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C Eaves

Publications and source records attributed to C Eaves.

At least 19 recordsLinked to original sources
PubMed2006

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34(+) CML cells using a strategy that combines a brief exposure to imatinib (0.5-1.0 microM for 72 h) and then mafosfamide (30-90 microg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 microM imatinib, 60 microg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL(+) cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

Antigens, CD↗
PubMed2005

BCR-ABL-transduced human cord blood cells produce abnormal populations in immunodeficient mice.

In this study, we describe the successful use of a gene transfer approach to demonstrate the ability of forced BCR-ABL expression to deregulate the growth and differentiation of primitive naive human hematopoietic cells after their transplantation into immunodeficient mice. Human CD34+ cord blood cells were exposed to an MSCV retrovirus containing a BCR-ABL-IRES-GFP (P210) cassette and then injected immediately into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) or NOD/SCID-beta2microglobulin-/- mice. P210- and control-transduced (GFP+) human hematopoietic cells were produced in the bone marrow of the mice at similar levels until termination of the experiments 5-6 months later. However, the P210-transduced cells produced a markedly different spectrum of progeny, with an increased ratio of myeloid to B-lymphoid cells and a frequently prolonged increase in erythroid and megakaryocytic cells. After 5 months, several of the mice transplanted with P210-transduced cells developed an increased WBC count and/or splenomegaly due to an expansion of the human GFP+ population. These findings demonstrate that forced expression of BCR-ABL in primitive transplantable human hematopoietic cells is sufficient to cause a rapid and persistent deregulation of their growth and differentiation in vivo with occasional evidence after several months of progression to an early stage of disease.

Animals↗
PubMed2002

Changes in the proliferative activity of human hematopoietic stem cells in NOD/SCID mice and enhancement of their transplantability after in vivo treatment with cell cycle inhibitors.

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [(3)H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-beta was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered G(o) and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.

Animals↗
PubMed2001

Characterization of human hematopoietic cells with short-lived in vivo repopulating activity.

Recent studies with purified hematopoietic stem cells in vitro support a model of stem cell self-renewal control that involves distinct mechanisms regulating permissiveness to and execution of lineage restriction. Such a model predicts the existence of phenotypically separable populations of hematopoietic cells that are pluripotent and either capable or incapable of extensive self-renewal. Such populations have been previously described in the mouse. We describe here the first evidence that such cells can now be identified in humans using different types of immunodeficient mice as hosts.

Animals↗
PubMed2001

The development and implementation of a crisis response team in a school setting.

Traumatic events and subsequent crises within the school setting can have a devastating effect on students, faculty, staff and paebts. Crises serve to compromise the most important mission of the school, i.e., learning. A school crisis response plan is a mandatory aspect of effective educational planning and administration. This paper reviews the need for and development of a school-based crisis intervention system.

Crisis Intervention↗
PubMed2000

Autonomous multi-lineage differentiation in vitro of primitive CD34+ cells from patients with chronic myeloid leukemia.

Neoplastic CD34+ cells from chronic myeloid leukemia (CML) patients proliferate in vitro in the absence of serum or defined growth factors due to an autocrine mechanism involving IL-3 and G-CSF (Jiang et al. Proc Natl Acad Sci USA 1999; 96: 12804). Detailed examination of the various cell types produced in such cultures has now demonstrated the rapid, factor-independent, generation of clonogenic progenitors for all lineages (granulocyte-macrophage, megakaryocyte and erythroid) with the additional appearance within 10 days of large numbers of mature granulocytes, macrophages, and megakaryocytes, as well as occasional erythroid cells. Inclusion of flt3-ligand, Steel factor, IL-3, IL-6, and G-CSF +/- erythropoietin (EPO) in the cultures enhanced only slightly the output of mature cells (except for the erythroid population which was much larger when EPO was added). Analogous subpopulations of normal CD34+ cells produced similar numbers and types of cells but, as expected, only when growth factors were added. Thus primitive CD34+ CML cells proliferating autonomously in vitro recapitulate the full spectrum of differentiation responses of normal CD34+ cells stimulated by IL-3 and G-CSF. These findings point to a role of autocrine IL-3 and G-CSF in the similar multi-lineage expansion of differentiating CD34+ CML cells that occurs in vivo.

Antigens, CD34↗
PubMed1999

Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia.

