Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4- hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.     

Differential sensitivity of adherent CFU-blast, CFU-mix, BFU-E, and CFU-GM to mafosfamide: implications for adjusted dose purging in autologous bone marrow transplantation.

The availability of an in vitro assay able to detect hematopoietic progenitor cells closely related to those responsible for marrow engraftment following autologous bone marrow transplantation (ABMT) prompted us to establish a procedure aimed at maximally increasing the concentration of the cyclophosphamide derivative mafosfamide used for marrow purging. It, therefore, was the aim of the present study to investigate in a group of patients with acute nonlymphoblastic leukemia (ANLL; n = 19) and acute lymphoblastic leukemia (ALL; n = 19) in complete remission the effect of mafosfamide at the level of adherent blast colony-forming units (blast colony-forming units, CFU-Blast), as well as multipotential (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM), erythroid (erythroid burst-forming units, BFU-E), and granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) progenitor cells. When nonadherent marrow mononuclear cells (MNCs) were incubated (30 min, 37 degrees C) with increasing doses of mafosfamide (30-120 micrograms/ml), a statistically significant (p less than or equal to 0.0005) dose-dependent suppression of CFU-Blast growth was observed. The mean (+/- 1 standard error of the mean [SEM]) values of 50% inhibition (ID50) of the CFU-Blast growth were not significantly different for ANLL (106 +/- 5) and ALL (107 +/- 5) patients. Analysis of CFU-Blast ID50 distribution demonstrated that ID50 ranged from 100 to 120 micrograms/ml in 17 cases (45%), whereas it ranged from 60 to 100 micrograms/ml in 12 cases and from 120 to 160 micrograms/ml in 9 cases. A statistically significant (p less than or equal to 0.05), dose-dependent suppression of colony growth from multi-potential and lineage-restricted progenitor cells was also observed. However, the value of CFU-Blast ID50 was significantly higher (p less than or equal to 0.05) than CFU-GEMM, BFU-E, and CFU-GM ID50 and ID95 values. In conclusion, our data demonstrate that: 1) the CFU-Blast assay allows to detect on an individual basis the doses of mafosfamide used for marrow purging, and 2) the concentrations of mafosfamide extrapolated by using the CFU-Blast assay are significantly higher than those obtained with the CFU-GM assay. The absence of any detrimental effect on marrow engraftment in vivo supports the safety of the CFU-Blast assay to evaluate the dose of mafosfamide used for marrow purging before ABMT.     

Guideline for investigation and management of adults and children presenting with a thrombocytosis

Acute lymphoblastic leukaemia (ALL) remains the most frequent cause of cancer‐related mortality in paediatrics and outcome is poor for patients who have high‐risk ALL or relapse. HA22 (CAT‐8015) is an immunotoxin composed of an anti‐CD22 variable fragment linked to a 38 kDa truncated protein derived from Pseudomonas exotoxin A. Using a bone marrow mesenchymal cell culture assay to support ALL cell viability, we investigated the in vitro cytotoxicity of HA22 against ALL blasts from newly diagnosed (n = 13) and relapsed patients (n = 22). There was interpatient variability in sensitivity to HA22. Twenty‐four of 35 patient samples tested were sensitive (median 50% lethal concentration 3 ng/ml, range 1–80 ng/ml). Blasts from the other 11 patients were not killed by 500 ng/ml HA22. The median 50% lethal concentration was 20 ng/ml for all patients. There was no significant difference in HA22 sensitivity between diagnosis and relapse samples but peripheral blood ALL blasts were more sensitive to HA22 than those from bone marrow (P = 0·008). Thus, HA22, at concentrations achievable in patients, is highly cytotoxic to B‐lineage ALL cells. These results provide a strong rationale for clinical testing of this agent in children with drug‐resistant ALL and offers the potential to reduce morbidities of treatment while improving outcome.     

