovariectomy was performed on G-Tg, it did not further reduce bone mass as revealed by radiography, dual-energy X-ray absorptiometry, and peripheral quantitative computed tomography. Ovariectomy increased the amount of colony-forming units of interleukin 7 (CFU-IL-7) by threefold in the marrow of normal mice in association with an increase in the number of B220-positive cells expressing the receptor activator of nuclear factor-B ligand (RANKL). In contrast, the number of B220-positive cells expressing RANKL and CFU-IL-7 remarkably decreased in the marrow of G-Tg. Ovariectomy induced neither CFU-IL-7 nor B220-positive cells expressing RANKL in the marrow of G-Tg. Strong inhibition of B lymphopoiesis by G-CSF resulted in depletion of B cells expressing RANKL from the marrow, which may lead to resistance to bone loss due to ovariectomy. This observation suggests that B lymphopoiesis plays a possible role in bone loss in a condition of acute estrogen deficiency.     

粒细胞集落刺激因子和干细胞因子对骨髓单个核细胞的动员效率及其机制

目的 联合应用粒细胞集落刺激因子(G-CSF)和干细胞因子(SCF)动员骨髓单个核细胞,评价其动员效果,探讨CXCL12/CXCR4信号通路在骨髓单个核细胞动员中的作用及机制.方法 将昆明小鼠随机分为两组,治疗组皮下注射重组鼠G-CSF 100μg/(kg·d)和重组鼠SCF25μg/(kg·d),连续使用5d;对照组皮下注射等剂量的生理盐水.每组于不同时间点取小鼠骨髓,分离培养骨髓单个核细胞,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪分选CD34+ CXCR4+单个核细胞(MNCs);应用酶联免疫吸附试验(ELISA)法测定骨髓细胞外液CXCL12a的水平;采用逆转录-聚合酶链反应(RT-PCR)检测骨髓CXCL12 mRNA表达变化.结果 应用G-CSF/SCF后,骨髓及外周血中单个核细胞计数较对照组明显增加(P<0.01),CFU-F形成能力显著增强(P<0.05);流式分选表明CD34+ CXCR4+细胞占骨髓CD34+单个核细胞总数的(44.6±8.7)%;RT-PCR和EUSA检测示骨髓CXCL12 mRNA表达下降,骨髓细胞外液CXCL12蛋白也显著下降,两者变化一致.结论 G-CSF/SCF可有效地诱导骨髓单个核细胞动员,其机制可能是通过破坏骨髓CXCL12/CXCR4信号通路平衡,下调CXCL12/CXCR4间相互作用,以促进骨髓单个核细胞动员.     

Recombinant granulocyte-macrophage colony-stimulating factor improves hematopoietic recovery after 5-fluorouracil

Antitumor chemotherapy is often limited by hematopoietic toxicity. In an attempt to determine if it is possible to attenuate the myelosuppressive effects of chemotherapy, we administered recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF), a multilineage hematopoietic growth factor, to mice receiving 5-fluorouracil (5-FU). Mice receiving injection of 5-FU followed 24 hr later by a single 1-microgram injection of rmGM-CSF had significantly increased femoral bone marrow granulocyte-macrophage colony-forming cells (GM-CFC) 48 hr after 5-FU injection compared to animals receiving 5-FU alone. Animals receiving rmGM-CSF twice daily beginning 24 hr after 5-FU had significantly elevated white blood cell counts and increased granulocyte and monocyte counts at Day 7 following 5-FU injection, compared to those of 5-FU animals. The total reserve of GM-CFC was also expanded initially in the femoral marrow and later in the spleen of animals receiving rmGM-CSF following 5-FU. A means of accelerating bone marrow recovery and restoration circulating granulocytes and monocytes could allow more frequent doses of chemotherapy to be administered or shorten the time period that patients are leukopenic.     

