The role of haemopoietic growth factors in bone marrow transplantation.

The availability of recombinant haemopoietic growth factors for clinical use has led to a proliferation of trials in the setting of bone marrow transplantation. Early results from these studies, using GM-CSF and G-CSF, show that these factors are able to reduce the period of cytotoxic induced neutropaenia but have little effect on platelet recovery. Toxicity has been relatively mild unless very high doses are administered. Randomised controlled trials are in progress and will help to define the exact role of growth factors in this setting. Future prospects include the increasing availability of other growth factors for clinical use and the potential for combination growth factor therapy to provide a more optimal haemopoietic response.     

Detection of host cells following sex-mismatched bone marrow transplantation by fluorescent in situ hybridization with a Y-chromosome specific probe

Fluorescent in situ hybridization (FISH) with a biotinylated Y-chromosome specific repetitive DNA probe was applied to detect Y-bearing cells in blood and bone marrow samples from patients with hemopoietic malignancies after a sex-mismatched bone marrow transplantation. The sensitivity of this method is in the order of 0.1% Y-bearing nuclei in male recipients transplanted with female marrow. In female recipients of male marrow, the detection of low numbers of non-Y-bearing nuclei is less sensitive. The presence of host cells in blood and bone marrow of seven patients (four males, three females) was investigated with respect to successful engraftment or recurrence of the disease. The results obtained were compared with cytology (all seven cases) and with conventional cytogenetics (five cases). In five patients, the results of Y-FISH and cytology were identical. In two patients, low numbers of male host cells were detected in the marrow by Y-FISH, whereas cytology indicated complete remission of the disease. In three patients Y-FISH and cytogenetic data were similar, but in two patients Y-FISH revealed the presence of 0.2% and 7% male host cells, respectively, in bone marrow, whereas cytogenetics indicated a 100% female marrow in both cases. Because the hybridization was performed in situ, the morphology of the nuclei was preserved. To differentiate between normal and leukemic cells, the size of the blast cell nuclei appeared to be a very useful indicator. Our data suggest that fluorescent in situ hybridization with a Y-chromosome specific probe is a fast and sensitive technique to identify the host cells after sex-mismatched bone marrow transplantation, in particular in case of male recipient and female donor combinations.     

Donor marrow progenitors (CFU-Mix, BFU-E and CFU-GM) and haemopoietic engraftment following HLA matched sibling bone marrow transplantation

Bone marrow multipotent (CFU-Mix) and unipotent (CFU-GM and BFU-E) progenitor cells in the donor marrow inoculums were measured in 24 histocompatible sibling bone marrow transplants. The number of donor marrow nucleated cells, CFU-Mix, CFU-GM and BFU-E given per kilogram (kg) of recipient's body weight were 2.4 +/- 0.6 X 10(8), 3.6 +/- 4.2 X 10(3), 4.9 +/- 3.3 X 10(-4) and 4.3 +/- 4.1 X 10(4) respectively (mean +/- S.D.). Fast engraftment patients, as assessed by rise in peripheral blood neutrophils (greater than or equal to 0.5 and greater than or equal to 1.0 X 10(9)/l) and platelets (greater than 20 and greater than 50 X 10(9)/l), received a significantly greater amount of CFU-Mix/kg (greater than 3 X 10(3)/kg, p less than 0.025) and CFU-GM/kg (greater than 3 X 10(4)/kg, p less than 0.05 except for plat greater than or equal to 20 X 10(9)/l) than the slow recovery patients. Significant correlations were found between the donor CFU-Mix/kg infused and neutrophil recovery to 1 X 10(9)/l and platelet to 50 X 10(9)/l (Spearman's rank correlation coefficient r = 0.38, p = 0.04 and r = 0.58, p = 0.003, respectively). The amount of donor CFU-GM/kg given also correlated significantly to neutrophil (1 X 10(9)/l) and platelet (50 X 10(9)/l) recovery, (r = 0.33 and r = 0.37, respectively, p less than or equal to 0.05). There was no association between BFU-E, and marrow nucleated cells infused per kg and haemopoietic recovery. A number of clinical parameters were also examined to determine other factors that may influence the rate of engraftment. Acute graft vs host disease (greater than or equal to grade II) and methotrexate therapy post-transplant delayed the platelet regeneration. The results of the present report indicate that in vitro measurement of donor CFU-Mix and CFU-GM progenitors infused, correlate with the speed of granulocyte and platelet recovery in clinical allogeneic bone marrow transplants.     

