ALBERT

Description: To determine if elemental carbonyl iron powder is safe and effective therapy for iron deficiency anemia, 20 nonanemic and 32 anemic volunteers were studied. Single doses of 1,000 to 10,000 mg of carbonyl iron (15 to 150 times the 65 mg of iron in the usual dose of ferrous sulfate) were tolerated by nonanemic volunteers with no evidence of toxicity and only minor gastrointestinal side effects. Anemic volunteers (menstruating women who had previously donated blood) were treated with several regimens providing 1,000 to 3,000 mg of carbonyl iron daily in one to three doses for 8 to 28 days. After 12 weeks anemia was corrected in 29 of 32 patients, and serum ferritin was greater than 12 micrograms/L in 14. Hemoglobin regeneration proceeded at a rate similar to that described for therapy with oral iron salts and parenteral iron dextran. There was no evidence of hematologic, hepatic, or renal toxicity, but mild gastrointestinal side effects occurred in a majority of anemic volunteers. Carbonyl iron is an effective, inexpensive treatment for iron deficiency anemia, is accompanied by tolerable side effects and may have an advantage over therapy with iron salts by substantially reducing or eliminating the risk of iron poisoning in children.

Description: Marrow stromal cells are a heterogeneous population, comprising a variety of lineages including osteogenic cells. In the presence of ascorbic acid, sodium beta-glycerophosphate, and dexamethasone, rat bone marrow stromal cells form discrete nodules of mineralized, bonelike tissue. We used nodule formation by rat bone marrow stromal cells to assay for the self-renewal capacity of osteogenic progenitor cell populations. Cultures were subcultured every 5 days up to six times. Osteogenesis was assayed from second to sixth subcultures by counting the number and measuring the areas of mineralized nodules formed in cultures grown with 10(-8) mol/L dexamethasone. Nodule number and area decreased progressively between second and sixth subcultures. Alkaline phosphatase activity associated with individual cells and measured videodensitometrically decreased exponentially between the second and sixth subculture. The number of cells with alkaline phosphatase activity also decreased with progressive subculturing. The proportions of 3H-thymidine-labeled cells after continuous labeling from the beginning of the culture period showed 90% labeling for cells with alkaline phosphatase activity and fibroblastlike cells. Cultures labeled for only the first 3 days exhibited higher labeling of alkaline phosphatase-positive cells than fibroblastlike cells (P less than .05). Cultures that were flash-labeled at the end of the culture period demonstrated low labeling indices for cells with alkaline phosphatase activity and up to 10-fold higher labeling indices for fibroblastlike cells. Separate cultures treated with a cytocidal dose of high specific activity 3H-thymidine did not form nodules. These results indicate that osteogenic progenitor cells or another cell type required for nodules to develop must divide early in culture if nodule formation is to occur, and that osteoprogenitor cells express a limited capacity for self-renewal.

Description: We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia- positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.

Description: To determine if elemental carbonyl iron powder is safe and effective therapy for iron deficiency anemia, 20 nonanemic and 32 anemic volunteers were studied. Single doses of 1,000 to 10,000 mg of carbonyl iron (15 to 150 times the 65 mg of iron in the usual dose of ferrous sulfate) were tolerated by nonanemic volunteers with no evidence of toxicity and only minor gastrointestinal side effects. Anemic volunteers (menstruating women who had previously donated blood) were treated with several regimens providing 1,000 to 3,000 mg of carbonyl iron daily in one to three doses for 8 to 28 days. After 12 weeks anemia was corrected in 29 of 32 patients, and serum ferritin was greater than 12 micrograms/L in 14. Hemoglobin regeneration proceeded at a rate similar to that described for therapy with oral iron salts and parenteral iron dextran. There was no evidence of hematologic, hepatic, or renal toxicity, but mild gastrointestinal side effects occurred in a majority of anemic volunteers. Carbonyl iron is an effective, inexpensive treatment for iron deficiency anemia, is accompanied by tolerable side effects and may have an advantage over therapy with iron salts by substantially reducing or eliminating the risk of iron poisoning in children.

