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D H Ryan

Publications and source records attributed to D H Ryan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Cytokine regulation of early human lymphopoiesis.

An in vitro culture system in which bone marrow-derived fibroblast-like cells support the growth of TdT+ colonies derived from CD34+/CD10- human bone marrow progenitor cells has recently been described. The regulatory role of cytokines during early B lineage commitment was investigated using this culture system. Expression of IL-7, a cytokine that induces proliferation of B cell precursors, was detectable in the adherent layer by PCR and bioassay. Lymphoid progenitor colonies were inhibited by neutralizing anti-IL-7 Ab, suggesting that IL-7 produced by the adherent layer was required even in the earliest recognizable stages of human B cell lymphopoiesis. IL-1 alpha, IL-4, and TNF-alpha inhibited lymphoid progenitor colonies in a dose-dependent fashion. Neutralizing Ab to IL-1 alpha, IL-4, or TNF-alpha did not increase lymphoid progenitor colonies, suggesting that inhibitory concentrations of these cytokines are not constitutively elaborated in the adherent layer. Recombinant Steel factor and IL-6 as well as neutralizing Abs to these cytokines did not significantly affect lymphoid progenitor colonies, arguing against an important role for these cytokines in early human B lymphopoiesis. These results indicate that IL-7 provided by the bone marrow microenvironment is a critical growth factor at the earliest recognizable stages of human lymphopoiesis. IL-1 alpha, IL-4, and TNF-alpha have been shown to indirectly stimulate release of myeloid growth factors. The inhibition of lymphopoiesis by these cytokines suggests a possible mechanism for the observed reciprocal relationship between lymphoid and myeloid supportive bone marrow microenvironments.

Adult↗
PubMed1994

Mortality before AIDS: a review of causes of death in young men in the city of Edinburgh (1979-88).

Mortality in young Edinburgh males aged 20-34 years was analysed over the period 1979-88. Proportional mortality was calculated from cause of death data: 23% of deaths were caused by accidents, 16% by infection, 15% by suicide and 9% of deaths were due to cancer. Area mortality rates were measured, and rates were significantly higher (P < 0.001) in sectors of low socio-economic status. Edinburgh has the highest HIV positive prevalence rate in the UK (111 per 100,000) and 10% of known UK HIV positive cases in 1988 were traced to Lothian. The Edinburgh rate for AIDS-related deaths in males aged 20-34 years was 0.57 per 100,000, and has been contrasted with the pattern of mortality in New York where the rate was 35.8 per 100,000. The pattern of mortality amongst young Edinburgh males is likely to register a significant change over the next ten years. The results presented may be used as a standard against which change can be measured.

Acquired Immunodeficiency Syndrome↗
PubMed1994

CD34+ progenitor cell isolation from blood and marrow: a comparison of techniques for small-scale selection.

The isolation and characterization of primitive hematopoietic cells and their purification in sufficient numbers is important in clinical and research marrow transplantation settings. As systems for large-scale isolation and amplification of such cells are developed, they may assume importance in transplantation, treatment of marrow failure and for gene therapy applications. Such cells have been isolated by numerous techniques and in this work, small-scale isolation of CD34+ cells by two immunoadsorption purification methods is compared with isolation by flow cytometry. While the immunoadsorption techniques allow for the processing of large numbers of density gradient-separated or unseparated cells for progenitor isolation, such techniques do not achieve the purity afforded by fluorescence activated cell sorter separation.

Antigens, CD↗
PubMed1994

Sodium bromide acts as a contrast material on DEXA scanning.

Subjects undergoing a series of procedures to determine body composition, including dual-energy x-ray absorptiometry (DEXA) scanning and extracellular water volume determination by bromide dilution, demonstrated abnormal DEXA scans in some cases. Oral doses of sodium bromide in the stomach were detected on total body DEXA scanning as contrast material. The physical principles that cause bromide to be imaged in a manner similar to calcium by the DEXA scanner are discussed so that other investigators may be aware of this potential pitfall in body composition analysis.

Absorptiometry, Photon↗
PubMed1993

Subpopulations of bone marrow fibroblasts support VLA-4-mediated migration of B-cell precursors.

