Silent polymorphisms within the coding region of human sodium/hydrogen exchanger isoform-1 cDNA in peripheral blood mononuclear cells of leukemia patients: A comparison with healthy controls.

We have examined the sequence of the cDNA encoding the sodium/hydrogen exchanger isoform 1 (NHE1), from 23 bases upstream of the start codon to 28 bases downstream of the stop codon. Template was prepared from (1) peripheral blood mononuclear cells (PBMC) isolated from 10 healthy unrelated Caucasian volunteers; (2) PBMCs isolated from 6 leukemic patients (acute lymphoblastic leukemia [ALL], n = 3; chronic lymphocytic leukemia [CLL], n = 1; chronic myelogenous leukemia [CML], n = 2); and (3) samples of 4 leukemic cell lines (ALL: CEM, MOLT4; AML: KG1a; CML: K562). NHE1 cDNA in normal PBMCs showed silent polymorphism of nucleotides 112 (N1: T, frequency 0.70; C, frequency 0.30; prevalence of heterozygosity 0.42); 2248 (N2: G, frequency 0.90; A, frequency 0. 10; heterozygosity 0.18); and 2493 (N3: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18). Deduced primary structure of NHE1 protein in all normal volunteers was identical to that previously published for NHE1 from renal and cardiac tissue. Similar to normal PBMCs, NHE1 cDNA from leukemic cells showed polymorphism of nucleotides N1, N2, and N3, but failed to demonstrate leukemia-specific sequence differences. We conclude that the coding region of NHE1 cDNA shows a greater level of polymorphism than is currently recognized, but that sequence mutation of NHE1 is not a key event in the pathogenesis of leukemia.

Premature infants require additional folate and vitamin B-12 to reduce the severity of the anemia of prematurity.

One hundred eighty-four premature infants, < 1800 g at birth and < 36 wk gestation, were entered into a study investigating the role of additional folate and vitamin B-12 supplementation of the anemia of prematurity. All patients initially received vitamin E and iron in accordance with accepted standards. Patients were randomly assigned to four groups to receive orally 0.1 mg folate/d for 4 mo, 100 micrograms vitamin B-12 intramuscularly monthly for 4 mo, both supplements, or neither. All other activities including parenteral nutrition were carried out according to established practices, irrespective of study group. By 10-12 wk, infants treated with vitamin B-12 alone or combined with folate had higher hemoglobin values than the untreated (P < 0.0005) or solely folate-treated (P < 0.01) groups. These findings held true irrespective of wide variations in treatment and feeding practices. The only uncontrolled hematologic nutritional factor, selenium, showed a similar pattern of decline for 10-12 wk in all study patients, whether or not they received additional vitamin supplements.

Estrogen blocks the cimetidine-induced suppression of CFU-GM.

Cimetidine, an H2-histamine antagonist used for the treatment of duodenal ulcers, has been shown to suppress granulocyte/macrophage colony forming cells (CFU-GM) in males. This study was initiated to examine the role of sex hormones on this cimetidine-induced suppression of CFU-GM. Preincubation of light-density, nonadherent bone marrow cells in male patients with 10(-6) M testosterone resulted in a modest decrease in the suppressive effect of cimetidine, whereas preincubation with 10(-6) M 17-beta-estradiol, for as little as 10 minutes, completely abolished the 50% reduction in colony numbers induced by cimetidine. Using supra-pharmacologic doses of cimetidine in order to detect CFU-GM suppression in female patients, identical results were obtained. Tamoxifen completely reversed this protective effect of estrogen and preincubation with hydroxyurea and the elimination of T cells from the system failed to alter any of these results, lending support to the likelihood that both cimetidine and estrogen directly affect marrow myeloid progenitor cells.

Suppression of bone marrow by low-density lipoproteins in renal disease patients.

Patients with chronic renal disease in whom erythropoietin production is inadequate invariably experience moderate to severe debilitation-induced fatigue. Unlike the direct humeral control of erythropoiesis, neutropenia in the same cohort of patients appears to be under indirect control, very likely brought about by the suppressive effect of increased levels of low-density lipoprotein (LDL) on granulocyte-monocyte colony formation. Markedly elevated LDL levels were identified in plasma samples obtained from a study population of 179 chronic renal disease patients. The effect of the elevated LDL levels in the plasma of these patients resulted in a greater than 60% decrease in granulocyte-macrophage colony-forming unit in comparison with age-matched plasma from normal individuals. Careful review of all nutritional and therapeutic events in these patients did not offer any evidence, other than the elevated LDL levels, in support of the etiology of the chronically low absolute neutrophil counts.

