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Biomedical subjects

T M Dexter

Publications and source records attributed to T M Dexter.

At least 19 recordsLinked to original sources
PubMed1992

Expression and downregulation of cytotoxic cell protease 1 or Granzyme 'B' transcripts during myeloid differentiation of interleukin-3-dependent murine stem cell lines.

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

Amino Acid Sequence↗
PubMed1992

Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors.

The effects of the c-kit ligand (stem cell factor [SCF]) on the development of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. In soft agar assays, both in serum-containing and in serum-deprived cultures, SCF promoted the formation of colonies that contained predominantly granulocytic cells with some blast cells also present. The size of these colonies was far smaller than observed in the presence of interleukin-3 (IL-3). In serum-deprived conditions, no colonies were formed in the presence of macrophage colony-stimulating factor (M-CSF), but when M-CSF was combined with SCF, a marked change was noted in that large colonies were produced containing predominantly macrophages. When GM-CFC were cultured in the presence of IL-3 and SCF, colonies were formed that contained blast cells, granulocytes, and macrophages. A synergistic interaction was also seen using a combination of G-CSF plus SCF in either serum-containing or serum-deprived cultures. The addition of SCF to colony-forming assays markedly reduced the concentration of IL-3 or G-CSF required for optimal levels of colony formation. Furthermore, SCF was capable of promoting the survival of GM-CFC for several days, after which large colonies containing mature cells were formed upon the addition of a secondary growth factor such as G-CSF or IL-3. Thus, SCF can directly act on highly enriched committed progenitor cells in serum-deprived conditions to promote survival, proliferation, and development.

Animals↗
PubMed1992

Growth of human umbilical-cord blood in longterm haemopoietic cultures.

Cryopreserved human umbilical-cord (HUC) blood is an alternative to bone marrow as a source of haemopoietic "stem" cells for HLA-identical transplantation of children with leukaemia or Fanconi's anaemia. We have studied the in-vitro growth potential of HUC blood in clonogenic assays and in longterm haemopoietic cultures. Clonogenic assays showed that HUC blood produced as many haemopoietic-cell colonies as normal adult bone marrow and a higher proportion of primitive-cell colonies. In longterm culture on preformed irradiated marrow stroma, both progenitor-cell production and lifespan of cultures were significantly greater in HUC blood than in normal bone marrow (p = 0.0007). Our findings indicate that the quality and quantity of HUC-blood-derived haemopoietic "stem" cells are better than those of normal bone marrow. Therefore, single HUC-blood donations are probably sufficient for adults requiring transplantation for leukaemia and other haemopoietic disorders. Banking of HLA-typed HUC blood to facilitate transplantation of patients who lack a family donor should be considered.

Blood Transfusion↗
PubMed1992

"Stem cell" origin of the hematopoietic defect in dyskeratosis congenita.

We have used the long-term bone marrow culture (LTBMC) system to analyze hematopoiesis in three patients with dyskeratosis congenita (DC), two of whom had aplastic anemia, and the third had a normal blood count (apart from mild macrocytosis) and normal BM cellularity. Hematopoiesis was severely defective in all three patients, as measured by a low incidence of colony-forming cells and a low level of hematopoiesis in LTBMC. The function of the marrow stroma was normal in its ability to support the growth of hematopoietic progenitors from normal marrows seeded onto them in all three cases, but the generation of hematopoietic progenitors from patients marrow cells inoculated onto normal stromas was reduced, thus suggesting the defect to be of stem cell origin. The parents and unaffected brother of one of the families have also been studied in LTBMC and all showed normal hematopoietic and stromal cell function. From this study we speculate that there are some similarities between DC and the defect in the W/Wv mouse.