Comparative regimens for the ex vivo chemopurification of B cell lymphoma-contaminated marrow

The success of autologous bone marrow transplantation for B cell lymphoma may depend on the efficacy of in vitro purification of patients' tumor cell-contaminated marrow. In this study, we tested the toxicity of seven different chemotherapeutic agents against two B cell lymphoma lines (LY-16 and SK-DHL-2) as compared to normal human bone marrow granulocyte-macrophage progenitor cells (CFU-GM). 4-Hydroperoxycyclophosphamide (4-HC), VP-16-213 (VP-16), nitrogen mustard, and vincristine showed a highly selective toxicity against cultured lymphoma cells; i.e., at doses sufficient to induce a 4-log clonogenic tumor cell reduction (4-HC 21 micrograms/ml, VP-16 50 micrograms/ml, nitrogen mustard and vincristine 5 micrograms/ml), 10.0 +/- 6.7, 3.0 +/- 3.2, 23.2 +/- 22.7 and 24.0 +/- 17.0% (mean +/- 1 SD), respectively, of normal bone marrow CFU-GM were preserved. The differential sensitivity of tumor cells and normal hematopoietic precursors was less prominent after exposure of cells to cis-diamminechloroplatinum II (cis-platinum); thus, at a drug dose of 100 micrograms/ml, all detectable lymphoma cells could be eradicated (i.e., greater than or equal to 4 log reduction) while a CFU-GM recovery of only 0.2 +/- 0.2% was observed. In contrast, adriamycin and bleomycin, at the highest tumoricidal concentrations tested (5 and 100 micrograms/ml, respectively) did not exhibit a selective toxicity toward lymphoma cell lines. In summary, our results suggest that nitrogen mustard and vincristine, as well as 4-HC and VP-16, may be useful agents for the ex vivo treatment of bone marrow grafts form B cell lymphoma patients.     

Effect of auranofin on cytokine induced secretion of granule proteins from adherent human neutrophils in vitro.

The effect of auranofin on granule protein secretion from neutrophils was investigated by a haemolytic plaque assay which can detect release of lactoferrin and myeloperoxidase from single adherent neutrophils. Lactoferrin secretion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was enhanced at low (0.25-1.0 micrograms/ml) and inhibited at high concentrations of auranofin (50% inhibition (IC50) at 3.7 micrograms/ml). A similar biphasic effect was also seen on degranulation mediated by granulocyte-macrophage colony stimulating factor (GM-CSF) (IC50 1.8 micrograms/ml). In contrast, exocytosis mediated by tumour necrosis factor was inhibited even at low concentrations of auranofin (IC50 0.6 micrograms/ml). Secretion induced by phorbol 12-myristate 13-acetate and A23187 was only inhibited at very high auranofin concentrations (IC50 10 and 8 micrograms/ml respectively). The effect of auranofin on myeloperoxidase secretion was also assessed and the IC50 values for the respective agents were as follows: tumour necrosis factor 0.7 micrograms/ml, fMLP 1.6 micrograms/ml, and phorbol myristate acetate 7.6 micrograms/ml. When neutrophils were preincubated with auranofin (4 micrograms/ml) and then exposed to fMLP, tumour necrosis factor, or GM-CSF in the absence of auranofin, lactoferrin release was enhanced if the preincubation time was short (one to three minutes) and inhibited when the time of preincubation was longer. It was concluded that auranofin, at concentrations achieved in the serum of patients, is a potent inhibitor of cytokine induced release of granule proteins from adherent neutrophils. This finding may be of clinical importance and shed light on the mechanism by which auranofin acts in rheumatoid arthritis.     

Dipyridamole enhancement of drug sensitivity in acute lymphoblastic leukemia cells

The effect of dipyridamole (DPM) on cell sensitivity to anticancer drugs was examined in acute lymphoblastic leukemia (ALL) cell lines. We established two ALL cell lines (KMO-90 and KMO-R) from bone marrow samples of a 12-year-old girl with ALL. The drug concentrations needed to reduce optical density to 50% of that of control cells (IC₅₀) showed that KMO-R was about twofold more resistant to doxorubicin (DOX), mitxantrone (MIT), vincristine (VCR), and etoposide (VP-16) than was KMO-90. Considering that both KMO-90 and KMO-R were established from a patient with ALL at the time of presentation and relapse, respectively, these two cell lines might be novel and useful models for research into the acquisition of drug resistance in ALL cells. Although cytotoxicity of DPM in KMO-90 was about 6% at 1 μg/ml, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 at this concentration. Cytotoxicity of DPM in KMO-R was less than 5% at 1,5, and 10 μgg/ml. In KMO-R, DPM enhanced cell sensitivity to these four drugs in a dose-dependent manner. The plasma concentrations achieved by oral administration of DPM is about 1 μg/ml. At clinically achievable concentrations, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 in both KMO-90 and KMO-R, thus showing DPM to be a useful agent for potentiating anticancer chemotherapy of hematopoietic malignancy.     

