Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony-stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.     

Phosphatidyl serine-dependent antiprothrombin antibody is exclusive to patients with lupus anticoagulant

In attempt to develop a new chemotherapeutic regimen including carboplatin (CBDCA), epirubicin (EPI), and VP-16 in extensive small cell lung cancer, with a higher dose intensity compared with previous experience of our group, we determined the maximum tolerated dose (MTD) of VP-16 when administered in association with CBDCA (300 mg/ m2, i.v., day 1) and EPI (75 mg/m2, i.v., day 1), recycling chemotherapy every 3 weeks, with the support of granulocyte-colony-stimulating factor (G-CSF). A total of 15 patients received three dose levels of VP-16 (mg/m2, i.v., daily on days 1-3): 100 (three patients), 120 (six), and 140 (six). G-CSF was administered subcutaneously at the dose of 5 micrograms/kg/day on days 6-15 of each chemotherapy course. The MTD was established at 140 mg/m2 and myelotoxicity, grade 4 neutropenia with death for sepsis in one case and grade 3 thrombocytopenia in three cases, was dose limiting. The recommended dose of VP-16 for a phase II study is 140 mg/m2.     

Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene

We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.     

Recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor (G-CSF and GM-CSF) administered following cytotoxic chemotherapy have a similar ability to mobilize peripheral blood stem cells

The availability of hematopoietic growth factors has greatly facilitated the mobilization and collection of peripheral blood stem cells (PBSC). It was the aim of this double-blind study to compare the PBSC-mobilizing efficacy of recombinant human G-CSF and GM-CSF when administered post-chemotherapy. Twenty-six patients with relapsed Hodgkin's disease were included in the study. Their median age was 31 years (range, 22-59) and 14 patients were males and 12 were females. Patients were pretreated with a median of eight cycles of cytotoxic chemotherapy, while 18 patients had undergone extended field irradiation. The patients received dexamethasone 24 mg days 1-7, melphalan 30 mg/m2 day 3, BCNU 60 mg/m2 day 3, etoposide 75 mg/m2 days 4-7, Ara-C 100 mg/m2 twice daily days 4-7 (Dexa-BEAM). Twelve patients were randomized to receive 5/microg/kg/day G-CSF and 14 patients to receive 5 microg/kg/day GM-CSF, both administered subcutaneously starting on day 1 after the end of Dexa-BEAM. Primary endpoints of the study were the number of CD34+ cells harvested per kg body weight on the occasion of six consecutive leukaphereses and the time needed for hematological reconstitution following autografting. Twenty-one patients completed PBSC collection, and six patients of the G-CSF group and nine of the GM-CSF group were autografted. No difference was observed with respect to the median yield of CFU-GM and CD34+ cells: 32.5 x 10(4)/kg vs 31.3 x 10(4)/kg CFU-GM, and 7.6 x 10(6)/kg vs 5.6 x 10(6)/kg CD34+ cells, for G-CSF and GM-CSF, respectively (U test, P= 0.837 and 0.696). High-dose chemotherapy consisted of cyclophosphamide 1.7 g/m2 days 1-4, BCNU 150 mg/m2 days 1-4, etoposide 400 mg/m2 days 1-4. All patients transplanted with more than 5 x 10(6) CD34+ cells/kg had a rapid platelet recovery (20 x 10(9)/l) between 6 and 11 days and neutrophil recovery (0.5 x 10(9)/1) between 9 and 16 days, while patients transplanted with less than 5 x 10(6)/kg had a delayed reconstitution, regardless of the kind of growth factor used for PBSC mobilization. In conclusion, our data indicate that in patients with Hodgkin's disease G-CSF and GM-CSF given after salvage chemotherapy appear to be not different in their ability to mobilize PBSC resulting in a similar time needed for hematological reconstitution when autografted following high-dose therapy.     

