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Biomedical subjects

T M Dexter

Publications and source records attributed to T M Dexter.

At least 127 records · Page 7Linked to original sources
PubMed1991

The biology of long-term bone marrow cultures and its application to bone marrow transplantation.

Long-term bone marrow cultures have been applied to the study and to the treatment of hemopoietic disease. The incidence of murine primitive (stem) cells with the ability to repopulate irradiated bone morrow stroma is similar to that of cells able to repopulate the hemopoietic system of potentially lethally irradiated mice. In human bone marrow, cells with in vitro repopulating ability have been estimated as 0.5% to 1% of the CD34+ cells. The increase in primitive cells in the circulation seen after administration of growth factors has been exploited by harvesting those cells for use together with bone marrow in autologous transplants. The ability of primitive cells (harvested after granulocyte colony-stimulating factor plus chemotherapy) to reconstitute irradiated marrow stromas indicates that their repopulation capacity in vitro is at least as good as that of bone marrow. This is confirmed by experimental data in mice showing that small volumes of peripheral blood (after granulocyte colony-stimulating factor administration) are capable of long-term repopulation. The regulator capacity of marrow stroma has been exploited to purge bone marrow cultures from patients with acute myeloblastic leukemia in first remission and from treated patients with chronic myelogenous leukemia before using the cultured cells for autologous transplant. The results to date, although preliminary, indicate a role for this methodology in the treatment of leukemia.

Animals↗
PubMed1991

In vitro assessment of marrow 'stem cell' and stromal cell function in aplastic anaemia.

An in vitro model system is described that allows separate assessment of 'stem cell' and stromal cell function in aplastic anaemia (AA). Seven patients with non-severe AA, who had responded to immunosuppressive therapy and had haematological evidence of residual marrow function, were studied. Of these, three with otherwise typical AA had an acquired clonal cytogenetic marker. Purified bone marrow haemopoietic progenitors labelled with CD34 monoclonal antibody were positively selected using the fluorescence activated cell sorter (FACS) from both normal subjects and from patients with AA. The generative capacity of the CD34 positive cells was assessed by monitoring the output of granulocyte/macrophage colony forming cells (CFU-GM) in the non-adherent layer after inoculation onto irradiated performed long-term marrow culture (LTBMC) stromas. Stromal function in AA was assessed by inoculating CD34 positive cells from normal bone marrow onto performed irradiated stromas from patients with AA. Haemopoietic cell ('stem cell') function in AA was assessed by inoculating CD34 positive cells from AA patients onto confluent irradiated normal marrow stromas. Using these crossover/LTBMC experiments, all patients exhibited severe defects in haemopoietic cell function with normal functioning stroma. The proportion of CD34 positive cells present in bone marrow from these patients was reduced compared with controls, they comprised fewer small primitive 'blast-like' cells which in normal bone marrow are known to possess marrow repopulating ability, and demonstrated reduced clonogenic potential in short-term colony assays.

Adult↗
PubMed1991

Development of multipotential haemopoietic stem cells to neutrophils is associated with increased expression of receptors for granulocyte macrophage colony-stimulating factor: altered biological responses to GM-CSF during development.

Interleukin-3 (IL-3) dependent multipotent haemopoietic stem cells FDCP-Mix A4 (A4) were induced to differentiate and develop into mature neutrophils in response to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) plus granulocyte CSF (G-CSF). This resulted in an increase in cell number over seven days of culture, following which the cells lost the ability to undergo further proliferation. The effect of GM-CSF on these cells has been assessed at various stages of development. Clonogenic cells, able to respond to GM-CSF, were generated only at days 3, 4 post-induction. From day 5 onwards, mature post-mitotic neutrophils are produced and clonogenic cells are lost. Loss of proliferative potential, in response to GM-CSF, was confirmed using [3H]-thymidine incorporation. Receptors for GM-CSF, were also measured during development using [125I]-GM-CSF binding assays. Although the dissociation constant for GM-CSF binding sites did not vary considerably, the number of such sites increased dramatically from about 20 (day 0, when the cells have a primitive morphology) to about 1000 by day 6 (when the cells are predominantly mature neutrophils). GM-CSF-stimulated Na+/H+ antiport activation was also determined. Although few GM-CSF receptors are expressed at day 0, there is a significant response (63% of maximal) to GM-CSF in terms of intracellular alkalinisation: this response increased markedly until, by day 4 (700 GM-CSF binding sites/cell), there is a maximal activation of the antiport by GM-CSF. By day 7 (greater than 900 GM-CSF binding sites/cell), however, there is significant reduction in activation of the Na+/H+ antiport by GM-CSF. Nonetheless, increased viability of these mature cells is still seen in response to GM-CSF. These results suggest that not only does expression of GM-CSF receptors alter during development of multipotential cells to mature neutrophils, but that these receptors are coupled to different intracellular effector mechanisms as the cells progressively mature.

