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Biomedical subjects

A D Ho

Publications and source records attributed to A D Ho.

At least 109 records · Page 6Linked to original sources
PubMed1999

Correlation between IL-3 receptor expression and growth potential of human CD34+ hematopoietic cells from different tissues.

CD123 (alpha-subunit of IL-3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single-cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34+ cells were defined by the levels of surface CD123: CD123negative, CD123low, and CD123bright. Coexpression of lineage markers showed that a majority of CD34+CD123bright cells were myeloid and B-lymphoid progenitors, while erythroid progenitors were mainly in the CD34+CD123negative subset. The CD34+CD123low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34+ cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell factor, interleukin 3 (IL-3), IL-6, GM-CSF, erythropoietin, insulin-like growth factor-1, and basic fibroblast growth factor. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34+CD123negative and CD34+CD123low fractions (37+/-23% and 44+/-23%, respectively) than in the CD34+CD123bright fraction (15+/-21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed growth pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34+CD123negative and CD34+CD123low subsets. When CD38 and CD90 (Thy-1) were used for further characterization of CD34+ cells from FLV, CE increased from 37+/-23% in CD123negative to 70+/-19% in CD123negativeCD38- and from 44+/-23% in CD123low to 66+/-19% in CD123lowCD38-. No significant increase in CE or DGP progenitors was observed when CD34+ cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B-lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123negative/low expression.

ADP-ribosyl Cyclase↗
PubMed1999

Favorable therapeutic index of a p210(BCR-ABL)-specific tyrosine kinase inhibitor; activity on lineage-committed and primitive chronic myelogenous leukemia progenitors.

PURPOSE: In order to assess the effect of the tyrosine kinase inhibitor CGP57148B on lineage-committed and primitive chronic myelogenous leukemia (CML) progenitor cells, peripheral blood progenitor cells (PBPC) mobilized in chronic phase CML were exposed to this compound in vitro. METHODS: Both short-term (</=1 week) and long-term exposure (>/=2 weeks) to CGP57148B were investigated using suspension culture, semisolid (methylcellulose) assay or stroma-dependent long-term culture (LTC). The proportion of bcr/abl-positive progenitors was determined after direct plating [2 weeks in colony-forming cell (CFC) assay] as well as after 2 or 6 weeks LTC (LTC always followed by CFC replates). RESULTS: Incubation of CML PBPC over 48 h in suspension culture with 100 microM CGP57148B reduced the proportion of bcr/abl-positive colonies to 4.4 +/- 4.3% (n = 5) after direct plating, 6.6 +/- 4.2% (n = 5) after 2 weeks LTC and 5 +/- 5.6% (n = 2) after 6 weeks LTC. At this dose, survival of drug-exposed normal PBPC was 53 +/- 4.2%, 51 +/- 2.8% and 54.5 +/- 4.9% (n = 2), respectively. Incubation with CGP57148B at a concentration of 10 microM over 1 week under LTC conditions reduced the number of bcr/abl-positive colonies to 11.8 +/- 6.1% (n = 5) after direct plating, 12 +/- 6.4% (n = 4) after 2 weeks LTC and 14.3 +/- 11.4% (n = 3) after 6 weeks LTC; survival of normal PBPC was 84.5 +/- 2.1%, 93 +/- 4.2% and 86 +/- 1.4% (n = 2), respectively. Following long-term exposure to CGP57148B at a concentration of 1 microM, the proportion of remaining bcr/abl-positive colonies was 35%, 9% and 25% of untreated CML samples after direct plating as well as after 2 and 6 weeks LTC, respectively. The respective values for 10 microM CGP57148B were 10%, 11% and 19%. Long-term exposure of normal progenitors to CGP57148B yielded a survival of 98%, 100% and 93% (1 microM) or 77%, 86% and 80% (10 microM), respectively. CONCLUSION: The results support the use of CGP57148B either for short-term exposure in vitro (e.g. purging) or for continuous treatment of CML in vivo.