Immune recovery following allogeneic bone marrow transplantation

A total of 144 evaluations of cell surface markers and cellular immune functions were carried out in 57 patients undergoing allogeneic bone marrow transplantation for acute leukemia in remission and relapse and for aplastic anemia. The periods tested were pretransplant, and 1-3, 4-6, 7-12 and more than 12 months posttransplant. The determination consisted of lymphocyte counts; lymphocyte surface marking using OKT3, OKT4, and OKT8 antibodies; and determination of adherent cells, lysozymes and antibody dependent cellular cytotoxicity (ADCC) against chicken red blood cells, human red blood cells, and CEM cells. Natural killer cells were determined against K562 target cells. Lymphoblastic responses were tested after stimulation with pokeweed mitogen (PWM), concanavalin-A (Con-A), and phytohemagglutinin (PHA). We found that the progression in the leukemic state (first remission, second remission, and relapse), prior to transplantation was paralleled by a decrease in T4 lymphocytes (976/microliter +/- 462; 411/microliter +/- 222; 372/microliter +/- 419; P = .04). There was a lack of helper cells and an inverted T4:T8 ratio beyond one year posttransplant independent of graft-versus-host disease status. Lymphocyte functions persisted to be depressed for more than one year. We found a direct correlation of T4 helper cells and an inverse correlation of T8 suppressor cells with lymphoblastic responses to mitogens. It is hoped that the longitudinal evaluations of immune functions after allogeneic bone marrow transplantation, and the characterization of the immune defects seen may lead to better immunorestorative treatments.     

Effect of granulocyte colony-stimulating factor (G-CSF) on chemotherapy-induced oral mucositis

In this study, the ability of granulocyte colony-stimulating factor (G-CSF) to treat or prevent chemotherapy-induced oral mucositis in patients with advanced breast cancer was evaluated. A total of 14 patients who received intra-arterial (i.a.) adriamycin (ADM) preoperatively were divided into two groups according to whether or not G-CSF was given. Thus, group A (n=7) was given G-CSF and group B (n=7) was not. G-CSF therapy reduced both the incidence and duration of ADM-induced oral mucositis, and a positive correlation was also seen between the incidence of mucositis and ADM-induced leukopenia (<2,000/mm³). Group A was further divided into two subgroups according to whether G-CSF was given after or before the leukopenia had dropped below 2,000/mm³: group A-1 (n=3) and group A-2 (n=4), respectively. ADM-induced mucositis was observed in two of the three patients in group A-1, but in none of the four patients in group A-2. These results strongly support the idea that G-CSF can effectively treat and prevent ADM-induced oral mucositis.     

Predictability before transplant of hepatic complications following allogeneic bone marrow transplantation

This study was undertaken to evaluate the occurrence of VOD and other liver diseases following BMT in a patient population with a high incidence of hepatitis before conditioning regimen. We prospectively reviewed 186 consecutive patients undergoing BMT from 1976 to 1986 to determine incidence and type of liver disease after BMT and predisposing factors. Two of 186 patients experienced VOD (1.07%). Acute and chronic liver GVHD were found in 25.8% and 36% of the patients, respectively. Acute hepatitis (AH) was diagnosed in 29.4% and chronic hepatitis (CH) in 42.6% of the patients. Statistical analysis showed no influence of pretransplant variables on the occurrence of acute GVHD and AH; there was a weak correlation (P = 0.01) between pre-BMT abnormal transaminases and occurrence of chronic GVHD. Contingency table and Cox analysis showed a greater risk of CH for patients with abnormal pretransplant SGPT levels (P = 0.0004 and P = 0.0022). No other variables could be associated with posttransplant CH. Actuarial survival was 71% versus 69% for patients with normal versus abnormal transaminases (P = 0.2). As VOD was a rare event, despite 53% of patients having abnormal transaminase values before transplant, we suggest that a lower and slower TBI is more important than pretransplant normal transaminases in preventing this complication. We conclude that evidence of compensated hepatitis is not a relative contraindication for BMT.     

Use of granulocyte colony-stimulating factor for neutropenia after orthotopic liver transplantation: report of two cases

Early postoperative infections are one of the major causes of morbidity and mortality following orthotopic liver transplantation. The severity of these infections may be increased in patients with neutropenia. There are no guidelines on the use of granulocyte colony-stimulating factor (G-CSF) for the treatment of neutropenia in posttransplant liver recipients. However, it has been recommended by several authors. We have herein presented two patients who were treated effectively with G-CSF. Both patients developed severe neutropenia (<500/mm³) on the third postoperative day, and received intravenous G-CSF administration for 3 days. The neutrophil counts gradually increased and additional infusions were not needed. The immunosuppressive and prophylactic treatments were not altered. G-CSF administration was used effectively for 3 days in our two patients. No evidence of infectious or acute rejection episode was encountered during or following G-CSF treatment.     