Recovery of blood and bone marrow stem cells following intense chemotherapy and autologous bone marrow transplantation

Sixteen patients with advanced (stage III) malignant melanoma were treated with escalating doses of intravenous BCNU and melphalan starting at 400 and 35 mg/m2, respectively, and escalating to 1,000 and 110 mg/m2, respectively, combined with autologous marrow transplantation. The duration of granulocytopenia and time to granulocyte recovery was similar in all groups regardless of chemotherapy dose. Platelet recovery was delayed in patients receiving the highest doses of chemotherapy. This study showed that bone marrow colony-forming units in culture took as long as 6 months to recover. This was adequate to bring peripheral blood counts to normal but not to pretreatment levels. These studies indicate that autologous bone marrow transplantation is beneficial in enhancing short-term recovery, but may not be beneficial in the long-term hematopoietic recovery.     

Extra-medullary relapse of leukemia following allogeneic bone marrow transplantation.

The curative effect of allogeneic bone marrow transplantation (BMT) for acute and chronic leukemia is attributed to the intensive conditioning chemotherapy with or without radiotherapy, as well as an immune-mediated graft versus leukemia (GVL) effect. A different pattern of relapse has been observed after allogeneic BMT for patients with leukemia. Compared with treatment using conventional chemotherapy alone, isolated extra-medullary relapse of disease appears to be seen more commonly after allogeneic BMT. While a full donor's hematopoiesis may be retained, prolonged morphological remission has been observed in the recipient's bone marrow. There appears to be a population of leukemic cells with an affinity to extra-medullary tissues. The failure of the leukemic clone to repopulate the recipient's marrow suggests the presence of a more profound GVL effect in the marrow environment. The optimal treatment for extra-medullary relapse of leukemia following allogeneic BMT remains uncertain. In the case of isolated extra-medullary relapses following BMT, the leukemia may still be responsive to further treatment with chemotherapy and/or radiotherapy. The prognosis is poor in general, but prolonged survival has been observed in some of these patients. With the preservation of donor's hematopoiesis in the recipient's marrow, the use of intensive chemotherapy followed by donor lymphocyte or stem cell re-infusion is a promising option.     

Histomorphologic study of bone marrow in acute leukemia following chemotherapy and autologous bone marrow transplantation

Twenty-six patients with acute myeloid leukemia, acute lymphoid leukemia and chronic granulocytic leukemia in blast crisis were studied by means of multiple biopsies during a polychemotherapeutic or autologous bone marrow transplant protocol. Following chemotherapy, 3 main phases were observed: leukemic cellular depletion, stromal bone marrow reconstruction, and bone marrow hemopoietic restoration. Following intensive chemotherapy (in 2 patients after cyclophosphamide and total body irradiation) and autologous bone marrow transplantation, the 3 phases appeared to be shorter. A focal or diffuse increase in marrow fibrosis was a common finding in leukemia. An effective antileukemic therapy resulted in a decrease in fibrosis, whereas in some cases a further increase was a precocious sign of leukemia relapse.     

