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Biomedical subjects

R Hoffman

Publications and source records attributed to R Hoffman.

At least 325 records · Page 18Linked to original sources
PubMed1986

Effects of recombinant human tumor necrosis factor alpha, recombinant human gamma-interferon, and prostaglandin E on colony formation of human hematopoietic progenitor cells stimulated by natural human pluripotent colony-stimulating factor, pluripoietin alpha, and recombinant erythropoietin in serum-free cultures.

The influences of pure human pluripotent colony-stimulating factor, highly purified pluripoietin alpha, pure recombinant human tumor necrosis factor alpha, pure recombinant human gamma-interferon, and natural prostaglandin E1 (PGE1) were evaluated on colony formation of multipotential and erythroid progenitor cells in the presence of recombinant erythropoietin and hemin and on colony formation of granulocyte-macrophage progenitors in normal human marrow cultured in the presence or absence of serum. Serum was replaced by bovine serum albumin, iron-saturated transferrin, cholesterol, and calcium chloride. Increasing concentrations of pluripotent colony-stimulating factor and pluripoietin alpha stimulated increasing numbers of colonies from nonadherent low-density T-lymphocyte-depleted cells in the absence and presence of serum. Growth was usually greater in the presence of serum and on a unit basis pluripoietin alpha was more active than pluripotent colony-stimulating factor. Recombinant human tumor necrosis factor alpha and recombinant human gamma-interferon suppressed colony formation colony forming unit-granulocyte-macrophage, burst forming unit-erythroid, and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte; PGE1 suppressed colony formation by colony-forming unit-granulocyte-macrophage, stimulated colony formation by burst forming unit-erythroid, and had no effects on colony formation by colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte in both serum-containing and serum-free medium. The PGE1 enhancing effects on erythroid colony formation required T-lymphocytes. Thus, results are similar using serum-containing and serum-free cultures of human bone marrow cells and serum-free defined culture medium can be used to study the mechanism of action of purified natural and recombinant growth and suppressor molecules in vitro.

Bone Marrow Cells↗
PubMed1986

Terminal cytoplasmic maturation of human megakaryocytes in vitro.

Several studies suggest that serum factors (thrombopoietins) regulate thrombopoiesis by altering the number, size, ploidy, and maturation rate of megakaryocytes (MK). Various in vivo systems have been used to quantitate these events. In this study, an in vitro system was developed to monitor terminal cytoplasmic maturation of isolated human MK. MK enriched by elutriation, which eliminated the MK progenitors, were suspended in culture with serum from either normal donors (NABS) or patients with aplastic anemia (AAS). In cultures composed of small platelet glycoprotein-positive mononuclear cells and morphologically immature MK, development was characterized by sequential shifts in MK through morphologically recognizable maturation stages I, II, III, and IV over eight days of incubation (I and II only; then I, II, III; II, III, IV; III and IV; then IV only). Platelet formation coincided with the appearances of stage IV cells. Cultures composed of a mixture of all stages followed a similar maturation sequence, only at an accelerated rate. AAS resulted in the more rapid appearances of the mature cells in either system. This study indicates that human MK can undergo terminal cytoplasmic maturation in vitro, and that altering culture conditions (AAS for NABS) can accelerate the rate of maturation. Three major events occur during megakaryocytopoiesis: proliferation of the progenitor cells, polyploidization, and cytoplasmic maturation. Now it is possible to study the terminal steps of differentiation independent of proliferative events.

Anemia, Aplastic↗
PubMed1985

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis.

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.

Bisbenzimidazole↗
PubMed1985

Evidence that the major surface proteins of three Leishmania species are structurally related.

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.

Animals↗
PubMed1985

Current considerations of the etiology of aplastic anemia.

Aplastic anemia is a disorder characterized by marrow aplasia and pancytopenia. The pathogenetic mechanisms that lead to bone marrow aplasia have been intensively studied. Data obtained from these studies suggest that aplastic anemia is a heterogeneous disorder with regards to pathogenesis. Bone marrow aplasia may result from a number of abnormalities including qualitative or quantitative abnormalities of hematopoietic stem cells, abnormal interaction between bone marrow accessory cells (lymphocytes and macrophages) and hematopoietic stem cells, cytotoxic humoral inhibitors of hematopoiesis, and abnormalities of the bone marrow microenvironment. A number of new therapeutic options have improved the survival of patients with aplastic anemia. Allogeneic bone marrow transplantation has actually resulted in the cure of patients. Unfortunately, only a minority of patients have a suitable bone marrow donor and alternate modes of therapy have been sought. Encouraging results have been reported from several centers concerning the use of antilymphocyte serum in patients with aplastic anemia. Certainty of the ultimate long-term benefit of this type of immunosuppressive therapy is not possible until careful, randomized, prospective studies of its use are completed.

