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CD34+ cell selection significantly improves GvHD-free survival in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, specific information regarding long-term prognosis and risk factors for late mortality after CD34+ cell-selected allo-HSCT is lacking. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT for AML (n=164), ALL (n=33) or myelodysplastic syndrome (n=79). At 5 years' follow-up after the 1-year landmark (range 0.03-13 years), estimated relapse-free survival (RFS) was 73% and overall survival (OS) 76%. The 5-year cumulative incidence of relapse and non-relapse mortality (NRM) were 11% and 16%, respectively. In multivariate analysis, Hematopoietic Cell Transplantation Comorbidity Index score⩾3 correlated with marginally worse RFS (hazard ratio (HR) 1.78, 95% confidence interval (CI) 0.97-3.28, P=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26-5.08, P=0.004). Despite only 24% of patients with acute GvHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GvHD associating with increasingly poorer survival on multivariate analysis (P<0.0001). Of 63 deaths after the landmark, GvHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. Although prognosis is excellent for patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GvHD.

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High-dose chemotherapy (HDC) followed by autologous hematopoietic reconstitution is an experimental treatment option for patients with epithelial ovarian cancer. However, the incidence of occult ovarian tumor cell involvement in autologous bone marrow (BM) or peripheral blood stem cell (PBSC) autografts has not been widely investigated. We used a highly sensitive immunocytochemical (ICC) procedure that detects occult blood-borne tumor micrometastases. We analyzed 24 BM specimens (15 obtained during therapy and 9 harvest samples) and seven PBSC specimens from 22 patients with ovarian cancer. Overall, ICC analysis detected immunostained tumor cells in 10 of 23 evaluable BM specimens (43%) from 9 of 19 patients (47%). One of 9 (11%) harvest samples contained tumor cells. Only one of the 10 ICC-positive BM specimens had tumor cells detected by routine histopathological analysis. ICC-detectable tumor cells were cleared from the marrow of two patients during chemotherapy. None of the seven PBSC specimens contained tumor cells. We conclude that ovarian cancer micrometastases have the potential to contaminate BM, as is also the case in patients with other epithelial malignancies. In the limited number of specimens analyzed, PBSC harvests appeared to provide a less tumor-contaminated source of hematopoietic stem cells for autologous transplantation.

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Improved marrow processing techniques and in vitro marrow manipulations are revolutionizing the clinical application of both allogeneic and autologous bone marrow transplantation. The rapid evolution of clinically useful laboratory techniques now necessitates more sophisticated laboratory support of bone marrow transplantation.

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This study demonstrated for the first time that bone marrow is a target of enhanced in vivo monocytopoiesis in hyperlipemia. A significantly greater number of bone marrow cells (BMC) were recovered per femur in swine fed a hyperlipemic (HL) diet compared with swine fed a normal (N) diet. In addition, a significantly elevated number of monocytic precursors proliferated in HL-swine compared with N-swine BMC cultures grown in standardized media in the absence of an exogenous source of colony stimulating factor (CSF). HL-swine sera stimulated a significant enhancement in the number of proliferating monocytic precursor cells, regardless of whether or not the BMC were from HL- or N-swine. In addition, HL-swine compared with N-swine BMC demonstrated an enhanced intrinsic capacity to form monocytic colonies in culture, irrespective of the source of swine sera used to stimulate growth.