A case of bladder tumor producing granulocyte colony-stimulating factor

A case of bladder tumor producing granulocyte colony-stimulating factor (G-CSF) is reported. A 78-year-old male presented with macroscopic hematuria. Cystoscopy demonstrated a large bladder tumor. Drip infusion pyelography and computerized tomography of the chest, abdomen and pelvis revealed right hydronephrosis, but did not reveal any metastasis. Total cystectomy and construction of bilateral cutaneous ureterostomy was performed. Histologically, the tumor consisted of sarcomatoid carcinoma with foci of transitional cell carcinoma (G3 > G2), showing marked infiltration of polymorphonuclear leukocytes. Immunohistochemical examination revealed a high concentration of G-CSF in the tumor specimen. Preoperatively, the white blood cell, count in the peripheral blood and serum G-CSF concentration were elevated, 45,400/mm3 and 73.4 pg/ml, respectively. After surgery these values became normal. This is the 19th reported case of G-CSF producing bladder tumor in Japan. The prognosis of the reported cases of G-CSF producing bladder tumor has been very poor. Fourteen of the reported 19 cases died within one year. This patient should be carefully followed up.     

粒细胞-单核巨噬细胞集落刺激因子对人皮肤成纤维细胞血管内皮细胞生长因子表达的影响

目的探讨粒细胞-单核巨噬细胞集落刺激因子（granulocyte-macrophage colony stimulating factor,GM—CSF）对人皮肤成纤维细胞血管内皮细胞生长因子（vascular endothelial cell growth factor,VEGF）表达的影响。方法分别用含GM—CSF（GM—CSF组）和不含GM—CSF（对照组）培养液,孵育离体培养的人皮肤成纤维细胞,作用不同时间后,采用逆转录-聚合酶链反应（RTPCR）、蛋白质印迹法（Western印迹法）、酶联免疫吸附试验（EI,1SA）分别检测人皮肤成纤维细胞VEGF mRNA表达和蛋白表达。结果GM—CSF作用1、3、6、12h后,人皮肤成纤维细胞VEGF mRNA表达均较对照组显著增强（P〈0．05）,其中GM—CSF作用6h后VEGFmRNA表达最强;与对照组比较,GM—CSF作用12、24、48h后。成纤维细胞内VEGF蛋白表达旺著增强,其中24h表达最强;细胞培养上清液中VEGF蛋白分泌堪随时间延长逐渐增加,各时相点GM-CSF组VEGF蛋白分泌量高于对照组（P〈0．01）。结论GM—CSF可促进人皮肤成纤维细胞VEGF的表达,这可能是GM-CSF加速创面血管新生化的机制之一。     

A case of bladder tumor producing granulocyte colony-stimulating factor

A 75-year-old male was admitted with high grade fever, general fatigue, and appetite loss. The laboratory examinations revealed leukocytosis of 31,700/mm3 (neutrophils: 88%) in the peripheral blood. Cystoscopy demonstrated multiple bladder tumor. Computed tomography revealed bilateral hydronephrosis due to bladder tumor. Bilateral nephrostomy and transurethral resection bladder tumor were performed. Histological diagnosis was squamous cell carcinoma and immunohistochemical staining of the resected tumor using antihuman granulocyte colony-stimulating factor (G-CSF) antibody showed positive staining in the cytoplasm of the tumor cells. Serum analysis revealed a high level of G-CSF 126 pg/ml (normal: less than 18.1 pg/ml). Total cystectomy and bilateral cutaneous ureterostomy were performed. He died of cancer 3.5 months after admission. This is the 48th case in Japanese literature.     

Treatment of intra-abdominal infection with granulocyte colony-stimulating factor.