Transplant of rhesus-positive bone marrow in a rhesus-negative woman having anti-rhesus D alloantibodies

A woman affected by acute myeloblastic leukemia was grafted with HLA A, B and D compatible rhesus-positive bone marrow from her brother. Before grafting, she had anti-D alloantibodies (1/512 IAT, 2.9 micrograms/ml). To prevent the destruction of donor red blood cells, four plasma exchanges and a conditioning regimen (total-body irradiation 800 rad, cyclophosphamide, methotrexate) were carried out to decrease anti-D from 2.9 to less than 0.02 micrograms/ml on day 0. The anti-D level was 0.8 micrograms/ml on day 12 and was decreased to 0.2 micrograms/ml by eight plasma exchanges until day 35. Anti-D antibodies were undetectable with Lalezari's technique on day 45. Engraftment was obtained on day 25 (3,000 leukocytes/mm3 and 50% erythroblasts in bone marrow). The patient died from aspergillosis and graft-versus-host disease on day 54. This observation shows that an engraftment of rhesus-positive bone marrow in a recipient with anti-D antibody is possible.     

Mechanism of aprindine induced agranulocytosis: direct toxicity on CFU-C and CFU-GEMM

The in vitro bone marrow growth of 2 patients with aprindine-induced agranulocytosis was studied. Granulocyte-macrophage committed stem cell (CFU-C) growth is inhibited by aprindine in a dose dependent manner. 50% inhibition of CFU-colony growth (TD50) was seen at 5.1 and 3.4 micrograms aprindine/ml medium respectively. The TD50 of control marrow CFU-C was 3.2 micrograms/ml. 100% inhibition was seen at 16 micrograms aprindine/ml, both in patients and controls. Pluripotential stem cell growth (CFU-GEMM) in control marrow was equally inhibited in a dose dependent manner by aprindine, though to a lesser extent (TD50: 9.1 micrograms/ml) and with relative sparing of pure megakaryocyte and erythroid colonies. Co-culturing of patients marrows with their respective acute phase serum did not inhibit CFU-C growth.     

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) on 3'-azido-3'-deoxythymidine (AZT)-mediated biochemical and cytotoxic effects on normal human myeloid progenitor cells

Administration of 3'-azido-3'-deoxythymidine (AZT) to patients with acquired immunodeficiency syndrome (AIDS) causes significant anemia and neutropenia. The bone marrow cytotoxicity of AZT has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined the effect of human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) on AZT-mediated biochemical perturbations and in vitro growth inhibition of normal bone marrow myeloid progenitor cells. Exposure of nonadherent, bone marrow mononuclear cells (BMMC) to 100 ng/ml of rGM-CSF for 6 h resulted in approximately twofold increments in the mean intracellular deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) levels. Administration of 10 microM AZT alone to BMMC for 6 h markedly reduced dCTP and dTTP levels and generated significant levels of AZT triphosphate (AZT-TP). Coadministration of rGM-CSF (100 ng/ml) along with AZT (10 microM) partly restored dCTP and dTTP levels and significantly reduced AZT-TP levels. Furthermore, simultaneous exposure of BMMC for 4 h to 100 ng/ml of rGM-CSF reduced the mean DNA incorporation of [3H]AZT (10 microM) from 27.2 to 19.1 pmol/micrograms of DNA. Additionally, the inhibitory effects of AZT (10 microM) on granulocyte-macrophage colony-forming unit (CFU-GM) colony growth were significantly reduced in the presence of 100 ng/ml of rGM-CSF. These in vitro studies suggest that rGM-CSF partly corrects AZT-mediated biochemical perturbations as well as reduces the cytotoxicity of AZT in normal human bone marrow myeloid progenitor cells.     

Difference in kinetics of hematopoietic reconstitution between ALL and ANLL after autologous bone marrow transplantation with marrow treated in vitro with mafosfamide (ASTA Z 7557)