In vitro manipulation of bone marrow cells for bone marrow transplantation

Recently, in vitro manipulations of bone marrow cells have been developed for the prevention of relapse in autologous bone marrow transplantation (BMT) and for the prevention of graft versus host disease in allogeneic BMT. Two methods were mainly employed clinically for the depletion of cells from transplanted bone marrow cells. Firstly, bone marrow cells were treated in vitro with monoclonal antibodies reactive to leukemia cells or T cells using complement, immunotoxin or magnetic bead. Secondly, bone marrow cells were incubated with cancer drugs such as 4-HC or mafosfamide. Our results and other reports using in vitro purging of leukemia or lymphoma cells suggest that these autologous BMTs are effective modes of cancer therapy in patients with hematological malignancies.     

Absorption of oral aminoglycosides following bone marrow transplantation

Four patients with severe gastrointestinal reactions receiving oral "nonabsorbable" antibiotics for gut sterilization following bone marrow transplantation absorbed clinically significant amounts of aminoglycoside (gentamicin and/or tobramycin). Serum concentrations of 2.2, 2.6, 5.8, and 12.0 micrograms/ml were measured. Two of these patients had acute graft versus host reactions and two had severe mucositis following cytoreduction with intensive chemotherapy and irradiation. Nephrotoxicity occurred in the latter patients. One patient was studied in detail. Her hospital course and investigative results are presented. Four additional patients with mild gastrointestinal reactions following cytoreduction did not absorb gentamicin when their toxicity was maximal. Serum aminoglycoside determinations are necessary in patients receiving oral aminoglycosides for gut sterilization following bone marrow transplantation if moderate to severe gastrointestinal reactions occur.     

甲异靛对白血病骨髓基质细胞调节白血病细胞增殖的影响

 【摘要】　目的　探讨甲异靛对白血病骨髓基质细胞干预白血病细胞增殖的影响。方法 　利用白血病骨髓单个核细胞培养骨髓基质细胞,并建立白血病细胞和骨髓基质的共培养体系。用锥虫蓝拒染实验测定甲异靛对共培养体系中白血病细胞增殖的影响;流式细胞术检测白血病细胞膜上CXCR4的表达。结果　白血病骨髓基质细胞可抑制白血病细胞的增殖。低浓度的甲异靛（5 μmol／L）可促进白血病和骨髓基质细胞共培养体系中白血病细胞的增殖。白血病细胞膜异常高表达CXCR4,甲异靛可明显抑制HL-60细胞和原代白血病细胞膜上CXCR4的表达,骨髓基质细胞可以促进白血病细胞膜上CXCR4的表达。结论　甲异靛可能通过下调白血病细胞膜上CXCR4的表达起抗白血病作用。     

同种自然杀伤细胞在移植与免疫治疗中的研究进展

自然杀伤（NK）细胞是具有多种免疫学功能的淋巴样细胞,处于机体防御体系的第一道防线。输注活化同种NK细胞是近年来肿瘤免疫治疗的方法之一,尤其配合骨髓移植治疗显示出了强大的抗瘤效应。现综述同种NK细胞识别、作用机制及治疗方面的研究进展。     

Multipotent and committed CD34+ cells in bone marrow transplantation.

In order to study the role of CD34+ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34+ cells were also performed to evaluate their in vitro hematopoietic function. CD34+ cells were detectable in bone marrow cells on day 14. More than 80% of CD34+ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34+ cells. In normal bone marrow cells, CD34+, CD33+ cells amounted to about 40% of CD34+ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34+, CD33- cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34+, CD33+ cells as committed progenitors and CD34+, CD33- cells as multipotent stem cells have distinctive biological behaviors in BMT.     

Risk factors in interstitial pneumonitis following allogenic bone marrow transplantation

Total body irradiation is part of the preparatory regimen for allogeneic bone marrow transplantation because of its cytotoxic and immunosuppressive properties. A major toxicity of bone marrow transplantation has been interstitial pneumonitis, which may be, in part, related to the lung irradiation. One hundred and sixty-one consecutive patients receiving allogeneic bone marrow transplantation for leukemia and aplastic anemia at Johns Hopkins Hospital (1968-1979) were retrospectively studied. The present study demonstrated that lung shielding to 600 rad maximum in single dose total body irradiation, fractionation of total body irradiation in comparison to single dose total body irradiation, and absence of graft versus host disease in the leukemia patients, each reduced the risk of interstitial pneumonitis. Total body irradiation significantly reduced the leukemia recurrence rate and/or the failure of remission induction.     

