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D T Scadden

Publications and source records attributed to D T Scadden.

At least 73 records · Page 4Linked to original sources
PubMed1995

Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells.

Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell.

Adipose Tissue↗
PubMed1995

Thrombopoietin receptor expression in human cancer cell lines and primary tissues.

c-mpl is the receptor for the recently identified megakaryocyte growth and differentiation factor thrombopoietin. Thrombopoietin has been shown to be capable of raising platelet counts in animals and is about to enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of human c-mpl by reverse transcription PCR on 39 human cell lines and 20 primary human tissue samples derived from both normal and malignant sources. c-mpl transcripts were found in all megakaryocytic cell lines tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia cell line KMT-2, and a hepatocellular carcinoma cell line (Hep3B). Among primary tissues, fetal liver cells and brain had detectable levels of c-mpl message, and among primary tumors, none were found to express c-mpl. These data support the conclusion that c-mpl has restricted expression that is primarily, but not exclusively, related to megakaryocytopoiesis. These observations suggest that thrombopoietin is unlikely to have direct effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia.

Amino Acid Sequence↗
PubMed1995

Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma.

Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis.

B-Lymphocytes↗
PubMed1995

Functional isolation and characterization of human hematopoietic stem cells.

Hematopoietic cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Human bone marrow cells that were responsive to the early-acting cytokines Kit ligand and interleukin-3 were forced to a metabolic death. The subfraction remaining represented 1 in 10(5) bone marrow mononuclear cells, were determined to be quiescent by cell cycle analysis, and had a stem cell immunophenotype. The cells were highly enriched for long-term culture-initiating cells, were capable of secondary colony formation, and produced both myeloid and lymphoid progeny. Thus, this technically simple strategy led to the efficient purification of cells with characteristics of hematopoietic stem cells.

Antigens, CD↗
PubMed1995

Cytotoxic CD8+ T lymphocytes reactive with human immunodeficiency virus-1 produce granulocyte/macrophage colony-stimulating factor and variable amounts of interleukins 2, 3, and 4 following stimulation with the cognate epitope.

Infection with human immunodeficiency virus type 1 (HIV-1) induces vigorous and persistent cytotoxic CD8+ T cell responses. CTL clones were derived from peripheral blood or cerebrospinal fluid of three HIV-1 patients, with depressed CD4+ T cell counts. When stimulated with HLA-compatible target cells (B-LCL) presensitized with cognate HIV-1 peptides, all clones produced GM-CSF, TNF-alpha, and IFN-gamma and most produced low amounts of IL2, IL3, and IL4. After nonspecific stimulation with a phorbol ester and calcium ionophore, the clones secreted cytokines at levels similar to those from CD4+ lines from an HIV-1 infected donor. The ability of supernatants from the stimulated CTL clones to support the formation of granulocyte-macrophage colonies in normal bone marrow suggests that the GM-CSF was biologically active. Release of cytokines by activated CTL may influence the immunopathogenesis of HIV disease.

CD4-Positive T-Lymphocytes↗
PubMed1995

In vivo effects of interleukin 3 in HIV type 1-infected patients with cytopenia.

OBJECTIVE: To determine the safety, tolerance, and hematological and virological effects of the recombinant hematopoietic growth factor interleukin 3 (IL-3) in HIV-1-infected individuals with cytopenia. DESIGN AND METHODS: A phase I single-center trial was conducted with patients in cohorts of three receiving one of four dose levels of self-administered, subcutaneously injected IL-3 (0.5, 1.0, 2.5, or 5.0 micrograms/kg/day). Toxicities, hematological effects, and virological effects were recorded. Viral studies included serum HIV p24 antigen levels, quantitative plasma and peripheral blood mononuclear cell cultures, and quantitative, competitive polymerase chain reaction of patient plasma. RESULTS: Increases in white blood cell counts (WBC) and absolute neutrophil counts (ANC) were noted at the higher dose levels while absolute eosinophil counts (AEC) increased in all patients. The percent changes in WBC from baseline ranged from 52 to 309 and in ANC from 20 to 262 in the 2.5- and 5.0-micrograms/kg/day groups. The mean AEC change was 17-fold (range, 2- to 59-fold). Hemoglobin, hematocrit, platelets, and CD4 and CD8 counts were generally unaffected although individual patients demonstrated increases in hemoglobin and platelet levels. Toxicities were generally mild, but one patient developed a transient local erythematous rash at the sites of IL-3 injection which pathologically demonstrated hypersensitivity vasculitis. Of note, viral studies did not demonstrate any consistent changes in HIV-1 activity. CONCLUSION: These data demonstrate limited hematological effects of IL-3 monotherapy in HIV-1-infected patients with cytopenia. However, should IL-3 be incorporated into combination cytokine therapies for HIV disease, these data suggest that IL-3 does not enhance in vivo HIV-1 activity.