The kinetics of hematopoietic recovery after autologous bone marrow transplantation (ABMT) reflect the hematopoietic capacity of the infused marrow. In vitro treatment of marrow with high doses of mafosfamide (ASTA Z 7557) alters the hematopoietic regenerative capacity of the graft. Thirty-two patients with acute leukemia (12 acute lymphoblastic leukemia (ALL) and 20 acute non-lymphoblastic leukemia (ANLL] with 27 in complete remission and five in partial remission were consolidated with cyclophosphamide (60 mg/kg x 2) and total body irradiation (10 Gy), followed by reinfusion of autologous marrow treated in vitro with mafosfamide. The marrow of each patient had been incubated with the highest tolerable dose of mafosfamide, individually predetermined from a preincubation test. We report here that the kinetics of engraftment are strikingly different in ANLL and ALL patients. In the ANLL group recovery to 0.1% reticulocytes took a median of 20.5 days (range 14-32) versus 15 (11-28) in the ALL group; 33.5 days (18-45) versus 19 (15-30) for leukocytes to reach 1.0 x 10(9)/l; 35 (19-60) versus 20.5 (15-30) for neutrophils to reach 0.5 x 10(9)/l; 110+ (45-480+) versus 50 (23-90) for platelets to reach 50 x 10(9)/l (p less than 0.01 and p less than 0.05). Detection of granulocyte-macrophage progenitors (CFU-GM) regeneration in marrow aspirates post-ABMT was delayed in ANLL (p less than 0.05). Neither the nature of the previous induction therapy, nor the status of the blood or bone marrow at the time of collection (CFU-GM and erythroid burst-forming units/ml) nor the stem cell sensitivity to mafosfamide, nor the doses of progenitor cells infused could explain these differences. We interpreted these observations as suggesting that the engraftment potential has been more severely altered in ANLL than in ALL, which may reflect both the intensity of the in vitro treatment and the intrinsic fragility of the stem cell pool in ANLL.     

Treatment of chronic pseudomonas aeruginosa infection in cystic fibrosis patients with ceftazidime and tobramycin

50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) of ceftazidime, cefsulodin, cefotaxime, moxalactam, azlocillin, carbenicillin, netilmicin and tobramycin against 90 strains of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients were determined by means of the agar plate dilution method. Ceftazidime was most active of the antibiotics in vitro; the geometric mean of IC50 was 0.2 and of MIC 0.4 micrograms/ml. 11 CF patients suffering from P. aeruginosa infection were treated with 14 day courses of ceftazidime (100 mg/kg/24 h) and tobramycin (10 mg/kg/24 h). P. aeruginosa was only temporarily eradicated in one of the patients, but a significant improvement of respiratory function and a significant fall in white blood cell count was recorded in the patients during chemotherapy. There was no development of resistant strains against ceftazidime during treatment and no side-effects were observed. Ceftazidime is a promising new antimicrobial agent with high in vitro activity which deserves further in vivo evaluation in CF patients.     

Immunoassay of circulating terminal transferase in patients with acute lymphoblastic leukemia: A new technique for diagnosis

The measurement of circulating terminal deoxynucleotidyl transferase (TdT) antigen can be accomplished by the use of an ELISA immunoassay system. TdT was present in either peripheral or bone marrow plasma of five patients with acute lymphoblastic leukemia (ALL) and absent from normal controls using ELISA. The assay was sensitive to a level of 20 ng/ml. The ALL patient values ranged from 160 ng/ml to 420 ng/ml. Use of ELISA for quantitation of TdT may have important diagnostic implications in patients with ALL.     

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.     

Effect of iodide treatment on iodine concentration and volume of endemic non-toxic goitre in childhood

In a total of 195 children and adolescents of both sex (mean age 12.9, range 5-17 years) with endemic non-toxic goitre the thyroidal iodine concentration (IC) was determined using X-ray fluorescent scanning on admission and during iodine (100 micrograms daily) and L-thyroxine (3 micrograms/kg body weight daily) treatment respectively. Additionally the thyroid volume was measured sonographically in a longitudinal study including 46 patients before and after 4-8 months of iodine supplementation (100 micrograms daily). The IC was 305 +/- 144 micrograms/g. It compared well with that of adult goitre patients (288 +/- 109 micrograms/g) and was significantly inferior to the value of normal controls (389 +/- 170 micrograms/g). Under L-thyroxine therapy the IC further decreased (243 +/- 144 micrograms/g), whereas patients receiving iodide showed an increase of the IC (570 +/- 197 micrograms/g). The mean TSH level fell from 2.3 +/- 0.9 microU/ml to 1.4 +/- 0.6 microU/ml. The average T4/TBG (thyroxine binding globulin) ratio showed a slight increase which, however, was not significant. The mean goitre volume decreased by 40%. It was evidenced that iodide is useful not only in the prophylaxis of non-toxic goitre but also as a more physiologic treatment than thyroid hormones, at least for young subjects with simple diffuse goitres.     

Circulating transferrin receptor in acute leukemias.

