Growth hormone: species-specific stimulation of erythropoiesis in vitro.

The effects of purifed bovine and human growth hormone were tested in vitro with murine and human bone marrow by means of granulocyte-monocyte and erythroid progenitor cloning techniques. Nanogram concentrations of the growth hormones potentiated erythropoietin-stimulated erythropoiesis, but not granulopoiesis, in a species-specific manner.

Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity.

A permanent human cell line that maintains the granulocytic characteristics of acute myelogenous leukemia cells has been established. The cells of this line form myeloid colonies in soft gel culture in the presence of human colony-stimulating activity. The cell line may be useful for studying human acute myelogenous leukemia and the mechanism of response to colony-stimulating activity.

Bone marrow origin of hepatic macrophages (Kupffer cells) in humans.

Hepatic macrophages (Kupffer cells) from two male recipients of bone marrow transplants from females were studied for fluorescent Y body staining and sex chromatin (Barr body). After the transplant, macrophages had the sex karyotype of the donor, indicating that human hepatic macrophages originate in bone marrow.

Expression of the 3' terminal region of human T-cell leukemia viruses.

Human T-cell leukemia viruses (HTLV) are closely associated with some human T-cell leukemias and lymphomas. A unique 3' region of the HTLV genome is believed to be involved in HTLV-induced cellular transformation, although the function of this region has yet to be determined. A subgenomic messenger RNA transcribed from this region of HTLV has now been characterized. These results provide direct evidence for the expression of a novel gene in HTLV.

Identification of the putative transforming protein of the human T-cell leukemia viruses HTLV-I and HTLV-II.

The human T-cell leukemia viruses HTLV-I and HTLV-II are unique among the transforming retroviruses of vertebrates in their ability to transform human T cells in vitro and in their close association with human malignancies (T-cell lymphomas and leukemia). Their genomes are relatively simple, containing the genes gag, pol, env, and a 3' region termed "X." This 3' region may be responsible for the transforming potential of the viruses. The existence of proteins encoded by the 3' region has been postulated on the basis of multiple open reading frames. In the present study this region is shown to contain a gene encoding a protein of 40 kilodaltons in HTLV-I and 37 kilodaltons in HTLV-II. It is proposed that these proteins be called, respectively, p40xI and p37xII.

Proliferation of astroglia and oligodendroglia in response to human T cell-derived factors.

Human T lymphocytes transformed by human T cell leukemia-lymphoma viruses or activated by lectins were found to produce stimulating factors that promoted both proliferation and maturation of oligodendroglial and astroglial cells in vitro.

The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

HTLV x-gene product: requirement for the env methionine initiation codon.

The human T-cell leukemia viruses (HTLV) are replication-competent retroviruses whose genomes contain gag, pol, and env genes as well as a fourth gene, termed x, which is believed to be the transforming gene of HTLV. The product of the x gene is now shown to be encoded by a 2.1-kilobase messenger RNA derived by splicing of at least two introns. By means of S1 nuclease mapping of this RNA and nucleic acid sequence analysis of a complementary DNA clone, the complete primary structure of the x-gene product has been determined. It is encoded by sequences containing the env initiation codon and one nucleotide of the next codon spliced to the major open reading frame of the HTLV-I and HTLV-II x gene.

Direct evidence for a bone marrow origin of the alveolar macrophage in man.

Alveolar macrophages were obtained from 23 patients who had received marrow transplants for hematologic disorders. The presence of a Y body in macrophages of male origin was demonstrated by fluorescence microscopy. In those patients with a marrow donor of opposite sex the alveolar macrophages were shown to be of donor origin. The disappearance with time of host macrophages indicates a life-span, under the conditions, of approximately 81 days.

Cytokines alter production of HIV-1 from primary mononuclear phagocytes.

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.

Studies of the putative transforming protein of the type I human T-cell leukemia virus.

The putative transforming protein of the type I human T-cell leukemia virus (HTLV-1) is a 40-kilodalton protein encoded by the X region and is termed p40XI. On the basis of both subcellular fractionation techniques and immunocytochemical analysis, it is now shown that p40XI is a nuclear protein with a relatively short half-life (120 minutes). It is synthesized de novo in considerable quantities in a human T-cell line infected with and transformed by the virus in vitro, and it is not packaged in detectable amounts in the extracellular virus.