Adolescent↗
PubMed1992

Macrophage-inflammatory protein protects multipotent hematopoietic cells from the cytotoxic effects of hydroxyurea in vivo.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has been assessed for its potential in vivo to protect hematopoietic progenitor cells from the cytotoxic effects of a cycle-specific drug--in this case hydroxyurea (HU). Two doses of HU, 7 hours apart, were administered to mice to induce spleen colony-forming unit (CFU-S) cycling and then to kill them during DNA-synthesis. MIP-1 alpha, in a variety of dose and time combinations, was injected before the second dose of HU in an attempt to prevent recruitment or maintain CFU-S quiescence, and thus protect them from the second dose of HU. Without MIP-1 alpha, recovery of the CFU-S population was complete in 7 days. In a dose-dependent manner, MIP-1 alpha either reduced the initial kill and accelerated recovery, or completely protected the CFU-S population. We conclude that MIP-1 alpha does protect multipotent progenitor cells in vivo and that these observations provide a base from which to build practical clinical applications.

Animals↗
PubMed1992

The mouse M-lysozyme gene domain: identification of myeloid and differentiation specific DNasel hypersensitive sites and of a 3'-cis acting regulatory element.

The mouse M-lysozyme gene is exclusively expressed in myeloid cells of the blood system being progressively turned on upon cell differentiation. In this study the mechanism controlling this tissue- and differentiation stage-specific gene expression was analyzed at the level of chromatin structure. A complex pattern consisting of constitutive and differentiation dependent DNasel hypersensitive sites (HSs) was found in a set of various myeloid cell lines, representing different stages of maturity. The chromatin of a lymphoid cell line, which does not express the lysozyme gene, is completely insensitive to DNasel digestion. Chromatin analysis of two multipotent hematopoietic stem cell lines which can be differentiated in vitro to mature myeloid cells confirmed that these identified DNasel HSs are specific for distinct differentiation stages, rather than being a characteristic feature of the cell lines. Additionally, the stem cell studies revealed that the hypersensitivity of the chromatin domain is already established at the multipotent stage. DNA fragments spanning a cell type- and differentiation stage-specific cluster of HSs in the 3' region of the gene showed enhancer activity in all cell types tested. In the light of this lack of specificity, we suggest that cell type-specific modification of the chromatin structure in this region may play a role in determining the binding of a widespread transcription factor, and hence contribute to the time specificity of lysozyme M gene expression.

Animals↗
PubMed1992

Haemopoietic cell kinetics in humans treated with rGM-CSF.

We have investigated the kinetics of myeloid cell proliferation in the marrow of patients with small-cell lung cancer and treated with 10 daily subcutaneous injections of granulocyte/macrophage colony-stimulating factor (GM-CSF). Bone marrow, obtained before and during treatment with the growth factor, was labelled in vitro with tritiated thymidine (3H-TdR). A 3rd bone-marrow sample was obtained 1 hr following an intravenous injection of 3H-TdR. Subsequent daily blood samples were also collected, and 3H-TdR labelling was assessed on these and the marrow preparations by autoradiography. GM-CSF treatment increased the peripheral granulocytic cells nearly 5-fold, but this included significant eosinophilia, so that the neutrophilic granulocytes increased only 3.3-fold. These cells were released from the marrow over a normal time scale, but their peripheral half-life was about 6 times longer than normal and they were probably functionally defective. Furthermore, significant numbers of immature cells were released from the marrow. Neutrophil production stimulated by GM-CSF was thus overestimated by measurement of the apparent peripheral granulocytosis. Increased labelling indices and grain counts in the proliferating granulocytic cells of the marrow indicate shortened cell-cycle times, and the excess granulocyte production appears to be the result of extra amplification divisions in the proliferative compartments.

Carcinoma, Small Cell↗
PubMed1992

Erythropoietin and myeloid colony stimulating factors.

The production of blood cells in the body is controlled by at least 20 polypeptide growth factors. Most of these factors have been cloned and many expressed in bacterial and eukaryotic systems to give biologically active proteins. Currently, these recombinant human proteins are undergoing intensive evaluation for their use in treating primary haemopoietic diseases, or stimulating normal haemopoiesis following drug-, radiation- or virus-induced trauma of the bone marrow. Erythropoietin (EPO) and the myeloid colony stimulating factors (IL-3, G-CSF, GM-CSF and M-CSF) were among the first to be cloned and expressed.

Animals↗
PubMed1992

Colony-stimulating factors in the clinic.

