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Biomedical subjects

J L Spivak

Publications and source records attributed to J L Spivak.

At least 19 recordsLinked to original sources
PubMed1992

Impaired erythropoietin response to anemia after bone marrow transplantation.

Delayed erythroid recovery is common after bone marrow transplantation (BMT), with some patients continuing to require red blood cell (RBC) transfusion support for as long as 1 year. While the etiology is multifactorial, inadequate stimulation of erythroid progenitors by the erythroid growth factor, erythropoietin, may play a role. In this study, the erythropoietin response to anemia of 70 consecutive patients undergoing BMT at the Johns Hopkins Oncology Center was compared with the erythropoietin response in uncomplicated iron deficiency anemia. Erythropoietin levels were elevated for the degree of anemia early after BMT; however, at the time of marrow recovery, erythropoietin levels were significantly suppressed in both allogeneic and autologous BMT patients compared with the iron-deficient patients. Patients with acute graft-versus-host disease (GVHD) had a more marked suppression of the erythropoietin response to anemia. In the patients who remained anemic for extended periods of time (up to 12 months after BMT), an inadequate erythropoietin response to anemia persisted. Delayed erythroid recovery after BMT is associated with inadequate erythropoietin levels. Therefore, recombinant human erythropoietin may be useful in the treatment of the anemia associated with both autologous and allogeneic BMT.

Anemia↗
PubMed1992

Protein kinases and phosphatases are involved in erythropoietin-mediated signal transduction.

To study the role of protein phosphorylation in erythropoietin (EPO)-mediated signal transduction, we examined the effects of tyrosine phosphatase and tyrosine and serine-threonine kinase inhibitors as well as activators of serine kinases on DNA synthesis and cell proliferation in the murine EPO-dependent cell line HCD-57. HCD-57 cells were obtained synchronized in G0 by centrifugal elutriation, and DNA synthesis was measured by incorporation of labeled thymidine into DNA. Half-maximal DNA synthesis was stimulated by 0.001 U/ml of EPO. Sodium orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor, at 5 microM potentiated a subsaturating concentration of EPO. Na3VO4 alone stimulated HCD-57 DNA synthesis at concentrations of 0.1-20 microM. Zinc chloride, another tyrosine phosphatase inhibitor, also stimulated HCD-57 DNA synthesis at concentrations of 50-100 microM. Genistein, a tyrosine kinase inhibitor, blocked the effect of EPO at a concentration of 5 micrograms/ml. Bryostatin, a protein kinase C (PKC) activator, stimulated DNA synthesis in HCD-57 cells at concentrations of 10(-9)-10(-10) M, whereas the phorbol ester, phorbol 12,13-dibutyrate (PDBu), was stimulatory only at a concentration of 10(-11) M. Staurosporine, a PKC inhibitor, blocked the effect of EPO at a concentration of 10(-7) M, and H-7, a nonspecific protein kinase inhibitor, was not inhibitory. These agents also had similar effects on the in vitro proliferation of HCD-57 cells. Taken together, the data indicate that the EPO-mediated transition from G0 to S phase in HCD-57 cells involves the activation of both tyrosine and serine-threonine kinases and is modulated by tyrosine phosphatase activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Hexamethylene bisacetamide in myelodysplastic syndrome: effect of five-day exposure to maximal therapeutic concentrations.