Lack of efficacy of thrombopoietin and granulocyte colony-stimulating factor after high dose total-body irradiation and autologous stem cell or bone marrow transplantation in rhesus monkeys

The efficacy of recombinant human thrombopoietin (TPO) and recombinant human granulocyte colony stimulating factor (G-CSF) in stimulating platelet and neutrophil recovery was evaluated in a placebo-controlled study involving transplantation of limited numbers (1-3 x 10(4)/kg) of highly purified autologous stem cells (CD34++/RhLA-DR[dull]) into rhesus monkeys after the animals were subjected to 8 Gy of total body irradiation (TBI) (x-rays). The grafts shortened profound TBI-induced pancytopenia from 5 to 6 weeks to 3 weeks. Daily subcutaneous (sc) injection of TPO (10 microg/kg/day, days 1-21 after TBI) did not stimulate platelet regeneration after transplantation either alone or in combination with G-CSF (5 microg/kg/day sc, days 1-21 after TBI). G-CSF treatment failed to prevent neutropenia in the monkeys and did not stimulate recovery to normal neutrophil levels. Simultaneous administration of TPO and G-CSF did not influence the observed recovery patterns. To test the hypothesis that the limited number of cells transplanted or the subset chosen was responsible for the lack of effectiveness of TPO, three additional monkeys were transplanted with 10(7)/kg unfractionated autologous bone marrow cells. Two of these animals received TPO and the other served as a control. In this setting, as well, TPO treatment did not prevent thrombocytopenia. This study demonstrates that treatment with TPO does not accelerate platelet reconstitution from transplanted stem cells after high-dose TBI. These findings contrast with the rapid TPO-stimulated platelet recovery in myelosuppression induced by 5 Gy of TBI in rhesus monkeys; we conclude from this that the clinical effectiveness of the TPO response depends on the availability of TPO target cells in the first week after TBI, that is, before endogenous TPO levels reach the saturation point. In addition, protracted isolated thrombocytopenia was observed in two G-CSF-treated monkeys, one of which also received TPO. Furthermore, TPO treatment for 7 days in the 6th week after TBI during severe thrombocytopenia in one monkey produced prompt clinical improvement and an increase in platelet counts.     

Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area, CA) beneath the stromal layer was weekly determined for at least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size, but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases.     

2-Chloroethyl-3-sarcosinamide-1-nitrosourea, a novel chloroethylnitrosourea analogue with enhanced antitumor activity against human glioma xenografts

Nitrosoureas are among the most widely used agents used in the treatment of malignant gliomas. Here, the activity of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) was compared with that of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), in vivo against s.c. implanted SF-295 and U-251 central nervous system (CNS) tumor xenografts. When given i.v., q4d for 3 doses, to athymic mice bearing s.c. SF-295 tumors, SarCNU, at an optimum of 167 mg/kg/dose, produced 9 tumor-free animals of 10 total animals, 1 regression, and no evidence of overt toxicity (> or =20% body weight loss). With a similar dosing schedule, BCNU produced no tumor-free animals, six regressions, and one drug-related death at its optimum of 30 mg/kg/dose. Furthermore, SarCNU retained high antitumor activity at two lower dose levels, 66 and 45% of the optimal dose, whereas BCNU demonstrated a progressive loss of antitumor activity at lower doses. Following p.o. administration, SarCNU similarly demonstrated antitumor activity that was superior to that of BCNU. In the U-251 CNS tumor model, SarCNU yielded six of six tumor-free animals at 80 mg/kg/dose with i.p. administration q.d. for 5 days, starting on day 14, whereas BCNU, at 9 mg/kg/dose, yielded three of six tumor-free mice and one drug-related death. Again, SarCNU resulted in tumor-free animals at 66 and 45% of its optimal dose and was relatively nontoxic, in contrast to BCNU. Results of testing to date indicate that SarCNU is clearly more effective than BCNU against the human CNS tumors SF-295 and U-251 in vivo. These results encourage the initiation of clinical trials for SarCNU, in an effort to improve therapeutic approaches to glioma, but clinical trials must determine whether superiority of SarCNU in preclinical models can be extrapolated to patients.     