Recombinant purified human haemopoietic growth factors are available for clinical trials and some have been licensed for therapeutic use. Some haemopoietic lineages (erythroid, neutrophilic, monocyte-macrophagic) can be selectively stimulated in order to ameliorate the cytopenias that follow cytotoxic treatment, or that characterize some haematological syndromes, and to stimulate mature cell function. Advances in the knowledge of receptor-ligand interactions and of transduction mechanisms, plus the production of synthetic or mutant molecules that may mimic, potentiate or antagonize the effects of the natural growth factors, should make novel therapeutic approaches possible.

Animals↗
PubMed1992

Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).

Animals↗
PubMed1992

Cell adhesion in the stromal regulation of haemopoiesis.

Haemopoiesis occurs in close physical contact with elements of the bone marrow stroma. This review examines some adhesion molecules which are expressed in haemopoietic environments. These molecules may act to maintain cell contact between haemopoietic progenitors and the cellular and extracellular matrix elements of the marrow stroma. A review is undertaken of several families of adhesion molecules and their ligands, including integrins, CD44, LEC-CAMs, CAMs and variant glycosylation patterns expressed by haemopoietic cells. Both CD44 and integrin receptors have been implicated in mediating important cellular interactions within the haemopoietic system and the significance of these and other molecules in haemopoiesis is discussed.

Amino Acid Sequence↗
PubMed1992

The capacity of peripheral blood stem cells mobilised with chemotherapy plus G-CSF to repopulate irradiated marrow stroma in vitro is similar to that of bone marrow.

After treatment of patients with intermediate or high grade non-Hodgkin lymphoma with chemotherapy plus G-CSF the numbers of haemopoietic progenitor cells in the circulation increased to a mean of 226-fold for mixed CFC (Mix-CFC), 278-fold for GM-CFC and 29-fold for erythroid burst forming unit (BFU-E). The mean increase was modest (7-12-fold) for patients treated with chemotherapy alone. Peripheral blood mononuclear cells harvested at the time of the peak in the numbers of progenitors, or 2-4 days before the peak, seeded onto irradiated marrow stroma in vitro, repopulated the stroma and generated active haemopoiesis at least as effectively as bone marrow cells on a cell per cell basis. This is in contrast to the poor repopulating capacity of pretreatment blood. The results indicate that not only the progenitor cells, but also the repopulating stem cells migrated into the blood after chemotherapy plus G-CSF in sufficient numbers to allow harvesting and successful grafting without the possible complication of late haemopoietic failure.

Adult↗
PubMed1992

Recombinant human interleukin 4 (IL-4) given as daily subcutaneous injections--a phase I dose toxicity trial.

Recombinant Interleukin 4 was administered by subcutaneous injection at daily doses of 0.5, 1.0 or 5.0 micrograms kg-1 to nine patients as part of a Phase I Dose Toxicity Study. Dose limiting toxicity was reached at 5 micrograms kg-1 day-1. Symptoms of toxicity included fatigue, 'flu like symptoms and elevated liver enzymes. Modest but significant elevations of neutrophil and platelet counts occurred. No clear evidence of antitumour effects emerged although pain in metastatic lymph nodes and a small fall in myeloma paraprotein levels during dosing were observed. In vitro and murine in vivo studies indicate that patients with lymphoproliferative disease should be selected for Phase II trials.

Bone Marrow↗
PubMed1992

Human cord blood: a source of transplantable stem cells?

In a preliminary study we have shown that optimally collected human umbilical cord (HUC) blood cells grow significantly better in long term cultures (LTC) than normal adult marrow cells (NBM) p = 0.0007. The LTC findings are supported by the observation in clonogenic assay that a similar number of GM-CFC colonies can be grown from HUC blood and NBM mononuclear cells (MNC). Also there is a trend towards a higher proportion of primitive erythroid (BFU-E) and primitive megakaryocyte colonies (MK-CFC containing greater than 20 cells) in HUC blood compared with NBM. We suggest that further work on the feasibility of a HLA typed, cryopreserved HUC blood bank as a source of unrelated haemopoietic 'stem' cells for clinical transplantation is indicated.