The efficacy of hexamethylene bisacetamide (HMBA), a potent polar-planar solvent which is capable of differentiating leukemias and solid tumors in vitro at clinically achievable concentrations, was studied in 16 patients with severe myelodysplastic syndromes (MDS). An adaptive control dosing algorithm was used to maintain HMBA steady-state concentrations (Css) within a narrow therapeutic window (1-2 mM) for five days every four weeks. Despite achieving the target HMBA Css during at least two courses in each of 15 patients, HMBA did not produce clinically relevant improvements in blood cell counts nor in other functional indices. Instead, HMBA induced cytopenias in the majority of these patients, most of whom had preexisting cytopenias and limited hematopoietic reserves. These disappointing results correlated with concurrent in vitro bone marrow studies from these patients in which both the HMBA concentrations that were optimal for differentiation in vitro (2-5 mM) and the HMBA Css that were achieved in this study (1-2 mM) substantially inhibited the growth of granulocyte-macrophage colony-forming units and erythroid burst-forming units. Although the mechanism responsible for the anti-proliferative effects of HMBA on hematopoietic progenitors (cytotoxicity versus terminal differentiation) could not be determined, the induction of cytopenias and lack of significant clinical improvements suggest that HMBA is cytotoxic and will not be useful alone as a differentiating agent on this schedule of administration in the treatment of MDS.

Acetamides↗
PubMed1992

The application of recombinant erythropoietin in anemic patients with cancer.

Recombinant human erythropoietin (r-HuEPO) has been shown to correct anemia and alleviate transfusion dependency in patients with end-stage renal disease, to ameliorate anemia and reduce transfusion requirements in anemic patients with acquired immunodeficiency syndrome, to correct anemia in patients with rheumatoid arthritis, and to facilitate predeposit autologous blood donation. Cancer is frequently complicated by anemia, and a survey of serum erythropoietin concentrations in anemic cancer patients showed inappropriately low concentrations for the degree of anemia. Initial clinical trials suggest that r-HuEPO can correct the anemia associated with malignancy, but the exact role of the recombinant hormone in this situation remains to be defined.

Anemia↗
PubMed1991

Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation.

Erythropoietin mediates the rapid phosphorylation of Raf-1 in the murine cell lines HCD-57 and FDC-P1/ER, which proliferate in response to this cytokine. Phosphorylation occurs at both serine and tyrosine residues and as such is similar to the Raf-1 phosphorylation seen after interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and interleukin-2 stimulation in other murine cell lines. Such data suggest that these growth factors may share a common mechanism(s) of Raf-1 phosphorylation. Furthermore, in association with Raf-1 phosphorylation, erythropoietin induces a 2-3-fold increase in Raf-1 kinase activity as measured in immune complex kinase assays in vitro. Finally, a c-raf antisense oligodeoxyribonucleotide, which specifically decreases intracellular Raf-1 levels, also substantially inhibits both erythropoietin and IL-3-directed DNA synthesis. Together, these results provide evidence that activated Raf-1 is a necessary component of erythropoietin and IL-3 growth signaling pathways.

Animals↗
PubMed1991

Erythropoietin is both a mitogen and a survival factor.

Erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells, but whether it functions solely as a survival factor or also acts as a mitogen is unresolved. Because late erythroid progenitor cells (CFU-E) are largely in cell cycle, we examined this issue by using an Ep-dependent, murine erythroleukemia cell line, HCD-57. In the presence of human Ep and fetal calf serum, HCD-57 cells had a doubling time of approximately 24 hours, and during log-phase growth approximately 36% of the cells were in G1, 45% in S, and 19% in G2/M. With Ep deprivation, there was a gradual loss of viability and an arrest of proliferation with a 44% increase in the G0/G1 population, which could be reversed by reexposure to Ep even after 72 hours of hormone withdrawal. As little as 2 hours of exposure to Ep was sufficient to stimulate DNA synthesis, and the lag time for initiation of DNA synthesis after exposure to the hormone was approximately 10 hours as measured by either incorporation of labeled thymidine into DNA or cell cycle analysis by flow cytometry. RNA synthesis, by contrast, was initiated within 2 hours after exposure to Ep and did not require DNA synthesis. Total cell DNA content increased after exposure to Ep, indicating that it was acting as mitogen in HCD-57 cells. Ep was also able to stimulate DNA synthesis in the absence of serum as well as in its presence, indicating that the hormone could act as both a competence and a progression factor. Qualitative analysis of the integrity of HCD-57 DNA by electrophoresis in agar as well as direct measurement of DNA fragmentation after metabolic labeling with radioactive thymidine indicated that programmed cell death was occurring that could be reduced but not completely prevented by Ep. These data indicate that Ep acts as both a mitogen and a survival factor for HCD-57 cells.

