Biomedical subjects

Shelly Heimfeld

Publications and source records attributed to Shelly Heimfeld.

12 recordsLinked to original sources

PubMed2007

Connective tissue growth factor (CTGF) expression and outcome in adult patients with acute lymphoblastic leukemia.

We compared the gene expression profile of adult acute lymphoblastic leukemia (ALL) to normal hematopoietic and non-ALL samples using oligonucleotide arrays. Connective tissue growth factor (CTGF) was the highest overexpressed gene in B-cell ALL compared with the other groups, and displayed heterogeneous expression, suggesting it might have prognostic relevance. CTGF expression was examined by quantitative reverse transcriptase-polymerase chain reaction (ORT-PCR) on 79 adult ALL specimens. CTGF expression levels were significantly increased in ALL cases with B-lineage (P < .001), unfavorable cytogenetics (P < .001), and blasts expressing CD34 (P < .001). In a multivariate proportional hazards model, higher CTGF expression levels corresponded to worsening of overall survival (OS; hazard ratio 1.36, for each 10-fold increase in expression; P = .019). Further studies are ongoing to confirm the prognostic value of CTGF expression in ALL and to investigate its role in normal and abnormal lymphocyte biology.

Adolescent↗

PubMed2006

HES1 inhibits cycling of hematopoietic progenitor cells via DNA binding.

Notch signaling is implicated in stem cell self-renewal, differentiation, and other developmental processes, and the Drosophila hairy and enhancer of split (HES) 1 basic helix-loop-helix protein is a major downstream effector in the Notch pathway. We found that HES1 was expressed at high levels in the hematopoietic stem cell (HSC)-enriched CD34+/[CD38/Lin](- /low) subpopulation but at low levels in more mature progenitor cell populations. When CD34+ cells were cultured for 1 week, the level of HES1 remained high in the CD34+ subset that had remained quiescent during ex vivo culture but was reduced in CD34+ cells that had divided. To investigate the effects of HES1 in human and mouse hematopoietic stem-progenitor cells (HSPCs), we constructed conditional lentiviral vectors (lentivectors) to introduce transgenes encoding either wild-type HES1 or a mutant lacking the DNA-binding domain (BHES1). We found that lentivector-mediated HES1 expression in CD34+ cells inhibited cell cycling in vitro and cell expansion in vivo, associated with upregulation of the cell cycle inhibitor p21(cip1/Waf1) (p21). The HES1 DNA-binding domain was required for these actions. HES1 did not induce programmed cell death or alter differentiation in HSPCs, and while short-term repopulating activity was reduced in HES1-transduced mouse and human cells, long-term reconstituting HSC function was preserved. Our data characterize the complex, cell context-dependent actions of HES1 as a major downstream Notch signaling regulator of HSPC function.

Animals↗

PubMed2006

Reduced incidence of acute and chronic graft-versus-host disease with the addition of thymoglobulin to a targeted busulfan/cyclophosphamide regimen.

To reduce the incidence of graft-versus-host disease (GVHD), we added Thymoglobulin (THY) to dose-adjusted oral busulfan plus cyclophosphamide (targeted BUCY). The starting dose of THY was 4.5 mg/kg given over days -3, -2, and -1, escalated in steps of 1.5 mg/kg in cohorts of 15 evaluable patients. Escalation was dependent on acute GVHD incidence and Epstein-Barr virus reactivation. Fifty-six patients with myelodysplastic syndrome and other myeloid disorders underwent transplantation with peripheral blood progenitor cells from related (n=30) or unrelated (n=26) donors. All but 2 patients achieved engraftment, and 56% survived in remission beyond 1 year. The incidence of acute GVHD was 50%, and that of chronic GVHD was 34%. The highest THY dose was 6.0 mg/kg, a dose at which 1 patient experienced Epstein-Barr virus reactivation. Nine patients did not receive the prescribed THY dose. Results were comparable for related and unrelated transplants and for patients given 4.5 or 6.0 mg/kg THY. Among 27 myelodysplastic syndrome patients (14 with related and 13 with unrelated donors) who underwent transplantation concurrently with targeted BUCY without THY, the incidence of acute and chronic GVHD was 82%. Thus, THY 4.5 to 6.0 mg/kg seemed beneficial for GVHD prevention in BUCY-conditioned patients who underwent transplantation with peripheral blood progenitor cells, although relapse-free survival did not differ significantly from that in comparable historical controls not given THY.

