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C Eaves

Publications and source records attributed to C Eaves.

31 records · Page 2Linked to original sources
PubMed1996

Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo.

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

Animals↗
PubMed1996

Comparison of natural killer activity of human bone marrow and blood cells in cultures containing IL-2, IL-7 and IL-12.

Previous studies have suggested that autologous bone marrow or mobilized peripheral blood progenitor cell transplants activated by prior culture of the cells in IL-2 may capture some of the beneficial graft-versus-leukemia effects obtained with unmanipulated allogeneic, but not autologous, transplants. To investigate ways of improving this approach,we have compared the ability of two other immunomodulating cytokines, IL-7 and IL-12, either alone or in combination with IL-2, to stimulate human bone marrow cells (BMC) or peripheral blood cells (PBC) to acquire the potential to lyse K562 or Daudi cells. For these studies, we measured the cytotoxic activity of BMC or PBC both before and at the end of their incubation with various cytokine(s) using a standard 51-chromium release assay. Results suggest that IL-2 at optimal concentration induces cytotoxicity significantly higher than IL-7 or IL-12 when tested alone. At optimal concentration, the combination of IL-2 and IL-12 showed a synergistic effect for BMC. Such a synergistic effect could be observed for PBC only when suboptimal concentrations of IL-2 were used. In addition, the ability of the hematopoietic cells to reduce the number of K562 cells remaining at the end of various culture periods in the presence of the cytokines was measured. This was made possible by the use of a G418-resistant K562 cell line which could, in contrast to normal human BMC or PBC, form colonies that wer detectable after 1 week in methylcellulose cultures containing the neomycin analog G418. Normal human PBC, stimulated by either IL-7 or IL-12 alone effectively suppressed K562 proliferation in both of these assays, whereas no activity could be detected when BMC were incubated under the same conditions. On the other hand, cells from both sources displayed anti-leukemic activity when incubated with IL-2 and IL-12 together, although IL-2/IL-12-activated PBC suppressed the growth of co-cultivated K562-neor cells about eight-fold more efficiently than IL-2/IL-12-activated BMC. Cryopreservation and subsequent stimulation of BMC and PBC with cytokines did not cause a significant decrease in cytotoxicity or their ability to inhibit the growth of co-cultivated K562 cells compared to fresh cells. However, the synergistic effect observed with the combination of IL-2/IL-12 was no longer detectable for BMC. These results suggest that (1) PBC are superior to BMC with respect to developing effective natural killer (NK) activity after culture in cytokines and that, (2) the combination of IL-2 and IL-12 may be more effective than IL-2 alone to inhibit proliferation/growth of K562 cells.

Blood Cells↗
PubMed1995

Quantitation of the quiescent fraction of long-term culture-initiating cells in normal human blood and marrow and the kinetics of their growth factor-stimulated entry into S-phase in vitro.

A method for quantitating the proportion of cycling long-term culture-initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions, as long as 3H-Tdr is not present. In addition, we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally, any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However, within 72 hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF, most had entered S-phase, although there was no net change in their numbers. This suggests that, under certain conditions in vitro, self-renewal divisions of LTC-IC can occur and, at least initially, balance any losses of these cells due to their differentiation or death. In contrast, many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating, although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications.

Blood Cells↗
PubMed1994

Differential manipulation of normal and chronic myeloid leukemia stem cell proliferation in vitro.

The long-term marrow culture (LTC) system allows the sustained production of primitive normal and neoplastic (chronic myeloid leukemia [CML]) hematopoietic cells in vitro for many weeks. This is achieved in the absence of exogenously added hematopoietic growth factors because of the presence in the cultures of supportive "stromal" cells of the fibroblast-endothelial-adipocyte lineages. These latter cells form a confluent adherent layer with which the most primitive hematopoietic cells become associated and which locally regulates their behavior. The LTC system has thus been considered as a model of the microenvironment of the bone marrow and used to delineate potentially physiologically relevant mechanisms that regulate the proliferation, self-renewal and differentiation of primitive normal hematopoietic cells. It has also been used to analyze the molecular basis of the altered proliferative behavior that characterizes primitive neoplastic cells from patients with CML. Most of the information obtained to date has emerged from experiments designed to shift the balance of stimulatory and inhibitory factors present in order to favor either the cycling or quiescence of primitive normal or CML cells in LTC. This has been achieved either by addition of soluble factors (or antagonists) to the LTC medium or by the use of genetically engineered factor-producing stromal cells. Such experiments have allowed the identification of a number of cytokines that promote one or the other of these responses (i.e., primitive progenitor cycling or quiescence), including some that are involved in control mechanisms endogenous to the LTC system. Recent studies suggest that the retention of primitive normal cells in a reversible G(o) state in this system is mediated by the cooperating action of limiting concentrations of at least two endogenously produced inhibitory factors (transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha)), either of which, however, if added exogenously at a sufficient concentration, can exert this action on its own. Interestingly, the heightened turnover characteristic of primitive CML cells appears to be due to a selective unresponsiveness to only one of these two inhibitors (MIP-1 alpha). These findings are consistent with a complex model of the extrinsic regulation of primitive hematopoietic cells in which a multiplicity of intracellular signaling intermediates within the target cells converge at different points ultimately to control their entry into S phase. Our findings further suggest that only some of these pathways may be affected by intracellular expression of the BCR-ABL fusion gene.

