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A A Fauser

Publications and source records attributed to A A Fauser.

At least 91 records · Page 5Linked to original sources
PubMed1988

Analysis of human hemopoietic progenitor cells for the expression of glycoprotein IIIa.

Human hemopoietic progenitor cells were examined for the expression of glycoprotein IIIa (GPIIIa). This protein, which forms the beta-subunit of the GPIIb/IIIa receptor for cytoadhesive proteins as well as the beta-subunit of the vitronectin receptor, represents the most sensitive cell surface marker so far identified for the megakaryocytic lineage. Bone marrow cells were fractionated by a discontinuous Percoll gradient to separate cells that form megakaryocytic colonies in culture (1.05 greater than rho less than 1.077 g/ml). Density centrifugation was followed by indirect immunopanning to select for an enriched population of progenitor cells depleted of most of the mature cells of the myeloid, lymphoid, and erythroid lineages. This cell suspension was labeled with antibodies directed against determinants of GPIIIa and analyzed using a fluorescence-activated cell sorter (FACS). Fractions of cells were sorted and analyzed for the ability to form hemopoietic colonies in culture. Our study demonstrated that megakaryocytic progenitor cells (CFU-M) as well as granulocyte-macrophage colony-forming units (CFU-C), erythroid colony-forming units (BFU-E), and mixed lineage colony-forming units (CFU-GEMM) express HLA-DR antigens but lack GPIIIa. Therefore GPIIIa represents a marker that is not present on hemopoietic progenitor cells, but is expressed on the progenies of CFU-M. In view of the importance of GPIIIa as a component of receptors for cytoadhesive proteins, this finding may help to elucidate the adhesive interactions between early hemopoietic cells and bone marrow interstitium.

Bone Marrow↗
PubMed1988

T-cell depletion with ricin A-chain T101 in allogeneic bone marrow transplantation to prevent severe graft-versus-host disease.

Bone marrow cells from 10 marrow transplant donors were treated with an immunotoxin, which couples A-chain of ricin with a monoclonal anti-T-cell antibody T101 to prevent graft-versus-host disease by the elimination of mature T-cells. Marrow cells treated with the anti human T-cell immunotoxin (IT101) were cultured for erythropoietic colonies, granulocytic colonies, and multilineage hematopoietic colonies (CFU-GEMMT) containing myeloid cells and T-cells, and optimal conditions were defined for the elimination of T-cells present in the harvested donor marrow prior to marrow transplantation. Marrow samples purged with IT101 were examined for residual T-cells by fluorescence activated cell sorting, using anti-T-cell antibodies, [3H]-thymidine incorporation after PHA stimulation, and an assay for clonogenic T-cells. The number of T-cell colonies observed in the treated marrows was less than 5% of the number in comparable unpurged donor marrows. Treatment with IT101 did not alter the plating efficiency of hematopoietic colonies compared to untreated donor marrow cells. These data suggest that multilineage progenitors responsible for the reconstitution of the recipient hematopoietic system are not affected by marrow IT101 purging. The clinical data on 10 patients indicate that the depletion of T-cells in the donor marrow with IT101 is effective in decreasing the severity of acute graft-versus-host disease in allogeneic marrow transplantation and warrants continued investigation.

Adult↗
PubMed1988

High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors.

Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.

Drug Resistance, Microbial↗
PubMed1988

Detection of messenger RNAs within single hemopoietic cells by in situ hybridization on small slide areas.

In situ hybridization provides a powerful tool to detect specific mRNA sequences at the cellular level. We have applied a modified in situ hybridization technique using specifically prepared regular glass microscope slides to evaluate mRNA levels in cells of small samples. Cells were derived from in vitro colonies or isolated by fluorescence-activated cell sorting and deposited on the slides. These slides were coated with polysiloxane, sparing small circular areas where adherent cells attach and can be grown directly; after preincubation of the collection areas with fibronectin, the slides can also be used to deposit and to grow nonadherent cells. In situ hybridization was performed with 35S-labeled probes. Acetylation of the slides and the cells prior to hybridization, the addition of vanadyl-ribonucleoside complexes, and a prehybridization step were found to be necessary to optimize signal-to-noise ratios, as shown by evaluation of c-myc-specific mRNA in phytohemagglutinin-stimulated T4-lymphocytes. This technique might be very useful to study mRNA expression in small samples of hemopoietic cells.

Cell Separation↗
PubMed1987

Human megakaryocytic progenitor cells.