Malignant gliomas actively recruit bone marrow stromal cells by secreting angiogenic cytokines

The transplantation of progenitor cells is a promising new approach for the treatment of gliomas. Marrow stromal cells (MSC) are possible candidates for such a cell-based therapy, since they are readily and autologously available and show an extensive tropism to gliomas in vitro and in vivo. However, the signals that guide the MSC are still poorly understood. In this study, we show that gliomas have the capacity to actively attract MSC by secreting a multitude of angiogenic cytokines. We demonstrate that interleukin-8 (IL-8), transforming growth factor-ss1 (TGF-ss1) and neurotrophin-3 (NT-3) contribute to this glioma-directed tropism of human MSC. Together with the finding that vascular endothelial growth factor (VEGF) is another MSC-attracting factor secreted by glioma cells, these data support the hypothesis that gliomas use their angiogenic pathways to recruit mesenchymal progenitor cells.     

Monoclonal antibody to ganglioside GQ discriminates between haemopoietic cells and infiltrating neuroblastoma tumour cells in bone marrow

An immunological approach has been sought for the identification of minimal metastatic spread of neuroblastoma to bone marrow. Here we describe the reactivity of the monoclonal antibody A2B5 to human neuroblastoma cell lines and fresh tumour tissue. This reagent, raised against chick retinal cells, reacts with all human neuroblastoma lines assayed although quantitative differences in antigenic expression exist between cultures. Analysis of tumour cells in heavily infiltrated bone marrow aspirates indicates that only 70% of the samples reacted with A2B5, suggesting that the heterogeneity seen in the expression of antigen on cell lines is paralleled in fresh tumour material. A2B5 showed no reactivity to either a panel of human leukaemic cell lines or normal human bone marrow, although reactivity to an occasional leukaemic marrow aspirate was detected. We suggest that A2B5 could form part of a panel of monoclonal reagents necessary for detecting metastatic spread of all neuroblastoma cells to bone marrow. Such a group of reagents may be useful therapeutically in a programmed of autologous bone marrow transplantation for the removal of tumour cells prior to reinfusion of haemopoietic cells to patients receiving high-dose chemotherapy.     

Lung damage following bone marrow transplantation: I. The contribution of irradiation

High dose whole body irradiation is commonly included in conditioning regimens for bone marrow transplantation for treatment of patients with hematological malignancies. Interstitial pneumonitis is a major complication after BMT. About one-fourth of all BMT patients die from IP. In about half of these cases, an infectious agent, particularly cytomegalovirus, is involved. When no infectious cause is found, it is classified as idiopathic IP (IIP). Total body irradiation is often associated with the induction of IIP; however, extrapolation of animal data from the experiments presented indicates that this is not the only factor contributing to IIP in man. Brown Norway (BN/Bi) rats were bilaterally irradiated to the lungs with 300 kV X rays at a high dose rate (HDR; 0.8 Gy/min) and at a low dose rate (LDR; 0.05 Gy/min). The dose-response curves found were very steep. In the LDR group, lung function studies were performed. There was a strong correlation between the increase in ventilation rate and the death pattern. The LD50 at 180 days was 13.3 Gy for HDR and 22.7 Gy for LDR. The ratios of LD50/180 at 0.05 Gy/min to that at 0.8 Gy/min is 1.7, which indicates a great repair capacity of the lungs. Extrapolation of animal data to patient data leads to an estimated dose of about 15-16 Gy at a 50% radiation pneumonitis induction for low dose rate TBI. As the absorbed dose in the lungs of BMT patients rarely exceeds 10 Gy, additional factors such as remission-induction chemotherapy, cyclophosphamide, methotrexate, cyclosporin A, graft-versus-host disease, etc., might be involved in the high incidence of IIP in man after BMT.     