Age Factors↗
PubMed1985

An acute myeloproliferative disorder characterized by myelofibrosis and blast cells that express phenotypic properties associated with multiple hematopoietic lineages.

The authors investigated the blast cells obtained from two patients with acute myelofibrosis with the use of recently developed immunochemical and ultrastructural markers. They intended to examine the expression of megakaryoblastic, erythroblastic, myeloblastic, and monoblastic properties in these cells. Respectively, 20% and 15% of the blasts from patient 1 and patient 2 expressed a megakaryoblastic phenotype as determined by reactivity with a polyclonal platelet glycoprotein antisera (PGP). However, 55% and 73% of these patients' blasts also exhibited myeloid/monocytic properties. Ultrastructural studies clearly demonstrated findings consistent with the presence of myeloblasts, monoblasts, and erythroblasts, as well as undifferentiated agranular blasts. These findings demonstrate the existence of a disorder in which blast cells simultaneously express properties associated with multiple hematopoietic lineages. This disorder is characterized by impaired terminal differentiation.

Acute Disease↗
PubMed1985

Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X 10(-8) M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethane-sulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

Colony-Stimulating Factors↗
PubMed1985

Hematologic malignancies associated with primary mediastinal germ-cell tumors.

Three men with primary mediastinal germ-cell tumors subsequently developed a malignant hematologic disorder characterized by pancytopenia and marrow infiltration with hematopoietic blast cells. Two of these patients were classified as having acute megakaryocytic leukemia and the third was believed to have a myelodysplastic syndrome with a prominent megakaryocytic component. Analysis of clinical characteristics of these patients and review of the literature suggest that the proximate association of mediastinal germ-cell tumors with malignant hematologic disorders is neither a coincidence nor a consequence of chemotherapy given for the germ-cell tumor. We believe this association represents the evolution of a neoplastic disorder that initially involves a totipotent germ cell. These germ cells, when located in the mediastinum, apparently acquire hematologic phenotypes and are manifested clinically as a hematologic malignancy.

Acute Disease↗
PubMed1985

Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture.

Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. As previously reported, sideroblastic anemia bone marrow cells grew large numbers of CFUE in methylcellulose culture in the absence or presence of erythropoietin. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and 14C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of 14C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

5-Aminolevulinate Synthetase↗
PubMed1985

In vitro hematopoiesis following induction chemotherapy for acute leukemia.

The treatment of acute nonlymphocytic leukemia results in predictable bone marrow hypoplasia and eventual cellular repopulation. In order to study this postchemotherapy repopulation, assays for hematopoietic progenitor cells were performed on bone marrow samples obtained from seven patients with acute nonlymphocytic leukemia who had received similar chemotherapeutic induction regimens. Burst-forming units (erythrocyte), colony-forming units (megakaryocyte), colony-forming units (granulocyte-macrophage), and colony-forming units (granulocyte-erythrocyte-megakaryocyte-macrophage) were cloned from human bone marrow mononuclear cells 5 and/or 10 days following completion of chemotherapy. All patients were pancytopenic and had hypocellular marrows when studied. Assays were performed 7 to 30 days prior to complete remission. Colony-forming units (granulocyte-macrophage) were equivalent to control values 5 days following chemotherapy, while burst-forming units (erythrocyte) and colony-forming units (granulocyte-erythrocyte-megakaryocyte-macrophage) were not assayable at that time. Ten days following chemotherapy, colony-forming units (granulocyte-erythrocyte-megakaryocyte-macrophage) and colony-forming units (granulocyte-macrophage) were 200 and 250% of normal controls, respectively, while burst-forming units (erythrocyte) were 29% of control values. Colony-forming units (macrophage) were 10 to 15 times normal values 10 days following chemotherapy. In contrast to colonies from normal individuals, those grown from marrow obtained following chemotherapy were frequently macroscopic and were composed of thousands of cells. Patient marrow had larger proportions of progenitor cells in S phase of the cell cycle than did normal controls. These studies suggest the presence of a stem cell in human bone marrow which is resistant to chemotherapeutic agents and has a high capacity to regenerate hematopoietic progenitor cells. The period following completion of chemotherapy for acute nonlymphocytic leukemia appears suitable for the study of the hierarchical nature of human hematopoiesis.

Acute Disease↗