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Evidence for the involvement of GM-CSF and FMS in the deletion (5q) in myeloid disorders.

By in situ chromosomal hybridization, the GM-CSF and FMS genes were localized to human chromosome 5 at bands q23 to q31, and at band 5q33, respectively. These genes encode proteins involved in the regulation of hematopoiesis, and are located within a chromosome region frequently deleted in patients with neoplastic myeloid disorders. Both genes were deleted in the 5q-chromosome from bone marrow cells of two patients with refractory anemia and a del(5)(q15q33.3). The GM-CSF gene alone was deleted in a third patient with acute nonlymphocytic leukemia (ANLL) who has a smaller deletion, del(5)(q22q33.1). Leukemia cells from a fourth patient who has ANLL and does not have a del(5q), but who has a rearranged chromosome 5 that is missing bands q31.3 to q33.1 [ins(21;5)(q22;q31.3q33.1)] were used to sublocalize these genes; both genes were present on the rearranged chromosome 5. Thus, the deletion of one or both of these genes may be important in the pathogenesis of myelodysplastic syndromes or of ANLL.

Identification of the colony-stimulating cell in human peripheral blood.

Bone marrow colony formation in soft gel culture may be stimulated by substances elaborated by human peripheral blood leukocytes. In order to determine the cell type responsible for colony stimulation, peripheral leukocytes were separated by Ficoll-Hypaque gradients and differential glass adhesion. Morphologic, histochemical, and functional criteria were applied to determine the purity of the monocyte, lymphocyte, and neutrophil fractions. Using these cells as feeder layers and as a source of conditioned medium, evidence was obtained that the monocyte is the colony-stimulating cell of human peripheral blood. Activity greater than that of mixed white cells was obtained with monocyte underlayers, and only monocyte- and macrophage-conditioned media were shown to have significant colony-stimulating activity.

Differentiation of macrophages from normal human bone marrow in liquid culture. Electron microscopy and cytochemistry.

To study the various stages of human mononuclear phagocyte maturation, we cultivated bone marrow in an in vitro diffusion chamber with the cells growing in suspension and upon a dialysis membrane. At 2, 7, and 14 days, the cultured cells were examined by electron microscopy and cytochemical techniques for peroxidase and for more limited analysis of acid phosphatase and arylsulfatase. Peroxidase was being synthesized in promonocytes of 2- and 7-day cultures, as evidenced by reaction product in the rough-surfaced endoplasmic reticulum, Golgi complex, and storage granules. Peroxidase synthesis had ceased in monocytes and the enzyme appeared only in some granules. By 7 days, large macrophages predominated, containing numerous peroxidase-positive storage granules, and heterophagy of dying cells was evident. By 14 days, the most prevalent cell type was the large peroxidase-negative macrophage. Thus, peroxidase is present in high concentrations in immature cells but absent at later stages, presumably a result of degranulation of peroxidase-positive storage granules. Clusters of peroxidase-negative macrophages with indistinct borders (epithelioid cells), as well as obvious multinucleated giant cells, were noted. Frequently, the interdigitating plasma membranes of neighboring macrophages showed a modification resembling a septate junction--to our knowledge, representing the first documentation of this specialized cell contact between normal macrophages. We suggest that such junctions may serve as zones of adhesion between epithelioid cells.

Potentiation of erythropoiesis in vitro by dexamethasone.

The effect of dexamethasone on erythropoiesis was examined in vitro. Hematopoietic cells from 13-day mouse fetal livers were cultured for 48 h in the presence or absence of erythropoietin and erythroid colonies enumerated. Colony formation occurring in cultures containing no added erythropoietin was inhibited by the incorporation of antierythropoietin antibody, suggesting that these colonies formed in response to endogenous hepatic erythropoietin. Maximal colony formation was observed with 0.5 U/ml of sheep erythropoietin. Dexamethasone increased erythroid colony formation with peak stimulation at 10(-9) M. Dexamethasone potentiation was most marked in cultures containing less than maximally stimulating concentrations of erythropoietin. The cells required only a brief exposure to glucocorticosteroid to exhibit the augmented cloning capacity, and dexamethasone stimulation was inhibited by progesterone (10(-6) M). A comparable response to dexamethasone was observed in cultures of adult murine and human bone marrow erythroid progenitors, implying that the phenomenon is not peculiar to fetal cells and is not dependent on the presence of fetal hepatocytes. These data suggest that erythroid progenitor cells possess a glucocorticoid receptor mechanism that can modulate the response to erythropoietin in vitro.