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One limitation of autologous bone marrow transplantation for patients with cancer has been the presence of tumor cells in the bone marrow. Methods to eliminate tumor cells while preserving hematopoietic stem cells have been sought. The present study was performed to analyze the in vitro effectiveness of light-activated merocyanine 540 phototreatment (LAMP) and an aminothiol (ethiofos) as a marrow-purging regimen for small cell lung cancer (SCLC). Two human SCLC cell lines (ATCC HTB-119 and HTB-120) were treated with LAMP and exposed to light for varying periods of time up to 120 min. LAMP reduced SCLC cell proliferation and colony formation in a light exposure-dependent manner; colony formation was not totally inhibited until light exposure of 120 min was used. At this light exposure interval, multipotential hematopoietic progenitors, colony-forming units-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), were substantially reduced. In an attempt to diminish hematopoietic toxicity, SCLC cells were incubated with ethiofos (formerly WR-2721) for 1 hour before LAMP. SCLC colony formation was eliminated at light exposure intervals (90 min or less) which had no inhibitory effect on CFU-GEMM. Ethiofos did not protect CFU-GEMM from LAMP inhibition at 120 min. Ethiofos alone had no effect on the SCLC or hematopoietic cells. When normal bone marrow was contaminated with 1 or 5% SCLC cells, ethiofos plus 60 min of LAMP eliminated SCLC cells but had no effect on CFU-GEMM. The results suggest that ethiofos sensitized SCLC cells to LAMP; thus ethiofos-enhanced LAMP may be an effective method for removing metastatic SCLC cells from bone marrow used for autologous marrow transplantation after high dose chemotherapy.

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Pokeweed mitogen-stimulated rat spleen cells were identified as a reliable source of rat burst-promoting activity (PBA), which permitted development of a reproducible assay for rat bone marrow erythroid burst-forming units (BFU-E). Optimum BPA dose, assay time, cell dose, and erythropoietin requirements for rat BFU-E were identified. A serum-free assay and a method for stimulating endogenous bone marrow BPA were developed. Identification of sources of rat BPA and characterization of the rat BFU-E assay makes this species more useful for hematopoietic studies. The similarity of rat BFU-E to human and mouse BFU-E strengthens the validity of rat models for erythropoiesis.

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Adenosine is an important regulatory molecule that increases in hypoxic and ischemic tissues and has been proposed to mediate blood flow in response to oxygen availability. The current study ascribes another oxygen-responsive role to adenosine, that of regulating synthesis of the erythropoiesis-stimulating hormone, erythropoietin. When perfused through isolated rat kidneys, exogenous adenosine in nanomolar concentrations increased erythropoietin production, whereas inosine, the deaminated nucleoside, had no effect. In addition, an adenosine antagonist, and adenosine deaminase, diminished erythropoietin titers in renal perfusates. In intact rats, adenosine deaminase injections followed by a hypoxic stimulus slightly reduced erythropoietin serum concentrations, whereas an adenosine deaminase inhibitor sharply increased erythropoietin titers. The results suggest that adenosine may function as a mediator to link oxygen supply with erythropoietin production.

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To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light-density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.

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An inhalation method of ethanol administration was used to study the effects of 14 days of ethanol administration on the immune and hematopoietic systems of the rat. A decrease in cellularity was found in the spleen, thymus, and bone marrow of ethanol-treated rats. Although the red blood cell count, white blood cell count, and hemoglobin concentration were not significantly different between treatment and control groups, treatment with ethanol altered the relative proportion of lymphocytes and polymorphonuclear leukocytes in the peripheral blood. The granulocyte-macrophage progenitor cells in the bone marrow were unaffected by ethanol treatment, but a significant decline in the number of erythroid progenitor cells was noted in ethanol-treated rats. Splenic lymphocytes, although fewer in number in the ethanol-treated rats, showed no significant difference in ability to proliferate when stimulated by nonspecific mitogens.

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Chronic ethanol abuse causes thrombocytopenia but the underlying mechanism is unknown. To determine the target cells involved, we examined the effects of the drug in vitro on both megakaryocyte progenitor cells (CFU-Meg) and isolated, maturing megakaryocytes. In the presence of ethanol concentrations of 0.05-2.0 g/dl, megakaryocyte colony formation by mouse CFU-Meg in soft agar was normal. At 5 g/dl ethanol, colony formation was reduced by 50%; with 7 g/dl ethanol, no megakaryocyte colonies were formed. Acetaldehyde did not inhibit colony formation unless very high concentrations (100 mg/dl) were employed. Isolated guinea-pig megakaryocytes can maintain their viability and incorporate 3H-leucine into TCA-precipitable protein for at least 24 h. Incubation of these maturing megakaryocytes with ethanol did not affect their viability, but at concentrations greater than 120 mg/dl ethanol progressively inhibited protein synthesis. At 0.5 g/dl ethanol, protein synthesis was decreased by 23% while viability was still 93% of control. Like CFU-Meg, maturing megakaryocytes were resistant to the toxic effects of acetaldehyde. To determine the in vivo correlates of these results, guinea-pigs were fed 5 g/dl ethanol in a liquid diet. By 11 d, when blood ethanol levels were 20-150 mg/dl, platelet counts in the animals were reduced by 17-29%, while the number of marrow megakaryocytes was unaltered. These data indicate that the site of action of ethanol in suppressing thrombopoiesis is at the level of the maturing megakaryocyte.