Synergy of growth factors during mobilization of peripheral blood precursor cells with recombinant human Flt3-ligand and granulocyte colony-stimulating factor in rabbits

The potential of recombinant human (rh)Flt3 ligand (FL), alone or in combination with other recombinant growth factors, to mobilize peripheral blood precursor cells (PBPCs) was examined in an animal model. Adult outbred New Zealand White rabbits received subcutaneous injections daily for 14 days in a standardized protocol; whole blood cell counts and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were measured 3 times weekly during the injection period and for an additional observation period of 14 days. Two animals in each group were treated as follows: 200 or 500 microg/kg FL, 10 microg/kg granulocyte colony-stimulating factor (G-CSF), 10 or 75 microg/kg stem cell factor (SCF), 10 microg/kg G-CSF + 500 microg/kg FL, 10 microg/kg G-CSF + 75 microg/kg SCF + 500 microg/kg FL. Both G-CSF and FL induced a sustained and dose-dependent increase in the leukocyte count to a maximum of 5-fold. They were additive in combination, leading to a tenfold increase in white blood cell counts. No consistent pattern was observed for platelet counts or red blood cells. No toxic side effects were seen. Both G-CSF and FL mobilized CFU-GM in a dose-dependent fashion to a 59-fold increase for G-CSF and 116-fold for FL. Maximum mobilization occurred on day 4 with G-CSF and on day 11 with FL. G-CSF + FL in combination acted synergistically, inducing a 503-fold increase of CFU-GM over baseline. The addition of SCF to this combination did not alter leukocyte counts or CFU-GM mobilization. Our results indicate that FL is a potent and safe agent for the mobilization of PB-PCs and is synergistic with G-CSF.     

Marginal benefit/disadvantage of granulocyte colony-stimulating factor therapy after autologous blood stem cell transplantation in children: results of a prospective randomized trial. The Japanese Cooperative Study Group of PBSCT

In this prospective trial, a total of 74 children who were scheduled to undergo high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT) were prospectively randomized at diagnosis to evaluate the effectiveness of exogenous granulocyte colony-stimulating factor (G-CSF) treatment in accelerating hematopoietic recovery after PBSCT. The diagnosis included acute lymphoblastic leukemia (ALL) (n = 27), neuroblastoma (n = 29), and miscellaneous solid tumors (n = 18). Eligibility criteria included (1) primary PBSCT, (2) chemotherapy-responsive disease, and (3) collected cell number >1 x 10(5) colony-forming unit-granulocyte-macrophage (CFU-GM)/kg and >1 x 10(6) CD34(+) cells/kg patient's body weight. After applying the above criteria, 11 patients were excluded due to disease progression before PBSCT (n = 6) or a low number of harvested cells (n = 5), leaving 63 patients for analysis; 32 patients in the treatment group (300 microg/m2 of G-CSF intravenously over 1 hour from day 1 of PBSCT) and 31 in the control group without treatment. Two distinct disease-oriented high-dose regimens without total body irradiation consisted of the MCVAC regimen using ranimustine (MCNU, 450 mg/m2), cytosine arabinoside (16 g/m2), etoposide (1.6 g/m2), and cyclophosphamide (100 mg/kg) for patients with ALL, and the Hi-MEC regimen using melphalan (180 mg/m2), etoposide (1.6 g/m2), and carboplatinum (1.6 g/m2) for those with solid tumors. Five patients (two in the treatment group and three in the control group) were subsequently removed due to protocol violations. All patients survived PBSCT. The median numbers of transfused mononuclear cells (MNC), CD34(+) cells, and CFU-GM were, respectively, 4.5 (range, 1 to 19) x 10(8)/kg, 8.0 (1.1 to 25) x 10(6)/kg, and 3.7 (1.2 to 23) x 10(5)/kg in the treatment group (n = 30) and 2.9 (0.8 to 21) x 10(8)/kg, 6.3 (1.1 to 34) x 10(6)/kg, and 5.5 (1.3 to 37) x 10(5)/kg, respectively, in the control group (n = 28), with no significant difference. After PBSCT, the time to achieve an absolute neutrophil count (ANC) of >0.5 x 10(9)/L in the treatment group was less than that in the control group (median, 11 v 12 days; the log-rank test, P =.046), although the last day of red blood cell (RBC) transfusion (day 11 v day 10) and the duration of febrile days (>38 degrees C) after PBSCT (4 v 4 days) were identical in both groups. However, platelet recovery to >20 x 10(9)/L was significantly longer in treatment group than control group (26 v 16 days; P =.009) and >50 x 10(9)/L tended to take longer in the treatment group (29 v 26 days; P =.126), with significantly more platelet transfusion-dependent days (27 v 13 days; t-test, P =.037). When patients were divided into two different disease cohorts, ALL patients showed no difference in engraftment kinetics between the G-CSF treatment and control groups, while differences were seen in those with solid tumors. We concluded that the marginal clinical benefit of 1 day earlier recovery of granulocytes could be offset by the delayed recovery of platelets. We recommend that the routine application of costly G-CSF therapy in children undergoing PBSCT should be seriously reconsidered.     