Cell Differentiation↗
PubMed1993

The biology of normal and neoplastic stem cells in CML.

Chronic myeloid leukemia (CML) has long served as a prototype malignancy for basic as well as clinical studies aimed at developing curative cancer treatment protocols. Well established features of chronic phase CML are its origin in a pluripotent stem cell, a now well defined molecular genetic basis involving the creation of a BCR-ABL fusion gene and evidence of resultant abnormalities in the mechanisms that normally control primitive hemopoietic cell proliferation. We have recently shown how the long-term marrow culture system can be adapted to quantitate and characterize a very primitive cell type in normal blood and marrow samples, as well as their normal and leukemic counterparts in patients with CML. This system has also been used to dissect mechanisms of normal progenitor regulation and to identify specific anomalies affecting leukemic (CML) progenitors. Our studies show that cells detected by their ability to initiate long-term cultures (LTC) of leukemic cells (i.e., CML LTC-initiating cells or LTC-IC) are differently distributed between marrow and blood by comparison to LTC-IC in normal individuals and, although functionally similar in terms of the number and differentiation types of clonogenic cells they produce, CML LTC-IC exhibit defective self-maintenance. Phenotypically these primitive leukemic cells are heterogeneous; the majority display features of activated/proliferating cells but a significant proportion do not. We have also documented heterogeneity in primitive CML cell responses to two factors that specifically and reversibly arrest the cycling of primitive normal hemopoietic cells; i.e., TGF-beta and MIP-1 alpha, to which CML cells are normally responsive and abnormally unresponsive, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Cells↗
PubMed1988

Human granulocyte-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhemopoietic origin.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of a family of glycoprotein hormones that stimulate the proliferation and differentiation of hemopoietic cells in vitro and in vivo. We now report that human GM-CSF can also stimulate the proliferation of two osteogenic sarcoma cell lines, a breast carcinoma cell line, a simian virus 40-transformed marrow stromal cell line, and normal marrow fibroblast precursors. These findings suggest a more general regulatory function of GM-CSF on nonhemopoietic cell types than previously anticipated. They also raise the possibility of adverse side effects of GM-CSF therapy in patients whose malignant cells may be directly stimulated by this molecule and suggest a previously unanticipated role of GM-CSF gene activation in the evolution of solid tumors and in the pathogenesis of myelofibrosis.

Animals↗
PubMed1985

Long-term marrow culture of cells from patients with acute myelogenous leukemia. Selection in favor of normal phenotypes in some but not all cases.

Long-term cultures were initiated with leukemic marrow aspirate cells from each of 13 newly diagnosed acute myelogenous leukemia (AML) patients. Initial assessment of the clonogenic potential of the marrow suggested that normal hemopoietic progenitors were reduced in most cases and progenitors of abnormal colonies and clusters were present in 10 cases. Subsequent assays of both nonadherent and adherent fractions of long-term cultures revealed two patterns of progenitor cell behavior. The most common pattern (nine cases) featured the detection after 1-4 wk of near normal numbers of typical erythroid, granulopoietic, and mixed colony-forming progenitor cells. Progenitors of abnormal (blast) colonies and clusters initially demonstrable in eight of these nine cases were, in these cases, not sustained in long-term culture and could not be found after 4 wk. Conversion to cytogenetic normalcy in long-term culture was confirmed in two experiments in this group. The second pattern (four cases) was characterized by the failure of progenitors capable of normal differentiation to become detectable in long-term cultures, and the concomitant maintenance of blast progenitors in the two cases in this group where such cells were initially demonstrable. Although progenitors capable of producing abnormal (blast) colonies or clusters in methylcellulose were not detected in either of the other two experiments, the maintenance for 6 wk of a hypercellular nonadherent blast population in one of these suggested the persisting activity of an "adherent layer-dependent" leukemic progenitor cell. Taken together, these findings indicate a strong correlation between the presence of leukemic blasts and their progenitors and a decreased level of normal hemopoiesis. In addition, the failure of leukemic cells to be maintained in long-term marrow cultures from some (but not all) AML patients suggests new applications of this methodology for studies of early stages of leukemic cell development.

Adult↗
PubMed1983

Individual BFU-E in polycythemia vera produce both erythropoietin dependent and independent progeny.