Proliferation of normal human lymphoid progenitors in culture is dependent on interaction with bone marrow-derived fibroblast-like cells (BM-FB). To investigate possible heterogeneity in this lymphoid-supportive microenvironment, we studied the interaction of a human B-precursor cell line (NALM-6) with BM-FB. NALM-6 cells associate with BM-FB by either adhesion or migration underneath the fibroblast. Individual fibroblasts in the BM-FB layer showed significant variation in the number of migrating NALM-6 cells. Migration of NALM-6 cells was primarily VLA-4-dependent, although residual migration observable after blocking with anti-VLA-alpha 4 antibody was inhibited by anti-VLA-alpha 5 antibody. Migration was not inhibited by blocking either of the known VLA-4 counterreceptors (VCAM-1 or fibronectin), although slight inhibition was observed using a combination of blocking antibodies to VCAM-1 and fibronectin. In contrast, NALM-6 adhesion without migration was significantly inhibitable by anti-VCAM-1 antibody. VCAM-1 or fibronectin expression on individual BM-FB did not correlate with NALM-6 migration. These results indicate that the adhesion and migration of human B-lymphoid precursors in the bone marrow microenvironment are mechanistically separable events and suggest the possibility of novel VLA-4 ligand(s), which may be important in human lymphopoiesis. Subpopulations of cells in the bone marrow microenvironment may preferentially support important aspects of lymphoid progenitor development.

Adult↗
PubMed1993

Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells.

Adhesion of hematopoietic progenitor cells to marrow-derived adherent cells has been noted for erythroid, myeloid, and lymphoid precursors. In this report, we have characterized very late antigen (VLA) integrin expression on normal CD34+ marrow progenitors, on leukemic cell lines, and on blasts from patients with acute myelogenous or monocytic leukemias. CD34+ progenitor cells expressed the integrin beta 1 chain (CD29), VLA-4 alpha (CD49d), and VLA-5 alpha (CD49e). The myeloid lines KG1 and KG1a also expressed CD49d and CD49e as did the Mo7e megakaryoblastic line. CD29, CD18, and CD11a were also present on each of these cell lines. Only the Mo7e line expressed the cytoadhesins GPIIbIIIa or GPIb. Binding of KG1a to marrow stroma was partially inhibited by antibodies to CD49d and its ligand, vascular cell adhesion molecule (VCAM-1). The majority of leukemic blasts studied expressed CD49d and CD49e as well. Blasts from patients with acute myelomonocytic leukemia consistently bound to stroma at levels greater than 20%, and adhesion to stroma could in some cases be partly inhibited by anti-CD49d. No role for glycosylphosphatidyl-inositol (GPI)-linked structures was demonstrated in these binding assays because the adhesion of leukemic blasts to stroma was not diminished after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). These studies indicate that CD34+ myeloid progenitors, myeloid leukemic cell lines, and leukemic blasts possess a similar array of VLA integrins. Their functional importance individually or in combination with other mediators of attachment in adhesion, transendothelial migration, and differentiation has yet to be fully elucidated.

Antibodies↗
PubMed1993

Adherence of normal and neoplastic human B cell precursors to the bone marrow microenvironment.

The early development of B lineage cells occurs in the bone marrow in close association with fibroblast-like accessory cells. The integrin VLA-4 is preferentially expressed by B cell precursors and mediates adhesion of B cell precursors to bone marrow derived fibroblasts (BM-FB), which express a VLA-4 counter/receptor, VCAM-1. The functional importance of this adhesion interaction is suggested by the inhibition by anti-VLA-4 antibody of in vitro proliferation of B cell progenitor colonies grown with a BM-FB adherent layer. In order to compare adhesion requirements of normal versus neoplastic human B cell precursors, quantitative expression of adhesion proteins by primary human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells was studied by flow cytometry. BCP-ALL cells expressed VLA-4 and VLA-5 at similar levels as normal immature B cell precursors. However, other adhesion molecules, particularly CD44, were overexpressed by the leukemic cells relative to their normal counterparts. Similar to normal B cell precursors, adhesion of BCP-ALL cells to BM-FB was inhibited by antibodies to VLA-4 and VCAM-1. These results suggest that the ordered program of adhesion protein expression and functional activation during B cell development is at least partially intact in BCP-ALL cells, and may play a role in tissue localization and growth of the neoplastic cells. The focal pattern of spreading of adherent BCP-ALL cell lines on individual cells in the BM-FB adherent layer suggests functional heterogeneity in the bone marrow microenvironment that may be essential in regulating normal and neoplastic lymphopoiesis.

Antigens, CD↗
PubMed1992

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.

Antigens, CD↗
PubMed1992

Analysis of treatment failure in acute nonlymphocytic leukemia patients over fifty years of age. A Southwest Oncology Group study.