The role of granulocyte colony-stimulating factor in mobilization and transplantation of peripheral blood progenitor and stem cells

The article provides a review of the role of granulocyte colony-stimulating factor (G-CSF) for mobilization and transplantation of peripheral blood progenitor and stem cells. Recombinant gene technology has permitted the production of highly purified material for therapeutic use in humans. Progenitor cells can be assessed using semisolid and liquid culture assays or direct immunofluorescence analysis of cells expressing CD34. This antigen is found on lineage-determined hematopoietic progenitor cells as well as on more primitive stem cells with extensive self-renewal capacity. Administration of G-CSF during steady-state hematopoiesis or following cytotoxic chemotherapy leads to an increase of hematopoietic progenitor cells in the peripheral blood. The level of circulating CD34+ cells post-chemotherapy is greater compared with G-CSF administration during steady state. On the other hand, CD34+ cells harvested post-chemotherapy contain a smaller proportion of more primitive progenitor cells (CD34+/HLA-DR- or CD34+/CD38-) compared with G-CSF treatment alone. Independent of the mobilization modality, the amount of previous cytotoxic chemo- and radiotherapy adversely affects the yield of hematopoietic progenitor cells. While continuous subcutaneous administration of G-CSF between 5 and 16 micrograms/kg bodyweight is preferred, additional dose-finding studies may be helpful to optimize current dose schedules. Adhesion molecules like L-selectin, VLA (very late antigen)-4 and LFA (leukocyte function antigen)-1 are likely to play a role in mobilization, since these antigens are expressed on CD34+ cells from bone marrow in different densities compared with blood-derived CD34+ cells collected following G-CSF-supported cytotoxic chemotherapy. It is also relevant for transplantation that during G-CSF-enhanced recovery post-chemotherapy, peripheral blood is enriched with a greater proportion of CD34+ cells expressing Thy-1 in comparison with CD34+ cells from bone marrow samples obtained on the same day or before the mobilization therapy was started. The early nature of the CD34+/Thy-1+ cells is very likely since this phenotype has been found on stem cells from human fetal liver and bone marrow and on cord blood cells. As a result, G-CSF-mobilized blood stem cells provide rapid and sustained engraftment following high-dose therapy, including myeloablative regimens. Positive selection of CD34+ cells as well as ex vivo expansion using different cytokines are currently being investigated for purging and improvement of short-term recovery post-transplantation. Future developments include the use of blood-derived hematopoietic stem cells for somatic gene therapy. The availability of growth factors has been an important prerequisite for the development of these new avenues for cell therapy.     