Animals↗
PubMed1991

Effect of reversible androgen deprivation on hemoglobin and serum immunoreactive erythropoietin in men.

To examine the role of testosterone in the maintenance of hemoglobin levels, we studied the effect of reversible androgen deprivation on hemoglobin, serum immunoreactive erythropoietin, and serum testosterone in seven men treated with a luteinizing hormone-releasing factor (LHRH) agonist for 6 months and then followed for an additional 6 months. The mean serum testosterone level was 4.35 +/- 1.05 ng/ml initially and it decreased to castrate levels in all patients by 6 months. After stopping therapy, there was a rapid increase in serum testosterone such that by 12 months the mean concentration was normal. The pretreatment hemoglobin was 15.2 +/- 0.9 g/dl (mean +/- SD); after 6 months of androgen deprivation it had fallen to 14.1 +/- 0.4 g/dl (P less than 0.05). Six months after stopping therapy, the hemoglobin rose to pre-treatment levels. Before treatment, serum immunoreactive erythropoietin was 9.5 +/- 4.6 mu/ml (mean +/- SD) and did not change significantly during or after the 6 month period of androgen deprivation. No significant inhibition of burst-forming unit-erythroid (BFU-E) or colony-forming unit-granulocyte macrophage (CFU-GM) was observed at the serum level of nafarelin acetate obtainable in vivo. These data suggest that, within the normal range of hemoglobin in men, androgens are a determinant of the red cell mass.

Aged↗
PubMed1991

Erythropoietin response to anemia as a function of age.

The erythropoietin (EPO) response to anemia was assessed for 244 subjects aged 1-64 years (mean 45.2 years) and 121 subjects aged 65-94 years (mean 68.3 years). Subjects included non-anemic individuals as well as those with anemia of various etiologies, excluding renal disease and pregnancy. Significant inverse correlations between serum immunoreactive EPO and hematocrit were noted for both groups. Regression lines failed to show a significantly lower slope or y-intercept for older compared to younger subjects. EPO levels were not significantly lower for older compared to younger subjects when controlled for hematocrit level. These results suggest that the EPO response to anemia in older subjects is similar to that of younger subjects.

Adolescent↗
PubMed1979

Chemical modification of nuclear proteins by erythropoietin.

The spleen of the exhypoxic polycythemic mouse was employed as a model system to study the effect of erythropoietin on enzymes that chemically modify nuclear proteins. At selected time intervals after in vivo administration of erythropoietin, acetyltransferase and methyltransferase activity were measured in nuclei isolated from the spleens of treated mice. In addition, the incorporation of labeled methyl and acetate groups into individual histone proteins was also examined. A 36% increase in nuclear acetyltransferase activity was observed eight hours after administration of erythropoietin, whereas nuclear methyltransferase activity increased by 42% 24 hours after administration of the hormone. Selective acetylation or methylation of individual histone proteins was not observed, and it is concluded that activation of transcription by erythropoietin is not the result of acetylation or methylation of nuclear proteins.

Acetylation↗
PubMed1979

Erythropoiesis in vitro. Role of calcium.