Fourteen participating centers registered 33 patients on a Southwest Oncology Group Study of adults with acute non-lymphocytic leukemia (ANLL). Induction consisted of cytosine arabinoside 70 mg/m2 days 1-7 by continuous intravenous (i.v.) infusion, VP-16 50 mg/m2 i.v. over 1 hour days 1-3, and daunomycin 30 mg/m2 i.v. bolus days 1-3. Twenty-five patients (median age 69 years) were evaluable for response. Eleven (44%) achieved a remission marrow but only 8 fulfilled both blood and marrow criteria for complete remission. Of the 11 patients with a remission marrow, there were no patients over 70 years of age. Major coexisting disease data were evaluated. Only 5 patients had no major coexisting disease and 4 of those 5 achieved a remission marrow. The study illustrates and underscores the following problems of remission induction in the elderly: (a) increased susceptibility to the stress of the induction period, with 6 patients (24%) dying before treatment day sixteen; (b) disease resistance to antileukemic therapy with persistent ANLL in 6 patients (24%), despite two induction courses; and (c) hematopoietic stem cell sensitivity in the elderly with marrow regeneration failure documented in 2 patients (8%) following induction. Acute nonlymphocytic leukemia in the elderly has a poor prognosis, and novel therapeutic approaches are warranted.

Aged↗
PubMed1992

Molecular mechanisms of human melanocyte attachment to fibronectin.

In this report we show that fetal and neonatal melanocyte attachment to fibronectin (FN) is inhibited by antibodies to the beta 1 integrin subunit, suggesting a role for these molecules in melanocyte attachment to FN. The VLA-5 integrin was shown to be the predominant receptor for fetal melanocyte attachment to FN, in contrast with neonatal melanocytes in which the very late antigen (VLA)-5, VLA-3, and alpha v integrins each contributed to melanocyte attachment to FN. Peptides containing the arginyl-glycyl-aspartyl-serine (RGDS) sequence inhibited fetal and neonatal melanocyte attachment to FN by a maximum of 48% and 85%, respectively. The almost complete inhibition of neonatal melanocyte attachment to FN by RGDS-containing peptides suggests that the central cell-binding domain of FN is the primary recognition site for neonatal cell attachment to FN. Fetal and neonatal melanocytes showed a concentration-dependent attachment to two proteolytically derived fragments of the FN molecule: a 75-kD fragment, which contains the central cell-binding domain, and 33/66-kD fragments of the FN molecule, which encompass the heparin-binding domains V and VI. Antibodies to the beta 1 subunit inhibited fetal and neonatal melanocyte attachment to the 33/66-kD fragments by a maximum of only 15% and 24%, respectively, suggesting that other, non-integrin, receptors are involved in melanocyte recognition of this portion of the FN molecule. We propose that human fetal and neonatal melanocytes attach to FN by different complements of receptors and ligand target sequences, and that these differences may direct melanocyte interactions with FN during development.

Antibodies↗
PubMed1991

Macrophage-active colony-stimulating factors enhance human immunodeficiency virus type 1 infection in bone marrow stem cells.

To define the relationship between human immunodeficiency virus type 1 (HIV-1) infection in hematopoietic stem cells and virus production by their progeny, we performed kinetic studies infecting bone marrow (BM) stem cells and culturing them in the presence of hematopoietic growth factors. CD34-positive (CD34+), CD4-negative (CD4-) BM cells were isolated and infected in vitro with the monocytotropic HIV-1JR-FL strain or the laboratory-maintained HTLV-IIIB strain at a high multiplicity of infection. The cells were susceptible to productive infection only with HIV-1JR-FL, and virus production as measured by p24 protein release was markedly increased (more than fivefold) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). Macrophage CSF (M-CSF) was less stimulatory and granulocyte CSF (G-CSF) had no effect on virus production. Virus production coincided with proliferation of mononuclear phagocytes but was not related to granulocytic proliferation in G-CSF-treated BM cultures. Although peak virus production from GM-CSF-treated macrophages occurred 2 to 3 weeks after infection, peak virus production in infected stem cells was observed 5 to 6 weeks after. Enhancement in virus production had a more rapid onset when CD34+/CD4- cells were cultured in the presence of both GM-CSF and IL-3 for 7 or 14 days. Under these conditions there was a 10-fold enhancement in virus production after 7 days of preincubation and a 50-fold enhancement after 14 days. These data indicate that while the stem cell compartment may be susceptible to infection with a monocytotropic HIV-1 strain, productive and sustained infection is realized only after macrophage differentiation. The lack of effect of G-CSF on virus production is likely because of the limited effect of this hematopoietin on mononuclear phagocyte generation and function.

Antigens, CD↗
PubMed1991

Characterization of human alveolar macrophage Fc gamma receptor III: a transmembrane glycoprotein that is shed under in vitro culture conditions.