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph(+)/BCR-ABL(+) chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34(+) leukemic cells. When these cells differentiate into CD34(-) cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR-ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Base Sequence↗
PubMed1999

Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.

Animals↗
PubMed1999

Introduction to stem cell biology in vitro. Threshold to the future.

Transplantable hematopoietic cells with multilineage reconstituting ability can be quantitated in suspensions of human or murine cells using similar assay procedures. The incorporation into these assays of stringently defined functional endpoints ensures a high degree of specificity for the cells detected. Application of these assays to stem cell-containing suspensions after they have been stimulated for several days with defined cytokines in vitro, or by a mixture of defined and/or undefined factors in vivo, has shown that net amplifications in these populations can be obtained under both circumstances. Such studies have allowed cytokine conditions that support stem cell self-renewal divisions to be identified and have also provided evidence that stem cell regeneration can be manipulated both in vitro and in vivo by altering the molecular milieu of the responding cells. These observations pave the way to future delineation of mechanisms that control the normal behavior, pathology and future clinical exploitation of hematopoietic stem cell populations.

Animals↗
PubMed1998

Diphtheria toxin fused to granulocyte-macrophage colony-stimulating factor is toxic to blasts from patients with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10(-9) mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units-leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit-granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34(+) cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34(+) bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor and/or beta chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF-induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

Adult↗
PubMed1998

BCR-ABL accelerates C2-ceramide-induced apoptosis.

In patients with chronic myeloid leukemia (CML), the neoplastic (BCR-ABL+) progenitor cells are characterized by an increased proliferative activity. Whether these cells are also resistant to apoptosis and if so, under what conditions remains controversial. We now show that highly purified populations of very primitive neoplastic progenitor cells obtained directly from CML patients survive and proliferate in vitro for several weeks in the absence of any added growth factors (except insulin). In contrast, purified primary normal progenitors maintained under the same conditions die rapidly. Nevertheless, both primary CML cells and BCR-ABL+ BAF3 cells show the same dose-dependent sensitivity to TNF-alpha or ceramide-induced apoptosis as their respective normal counterparts. In fact, time course studies demonstrated an even faster onset of apoptosis in ceramide-treated BCR-ABL+ BAF3 cells as compared to normal controls. BCR-ABL+ cells treated with ceramide also showed a rapid and sequential increase in the tyrosine phosphorylation of p210(BCR-ABL), p46-56SHC and p120Cbl. These findings suggest growth factor deprivation and treatment with TNF-alpha or ceramide trigger different initial events both of which can lead to apoptosis in factor-dependent hematopoietic cells. However, in the first case, activation of apoptosis is blocked by the basal activity of p210(BCR-ABL), whereas in the second, the presence of p210(BCR-ABL) appears to accelerate the onset of apoptosis by a mechanism that may involve an activation of its kinase function.

Apoptosis↗
PubMed1998

Changes in the cytokine regulation of stem cell self-renewal during ontogeny.

The last 10 years have seen the development of a quantitative assay that is specific for transplantable totipotent murine hematopoietic cells with durable in vivo blood-forming ability. Recently, this assay has been successfully adapted to allow the detection and enumeration of an analogous population of human hematopoietic stem cells using myelosuppressed immunodeficient (nonobese diabetic/severe-combined immunodeficiency) mice as recipients. Characterization of the cells detected by this assay indicates their close relationship in both mice and humans with cells detected in vitro as long-term culture-initiating cells (LTC-IC). Culture conditions have now been identified that support a significant net expansion of these cells from both species. More detailed analyses of the cytokine requirements for this response indicate that the viability, mitogenesis and maintenance of LTC-IC function by human CD34+ CD38- cells can be independently regulated by exogenous factors. Superimposed on this uncoupling of hematopoietic stem cell "self-renewal" and proliferation control is a change during ontogeny in the particular cytokines that regulate their responses. These findings unite stochastic and deterministic models of hematopoietic stem cell control through the concept of a molecular mechanism that actively blocks stem cell differentiation and must be maintained when these cells are stimulated to divide by exposure to certain types and concentrations of cytokines.

Animals↗
PubMed1998

Defective regulation of leukemic hematopoiesis in chronic myeloid leukemia.