Transporting bone marrow for in vitro purging before autologous reinfusion.

We describe the procedures employed for transporting bone marrow to and from a central facility. Marrow has been harvested from 80 patients with neuroblastoma, at 16 centers which are geographically dispersed throughout North America. Marrow from the outside transplant centers was packed on wet ice or cold packs in insulated containers, and transported by commercial carriers or chartered aircraft to the central processing laboratory. Post processed marrows were frozen in liquid nitrogen and returned by commercial carrier to the referring institution. In comparing transported with non-transported but similarly treated marrows, no differences were found in any of the following parameters: (1) CFU-GM recovery, (2) fraction viable cells at thawing, or (3) time to engraftment in patients. We conclude the transportation of harvested marrows to a central purging facility is safe. Based on this experience, we propose a set of standards, which, if adhered to, will insure the continued safe processing, shipping, and storage of bone marrow in all centers so engaged.

In vitro studies with melphalan and pediatric neoplastic and normal bone marrow cells.

Chemotherapeutic agents are used in increasingly high dosages to treat patients with refractory cancers. An in vitro clonogenic assay was used to investigate the effects of melphalan on cultures of human neuroblastoma, Ewing's sarcoma, and osteosarcoma cells, as well as on normal bone marrow cells. A 1-hr incubation with 10(-5) M melphalan significantly inhibited tumor colony growth of both fresh neuroblastoma and osteosarcoma cells (p less than 0.01) while Ewing's sarcoma cells and normal bone marrow appeared resistant to melphalan even at a 100-fold higher concentration. Incubation with melphalan for 8 hr did not significantly increase the sensitivity of neuroblastoma and osteosarcoma or the relative resistance of Ewing's sarcoma cells; however, normal bone marrow which had remained resistant to melphalan after 1 hr of incubation, showed significant inhibition of colony growth after an 8-hr incubation with the agent. Repeated exposure to melphalan increased the degree of inhibition of both tumor and normal marrow colonies. Fresh neuroblastoma cells were significantly more sensitive than long-term cultured neuroblastoma cells at all drug doses tested. HPLC studies demonstrated that the loss of melphalan followed first-order kinetics with a half-life of 69 min, and that in addition to the 2 known breakdown products, mono- and dihydroxy-melphalan, several other peaks were present which were not attributable to the tissue culture medium or the buffer solutions.

Cimetidine suppression of CFU-C in males.

A presumed cimetidine-induced granulocytopenia in a 6-year-old male with acute lymphocytic leukemia (ALL) prompted an investigation of the effects of cimetidine on in vitro bone marrow granulocyte colony growth (CFU-C) in normal individuals and ALL patients either on maintenance chemotherapy or off all therapy for at least 1 year. The addition of cimetidine resulted in a significant reduction of colony numbers as compared to untreated controls beginning at 1 x 10(-6) M (P less than 0.05) and increasing in significance as the concentration of cimetidine increased. Analysis of sex differences showed that CFU-C derived from females were not adversely affected by the addition of cimetidine (except at levels far in excess of recommended dosages) whereas CFU-C in males declined significantly from controls beginning at 1 x 10(-7) M (P less than 0.05) and became of greater significance as the concentration of cimetidine increased. These findings were identical for both normal individuals and ALL patients either receiving maintenance chemotherapy or off all therapy.

The effects of PGF2 alpha, PGI2, and TxB2 on human CFU-C in healthy and leukemic patients.

This study was undertaken to test the effects of certain arachidonate derivatives, PGF2 alpha, PGI2 and TxB2 on in vitro bone marrow granulocyte colony growth (CFU-C) in leukemia patients receiving maintenance chemotherapy and in normal controls. The addition of PGF2 alpha did not result in increased numbers of colonies, but it did cause a shift in the size of the colonies so that there was a significant increase in larger colonies (P less than 0.001) and significantly fewer small colonies (P less than 0.05) as compared to untreated samples. Of the prostenoids tested in a Tris-buffered system, PGI2 affected the greatest increase in CFU-C (P less than 0.01) followed by PGF2 alpha (P less than 0.05) whereas 6-keto-PGF1 alpha (the stable hydrate of PGI2) did not affect colony growth. Time-response curves revealed a linear growth pattern for PGF2 alpha with a peak at 10 days, whereas there was a 6-day growth lag with PGI2 followed by linear growth with a peak at 13 days. TxB2 added to cultures significantly reduced the number of bone marrow CFU-C at all doses tested. The prostanoid effects on CFU-C derived from leukemic patients on maintenance chemotherapy and from normal individuals were identical in every respect.