Adult↗
PubMed1994

Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity.

Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase.

Adult↗
PubMed1994

Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

3T3 Cells↗
PubMed1994

Modulation of megakaryocytopoiesis by human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) may act to modulate hematopoiesis in addition to its effects on mesenchymal cells. We studied the effects of bFGF on human and murine primary marrow megakaryocytes. bFGF modestly enhanced the size of the human megakaryocyte colony-forming unit (CFU-MK) and cell numbers per colony, in combination with interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Adhesion of human megakaryocytes to bone marrow (BM) stromal fibroblasts was enhanced when either stromal fibroblasts or megakaryocytes were treated with bFGF. This resulted in significantly increased proliferation of megakaryocytes. In addition, bFGF augmented secretion of the cytokines tumor necrosis factor alpha and IL-6 by human primary BM megakaryocytes. Immature murine megakaryocytes showed a significant growth response to bFGF as measured by the single cell growth assay. This effect was abrogated by specific antibodies for bFGF and combination of anti-IL-6 and anti-IL-1 beta antibodies. bFGF has no effect on murine CFU-MK formation, but significantly potentiated CFU-MK formation in the presence of IL-3 or GM-CSF. These results indicate that the effect of bFGF on various megakaryocyte populations is different and that bFGF may affect megakaryocytopoiesis via modulation of megakaryocyte-stromal interactions and via augmentation of cytokine secretion from megakaryocytes.

Animals↗
PubMed1994

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

3T3 Cells↗
PubMed1994

Cloning of FRK, a novel human intracellular SRC-like tyrosine kinase-encoding gene.

We report the cloning of a novel tyrosine kinase (TyK)-encoding gene (TYK) from the human hepatoma cell line Hep3B. Using the polymerase chain reaction (PCR) and oligodeoxyribonucleotide primers based on conserved TYK motifs, a 180-bp fragment was cloned and used to obtain full-length cDNA clones of 2.9 kb, with an open reading frame of 505 amino acids (aa). Restricted expression was detected by Northern blotting or reverse-transcribed PCR in a broad range of cell lines. The predicted aa sequence contains characteristic TyK motifs without a transmembrane region, suggesting an intracellular localization. There was 49% aa sequence identity with human FYN product and 47% with human SRC product; however, several structural differences distinguish this clone from other SRC subfamily members. This clone, FYN-related kinase or FRK, is a novel member of the intracellular TYK gene family.

Amino Acid Sequence↗
PubMed1994

In vitro effects of stem-cell factor or interleukin-3 on myelosuppression associated with AIDS.

OBJECTIVE: To determine whether the early-acting hematopoietic growth factors stem-cell factor (SCF) or interleukin-3 (IL-3), are able to overcome the bone-marrow suppressive effects of cytokines or drugs involved in the hematologic abnormalities that accompany HIV-1 infection. DESIGN: In vitro colony formation assays of normal human bone-marrow cells exposed to the myelosuppressive drugs, zidovudine, interferon-alpha (IFN-alpha) and ganciclovir, or the myelosuppressive cytokines, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta (TGF-beta), implicated in HIV dysmyelopoiesis. RESULTS: SCF (10 ng/ml) enhanced the numbers of erythroid (BFU-E) colonies in the presence of zidovudine or ganciclovir (P < 0.05) and myeloid [colony-forming unit granulocyte macrophage (CFU-GM)] colonies in the presence of ganciclovir or IFN-alpha (P < 0.05) relative to controls. IL-3 (10 ng/ml) also improved erythroid colony numbers in the presence of zidovudine (P < 0.05) and CFU-GM in the presence of IFN-alpha (P < 0.05). Neither factor consistently altered the inhibition of TGF-beta or TNF-alpha. The 50% inhibitory concentration (IC50) of the myelosuppressive agents was altered in only one setting, using IL-3 in the presence of zidovudine. CONCLUSIONS: These data suggest that SCF or IL-3 may have a therapeutic application in overcoming hematopoietic abnormalities associated with drugs commonly used in the care of AIDS patients. However, they may have less capacity to overcome the bone-marrow inhibitory effects of the endogenous cytokines TNF-alpha and TGF-beta.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Viral activity in early HIV disease.