Serum transferrin receptor (s-TR) levels in acute leukemia patients were measured by a recently developed sandwich radioimmunoassay. The mean s-TR level for normal subjects (n = 205) was 246 +/- 79 (mean +/- 1 SD) ng/ml. The values for patients with untreated acute myelocytic leukemia (AML, n = 18) and untreated acute lymphocytic leukemia (ALL, n = 14) were 398 +/- 175 ng/ml and 479 +/- 176 ng/ml, respectively, both of which were significantly higher than those for the normal subjects (AML, p < 0.02; ALL, p < 0.05). When complete remission was achieved with initial remission induction therapy, s-TR decreased to 262 +/- 47 ng/ml (n = 22, 12 AML and 10 ALL), returning to normal levels. There was a good correlation between s-TR levels and the number of leukemic cells in peripheral blood (r = 0.743, n = 32, p < 0.01). In two patients with AML, serial changes of s-TR values and numbers of blast cells in the peripheral blood and bone marrow occurred in a parallel manner. If, therefore, this assay for s-TR can be made more sensitive, it may become useful for assessing acute leukemia activity and for monitoring the effects of therapy.     

Recombinant human granulocyte-macrophage colony-stimulating factor accelerates engraftment kinetics after allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia

BACKGROUND AND OBJECTIVE: The use of recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) has been shown to be well-tolerated and to reduce post-transplantation morbidity in adults undergoing HLA-identical allogeneic bone marrow transplantation (BMT). There is however, limited experience in children. DESIGN AND METHODS: We performed a prospective, comparative multicenter trial using rhGM-CSF after allogeneic BMT in children with acute lymphoblastic leukemia (ALL). The study comprised 24 patients with ALL who received rhGM-CSF and 22 patients with ALL who did not receive rhGM-CSF. There were no statistically significant differences in the demographic characteristics between the rhGM-CSF-treated and untreated groups. rhGM-CSF was given at a dose of 10 micrograms/kg/day infusion over 4 hours from day +1 until +28 or until the absolute neutrophil count (ANC) was > or = 1 x 10(9)/L. All patients received HLA-identical sibling marrow and cyclosporine alone for graft-versus-host disease (GvHD) prophylaxis. The number of cells infused was similar in both groups. A software program (Statview 4.0, Abacus Concept, Inc., Berkeley, CA, USA) was used for statistical analysis. RESULTS: The median of days to achieve ANC > or = 0.5 x 10(9)/L was shorter in the rhGM-CSF-treated patients (14 days vs 18.5 days; p < 0.0001). Patients who received rhGM-CSF had a lower incidence of grade III-IV mucositis. The duration of hospital stay was significantly shorter in patients who received rhGM-CSF (31 days vs 45 days; p < 0.005). No differences in GvHD severity, relapse or survival were observed. At the dose and schedule used in the present study, rhGM-CSF was well-tolerated and no side effects were observed. INTERPRETATIONS AND CONCLUSIONS: rhGM-CSF at a dose of 10 micrograms/kg/day in children with ALL undergoing allogeneic BMT is well tolerated, accelerates neutrophil and platelet engraftment, reduces the intensity and severity of mucositis and permits a more rapid discharge from hospital.     

Serum ferritin in non-dialysis patients with chronic renal failure: relation to bone marrow iron stores

Serum ferritin and bone marrow haemosiderin iron was studied in 50 non-dialysis patients with chronic renal failure, and in 53 healthy subjects. S-ferritin was correlated to marrow iron both in patients with renal failure and in healthy subjects (P less than 0.001). Geometric-mean S-ferritin in patients with 0- (1+) marrow iron was 33 micrograms/l, 1+ marrow iron 166 micrograms/l, and 2+ marrow iron 519 micrograms/l. Healthy subjects with 0- (1+) marrow iron had a mean S-ferritin of 16 micrograms/l and those with 1+ marrow iron a value of 65 micrograms/l. S-ferritin levels were higher in patients than in healthy subjects at all marrow iron grades (P less than 0.001). Healthy subjects with S-ferritin less than 15 micrograms/l had absent or reduced marrow iron, while those with S-ferritin greater than 30 micrograms/l had normal marrow iron. Using a critical S-ferritin value of less than or equal to 20 micrograms/l, the diagnostic efficiency in terms of diagnosing absent or reduced marrow iron was 0.90 (PV pos = 0.85, Pv neg = 0.91). In patients with renal failure S-ferritin less than 60 micrograms/l indicated absent or reduced marrow iron, while values greater than 80 micrograms/l were associated with normal marrow iron. The diagnostic efficiency of S-ferritin using a critical value of less than or equal to 60 micrograms/l was 0.94 (PV pos = 0.93, PV neg = 0.97). S-ferritin is a useful indicator of marrow iron stores in patients with chronic renal failure.     