Thymus-dependent lymphocytes in human bone marrow.

Human bone marrow is known to contain significant numbers of bursa-dependent lymphocytes. The presence of thymus-dependent (T) cells is controversial. Bone marrow cells obtained from healthy volunteers was fractionated by density centrifugation. A lymphocte-enriched subpopulation was shown to be reactive to alloantigens in mixed lymphocyte culture and to contain substantial numbers of T lymphocytes. The T lymphocytes were detected by cell surface markers (rosette formation with sheep RBC) and by response to the mitogens phytohemagglutinin and concanavalin A. Bone marrow T cells exhibited functional characteristics quantitatively different from peripheral blood T cells, suggesting that they may represent a particular subpopulation of T cells. The lymphocyte-enriched fraction additionally contained committed granulopoietic stem cells capable of colony formation in semisolid gel. The presence of T cells in human bone marrow is consistent with findings in other mammals and may explain the high incidence of graft versus host disease in bone marrow transplant recipients.

Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters.

Vitamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.

Randomized study of the duration of treatment with interferon alfa-2B in patients with hairy cell leukemia.

Several previous studies have demonstrated that both partially purified and recombinant alpha-interferons (alpha-IFNs) have high response rates in advanced hairy cell leukemia. However, the optimal dose and duration of therapy have not yet been defined. In this study, 90 patients were randomized after 12 months of IFN alfa-2b therapy with a standard dose of 2 X 10(6) U/m2 sc three times weekly to either no further therapy or an additional 6 months of therapy (18 mo total). There was no significant difference in the peripheral blood cell counts between the two groups (when analyzed) dating from the end of IFN therapy rather than from the time of randomization. Eighteen evaluable patients relapsed and were re-treated with IFN: 11 in the no-further-therapy group and 7 in the treated group. No patient was resistant to re-treatment with IFN. There was a significantly greater incidence of fatigue in the treated group (44% vs. 21%; P = .02) during the first 6 postrandomization months. We conclude that the duration of IFN therapy does not influence the clinical course after therapy is discontinued, but responses are maintained while patients receive therapy. However, because of a high incidence of fatigue with prolonged therapy and the ability to reinduce a second response, we recommend that IFN therapy be discontinued after 12 months in asymptomatic patients.

Prostate cancer cell cycle regulators: response to androgen withdrawal and development of androgen independence.

BACKGROUND: Androgen withdrawal is a standard therapy for prostate cancer that results in a decrease in tumor volume and a decline in serum prostate-specific antigen in the majority of patients. To understand the factors associated with regression of prostate cancers after androgen withdrawal, we studied cell cycle regulator changes in the CWR22 human prostate cancer xenograft model. METHODS: Established tumors in nude athymic BALB/c mice were sampled at various times after androgen withdrawal and after the development of androgen independence. Changes in the expression of cell cycle regulators were categorized into early and mid-to-late events. RESULTS AND CONCLUSIONS: Early events included a decrease in androgen receptor expression, followed by a short-term increase in expression of the p53 and p21/WAF1 proteins and a marked decrease in the Ki67 proliferative index. Mid-to-late events included progressive and sustained increases in p27 and p16 protein expression, a decrease in retinoblastoma protein expression, and an increase in the transcription factor E2F1. Changes in apoptosis (programmed cell death) were not observed at any time after androgen withdrawal. These data suggest that androgen withdrawal results in a cell stress response, in which increased p53 protein produces a cell cycle arrest, without activation of p53-mediated apoptosis. The proliferative index is further decreased through the action of the cyclin-dependent kinase inhibitors p27 and p16. Androgen-independent sublines emerged 80-400 days after androgen withdrawal, and these sublines had variable growth phenotypes but were associated with mdm2 protein overexpression and increased expression of cyclin D1. These results indicate that tumor regression in this human prostate cancer model is due to cell cycle arrest rather than to apoptosis and that the emergence of androgen independence is associated with a release from cell cycle arrest.