Cell Differentiation↗
PubMed1991

Serum-free culture of enriched murine haemopoietic stem cells. I: Effect of haemopoietic growth factors on proliferation.

Using a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (termed the FACS-BM population), and a serum-free culture system, the requirements for development of multipotential cell have been investigated and compared to previous results using serum containing cultures. In both serum-free and serum supplemented cultures interleukin-3 (IL-3) was a potent colony stimulating factor, although it was more effective in serum free conditions. However, colony stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF) was markedly reduced in the absence of serum. Significantly, the ability of interleukin-1 (IL-1) and granulocyte-colony stimulating factor (G-CSF) to synergise with these two growth factors was retained in serum-free conditions, indicating that these growth factors act directly on the FACS-BM without serum co-factors. Furthermore synergistic interactions between IL-3 plus IL-1, and IL-3 plus M-CSF were only manifest in serum-free conditions. The significance of these results in relation to the ability of these growth factors to act directly on multipotential cells is discussed.

Animals↗
PubMed1991

Serum-free culture of enriched murine haemopoietic stem cells. II: Effects of growth factors and haemin on development.

A serum-free culture system was used to determine the effects of growth factors on the clonogenic development of a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (FACS-BM). Under these conditions, interleukin-3 (IL-3) was found to be primarily a proliferative stimulus, the progenitor cells developing in the clonal assay systems produced colonies of morphologically undifferentiated cells for up to 20 days. No such induction of proliferation without maturation was observed with other growth factors (eg. granulocyte-macrophage colony stimulating factor (GM-CSF)). However, combinations of IL-3 plus secondary growth factors such as GM-CSF, macrophage colony stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF) or interleukin-1 (IL-1) led to the formation of colonies containing mature haemopoietic cells of the granulocytic, megakaryocytic or monocytic lineages. In contrast, erythroid development did not occur unless the protoporphyrin, haemin, was added to the cultures. Under these conditions mature erythroid cells were produced in cultures containing either IL-3 or GM-CSF (with or without erythropoietin (epo)). In replating experiments it was determined that the FACS-BM cells were able to generate large numbers of clonogenic cells for up to 30-40 free cultures. Such cultures, therefore, may be useful for investigating the biological and basis of the generation of clonogenic cells and of haemopoietic cell differentiation and development in response to growth factors.

Animals↗
PubMed1991

Growth inhibitory effects of transforming growth factor-beta 1 in vivo.

Transforming growth factor-beta (TGF-beta) can reversibly inhibit the in vitro proliferation of murine and human haemopoietic progenitors and some of their more developmentally restricted progeny. Using an assay for measuring day 8 and day 11 CFU-S, TGF-beta caused a gradual decline in the number of CFU-S undergoing DNA synthesis so that after 5 days of daily treatment only quiescent cells were found. Release of this growth inhibition was seen within 24 hours post-treatment with recovery of all progenitors to normal levels. Similar inhibitory effects of TGF-beta were seen on the cells of the intestinal epithelium, indicating that TGF-beta is a general stem cell growth inhibitor. These results suggest that TGF-beta can be used as a cytostatic agent to protect normal stem cells in patients being treated with cell cycle-specific cytotoxic agents.

Animals↗
PubMed1991

Selective inhibition of primitive hemopoietic colony-forming cells by an extract from normal marrow: comparison with transforming growth factor-beta.

An extract from normal bone marrow (NBME) which inhibits proliferation of spleen colony-forming units CFU-S selectively inhibits interleukin 3 (IL-3)-driven colony formation by primitive hemopoietic progenitors. This activity is distinct from transforming growth factor-beta (TGF beta), which also inhibits development of primitive progenitors. There is evidence that the two activities inhibit proliferation of target cells by different mechanisms and that the bone marrow extract has a direct effect on cell cycling, whereas the effect of TGF beta to suppress proliferation is probably indirect.

Animals↗
PubMed1990

Role of endocytosis in the action of ether lipids on WEHI-3B, HL60, and FDCP-mix A4 cells.