The commonly accepted paradigm for the natural history of HIV disease has been an acute burst of virus replication followed by years of viral quiescence. A terminal phase of virus activity is accompanied by gradual immunologic failure. Recent studies of the tissue localization of HIV and developments in virus quantitation have prompted a revision of this model. Viral latency has been shown to be only a relative concept by the demonstration of virus accumulation in tissue sites during early disease and quantifiable circulating levels of virus throughout the course of HIV infection. The primacy of HIV infection in AIDS has been further reaffirmed by these studies, but new questions as to its pathogenic effects have been raised.

Dendritic Cells↗
PubMed1993

HIV-1 suppresses erythropoietin production in vitro.

Serum erythropoietin (Epo) levels are depressed in anemic AIDS patients relative to controls. The basis for abnormal regulation of Epo has not been defined. The hepatoma cell line Hep3B produces Epo in response to hypoxia and serves as a model for the study of Epo regulation. Hep3B cells are infectible with human immunodeficiency virus-1 (HIV-1) and were used as a model for evaluating potential direct effects of HIV on Epo expression. HIV-1 infected or transfected Hep3B cells produced Epo at significantly lower levels than uninfected Hep3B cells under low O2 conditions or following exposure to cobalt chloride. Epo production induced by hypoxia of HIV-1 infected Hep3B cells was depressed compared with non-HIV containing Hep3B cells when normalized for cell number, total cellular protein or albumin, but not depressed when normalized for alpha-fetoprotein production. The cellular levels of Epo mRNA were not diminished in the HIV-1+ Hep3B cells, indicating a probable posttranscriptional effect of HIV-1 on Epo production. Cellular protein production or secretion rates as measured by precipitable 3H-leucine were not affected by the presence of HIV-1. HIV-1 appeared to depress the production of Epo and some, but not all, other cellular proteins. These results suggest that impaired production of Epo may be a direct effect of HIV-1 infection possibly contributing to anemia in AIDS.

Carcinoma, Hepatocellular↗
PubMed1992

Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand.

Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre-mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryocytes and that soluble KL/SCF in combination with granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryocytes. Here we show that adhesion of human megakaryocytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast-stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhesion and proliferation of human megakaryocytes to an immortalized murine stromal cell line SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth-potentiating factor for megakaryocytes in the bone marrow.

Animals↗
PubMed1992

The use of GM-CSF in AIDS.

Hematopoietic growth factors may mitigate the cytopenias that frequently complicate HIV disease or its treatment. Clinical and in vitro studies have indicated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) or erythropoietin (EPO) to overcome the myelosuppression of HIV or many of the drug therapies used in the care of HIV-infected individuals. In addition, neutrophil or monocyte functional abnormalities observed in AIDS patients may be improved by the use of GM-CSF. Issues which may distinguish the use of hematopoietic growth factors in AIDS as compared with in other clinical settings include: 1) interaction of the growth factor with other cytokines which are aberrantly expressed, 2) direct effects of the growth factor on the replicative activity of HIV, and 3) potential interactions of the growth factor with other concurrently administered medications. This review focuses on the potential roles and limitations of growth factor use in AIDS and reviews the clinical studies using GM-CSF in HIV-infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Quantitation of plasma human immunodeficiency virus type 1 RNA by competitive polymerase chain reaction.

Clinical measures of human immunodeficiency virus (HIV) type 1 activity in vivo are limited and hinder the assessment of antiretroviral therapies. Reported here is a method for quantitating HIV-1 RNA in human plasma using the polymerase chain reaction (PCR). This method uses an internal cRNA standard generated from a cloned 113-bp deletion mutation of a highly conserved HIV-1 gag region sequence. The mutant cRNA (K4) was shown to amplify with efficiency equivalent to that of wild-type HIV-1. Known quantities of K4 cRNA added to wild-type HIV-1 in a competitive PCR strategy using a radiolabeled primer permitted quantitation of wild-type HIV-1 RNA over four orders of magnitude (10(3)-10(6) RNA copies). RNA isolated from plasma from AIDS patients yielded 10(3) to 8 x 10(4) HIV-1 RNA copies/ml of plasma with an average intrasample coefficient of variation of .26. This method offers a sensitive assay with a broad dynamic range for monitoring HIV-1 activity in the plasma of AIDS patients. It may provide a useful tool for assessing the effects of antiretroviral therapy.

Binding, Competitive↗