Blood Cell Count↗
PubMed1991

Interferon-gamma stimulates the survival and influences the development of bipotential granulocyte-macrophage colony-forming cells.

The effects of interferon-gamma (IFN-gamma) on a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. When added with myeloid growth factors (interleukin-3 [IL-3], granulocyte-macrophage colony-stimulating factor [GM-CSF], or macrophage-CSF [M-CSF]), IFN-gamma inhibited the formation of colonies in soft agar assays. Furthermore IFN-gamma stimulated an increase in the number of macrophages present in colonies formed in the presence of IL-3. IFN-gamma also inhibited M-CSF-, GM-CSF-, or IL-3-stimulated [3H]-thymidine incorporation in highly enriched GM-CFC. However, when added in the absence of hematopoietic growth factors, IFN-gamma promoted the survival of GM-CFC and had a modest stimulatory effect on DNA synthesis. The direct interaction of the IFN with GM-CFC was confirmed by showing its ability to rapidly activate the sodium/hydrogen antiport in GM-CFC, as do the mitogens GM-CSF, M-CSF, and IL-3. However, the effect of IFN-gamma on intracellular pH and DNA synthesis was transient and pretreatment with IFN markedly inhibited the ability of GM-CSF, M-CSF, and IL-3 to activate the sodium/hydrogen antiport. IFN-gamma has a dual effect on GM-CFC, decreasing the rate of cell death but also limiting the proliferative response to CSFs.

Animals↗
PubMed1991

The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor.

Stem cell factor (SCF) is the ligand for the receptor encoded by the c-kit proto-oncogene. Mutations of either c-kit or the SCF gene are responsible for the defects of W and SI mutant mice, which both suffer a macrocytic anemia, the former associated with defective stem cells and the latter with a defective hematopoietic microenvironment. PEGylated recombinant rat SCF was administered to normal or splenectomized mice for up to 21 days. SCF was found to be a modest stimulator of peripheral blood neutrophil numbers in both groups of animals. The peak in neutrophil numbers was higher and occurred earlier in splenectomized mice. Bone marrow and spleen cellularity changed little during treatment but the content of interleukin-3-responsive progenitor cells and spleen colony-forming cells (CFU-S) reached very high levels, particularly in the spleen. Using recombinant human granulocyte colony-stimulating factor (rhG-CSF), we have shown that SCF induces a greater than additive increase in both blood neutrophils and blood-borne CFU-S. This synergy was seen throughout the dose range and may indicate a clinical role for SCF either alone or in augmenting the activity of G-CSF upon blood neutrophils and transplantable stem cells.

Animals↗
PubMed1991

Myeloid cell kinetics in mice treated with recombinant interleukin-3, granulocyte colony-stimulating factor (CSF), or granulocyte-macrophage CSF in vivo.

Myeloid cell kinetics in mice treated with pure hematopoietic growth factors have been investigated using tritiated thymidine labeling and autoradiography. Mice were injected subcutaneously with 125 micrograms/kg granulocyte colony-stimulating factor (G-CSF) (in some cases 5 micrograms/kg), or 10 micrograms/kg of granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3) every 12 hours for 84 hours. 3HTdR labeling was performed in vivo after 3 days of treatment. G-CSF increased the peripheral neutrophil count 14-fold and increased the proportion and proliferation rate of neutrophilic cells in the marrow, suppressing erythropoiesis at the same time. Newly produced mature cells were released into the circulation within 24 hours of labeling, compared with a normal appearance time of about 96 hours. By contrast, GM-CSF and IL-3 had little effect on either marrow cell kinetics or on the rate of release of mature cells, although GM-CSF did stimulate a 50% increase in peripheral neutrophils. Monocyte production was also increased about eightfold by G-CSF and 1.5-fold by GM-CSF, but their peak release was only slightly accelerated. While the peripheral half-lives of the neutrophilic granulocytes were normal, those of the monocytes were dramatically reduced, perhaps due to sequestration in the tissues for functional purposes. The stimulated monocyte production in the case of G-CSF required an additional five cell cycles, a level that might have repercussions on the progenitor compartments.

Animals↗