Over the last two decades considerable knowledge has been acquired about the distribution of cell types within the dominant leukemic (Ph+/BCR-ABL+) clone that results in human chronic myeloid leukemia (CML). Evidence is now growing to indicate that three key biological changes affecting the development of such clones are: (1) an increased probability of differentiation at the level of the most primitive leukemic stem cells; (2) an increased turnover rate of the leukemic progenitors at all stages of differentiation: and (3) their increased ability to survive under conditions of factor-deprivation. Such a model explains the long latent period for the development of CML as well as why normal stem cells may persist in large numbers but still fail to compete in contributing to the daily output of mature blood cells in patients with disease. The recent development of new genetic and transplant models of human CML may now allow the molecular basis of these biological disturbances to be delineated and more effective therapeutic strategies developed.

Cell Count↗
PubMed1997

Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Animals↗
PubMed1997

Hematopoietic stem cells: inferences from in vivo assays.

Mice and humans both contain a population in their marrow which can permanently regenerate all of the hematopoietic lineages. This developmental potential was first demonstrated in myeloablated mice transplanted with genetically marked marrow obtained from congenic donors. More recently, this approach has been used to devise an in vivo limiting dilution assay for "competitive (lymphomyeloid) repopulating units" (CRU) that allows murine hematopoietic stem cells to be quantitated. Measurements of murine CRU have shown that this population expands concomitantly with the total hematopoietic system during ontogeny and to some extent post-transplant. During these periods of expansion, defective c-kit function can be seen to preferentially compromise CRU self-renewal more than early CRU detection (which requires differentiation and amplification of the progeny of CRU, but may not require extensive CRU self-renewal). In humans, a similar cell type with transplantable lymphomyeloid differentiation potential can be identified in cord blood on the basis of its ability to engraft sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice. Quantitation of these human CRU by limiting dilution analysis of unseparated, highly purified (CD34+CD38-) and cultured (CD34+CD38-) human cord blood cells indicates that their numbers (like the long-term culture-initiating cell [LTC-IC] population) can be slightly expanded in cytokine-supplemented serum-free media, although not as extensively as anticipated from analogous studies of human marrow LTC-IC cultured under the same conditions. Taken together, the results of our studies suggest that the self-renewal of mitotically activated hematopoietic stem cells can be enhanced by their interactions with particular cytokine combinations whose effectiveness in this regard may change during ontogeny.

Animals↗
PubMed1997

Both cycling and noncycling primitive progenitors continue to be mobilized into the circulation during the leukapheresis of patients pretreated with chemotherapy and G-CSF.

Colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) include a spectrum of progenitor types whose potential contributions to the haemopoietic recovery seen in patients transplanted with mobilized peripheral blood progenitor cells (PBPC) remains unclear. We evaluated both the number and cycling status of the circulating LTC-IC and CFC harvested from 12 patients treated with chemotherapy and G-CSF using a modified 6-week LTC-IC assay. The frequency of the LTC-IC and CFC in the mobilized PB samples were increased 45- and 750-fold, respectively. Interestingly, comparison of these values for PB samples, taken just prior to the start of the leukapheresis, with the progenitor content of the 3 h harvest, showed that, on average, the leukapheresis product contained 19 times more LTC-IC (P < 0.01) than had been detectable in the entire blood volume of the patients at the start of the collection, whereas the number of CFC collected was approximately the same as the number in the initial circulating pool of PBPC. Cycling studies showed many of the LTC-IC in the apheresis collections to be proliferating although not more so than in the steady-state marrow LTC-IC compartment (i.e. per cent kill of mobilized LTC-IC after 16 h in 3H-Tdr = 70 +/- 8%, n = 9). On the other hand, the majority of the CFC in the apheresis collections were initially quiescent (per cent kill after 16 h in 3H-Tdr = 37 +/- 6%, n = 12). These findings demonstrate the rapidity with which a primitive subset of LTC-IC may enter the circulation during the early phase of rebound haemopoiesis induced by chemotherapy plus G-CSF and provide evidence of differences in the mechanisms regulating LTC-IC and CFC mobilization.

Breast Neoplasms↗
PubMed1997

Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay.

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to > 100 Mk, detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions, the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However, optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors, of which a combination of IL-3, IL-6, GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large "pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+, approximately half of which are CD41+. Thus, CFC-Mk are more similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and CD71. Taken together, these findings support the concept that some erythroid and Mk progenitors may share a common developmental pathway. The availability of sensitive and reproducible procedures for isolating and detecting human Mk progenitors should facilitate future investigations of their biology and role in a variety of haematological conditions.

Cell Division↗