Three classes of Fc gamma receptors (FcR) have been identified on blood leukocytes: FcRI, FcRII, and FcRIII. Two forms of FcRIII have recently been characterized; a phosphatidylinositol linked form is found on neutrophils, whereas a transmembrane form of the molecule is found on a subset of peripheral blood lymphocytes. Peripheral blood monocytes express low levels of FcRIII on their surface, whereas FcRIII is readily expressed by tissue macrophages. The purpose of this investigation was to characterize the form of FcRIII expressed by normal human alveolar macrophages (AM) obtained from normal subjects by bronchoalveolar lavage. We found FcRIII expressed by AM has a molecular mass of 50 to 60 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis and migrates as a single band with a molecular mass of 35 kD after digestion with endoglycosidase F. Macrophage FcRIII was resistant to cleavage by phosphatidylinositol-specific phospholipase C. These results demonstrate that FcRIII expressed by AM is a transmembrane glycoprotein similar to the molecule found on peripheral blood lymphocytes. Scatchard binding analysis using 125I-labeled mAb 3G8 showed that AM express similar numbers of FcRIII as found on neutrophils (73,300 +/- 16,300 versus 69,300 +/- 8,500 receptor sites/cell, respectively; P = 0.73), whereas fewer binding sites were found on FcRIII-positive peripheral blood lymphocytes (35,300 +/- 13,900; P = 0.04). Of note, we found expression of FcRIII by AM was selectively and dramatically reduced during short term in vitro incubation at 37 degrees C. Receptor shedding as a result of proteolytic cleavage is probably responsible for the reduced expression that occurs during short-term in vitro culture.

Antibodies, Monoclonal↗
PubMed1991

Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment.

Adult↗
PubMed1991

Adhesive interactions of normal and leukemic human CD34+ myeloid progenitors: role of marrow stromal, fibroblast, and cytomatrix components.

Adhesive interactions between CD34+ myeloid progenitors, cytomatrix components, and marrow fibroblast and stromal monolayers are described and compared to the binding interactions of the CD34+ myeloid leukemic cell lines KG1a and KG1. Both normal precursors and their leukemic counterparts showed adhesion to marrow stroma and fibroblasts. CD34+ myeloid progenitors bound to the extracellular matrices of marrow stromal cell and fibroblast monolayers and to laminin and fibronectin to a lesser extent than to cellular stromal layers. These adhesive interactions were not inhibited by polyclonal antibodies to laminin or fibronectin, nor by 1 mM Arg-Gly-Asp-Ser (RGDS)-containing peptides. Also, although both normal and leukemic cells expressed the CD18 antigen, binding of these cells to stroma was not inhibited by blocking anti-CD18 monoclonal antibodies. Finally, KG1a adhesion was not blocked in the presence of anti-CD54 (ICAM) antibody, nor was it blocked when galactosyl or mannosyl pyranosides were added. KG1a binding was trypsin sensitive and enhanced in the presence of neuraminidase. These studies serve to characterize adhesive properties of normal and leukemic myeloid progenitors and begin to establish interactions important for the lodgement of early progenitor cells in human marrow.

Antigens, CD↗
PubMed1990

Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment.

Murine B cell precursors can be induced to proliferate in culture if allowed to bind to bone marrow derived adherent cells prepared under specific conditions. We studied the binding of human B cell precursor subpopulations to various in vitro microenvironments to determine which conditions may potentially be suitable models for human B precursor differentiation. Using the markers CD10, CD34, and CD20, B lineage populations of increasing maturation were quantitated: CD10+/CD34+, CD10+/CD20-, CD10+/CD20+, and CD10-/CD20+ cells in marrow, and CD10-/CD20+ mature B cells in peripheral blood. The adhesion of subpopulations of blood and marrow-derived light density cells to adherent cell layers or matrix was studied following a 2-h incubation in 24-well plates. The absolute number of bound B lineage cells was determined by cell counts and flow cytometry analysis. The adherence of B lineage cells to passaged human marrow fibroblasts (BM-FB) was highest in the most immature CD10+/CD34+ cells (34.3 +/- 4.2%), decreasing steadily with each stage of maturation to the peripheral blood B cells (11.2 +/- 2.4%). Increased adhesion of CD10+ B cell precursors relative to CD10-/CD20+ marrow B cells was confirmed by adhesion studies using sorted cells. The two most immature B lineage cells (CD10+CD34+ and CD10+/CD20-) showed more adherence to BM-FB than any other cell type tested, except for monocytes. Only B lineage precursor cells, erythroid precursors and CD10-/CD34+ cells showed significantly greater binding to BM-FB than to plastic. B lineage precursors bound equally well to primary and passaged human marrow fibroblasts, but bound significantly less well to passaged human foreskin fibroblasts, primary human marrow stroma, extracellular matrix of marrow fibroblasts, or fibronectin. These results suggest that specific binding to marrow fibroblasts is part of the differentiation program of early B lineage precursors. This binding activity gradually and predictably decreases during B lineage differentiation, in contrast to expression of other binding receptors, such as LFA-1 and CD44, which increase during B lineage maturation.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

Alveolar and peritoneal macrophages bear three distinct classes of Fc receptors for IgG.

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.

Antibodies, Monoclonal↗
PubMed1990

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis.

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.

Antibody-Dependent Cell Cytotoxicity↗