Acute Disease↗

PubMed2005

Collection of blood stem cells from patients with sickle cell anemia.

Prior to initiating gene therapy trials for sickle cell disease (SCD), methods to collect sufficient numbers of hematopoietic stem and progenitor cells will need to be developed. Bone marrow harvest entails significant morbidity that could be severe in patients with SCD. In addition, an ability to perform repeated stem cell collections so that several transfers of genetically modified cells could be attempted would be advantageous. In other settings, apheresis collection of mobilized blood stem cells has become the preferred source of stem cells for transplantation. Unfortunately, patients with SCD do not tolerate granulocyte-colony stimulating factor and therefore cannot be mobilized using these conventional methods. In this pilot study, we investigated whether withdrawal of hydroxyurea therapy results in an increase in circulating numbers of CD34+ cells and hematopoietic progenitors. In addition, we performed leukapheresis in one patient with severe SCD in an attempt to determine whether blood stem cell collection can be performed safely in patients who would be candidates for SC gene therapy trials. Our results highlight some of the potential difficulties in initiating gene therapy clinical trials for sickle cell disease.

Adult↗

PubMed2004

Microarray and serial analysis of gene expression analyses identify known and novel transcripts overexpressed in hematopoietic stem cells.

The human CD34(+)/CD38(-)/Lin(-) cell subset, comprising approximately 1-10% of the CD34(+) cell population, contains few of the less primitive hematopoietic (lineage-committed) progenitor cells (HPCs) but most of the primitive in vivo engrafting (lympho-)hematopoietic stem cells (HSCs). We analyzed gene expression in CD34(+)/CD38(-)/Lin(-) cell populations isolated from normal human adult donor bone marrow, neonatal placental/umbilical cord blood, and mobilized adult donor peripheral blood stem-progenitor cells. As measured by Affymetrix microarrays, 4746 genes were expressed in CD34(+)/CD38(-)/Lin(-) cells from all three tissues. We also determined the transcriptomes of the stem cell-depleted, HPC-enriched CD34(+)/[CD38/Lin](++) cell population from each tissue. Comparison of CD34(+)/CD38(-)/Lin(-) (HSC-enriched) versus CD34(+)/[CD38/Lin](++) (HPC-enriched, HSC-depleted) cells from each tissue yielded 81 genes overrepresented and 90 genes underrepresented, common to all three of the CD34(+)/CD38(-)/Lin(-) cell populations. These transcripts, which are selectively expressed in HSCs from all three tissues, include a number of known genes (e.g., transcription factors, receptors, and signaling molecules) that might play roles in key functions (e.g., survival, self-renewal, differentiation, and/or migration/adhesion) of human HSCs. Many genes/transcripts of unknown function were also detected by microarray analysis. Serial analysis of gene expression of the bone marrow HSC and HPC populations confirmed expression of most of the overrepresented transcripts for which reliable serial analysis of gene expression tags were detected and additionally suggested that current microarrays do not detect as many as 30% of the transcripts expressed in HSCs, including a number of previously unknown transcripts. This work is a step toward full definition of the transcriptome of normal human HSCs and may identify new genes involved in leukemogenesis and cancer stem cells.

ADP-ribosyl Cyclase↗

PubMed2004

Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning.

