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Thrombocytopenia is a dose-limiting toxicity of macrophage colony-stimulating factor (M-CSF) in preclinical and initial phase I trials. Modulation of macrophage-mediated platelet destruction in immune thrombocytopenic purpura (ITP) may be affected by M-CSF activity. In this study, plasma levels of M-CSF were determined by a sensitive radioimmunoassay in 23 patients with ITP. These were compared with control levels measured in 24 healthy subjects. M-CSF levels were significantly higher in the ITP patients than in the control subjects (218 v 179, P < .02); however, there was a great deal of overlap. The highest M-CSF levels (median = 299 U/mL) were observed in three patients with Evan's syndrome. Patients with severe ITP (platelets < 25,000/microL) had intermediate M-CSF levels (median = 231 U/mL) and those with mild thrombocytopenia (> 25,000/microL) had normal levels (median = 173 U/mL). Sixteen patients were treated with corticosteroids: 10 responded and 6 did not. Median M-CSF levels were higher in those who failed to respond compared with responders (272 v 202, P < .05). These findings suggest M-CSF may influence macrophage-mediated platelet destruction in ITP.

Phenylalanine methylester (PME), a lysosomotropic compound can be used to deplete monocytes and myeloid cells from peripheral blood and bone marrow (BM). The potential of PME for purging leukemic cells from BM was investigated using U937 and HL-60 cell lines as models. Optimal purging conditions for U937 cells were determined using an MTT assay (3-4, 5-dimethylthiazol-2, 5-diphenyl tetrazolium biomide; Sigma). Elimination of U937 cells was time-, temperature-, and dose-dependent. PME activity was optimal at 37 degrees C for 45 minutes. Depletion of U937 was > 2.8 logs for 50 mmol/L PME. Compared with another purging agent, 100 micrograms/mL 4-hydroperoxycyclophosphamide had activity comparable to 40 mmol/L PME. HL-60 cells were even more sensitive to PME than U937 cells. To support observations made with the MTT assay, clonogenic assays were performed. PME, 50 mmol/L at 37 degrees C resulted in total depletion (> 5 logs) of U937 colonies. Progressive depletion of normal progenitor cells occurred when BM was incubated with PME at concentrations from 5 to 100 mmol/L. At 37 degrees C, 50 mmol/L PME reduced colony-forming units-granulocyte-macrophage and burst-forming units-erythroid (BFU-E) recovery by 98%. Recombinant human mast cell factor augmented BFU-E after PME treatment but had no effect on HL-60 or U937. These studies suggest that PME deserves further study as an agent for ex vivo marrow purging.

Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L-phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment.

Ten patients, with bone marrow failure or malignant disorders, became refractory to platelet transfusions using random, as well as partial or fully HLA-matched, single-donor platelets. To determine its effect on platelet refractoriness, intravenous gamma globulin (IV IgG) was administered at 400 or 800 mg/kg/d for five days, and postinfusion platelet responses were monitored. Platelet transfusion responses following intravenous gamma globulin (IV IgG) were graded as follows: Excellent, 48-hour posttransfusion count greater than 50,000/microL; good, 48-hour count greater than 20,000 but less than 50,000/microL; Fair, increased increment, 48-hour count less than 20,000; and failed, no increased increment. Six of ten patients (60%) had improved responses to selected single-donor platelets (two were excellent, three were good, and one was fair). The time to achieve a platelet transfusion count greater than 25,000/microL ranged from one to nine days of IgG therapy. One individual had sustained benefit (greater than 1 year); the remaining responses persisted for 6 to 8 weeks. These results suggest that IV IgG may be useful in the management of platelet refractoriness, especially in patients receiving single-donor platelets.

In vitro coculture studies were performed in five patients with severe aplastic anemia (SAA) and their normal HLA-matched donors before and after allogeneic bone marrow transplantation (BMT) to determine whether the erythropoietic function of T cells is abnormal in this disorder. These coculture studies used fresh or cryopreserved marrow T lymphocytes with fresh or cryopreserved marrow T cell-depleted target cells. Four of five aplastic patients had little or no transfusion exposure before studies. The composite results showed that, in comparison to the erythropoietic effects of normal HLA-identical marrow T lymphocytes or engrafted T lymphocytes, T lymphocytes collected from the aplastic patients before BMT consistently suppressed or failed to support CFUE and BFUE growth optimally from autologous marrow, HLA-identical marrow, or engrafted aplastic T cell-depleted marrows. This T cell abnormality was not observed in four multiply transfused leukemics and three patients with myelodysplastic syndrome. Marker analyses of SAA marrow T lymphocytes performed before and after BMT suggested that the erythropoietic functional abnormality was due to abnormal marrow T cell composition reflecting an excess of activated Tac+, T3+, T11+ lymphocytes. Collectively, these in vitro studies provide firmer in vitro evidence implicating T cells in the pathogenesis of SAA. The erythropoietic T cells abnormalities in SAA are fully corrected by allogeneic BMT.