Graft-versus-ATLL effect induced by abrupt discontinuation of immunosuppression following allogeneic bone marrow transplantation

A 46-year-old man was admitted to our hospital with swelling of a neck lymph node in June, 2002, and was diagnosed with adult T-cell leukemia/lymphoma (ATLL). As ATLL cells were detected in the peripheral blood after two courses of multi-agent chemotherapy (LSG 15), the treatment was changed to biweekly CHOP therapy. After two courses, hematological remission was achieved. Allogeneic bone marrow transplant (allo-BMT) from HTLV- negative and HLA-matched sibling donor was performed (conditioned with cyclophosphamide 60 mg/kg x 2 and total body irradiation 12 Gy). Cyclosporine A (CsA) and short-term methotrexate (MTX) were used for graft-versus-host disease prevention. Though the HTLV- provirus DNA (Southern blot) disappeared, HTLV-I provirus DNA (real-time PCR) T-cell receptor ygammachain gene rearrangement DNA (Southern blot) were detected in bone marrow after allo-BMT. MRD disappeared after the withdrawal of CsA. After the allo-BMT transplant, a graft-versus-ATLL (GVATLL) effect may be induced by abrupt discontinuation of immunosuppression.     

Surgical implications of hepatic venocclusive disease following bone marrow transplantation

Hepatic venocclusive disease occurs with a spectrum of severity in an estimated 21% of bone marrow transplant patients. Clinical features include severe right upper quadrant pain, ascites, weight gain and initially minimal derangement of liver function. In contrast to hepatic graft versus host disease, venocclusive disease usually occurs within the first three weeks of engraftment and in autologous grafts. Urgent surgical consultation is requested when these features are prominent enough to mimic common acute processes requiring laparotomy. This condition must be included in the differential diagnosis in order to avoid an unnecessary laparotomy in this select group of patients who are usually severely thrombocytopenic and leukopenic. Clinical diagnosis alone is very reliable.     

Lung damage following bone marrow transplantation: II. The contribution of cyclophosphamide

The effect of high-dose cyclophosphamide (Cy), either alone or in combination with irradiation, upon the development of interstitial pneumonitis (IP) after bone marrow transplantation (BMT) was investigated in a Brown Norway rat model. The parameters that were examined included ventilation rate, mortality, and histopathology. No damage to the lungs was observed in rats given Cy alone in supralethal dosages plus BMT, and mortality resulted from severe aplasia of hemopoietic and lymphoid tissues with multifocal hemorrhages, secondary infections, and sepsis. Two separate periods of mortality were observed within the first 180 days following whole thorax irradiation with a high dose rate (HDR; 0.8 Gy/min) or a low dose rate (LDR; 0.05 Gy/min). The addition of Cy prior to irradiation resulted in an increased mortality in the first period (before day 100) in all experimental groups. The influence of Cy on mortality at 180 days however, was different for the HDR and LDR experiments. The LD50-180 after HDR irradiation, dose range 8 to 18 Gy, was not significantly altered by the addition of Cy (100 mg/kg) 1 day prior to irradiation, whereas Cy (100 mg/kg) 1 day prior to LDR irradiation, dose range: 16 to 24 Gy, caused an enhancement of radiation damage with a decrease of the LD50-180 by 1.33 Gy. The dose modification factor (DMF) was 1.07. This enhancement was no longer significant after splitting up the dose of Cy in two dosages of 50 mg/kg given on 2 consecutive days prior to irradiation with a LDR. The extrapolation of the data in this rat model to available dose-response curves on IP after BMT and radiation pneumonitis in humans, implied that non-infectious IP is a radiation pneumonitis that is only slightly enhanced by Cy.