We analyzed the kinetics of donor engraftment among various peripheral blood cell subpopulations and their relationship to outcomes among 120 patients with hematologic malignancies given hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning consisting of 2 Gy total body irradiation (TBI) with or without added fludarabine. While patients rapidly developed high degrees of donor engraftment, most remained mixed donor/host chimeras for up to 180 days after HCT. Patients given preceding chemotherapies and those given granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC) grafts had the highest degrees of donor chimerism. Low donor T-cell (P = .003) and natural killer (NK) cell (P = .004) chimerism levels on day 14 were associated with increased probabilities of graft rejection. High T-cell chimerism on day 28 was associated with an increased probability of acute graft-versus-host disease (GVHD) (P = .02). Of 93 patients with measurable malignant disease at transplantation, 41 achieved complete remissions a median of 199 days after HCT; 19 of the 41 were mixed T-cell chimeras when complete remissions were achieved. Earlier establishment of donor NK-cell chimerism was associated with improved progression-free survival (P = .02). Measuring the levels of peripheral blood cell subset donor chimerisms provided useful information on HCT outcomes and might allow early therapeutic interventions to prevent graft rejection or disease progression.

Adolescent↗

PubMed2004

Differences in F36VMpl-based in vivo selection among large animal models.

Animal models are indispensable tools for understanding physiological and pathological processes, as well as for developing new therapies. Ultimately, the results of animal experimentation must provide information that can guide the development of therapeutic approaches in humans. Significant differences have been reported comparing a gene therapy approach between different animal models. However, little information exists describing differences among the available large animal models. Here we evaluated, in the hemopoietic cells of baboons, a system of selection that has previously demonstrated activity in mice, in dogs, and in human cells ex vivo. This system employs a derivative of the murine thrombopoietin receptor (F36Vmpl), which is conditionally activated in the presence of a small-molecule drug called a chemical inducer of dimerization (CID). Whereas cultured mouse, human, and, to a lesser extent, dog hemopoietic cells all proliferate in response to the F36Vmpl signal, we observed only a minor and variable response to the F36Vmpl signal in the cultured cells of baboons. Similarly, we have noted significant rises in the frequency of transduced hemopoietic cells in mice and in dogs upon CID administration in vivo; however, here we show that responses to CID administration in three baboons were modest and variable. These findings have general implications for the evaluation and development of new strategies for gene therapy.

Animals↗

PubMed2004

CD34 cell dose and chronic graft-versus-host disease after human leukocyte antigen-matched sibling hematopoietic stem cell transplantation.

Chronic graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Recent reports suggest that chronic GVHD is more frequent after G-CSF--mobilized peripheral blood mononuclear cell (G-PBMC) transplantation compared to marrow transplantation from human leukocyte antigen (HLA)-matched siblings. Furthermore, higher numbers of CD34 positive cells in G-PBMC grafts were associated with an increased risk of chronic GVHD, whereas a correlation between CD34 cell numbers and chronic GVHD has not been reported after bone marrow transplantation. Potential mechanisms that might explain the association between G-PBMC CD34 numbers and chronic GVHD include enhanced antigen presentation to donor T cells by large numbers of transplanted CD34 cells or their dendritic cell progeny, which may enhance GVHD induction. However, these mechanisms remain highly speculative and are not supported by experimental data. This review discusses implications of CD34 cell dose adjustments in HLA-identical sibling G-PBMC transplantation and weighs the benefits and risks with respect to chronic GVHD, hematopoietic recovery, immune reconstitution and relapse.

Antigens, CD34↗

PubMed2003

Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate model.

Conditional suicide genes derived from pathogens have been developed to confer drug sensitivity and enhance safety of cell therapy, but this approach is limited by immune responses to the transgene product. We examined a strategy to regulate survival of transferred cells based on induction of apoptosis through oligomerization of a modified human Fas receptor by a bivalent drug (AP1903). Three macaques (Macaca nemestrina) received autologous T cells retrovirally engineered to express a Fas suicide-construct (LV'VFas). High levels of transduced cells were present in blood following cell transfer, but LV'VFas(+) cells declined rapidly after AP1903 administration. A small fraction of LV'VFas(+) cells resisted elimination by AP1903, in part due to insufficient levels of transgene expression in resting T cells, because reactivation of these cells in vitro enhanced sensitivity to AP1903. An immune response to the transgene product was observed, but epitope mapping indicated the response was directed to discrete components of human LV'VFas that were variant with the corresponding macaque sequences. These data demonstrate that chemically induced dimerization can be used to regulate survival of adoptively transferred T cells in vivo.