The in vitro plasma clot technique was employed to examine the role of calcium during the interaction of erythropoietin and mouse erythroid progenitor cells. Erythropoietin-induced erythroid colony formation was increased 24% by the carboxylic ionophore A23187 (10 nM), whereas a 35% increase was produced by the carboxylic ionophore Ro 2-2985/1 (1 nM). EGTA (3 mM) inhibited erythropoietin-induced erythroid colony formation. Inhibition of erythroid colony formation by EGTA could be reversed by Ca2+, but not by Mn2+, Mg2+, Zn2+, or Fe2+. At least 30 min exposure of marrow cells to erythropoietin in vitro was required for production of erythroid colonies. EGTA substantially inhibited erythropoietin-induced erythroid colony formation even when the marrow cells were exposed to the hormone for up to 2 h before addition of the chelator. Marrow cells incubated first in calcium-free medium with erythropoietin and then cultured in the presence of calcium but not erythropoietin, failed to form erythroid colonies although colony formation occurred when erythropoietin was provided. Taken together, the data indicate that calcium is required for both extracellular and intracellular events during the interaction of erythropoietin with its target cells.

Animals↗
PubMed1979

Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation.

Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.

Animals↗
PubMed1978

Sickle cell anemia and transposition of the great vessels.

A child with homozygous sickle cell disease and transposition of the great vessels had erythrocytosis associated with markedly increased plasma erythropoietin activity. Her clinical course was complicated by neurologic manifestations but not by recurrent sickle cell vasooculsive episodes. The fetal hemoglobin level which had been greater than 25% during the first two years of life gradually decreased to less than 10%. She died at 3 years of age of congestive heart failure and severe anemia. The only sickle cell painful crisis occurred during her terminal illness. It is likely that the high levels of fetal hemorglobin decreased sickling and thus allowed erythrocytosis to develop. Fetal hemoglobin may also have prevented frequent vaso-occlusive events despite the high hematocrit level.

Anemia, Sickle Cell↗
PubMed1977

Erythropoietin: isolation by affinity chromatography with lectin-agarose derivatives.

Affinity chromatography using agarose-bound lectins was used to isolate erythropoietin from crude preparations of sheep plasma and human urinary erythropoietin. On the basis of previous estimates of the sugar content of the hormone, six lectins (wheat germ agglutinin, phytohemagglutinin, Ricinus communis 120, soybean agglutinin, concanavalin A, and limulin) were chosen for study. Only wheat germ agglutinin-agarose and phytohemagglutinin-agarose derivatives had significant affinity for erythropoietin. By use of wheat germ aggutinin-agarose columns, erythropoietin could be separated from over 95% of the initial starting protein, resulting in an 8-to 100-fold purification and a recovery of at least 40% depending on the source of the hormone. Affinity chromatography with agarose-bound lectins provides a simple rapid method for isolating erythropoietin from crude preparations of the hormone.

Animals↗
PubMed1977

Felty's syndrome: an analytical review.

The clinical and laboratory features of 72 patients with Felty's syndrome described within the last ten years have been compared with Felty's five original patients. Felty's syndrome appears to be a variant of rheumatoid arthritis with extra-articular manifestations in which leukopenia (usually due to neutropenia) and splenomegaly occur, although not always at the same time. Both are manifestations of the underlying disease process and are not necessarily otherwise related. The mechanism of the leukopenia is complex and abnormalities in leukocyte function appear to be as important as the leukopenia in predisposing patients with Felty's syndrome to infection. Functional abnormalities of the leukocytes in this syndrome are due in part to immune complex formation. Hypocomplementemia associated with this process may be another cause for the increased susceptibility to infection. It is proposed, therefore, that therapy in Felty's syndrome be directed at the underlying disease process, and gold salts and penicillamine should be considered for this purpose. Splenectomy should be reserved for specific situations, such as hemolytic anemia, severe thrombocytopenia, leg ulcers, and infections associated with profound leukopenia that are not responsive to medical therapy.

Adult↗
PubMed1977

A simple hypoxic chamber.

A simple, normobaric hypoxic chamber constructed of easily obtainable parts is described. Important features include small size, low cost, simple construction, and easy maintenance. The chamber accommodates 50 mice and can be used for rats as well. Although designed to provide animals for the in vivo bioassay of erythropoietin, it is also useful for experimental studies requiring the acute induction of hypoxia.

Animals↗