Experimental hypercholesterolemia induces ultrastructural changes in the elastic laminae of rabbit aortic valve

The engraftment of hematopoietic stem and progenitor cells in lethally irradiated mice was evaluated following transplants of enriched hematopoietic cell populations which were defined by surface antigen and rhodamine-123 staining. Phenotypically defined long-term repopulating stem cells, short-term pluripotent progenitors, and committed myeloerythroid progenitors all rapidly reconstituted splenic cellularity and peripheral red blood cells after transplant into myeloablated animals. In contrast, marrow cellularity was reconstituted only after transplant of long-term repopulating stem cells. In addition, peripheral blood platelet and lymphocyte counts increased only after transplantation of the long-term repopulating population. Transplantation of highly enriched multipotent progenitors resulted in a transient increase in peripheral blood myeloid cells that occurred with kinetics similar to that seen after transplant of the primitive stem cell population. Erythroid reconstitution was similar in all groups, suggesting that the effect of myeloerythroid progenitor cells in mouse marrow transplants is primarily on reconstitution of the erythroid lineage due to splenic hematopoiesis. Collectively, these results suggest that the cells which function to rapidly reconstitute the nucleated blood cells in a transplant setting are more closely related to primitive, marrow-homing stem cells than to committed progenitor cells.     

Mobilization of peripheral blood stem cells in children using G-CSF after carboplatin containing myelosuppressive chemotherapy

PURPOSE: Peripheral blood stem cell (PBSC) apheresis provides an alternative to autologous marrow harvest as a source of hematologic stem cells for transplantation in children with solid tumors. PATIENTS AND METHODS: Eight children with metastatic or recurrent solid tumors underwent 27 apheresis procedures. Recovery from myelosuppressive chemotherapy occurred without continuous daily growth factor support prior to mobilization. Granulocyte colony stimulating factor (G-CSF) at 16 microgs/kg/day was used to increase stem cells in the peripheral circulation. CD 34 positive cells, mononuclear cells (MNC), and CFU-GM were measured in the apheresis products. Prior chemotherapy was examined as a clinical factor that affected PBSC yield. RESULTS: A significant correlation was found between CD 34+/kg and CFU-GM/kg of the products (r = 0.758, P < 0.001). Patients receiving cumulative doses of carboplatin over 1,600 mg/m2 produced adequate MNC (1 x 10(8)/kg) but yielded significantly less CD 34+ cells or CFU-GM than those patients receiving less carboplatin. Prior doses of etoposide and ifosfamide did not effect PBSC yield. CONCLUSIONS: The mobilization technique was well tolerated, and the products obtained produced trilineage engraftment in the patients that underwent peripheral blood stem cell transplantation. Peripheral blood stem cell apheresis in children can be optimized by selection of appropriate candidates and mobilization with G-CSF after an absence of hematopoietic growth factor support.     

Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.     

Pars plana vitrectomy for chronic pseudophakic cystoid macular edema

We report the effect of granulocyte colony stimulating factor (G-CSF) on neutropenia occurring during extended field radiotherapy in two groups of patients. The first group comprised 8 patients receiving craniospinal irradiation for a variety of central nervous system (CNS) neoplasms. None of these patients received cytotoxic chemotherapy. G-CSF was administered when the absolute neutrophil count (ANC) approached 1.5 x 10(9)/l. Neutropenia was promptly corrected in all cases, thereby avoiding unscheduled interruptions in radiotherapy. Following each G-CSF administration, ANC reached a peak on the following day and then declined steadily. Mean ANC rose from 1.33 x 10(9)/l on the day of G-CSF treatment to 7.07 x 10(9)/l the next day. Patients received 2-6 G-CSF injections during radiotherapy. Experiments were carried out in vitro to assess the risk of G-CSF causing increased CNS tumour cell proliferation. 11 human CNS tumour cultures (2 medulloblastomas, 2 primitive neuroectodermal tumours and 7 astrocytic tumours) were cultured in the presence of G-CSF at a range of concentrations up to 100 ng/ml. Their proliferation was compared with that of a G-CSF dependent murine leukemia cell line (NFS-60). None of the human tumour cultures demonstrated a significant increase in proliferation in response to G-CSF. 4 patients undergoing "mantle" type radiotherapy for Hodgkin's Disease or Non-Hodgkin's Lymphoma also received G-CSF treatment for neutropenia. All 4 had previously received cytotoxic chemotherapy. The number of G-CSF injections given per patient during radiotherapy ranged from 3-6. Mean ANC rose from 1.76 x 10(9)/l to 10.8 x 10(9)/l the next day. These results suggest that G-CSF is a reliable treatment for radiotherapy induced neutropenia and that an intermittent dosage schedule is effective.     