We investigated the effect of a number of platelet activating factor antagonists on cell killing by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphoryl choline (ET-18-OCH3). Of six platelet activating factor antagonists tested, four were found to protect WEHI-3B leukemic cells against cell death induced by ET-18-OCH3. Certain other compounds, not platelet activating factor antagonists, had similar protective effects. The protective compounds were all lipophilic weak bases. We describe experiments that indicate that these compounds protect by inhibition of endocytic uptake of ET-18-OCH3. Sensitive cells showed rapid endocytic uptake, whereas in resistant cells, uptake was slow. Uptake of ET-18-OCH3 could be suppressed by inhibitors of endocytosis such as chloroquine, monensin, and vinblastine. We conclude that one of the principal determinants of sensitivity or resistance to ether lipids may be the rate at which cells take them up by endocytosis.

Animals↗
PubMed1990

Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor.

The major effect of granulocyte colony-stimulating factor (G-CSF) is to induce neutrophilia in previously untreated animals or after chemotherapy or marrow transplantation in humans, primates and rodents. In addition, it has been reported that migration of committed progenitor cells to the blood occurs during G-CSF therapy. In this article, by using sex mismatched transplants and a molecular probe for Y-chromosome specific DNA sequences, we show that among the peripheral blood cells during G-CSF therapy are substantial numbers of primitive stem cells capable of (1) reconstituting the hematopoietic system in the long term, and (2) making a contribution to the lymphoid populations of the thymus, in radiation ablated recipients. These data suggest that blood from patients treated with G-CSF may provide a convenient source of the most primitive stem cells for autologous or allogeneic bone marrow transplantation.

Animals↗
PubMed1990

The hematopoietic defect in aplastic anemia assessed by long-term marrow culture.

Thirty-two patients with aplastic anemia (AA) have been studied using the long-term bone marrow culture (LTBMC) system. Of these patients, 26 had been treated with immunosuppressive therapy including antilymphocyte globulin (ALG) with or without androgens or high-dose methyl prednisolone. The remaining six patients either required no treatment or were studied before therapy was begun. Thirty-one of 32 patients (96%) had defective hematopoiesis in LTBMC with little or no evidence for the generation of primitive progenitor cells. The only exception was a patient with spontaneous recovery of aplasia in whom the defect was less marked. Crossover LTBMC experiments were performed in 23 cases by inoculating (1) patient marrow hematopoietic cells that had been depleted of adherent cells onto preformed, irradiated, normal stromas to assess the proliferative capacity of the hematopoietic cells, and (2) normal marrow hematopoietic cells that were depleted of adherent cells onto preformed, irradiated stromas from patients with AA to assess stromal function. Results of these experiments demonstrated a hematopoietic defect in all patients that was independent of the degree of hematologic recovery after ALG therapy. Only one patient had a probable stromal defect and this coexisted with a defect in the regenerative capacity of hematopoietic cells. We conclude that LTBMC is a sensitive method for detecting and defining the hematopoietic failure in AA. We suggest that the defective hematopoiesis present in all patients studied may be important in the pathogenesis of clonal evolution in AA.

Adolescent↗
PubMed1990

Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture.

We have studied the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), hG macrophage-CSF (hGM-CSF), and gibbon interleukin-3 (gIL-3) on cell proliferation and differentiation in human long-term bone marrow culture (LTBMC). hG-CSF induced a maximal increase of 2.3-fold in both total nonadherent cells and GM cluster-forming cells, but only an increase of 1.7-fold in GM-colony-forming cell (GM-CFC) numbers, influencing mainly neutrophil differentiation. Cultures treated with hGM-CSF demonstrated a peak of 12.8-, 21- and 3.2-fold elevations in total nonadherent cells, cluster, and GM-CFC, respectively, and influenced differentiation of neutrophils, monocytes, eosinophils, and lymphocytes. Cultures treated with gIL-3 demonstrated the largest expansion in the GM-CFC population, reaching a maximum of 5.3-fold in relation to that of unstimulated controls. IL-3 treatment also increased the numbers of GM clusters and mature cells (including all myeloid cells and lymphocytes) 7.8- and 4.8-fold, respectively. Similar quantitative and qualitative changes were induced by G-CSF, GM-CSF, and IL-3 in LTBMCs of patients in remission after treatment for acute lymphoblastic leukemia or Hodgkin's lymphoma. Overall, the expansion of GM progenitor cells in cultures treated with growth factors was larger in the adherent cell layer than in the nonadherent cell fraction. In addition, hGM-CSF, gIL-3, and hG-CSF to a less extent, increased the cycling rates of GM-CFC progenitors located in the adherent layer. These results indicate that hG-CSF is a much less potent stimulus of hematopoiesis in LTBMC than the other CSFs assayed, and that the increases in cell production after treatment with G-CSF, GM-CSF, or IL-3 may be achieved by primary expansion of different cell populations within the hierarchy of the hematopoietic system. The effects of the growth factors were transient and the longevity of hematopoiesis in the cultures was not altered, suggesting that treatment with IL-3, GM-CSF, or G-CSF had not compromised the ability of primitive cells to give rise to mature cells. This indicates that the stromal microenvironment in LTBMC can override potential differentiation-inducing activities of the CSFs.