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Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.

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Medium conditioned by mouse thymic cells cultured for 7 days in the presence of concanavalin A (Con A) and 5% human serum enhanced erythroid colony formation by CFU-E but not BFU-E. Medium conditioned by thymic cells for less than 7 days or more than 7 days inhibited erythroid colony formation. Enhancement of erythroid colony formation occurred at both suboptimal and saturating concentrations of erythropoietin. The thymus-conditioned medium also stimulated the growth of granulocyte/macrophage colonies by itself and in the presence of a saturating concentration of WEHI-3 conditioned medium. The conditioned medium contained neither burst-promoting activity nor erythropoietin. The erythroid colony-enhancing activity in the thymus-conditioned medium was heat stable, bound to immobilized Con A, lacked protease activity and migrated with an approximate molecular weight of 68,000 on gel filtration.

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To determine the extent intrinsic erythrocyte defects and/or extrinsic factors were involved in anemia of rats bearing Shay chloroleukemia (SCL), survival of 3H-DFP labeled erythrocytes was studied in leukemic and nonleukemic hosts. Red blood cells labeled before induction of leukemia, were rapidly lost from the peripheral circulation of SCL rats in terminal stages of disease. However, labeled erythrocytes from terminal SCL animals displayed normal lifespans when transfused into nonleukemic controls. Thus the anemia of this leukemia probably resulted from extrinsic factors associated with the leukemic process. Hemorrhage appeared to be primarily responsible for the anemia of this disease.

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The effects of alcohol on bone marrow are not well understood. We measured the influence of ethanol and its metabolite, acetaldehyde, on the in vitro proliferation of hematopoietic progenitor cells from mice and human beings. Colony formation by both early and late erythroid progenitor cells was suppressed by concentrations of ethanol (0.05 to 0.2 per cent) that are easily achieved in vivo. The corresponding suppressing concentration of acetaldehyde was 0.001 per cent. In contrast, suppression of granulocyte/macrophage progenitor cells required 3.0 per cent ethanol or 0.03 per cent acetaldehyde. Spleen colony formation by pluripotent stem cells was resistant to concentrations of ethanol and acetaldehyde that suppressed in vitro colony formation of committed myeloid and erythroid progenitor cells by 50 per cent. The suppression of both myeloid and erythroid colony formation was partially reversed by supplementing the cultures with folinic acid or pyridoxine. These data provide an explanation for the preferential suppression of erythropoiesis observed clinically in ethanol abuse. They also suggest that acetaldehyde has a role in ethanol-mediated bone-marrow suppression.

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In vivo and in vitro clonal assays of immature mouse blood cells showed that different populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%-4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.

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The pathogenesis of the anemia which occurs in rats bearing the Shay chloroleukemia (SCL) was investigated. Severe anemia, shown not to result from hemodilution, developed in the terminal stage of the disease. The rapid progression of the anemia suggested that reduced erythropoiesis was of no more than minor importance in the development of this anomaly. No evidence for a major hemolytic event was observed. Data are presented which suggest that hemostatic defects may be primarily responsible for the anemia of SCL. Because of many similarities with the pathogenesis of human myelogenous leukemia, SCL is proposed as a useful model for further studies on interreactions between the leukemic environment and the erythrocyte population.

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