Endogenous interleukin-8 (IL-8) surge in granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

The relationship between stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and the endogenous production of interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was studied in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. G-CSF was administered to 20 normal donors at a dose of 10 microg/kg/d for 5 days with aphereses on days 5 and 6 of G-CSF treatment. Cytokine serum levels were measured using an enzyme-linked immunosorbent assay (ELISA) before and during G-CSF treatment. Before treatment, the average level of IL-8 was 7.1 pg/mL, increasing to 207.0 pg/mL on day 5 and 189.1 pg/mL on day 6. Serum IL-8 levels correlated CD34(+) cell numbers (P =.0151 and P =.0005 on days 5 and 6, respectively) and colony-forming unit-granulocyte-macrophage (CFU-GM) numbers (P =.0019 and P =.0010 on days 5 and 6, respectively). Furthermore, preapheresis serum IL-8 levels correlated with the yield of CD34(+) cells (P =.0027). In contrast, before treatment, the average levels of MIP-1alpha, TNF-alpha, and IFN-gamma were 70.1, 4.03, and 3.84 pg/mL, respectively, and no significant changes in the levels of these cytokines were observed during G-CSF treatment. These studies suggest that IL-8 production may be critical to G-CSF-induced stem cell mobilization, although the underlying mechanism could not be clarified.     

Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells

One of the controversies surrounding the repopulating capacities of haemopoietic stem cells is whether or not the same or different populations are responsible for short-term and long-term repopulation after transplantation. To address this question, we analysed results obtained from an in vitro model for the clonal production of granulocyte-macrophage colony-forming cells (CFU-GM) by individual primitive multilineage precursors in adult human bone marrow. The primitive precursors adhere to plastic and produce CFU-GM in a 1-week long 'delta' type culture. The clones that form are classified as having short maturation pathways (clones containing predominantly day 7 CFU-GM) or long maturation pathways (clones containing predominantly day 21 CFU-GM). The results indicate that individual primitive (P delta) cells produce clones that reach full maturity after different periods of time so that cells corresponding to a range of maturational stages can become available simultaneously. Consequently, transplanted stem cells may be able to provide both rapid and long-term mature cell recovery whilst at the same time reconstituting the stem cell pool. These results suggest that it might be possible to use highly purified stem cell populations, devoid of committed progenitors, for clinical transplantation.     

The 15th anniversary of the Department of Therapy of the Military Medical Faculty in the Russian Medical Academy of Postgraduate Education

The relationship between dose intensity of cytotoxic agents and therapeutic results was examined in a retrospective analysis of 32 patients with non-metastatic high-grade osteosarcoma of the extremities. The average dose intensities of individual agents were 9.8 mg/m2/week for doxorubicin, 1.2 g/m2/week for methotrexate, and 10.5 mg/m2/week for cisplatinum. The dose intensities of doxorubicin and methotrexate were significantly correlated with the clinical results, while that of cisplatinum was not. These results indicate that maximal dose intensification of doxorubicin and methotrexate is an important determinant of treatment outcome for patients with osteosarcoma.     