Adoptive Transfer↗

PubMed2003

Male DNA in female donor apheresis and CD34-enriched products.

Increased risk of graft-versus-host disease (GVHD) has been described in recipients of hematopoietic stem cell transplantations when the donor is a parous woman. Cells from prior pregnancies are now known to persist in women and could contribute to GVHD. We asked whether male DNA (presumed fetal microchimerism) is present in apheresis products of female donors. A total of 50 samples were studied by using real-time quantitative polymerase chain reaction (PCR) for the Y chromosome-specific sequence DYS14. Among 29 growth factor-mobilized peripheral blood mononuclear cell (G-PBMC) products, 34% were positive for male DNA. Quantitative results, expressed as DNA genome equivalent of male cells per million host cells (gEq/mil), ranged from 0 to 35 gEq/mil. Among 21 CD34-enriched cell fractions, 48% were positive with a range of 0 to 357 gEq/mil. In summary, male DNA was frequently detected in G-PBMC and CD34-enriched products from female donors. Whether fetal microchimerism contributes to GVHD merits further investigation.

Antigens, CD34↗

PubMed2003

HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies.

A hematopoietic cell transplantation (HCT) approach was developed for elderly or ill patients with hematologic malignancies that employed nonmyeloablative conditioning to avoid common regimen-related toxicities and relied on graft-versus-tumor effects for control of malignancy. Eighty-nine patients, median age 53 years, were given fludarabine (90 mg/m2) and 2 Gy total body irradiation. Marrow (n = 18) or granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells (G-PBMCs; n = 71) were transplanted from unrelated donors matched for human leukocyte antigen A (HLA-A), -B, -C antigens and -DRB1 and -DQB1 alleles. Postgrafting immunosuppression included mycophenolate mofetil and cyclosporine. Donor T-cell chimerism was higher for G-PBMCs compared with marrow recipients. Durable engraftment was observed in 85% of G-PBMCs and 56% of marrow recipients. Cumulative probabilities of grade II, III, and IV acute graft-versus-host disease (GVHD) were 42%, 8%, and 2%, respectively. Nonrelapse mortality at day 100 and at 1 year was 11% and 16%, respectively. One-year overall survivals and progression-free survivals were 52% and 38%, respectively. G-PBMC recipients had improved survival (57% vs 33%) and progression-free survival (44% vs 17%) compared with marrow recipients. HLA-matched unrelated donor HCT after nonmyeloablative conditioning is feasible in patients ineligible for conventional HCT. G-PBMCs conferred higher donor T-cell chimerism, greater durable engraftment, and better progression-free and overall survivals compared with marrow.

Adolescent↗

PubMed2002

CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy.

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded products. Here we have developed a transduction and selection strategy for generating large numbers of polyclonal T cells transduced with a retroviral vector encoding the human low-affinity nerve growth factor receptor (LNGFR) for selection and a Fas-based suicide construct (LV'VFas). Ligation of CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to the generation of gene-modified T cells that retained clonal diversity, functional properties, and a homing receptor profile similar to untransduced peripheral blood lymphocytes. Microbeads conjugated directly to antibody specific to LNGFR significantly improved the immunomagnetic selection of LV'VFas-modified T cells and assisted in scaling of the selection procedure to therapeutic cell numbers. Thus, these studies identified a strategy that requires only a brief ex vivo culture and does not use drug selection to obtain large numbers of functional gene-modified polyclonal T cells that can be used for adoptive immunotherapy.

Animals↗