In vivo administration of recombinant human granulocyte/macrophage colony-stimulating factor in acute lymphoblastic leukemia patients receiving purged autografts [see comments]

Based on the recent reports that recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) accelerates the rate of engraftment in a variety of autologous bone marrow transplantation settings, we have investigated its effects on hematopoietic recovery of patients with acute lymphoblastic leukemia (ALL) undergoing autologous bone marrow transplantation. Our studies, which involved 25 autologous ALL recipients who received rhGM-CSF and 27 controls similar for disease status (remission or relapse) and disease type (B- or T-lineage) differed from previous studies in one important aspect: the bone marrows were purged with 4- hydroperoxcyclophosphamide (4HC) and anti-T or anti-B-cell lineage- specific antibodies before transplantation. Such treatments frequently lead to a reduction in the CFU-GM content of the transplanted marrow. Eighteen of 25 patients completed the entire course of rhGM-CSF. Of the 16 patients who received greater than or equal to 64 micrograms/M2/d for at least eight days, there were five patients who had an apparent rhGM-CSF response and 11 patients who did not respond. Of the parameters analyzed, only the number of CFU-GM progenitor cells infused per kilogram was significantly associated with an rhGM-CSF response. All patients receiving greater than or equal to 1.2 x 10(4) CFU-GM progenitors per kilogram achieved an absolute neutrophil count (ANC) greater than or equal to 1,000/microL by day 21 and had a greater than 50% decrement in ANC within 48 to 72 hours of discontinuing rhGM-CSF, as contrasted to none of the patients receiving less than or equal to 7.2 x 10(3) CFU-GM progenitors per kilogram.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cryopreservation on purging of leukemic marrow with alkyl-lysophospholipids

Alkyl-lysophospholipids (ALP) are reported to be selectively cytotoxic to neoplastic cells and have been demonstrated to be an effective purging agent for autologous marrow transplantation in a murine model, WEHI-3B. We studied the effect of in vitro treatment of normal bone marrow cells with ALP and of cryopreservation on spleen colony-forming units (CFU-s) in irradiated, syngeneic Balb/c mice. We found that exposure of cells to doses of ALP of 5 or 20 micrograms/ml followed by cryopreservation had no effect on CFU-s, but that a dose of 100 micrograms/ml was toxic. A simulated remission marrow was prepared by mixing normal murine bone marrow cells with WEHI-3B myelomonocytic leukemic cells to give a 1%-2% concentration of leukemic cells. These were exposed to 0- to 100-micrograms/ml concentrations of ALP for 24 h, cryopreserved, thawed, and injected into the tail veins of lethally irradiated syngeneic mice, and survival measured. It was found that concentrations of ALP from 10 to 50 micrograms/ml resulted in long-term disease-free survivors. These results established the fact that cryopreservation did not alter the effectiveness of ALP in purging marrows of residual leukemic cells.     

Protection of zidovudine-induced toxicity against murine erythroid progenitor cells by vitamin E

The ability of vitamin E (alpha-tocopherol) to stimulate erythroid progenitor cells was investigated in an attempt to identify ways to ameliorate zidovudine (azidothymidine, AZT)-induced anemia. In vitro, alpha-tocopherol acid succinate (ATS), upon incubation with murine bone marrow cells at concentrations of up to 4 micrograms/ml, caused a dose-dependent increase in erythroid colony-forming unit (CFU-E)-derived colonies. This increase was equivalent to the effect demonstrated by 50 mU of recombinant human erythropoietin (rhEpo) or 200 U of recombinant interleukin 3 (rIL-3). For in vivo studies, anemia was produced in CD-1 male mice by administering AZT in drinking water (1.5 mg/ml). Treatment with vitamin E (50 mg/kg body weight) or Epo (0.4 U per mouse) was initiated 24 h later and continued for five consecutive days. Seventh day bone marrow cells from femurs were assayed for CFU-E-derived colonies. Both vitamin E and Epo significantly increased the number of CFU-E-derived colonies by 75% and 86% of control, respectively, indicating that these agents were approximately similar in protecting the bone marrow from AZT-induced toxicity.