Induction of specific-locus and dominant lethal mutations in male mice by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) induced dominant lethal and specific-locus mutations in male mice. For both compounds the germ cell stage sensitive to the induction of dominant lethal mutations was dose dependent. A dose of 5 mg BCNU per kg b.wt. induced dominant lethal mutations primarily in spermatocytes, whereas higher doses of BCNU induced dominant lethals in spermatids and spermatocytes. Following doses of 5 and 10 mg CCNU per kg b.wt. dominant lethals were induced in spermatids and spermatocytes similar to the results for higher doses of BCNU. Higher dose exposure to BCNU and CCNU was associated with dominant lethals expressed as pre-implantation loss (reduction in total number of implants). In addition, higher doses of CCNU showed a cytotoxic effect in differentiating spermatogonia. Both compounds induced specific-locus mutations in post-spermatogonial germ cell stages of mice. However, CCNU increased also the specific-locus mutation frequency in spermatogonia in two out of three experiments. We conclude in analogy with criteria developed by IARC, that BCNU and CCNU are potential human mutagens.     

An investigation on plasma progesterone levels during pregnancy and at parturition in the Ivesi sheep

We have investigated the toxicity of dose-escalation of BCNU, etoposide and melphalan ('BEM') chemotherapy with autologous stem cell transplantation in patients with haematological malignancies. Seventy-two patients with haematological malignancies were treated with BCNU (600 mg/m2, 450 mg/m2 or 300 mg/m2), etoposide 2 g/m2 and melphalan 140 mg/m2 followed by autologous bone marrow transplantation (ABMT), n = 51, or autologous peripheral blood progenitor cell transplantation (APBPCT), n = 21. Liver and pulmonary function was monitored pretransplant and at regular intervals post-transplant. Mucositis was graded daily during in-patient stay. There was a significantly higher incidence of symptomatic pulmonary toxicity in the patients who received BCNU at 600 mg/m2 than in the other two groups, and there was a significant increase in the incidence of asymptomatic decrease in carbon monoxide (KCO) in the patients who received BCNU 450 mg/m2. There was no significant difference between the three groups in the incidence and severity of mucositis or in the incidence of transiently abnormal liver function. We conclude that etoposide at 2 g/m2 can be used without unacceptable mucositis. BCNU at 600 mg/m2 is associated with an unacceptably high incidence of lung toxicity, but at 450 mg/m2 there is minimal symptomatic lung toxicity.     

粒细胞集落刺激因子对供体造血干细胞移植物中树突状细胞的影响

目的 探讨粒细胞集落刺激因子(G-CSF)对正常异基因造血干细胞移植供者外周血与骨髓移植物中Ⅰ型树突状细胞(DC1)、Ⅱ型树突状细胞(DC2)的数量及DC2/DC1比例的影响.方法 以G-CSF每天10μg/kg动员5 d后,以流式细胞术(FCM)检测11例G-CSF动员的异基因外周血造血干细胞移植物及20例G-CSF动员的异基因骨髓移植物中的DC1、DC2数量及DC2/DC1比例,并与8例正常供者动员前外周血及10例健康者动员前骨髓进行比较.结果 动员前后骨髓DC2由14.37×106/L增至29.68×106/L(t=2.433,P=0.022),而骨髓DC1分别为13.77×10a/L和18.88×106/L(t=0.625,P=0.541);DC2/DC1比例在动员后为1.83±0.81,较动员前的1.12±0.32明显升高(t=2.685,P=0.013).正常供者以G-CSF动员前、后移植物中外周血DC2数量分别为14.92×106/L和26.76×106/L(t=2.390,P=0.029),DC2/DC1比例分别为1.00±0.37和2.02±1.43(t=2.158,P=0.044),但外周血DC1分别为14.21×106/L和18.02×106/L(t=0.625,P=0.541).结论 移植前以G-CSF动员正常异基因干细胞移植供者,可选择性提高外周血及骨髓移植物中DC2的数量,而DC1数量无明显增加.