Animals↗
PubMed1990

The role of growth factors in self-renewal and differentiation of haemopoietic stem cells.

Haemopoietic stem cells in vivo proliferate and develop in association with stromal cells of the bone marrow. Proliferation and differentiation of haemopoietic stem cells also occurs in vitro, either in association with stromal cells or in response to soluble growth factors. Many of the growth factors that promote growth and development of haemopoietic cells in vitro have now been molecularly cloned and purified to homogeneity and various techniques have been described that allow enrichment (to near homogeneity) of multipotential stem cells. This in turn, has facilitated studies at the mechanistic level regarding the role of such growth factors in self-renewal and differentiation of stem cells and their relevance in stromal-cell mediated haemopoiesis. Our studies have shown that at least some multipotential cells express receptors for most, if not all, of the haemopoietic cell growth factors already characterized and that to elicit a response, several growth factors often need to be present at the same time. Furthermore, lineage development reflects the stimuli to which the cells are exposed, that is, some stimuli promote differentiation and development of multipotential cells into multiple cell lineages, whereas others promote development of multipotential cell into only one cell lineage. We suggest that, in the bone marrow environment, the stromal cells produce or sequester different types of growth factors, leading to the formation of microenvironments that direct cells along certain lineages. Furthermore, a model system has been used to show the possibility that the self-renewal probability of multipotential cells can also be modulated by the range and concentrations of growth factors present in the environment. This suggests that discrete microenvironments, preferentially promoting self-renewal rather than differentiation of multipotential cells, may also be provided by marrow stromal cells and sequestered growth factors.

Animals↗
PubMed1990

A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induces leukocytosis in vivo in both intact and splenectomized mice. Full dose response data showed a plateau in this effect at doses over 500 micrograms rhG-CSF/kg body weight/d in intact mice. The effect is magnified in splenectomized mice, where leukocyte numbers reach 100 x 10(6) mL after 4 days' treatment at 250 micrograms/kg/d. Further hematopoietic precursor populations are also affected in both marrow and the spleen; in general, marrow parameters were depressed, while splenic populations were enlarged. In splenectomized mice, both blood-borne stem cells were enhanced, and foci of extramedullary hematopoiesis were enlarged in addition to the effects seen in intact mice. In the marrow of splenectomized and intact mice treated with a high dose of G-CSF, erythroid suppression in the marrow was confirmed with radioactive iron. Our studies confirm and extend previous work on the mode of action of G-CSF, and indicate that side effects of high dose G-CSF therapy might include erythroid suppression in the bone marrow.

Animals↗
PubMed1990

Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis.

The survival, differentiation, proliferation and development of haemopoietic precursor cells and the functional activity of mature blood cells are all influenced by colony stimulating factors (CSFs). As haemopoietic cells rapidly die in the absence of appropriate CSF, the promotion of cell survival mediated by CSFs, or growth factors, is fundamental to all the other effects exerted by these factors. This enhancement of cell survival is distinct from the stimulation of proliferation. Here we show that the death of haemopoietic precursor cells on withdrawal of the relevant CSF. is due to active cell death, or apoptosis, indicating that CSFs promote cell survival by suppression of the process of apoptosis. The existence of a positive control mechanism regulating precursor cell survival has important implications both for the regulation of normal haemopoiesis and for tumorigenesis.

Animals↗
PubMed1990

Stromal cells in haemopoiesis.

Stromal cells of the bone marrow can provide the growth-promoting and differentiation-inducing molecules which are necessary for haemopoiesis. While the nature of these stimuli is largely unknown, the development of haemopoietic cells in association with stromal cells requires intimate cell contact. Molecules of the extracellular matrix, such as heparan sulphate, are able to bind growth factors and in this way the stromal cells may form microenvironmental niches which preferentially promote development of multipotent and committed cells along discrete lineages. Cells from some patients with acute and chronic myeloid and lymphoid leukaemias are defective in their ability to interact with stromal cells and consequently cannot survive in stromal cell-mediated long-term marrow cultures. We have exploited this phenomenon to obtain normal haemopoietic cells from patients with leukaemia, and to use these cells for successful autografting in patients with acute and chronic myeloid leukaemias.

Animals↗
PubMed1990

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias.

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.

Bone Marrow↗