Megakaryocytopoiesis represents one of several differentiation pathways that hematopoietic stem cells may enter. Cells representing intermediate stages of differentiation between pluripotent stem cells and maturing megakaryocytes are called megakaryocytic progenitor cells. They are identified in human bone marrow and peripheral blood by their ability to proliferate in culture (colony forming unit-megakaryocyte, CFU-M); at some point they lose the capacity for cell division and acquire the ability for endoreduplication of DNA, a phenomenon that is unique to the megakaryocyte lineage. This review summarizes current understanding of the biology of human megakaryocytic progenitor cells, including characterization of their proliferation potentials, their antigenic determinants, and the mechanisms that govern their proliferation and maturation. Finally the involvement of CFU-M in various disorders of thrombopoiesis is discussed.

Cell Differentiation↗
PubMed1987

[Immune regulation of hematopoiesis].

Effective hematopoiesis is a multistep phenomenon. It consists in the presence of pluripotent hematopoietic stem cells (HSC), their proliferation and self-maintenance, their differentiation into various committed lineages of specific progenitors, their orderly maturation into functional cells that are released into the circulation in an orderly fashion in response to the body's demand. Increasing numbers of hematopoietic factors are being purified to homogeneity and/or cloned. The availability of sufficient quantities of these regulators promises a new area for research into the physiology and pathophysiology of the hematopoietic system. The purpose of this overview is to consider some newly-developed concepts in the field of hematopoiesis, with regard to regulatory control mechanisms and cellular interactions.

Animals↗
PubMed1986

Fluorescence-activated sorting of individual cells onto poly-L-lysine-coated slide areas.

Cells sorted by a fluorescence-activated cell sorter are collected onto small areas of a glass slide. These collection areas have been coated with poly-L-lysine to attach the cells firmly to the glass surface. This simple procedure proved to be suitable to sort single cells and small cell populations with preservation of cytomorphology and viability without modifying the cell sorter. Additional studies on sorted cells may be performed, as shown by peroxidase-anti-peroxidase analysis of cellular antigens and by mRNA in situ hybridization.

Bone Marrow Cells↗
PubMed1986

Reconstitution of hematopoiesis after bone marrow purging with ricin A chain immunotoxin.

Bone marrow cells from healthy individuals were treated with an antihuman T cell immunotoxin (IT101). The treated marrow cells were cultured for multilineage hematopoietic colonies (CFU-GEMMT) containing various myeloid cell lineages and T lymphocytes, erythroid colonies (BFU-E), and granulocytic colonies (CFU-C). Optimal conditions were defined for the elimination of clonogenic human T leukemic cells artificially admixed with bone marrow cells. Marrow purging with IT101 led to the restoration of hematopoietic colony formation which was abolished in the presence of T leukemic cells. Mixed colonies grown from bone marrow treated with IT101 contained cells that reacted with monoclonal anti-T-cell antibodies. This suggests that pluripotent stem cells are not affected by marrow IT101 purging and may be able to regenerate lymphoid as well as myeloid lineages.

Antibodies, Monoclonal↗
PubMed1986

Expression of c-myc in stimulated T lymphocytes of the helper/inducer phenotype producing lymphokine(s) supporting multilineage colony formation.

Phytohemagglutinin (PHA) renders human peripheral T lymphocytes competent to replicate their DNA and divide. The stimulation of peripheral T cells of the T4 phenotype by PHA, which appears to be a transcription-dependent event, leads to the production and release of lymphokines supporting proliferation and differentiation of human pluripotent stem cells (CFU-GEMMT). Supernatants of PHA-stimulated lymphocytes of the suppressor/cytotoxic phenotype (T8) failed to demonstrate reasonable activity to support the growth of CFU-GEMMT. Stimulation of T lymphocytes of the T4 but not of the T8 phenotype leads to an increased intracellular level of c-myc mRNA. This would be consistent with the c-myc gene product functioning as an intracellular mediator of the growth and lymphokine production response to PHA. Although the function of the c-myc gene product is not yet clear, it seems likely that it is involved in the control of cell proliferation. Such a contribution to control of cell proliferation by c-myc would probably be mediated by a family of genes inducing lymphokine production to stimulate proliferation of human pluripotent stem cells.

Gene Expression Regulation↗
PubMed1986

Lymphokine(s) from isolated T lymphocyte subpopulations support multilineage hematopoietic colony and megakaryocytic colony formation.

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.

Animals↗
PubMed1985

Identification of B cells in multilineage hematopoietic colonies derived from cells of patients with lymphocytic lymphoma.