Polymicrobial infection is a significant cause of mortality in critically ill patients. Antibiotics and surgical intervention are useful but limited in their effectiveness for combating mixed infections. New prophylactic and therapeutic approaches are required to improve survival in critically ill patients. Neutrophils are a known primary host defense mechanism against bacterial infection. We evaluated the use of a neutrophil growth factor, recombinant human granulocyte colony-stimulating factor (G-CSF), to improve survival in a well-established sepsis model, cecal ligation and puncture (CLP). When administered beginning 4 days before CLP with injections continuing for 14 days after CLP, mice that received 10, 100, or 1000 ng of G-CSF had significantly improved survival compared with the control group. When treatment began at the time of CLP and continued for 7 days after CLP, G-CSF treatment resulted in a dose-dependent improvement in survival in groups that received 100, 500, or 1000 ng. The interaction of G-CSF and conventional antimicrobial therapy was evaluated by administration of G-CSF plus gentamicin. Mice received 100 ng of G-CSF beginning on day 1 before CLP with injections continuing for 3 days after CLP. Gentamicin-treated mice received a single 15 mg/kg injection of gentamicin at the time of CLP. Mice that received G-CSF alone or gentamicin alone had significantly improved survival compared with controls. Mice that received G-CSF plus gentamicin had improved survival compared with control mice and compared with mice that received G-CSF alone but not compared with mice that received gentamicin alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophilia and granulocyte colony-stimulating factor levels after cardiopulmonary bypass

PURPOSE: The precise mechanism of neutrophilia after cardiac surgery is unknown. Granulocyte colony stimulating factor (G-CSF) can increase the number of leukocytes. The purpose of this study was to evaluate the relationship between serum G-CSF levels and peripheral blood leukocyte counts after cardiac surgery. METHODS: We prospectively studied 10 patients undergoing cardiac surgery (coronary artery bypass grafting) using cardiopulmonary bypass (CPB). Plasma G-CSF levels and neutrophil count were measured before induction of anaesthesia, at the end of surgery, and on the first postoperative day. These changes were compared with those in patients undergoing non-cardiac major surgery (control group). RESULTS: At the end of surgery, G-CSF levels increased (P < 0.01) in both groups, but were higher in the control than in the cardiac group (3,250 +/- 690 vs 194 +/- 29.5 pg ml(-1), respectively, mean +/- SEM, P < 0.01). On the first postoperative day, G-CSF levels were still high in both groups, and were still higher in the control (710 +/- 179 vs 122 +/- 19.9, respectively, P < 0.01). However, neutrophilia was greater in the cardiac group than in the control. G-CSF response correlated positively with neutrophilia in the control group (r = 0.656, P < 0.05) but not in the cardiac group. CONCLUSIONS: Our results indicate that changes in leukocyte count following cardiac surgery are unique to patients undergoing CPB. G-CSF plays an important role as the mediator of neutrophilia after non-cardiac surgery, but not after cardiac surgery with CPB.     

Pyoderma gangrenosum related to a new granulocyte colony-stimulating factor

A 23-year-old Caucasian man diagnosed with stage IVB Hodgkin's disease was referred to a university oncology section after completing 1.5 cycles of chemotherapy. His chemotherapy consisted of doxorubicin HCL, bleomycin, dacarbazine, and vinblastine, with prophylactic administration of a granulocyte colony stimulating factor. He had developed postchemotherapy complications of possible cellulitis and necrotizing fasciitis that required wound debridement. The wound and tissue cultures were negative. Biopsies taken at the time revealed a dense inflammatory infiltrate consistent with an abscess. Over the course of 2 months, the wound healed with systemic antibiotics. The patient was reluctant to resume chemotherapy for his Hodgkin's disease because of his previous presumed skin infections. However, positive emission tomographic scanning revealed disease progression. Doxorubicin, bleomycin, dacarbazine, and prophylactic pegfilgrastim (a granulocyte colony-stimulating factor), were administered. Vinblastine was excluded from the new regimen. Shortly after chemotherapy and an injection of pegfilgrastim, the patient developed poorly defined, rapidly progressive erythema, edema, and pain in his right forearm. He presented to the emergency room, was evaluated by the orthopedics service, and taken to the operating room for debridement of suspected necrotizing fasciitis. When the dermatology service consulted the following day, the patient had developed an erythematous, edematous, tender plaque on his chest. After developing two additional lesions that began to ulcerate despite treatment with imipenem, vancomycin, clindamycin, rifampin, and gentamicin, the patient consented to a skin biopsy. His wound cultures continued to be negative.