Description: High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

Description: We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony- stimulating factor and interleukin-1 beta or medium conditioned by a human marrow “stromal” cell line to medium conditioned by agar- stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.

Description: To identify possible secretory determinants of impaired hyperadherence and stimulated migration of neonatal granulocytes (NGs), we performed correlative studies of: (a) specific granule content and exocytosis, (b) secretago-gue-mediated upregulation of f-met-leu-phe (fMLP) receptors, (c) the chemotactic induction of the adhesive glycoproteins Mac-1 alpha (complement receptor 3) and beta, and (d) morphometric assessments of specific (peroxidase negative) granule depletion following chemotactic stimulation. Lactoferrin (LF) content of NG suspensions (cord blood or peripheral blood cells) was profoundly diminished (mean +/- SD 51% +/- 18% of normal) as compared with healthy adult granulocytes (AGs). Despite diminished cellular content, LF release by NG suspensions in response to fMLP was comparable to that of AGs. In contrast, LF release by NG suspensions was significantly diminished in response to phorbol myristate acetate (PMA) or calcium ionophor A23187 and/or during stimulated cell spreading, experimental conditions promoting overall greater LF depletion than chemotactic stimuli. In addition, NGs demonstrated an impaired capacity to upregulate fMLP receptors in response to PMA or A23187 when tested under the same experimental conditions. Baseline expression of the adhesive glycoproteins Mac-1 alpha and beta on NG surfaces was normal, but induction or upregulation of these proteins by chemotactic concentrations of fMLP, C5a as well as secretory (high) concentrations of PMA and A23187, was significantly diminished as compared with AGs. In contrast, chemotactic induction of the surface expression of the complement receptor-1 (CR-1) on NGs was normal. An impaired induction of Mac-1 alpha or beta was directly related to an impaired enhancement of adherence of NG in response to fMLP over a chemotactically relevant concentration range (10(-10) to 10(-7) mol/L). Moreover, in blocking- incubation experiments using anti-Mac-1 alpha/beta monoclonal antibodies (MAbs), significantly less inhibition of adherence by these MAbs was evident with fMLP-stimulated NG as compared with AG suspensions. Under selected chemotactic conditions, ultrastructural assessments of NGs demonstrated diminished peroxidase-negative granule loss in association with diminished granule-membrane fusion and the “addition” of plasma membrane. These studies suggest that abnormal expression of multiple surface determinants derived from peroxidase- negative granules or other intracellular pools may contribute to deficient chemotaxis or other inflammatory functions of NGs.(ABSTRACT TRUNCATED AT 400 WORDS)

Description: We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia- positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.

Description: We used the polymerase chain reaction (PCR) to detect residual leukemia- specific mRNA in blood and marrow from 37 patients in complete hematologic and cytogenetic remission after allogeneic bone marrow transplant (BMT) for chronic myeloid leukemia (CML). Our two-step PCR method involved the use of “nested primers” in the second step and could detect one K562 cell diluted into 10(5) normal cells. Elaborate measures were taken to exclude false-positive and false-negative results. In nine patients whose blood and marrow were studied simultaneously the results were concordant (two positive and seven negative). Twenty-three patients transplanted in chronic phase (CP) with unmanipulated donor marrow were studied. Blood cells from nine of these patients were studied 3 to 6 months post-BMT and six were PCR positive; three were negative on subsequent studies. Blood cells from 18 patients studied between 8 months and 8 years post-BMT were all PCR negative. Nine patients transplanted in CP with T-cell-depleted marrow cells were studied. Blood from five was positive 3 to 24 months post- BMT; blood from five was negative 3 to 6 years post-BMT. Four patients no longer in first CP were studied after BMT with unmanipulated donor marrow. Blood from all four was positive 5 to 19 months post-BMT. Based on the known clinical results of transplant in these three cohorts we conclude that PCR may be positive within 6 months of BMT in patients who can expect long-lasting remission, whereas PCR positivity later after BMT may indicate that the probability of cure is reduced. Thus, the technique may prove useful for early assessment of new transplant protocols that might inadvertently increase the risk of relapse.

Description: Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross- linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.