Pluripotent stem cells from human bone marrow can be identified in culture by their ability to form multilineage hematopoietic colonies containing different myeloid lineages and T cells of different phenotypes. The observation of a common progenitor of myeloid and lymphoid cells in normal and disturbed hematopoiesis prompted the question of whether B cells are part of the differentiation program of stem cells. The availability of hybridomas of azaguanine-resistant T-cell lines secreting monoclonal growth factors for B cells and clinical conditions that are considered to originate from malignant B cells might facilitate this investigation. We were able to identify surface immunoglobulin and B-cell-associated antigen-positive cells within such colonies, indicating that B cells are generated from a myelolymphopoietic stem cell. This report describes the presence of B cells in these colonies derived from bone marrow cells of patients with non-Hodgkin lymphoma.

B-Lymphocytes↗
PubMed1985

T cells and probably B cells arise from the malignant clone in chronic myelogenous leukemia.

Bone marrow cells from a patient with Ph' positive chronic myelogenous leukemia in chronic phase were cultured for multilineage hematopoietic colonies (CFU-GEMMT), erythroid bursts, and granulocytic colonies. With CFU-GEMMT colonies, T lymphocytes were identified by reaction with monoclonal antibodies Leu-5 and OKT-3; B cells were identified by reaction with B1. All CFU-GEMMT colonies examined contained the Ph' chromosome. Recloned secondary colonies of T cells reacted with Leu-5 and OKT-3 and were Ph' positive. This demonstrates that Ph' positive T lymphocytes were generated from the pluripotential stem cell of this patient. The presence of B cells in the mixed colonies indicates that these may also be derived from the neoplastic clone.

Adult↗
PubMed1985

Optimal elimination of leukemic T cells from human bone marrow with T101-ricin A-chain immunotoxin.

In view of bone marrow purging before autologous transplantation in T cell malignancies, an anti-human T cell immunotoxin (IT) has been prepared by coupling ricin A-chain to the monoclonal antibody T101 that binds the T1 differentiation antigen expressed by T lymphocytes as well as by T cell-derived hematologic malignancies. Using a sensitive and reliable clonogenic assay, optimal conditions were defined for the elimination of clonogenic human T leukemic cells among bone marrow cells. Maximal cytoreduction was obtained with IT at a dose of 2 micrograms/mL in the presence of 10 mmol/L NH4Cl. This treatment led to the reduction of more than six orders of magnitude of T101-positive clonogenic leukemic cells, with no harm to T101-negative cells. Moreover, we observed no toxicity of IT to human hematopoietic stem cells (CFU-GEMMT) derived from bone marrow of healthy volunteers. Thus, pretreatment of bone marrow samples with IT plus NH4Cl offers a safe, simple, reliable, and highly efficient means to eliminate undesirable leukemic T cells from the graft.

Ammonium Chloride↗
PubMed1985

Interaction of 3-deaza-adenosine, a phospholipid methyltransferase inhibitor, on the production of pluripoietins from human peripheral T cells.

The formation of mixed colonies is dependent upon the addition of leucocyte-conditioned medium prepared with the mitogenic lectin phytohemagglutinin (PHA). The activation of peripheral T cells by PHA revealed an increase in methylated phospholipids and subsequently led to the release of stimulatory activities into the media. Stimulatory activities supporting mixed colony formation could not be detected when peripheral T cells were preincubated with the methyltransferase inhibitor 3-deaza-adenosine for 60 min before PHA stimulation. The addition of 3-deaza-adenosine to the culture at day 0 demonstrated no inhibitory effect on multilineage colony formation. The data suggest that the activation of human peripheral T cells from healthy volunteers by the mitogenic lectin PHA causes an increase in methylated phospholipids and might be an important signal for the mitogenesis of T-lymphocytes, which subsequently lead to the production of stimulatory activities promoting the growth of pluripotent stem cells.

Humans↗
PubMed1984

Tumor cell colonies in bone marrow cultures from patients with small cell carcinoma of the lung.

Bone marrow specimens from 27 patients with small cell carcinoma of the lung (17 with limited and 10 with extensive disease) were plated in a culture system that supports the growth of multilineage haemopoietic progenitors CFU-GEMM. In five patients (three with extensive and two limited disease) atypical colonies could be observed that were not identifiable as haemopoietic colonies. Cytological staining and cytochemical examination as well as electronic micrographs suggest that these colonies are derived from metastatic carcinoma cells. The histological examination of marrow cells from three out of these five patients revealed no bone marrow involvement. Additional studies might provide further evidence whether bone marrow cultures are a useful probe in order to monitor bone marrow involvement in patients with small cell carcinoma of the lung.

Carcinoma, Small Cell↗