Erythropoietic progenitors from peripheral blood of normal individuals or patients with polycythemia vera (PV) were cultured in methylcellulose medium containing 2.5 U/ml of erythropoietin (Ep). After 7-9 days, colonies considered to be early stage large bursts were individually removed, resuspended in a small volume of fresh methylcellulose medium, and then divided between 2 dishes. To one of these secondary cultures, sufficient Ep was added to bring the concentration of Ep up to approximately 3 U/ml. To the other was added an equal volume of medium but no Ep. The final concentration of Ep in these cultures was determined to be less than 0.01 U/ml. Nine days later, both types of secondary cultures were scored for the presence of colonies containing 8 or more hemoglobinized erythroblasts. Of 90 primary colonies from 3 normal individuals assessed in this way, 59 gave secondary erythroid colonies in the high Ep cultures, while none gave secondary erythroid colonies in the low Ep cultures. Additional control experiments in which primary colonies from normal individuals were divided into duplicate high Ep cultures showed that on average, the procedure used divided primary colonies equally. Of 109 primary colonies from 5 PV patients that yielded secondary erythroid colonies in the high Ep cultures, 21 yielded no secondary erythroid colonies in the low Ep cultures. The other 88 yielded erythroid colonies in both, but the secondary colonies in the low Ep cultures were consistently smaller in size and significantly fewer in number. Similar results were obtained when primary colonies were generated in cultures to which no Ep was added. These findings indicate that primitive BFU-E in patients with PV can be subdivided into 2 populations: a minor population restricted to the production of erythroid colony-forming cells (Ep-dependent progenitors) that require Ep for their detection, and a major population that is not restricted in this way. In addition, these experiments show that most of the primitive BFU-E that generate Ep-independent progenitors also produce significant numbers of cells that are Ep-dependent.

Adult↗
PubMedDate not supplied

New insights in biology and current therapeutic options for patients with chronic myelogenous leukemia.

BACKGROUND AND OBJECTIVE: From the discovery of the Ph-chromosome, there has been an extraordinary progress in our understanding of chronic myeloid leukemia (CML). During the last three decades, new findings arising from dissection of the genetic abnormalities at a molecular level have received the most attention, but there have also been important new observations arising from studies of the biologic behaviour of normal and leukemic stem cells and, more recently, from clinical investigations. In this review we first report the most important observations relevant to understanding the oncogenic potential of the BCR-ABL chimeric gene, and the behaviour and the relationships of normal and leukemic stem cells. From a clinical point of view, allogeneic stem cell transplantation is the only procedure able to cure CML. The main issues are: who can receive this procedure, and when and how it can be given. The situation is more complex in unrelated transplants. In patients without HLA compatible donors, many large trials in different countries have demonstrated that interferon alpha therapy is indicated and effective in the majority of patients. On the other hand, autologous stem cell transplantation is still an experimental procedure. These aspects will be analyzed in detail and, at the end, a therapeutic algorithm of a possible approach to the patients with untreated CML is provided. EVIDENCE AND INFORMATION SOURCES: The method used for preparing this review was an informal consensus development. All the authors of the present review have been working in the field of chronic myeloid leukemia, and have contributed original papers in peer-reviewed journals. In addition, the material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline. STATE OF ART AND PERSPECTIVES: The oncogenic potential of BCR-ABL has been demonstrated in a number of in vitro and in vivo model systems. Current research efforts are focused on defining the mechanism by which BCR-ABL transforms primary hematopoietic cells. The fact that BCR-ABL contains tyrosine residues, an SH2 domain, an SH3 domain, and proline-rich sequences raises the possibility of multiple protein-protein interactions. Indeed, BCR-ABL is reported to bind and/or phosphorylate more than 20 proteins. The insights into the signal transduction pathways activated by BCR-ABL will hopefully provide a new basis for the treatment of CML patients. Clinical evidence of the existence of a transplantable CML stem cell population has recently been extended to xenogeneic recipients of transplanted CML cells and by retroviral marking to autograft recipients. The potential of using immunodeficient mice as recipients of CML stem cells to create an in vivo model of chronic phase CML should be invaluable for testing novel therapies designed to eliminate residual disease in the patient. Current therapeutic options include conventional chemotherapy, IFN-a and allogeneic stem cell transplantation as established procedures, and autografting as an experimental procedure. While IFN-a as a first line therapy does not seem to jeopardize further treatments, autografting, according to the Genoa approach or other procedures, i.e. Ph-positive cells collected at diagnosis without mobilization therapy, raises the question of an ideal sequential strategy in the management of CML patients. There seems to be a general agreement that a patient less than 50 years old, with an HLA identical sibling, should receive an allogeneic stem cell transplant. This approach should be offered also to younger patients (< or = 40 years) who are able to find an unrelated matched donor. Since it seems that the normal hematopoietic reservoir declines with time, it may be desiderable to mobilize and collect peripheral stem cells in order to store Ph-negative progenitors as soon after diagnosis as possible when the WBC count has been controlled by hydroxyurea while searchin

Animals↗