Antineoplastic Agents↗
PubMed1999

Mobilization of peripheral blood progenitor cells for human immunodeficiency virus-infected individuals.

Gene therapy is becoming one of the most promising modalities for the treatment of acquired immunodeficiency syndrome. The purpose of this study was to investigate the mobilization and collection of peripheral blood progenitor cells from human immunodeficiency virus (HIV)-infected individuals using granulocyte colony-stimulating factor (G-CSF). A total of 10 patients (9 male, 1 female; median age 36.5 years) with varying circulating CD4+ cell counts (13.9-1467/microL) were administered 10 microg/kg G-CSF daily for 6 days. Peripheral white blood cells (WBCs), CD34+ cell counts, lymphocyte subsets, and plasma viremia were monitored before each G-CSF injection. An average sixfold increase in WBCs was observed, which stabilized on day 4 or thereafter. The level of CD34+ cells was increased by 20-fold, and did not differ between days 5 and 6. Smaller increases in CD4+, CD8+, and CD4+CD8+ cells were observed. HIV viral load, as measured by RNA copy number in plasma, was not significantly altered by G-CSF administration. The leukapheresis product (LP), collected on day 7, contained an average of 6.25+/-4.52 (mean +/- standard deviation) x 10(10) WBCs and 3.08+/-2.98 x 10(6) CD34+ cells/kg. The levels of different CD34+ cell subsets were similar to those in the LPs of G-CSF-mobilized healthy individuals from an earlier study. Primitive hematopoietic cells (CD38- and CD38-HLA-DR+ cells) were detected in LPs (1.19+/-0.46% and 0.87+/-0.23%, respectively, of CD34+ cells). All parameters (WBC counts, lymphocyte populations, CD34+ cells, and HIV-1 RNA copies) measured 3 weeks after leukapheresis returned to baseline values. The administration of G-CSF was well tolerated by the HIV patients; side effects included bone pain, headache, flulike symptoms, and fatigue. There were no correlations between baseline CD4+ cell count and the WBCs, mononuclear cells, or CD34+ cells collected in the LP. Similarly, no correlation existed between baseline CD4+ and CD34+ cells, peak CD34+ cells, or days to achieve peak CD34+ cell counts after G-CSF mobilization. Our results showed that: (1) maximal mobilization can be achieved after 4 days of G-CSF administration; (2) therapeutic quantities of hematopoietic cells can be collected and used for gene therapy; and (3) G-CSF administration is well tolerated and does not cause a clinically significant increase in viremia.

Adult↗
PubMed1999

Bone marrow derived dendritic cells from patients with multiple myeloma cultured with three distinct protocols do not bear Kaposi's sarcoma associated herpesvirus DNA.

BACKGROUND: An association between Kaposi's sarcoma associated herpesvirus (KSHV) and the pathogenesis of multiple myeloma (MM) was postulated recently. The dendritic cells of patients with MM were proposed to be infected with the virus. PATIENTS AND METHODS: Bone marrow mononuclear cells (MNC) of 23 patients, 22 with MM and one with MGUS, were cultured according to three distinct protocols for the generation of dendritic cells. One was essentially the stromal cell culture protocol described by Rettig et al. (Science 1997; 276: 1851-4), while the two other protocols comprised growth factors. Cultured cells were characterised by FACS analysis and assessed for the presence of KSHV DNA with a highly sensitive and specific nested PCR assay detecting the KS 330233 sequence of the virus genome followed by hybridisation with a KSHV specific oligonucleotide. RESULTS: FACS analysis of the cells with the specific markers CD1a, CD86 and HLA-DR, characteristic for dendritic cells, revealed differences in the expression pattern depending on the protocol used. The proportion of CD1a+ cells was very low in the stromal cell cultures (median 0.4%), while a higher percentage of CD14+ cells could be observed (median 37.8%). Growth factor containing cultures revealed a distinctly higher median percentage of CD1a+ cells of 32.5%. The proportion of CD86+ cells varied between 10.4% and 78.5% and HLA-DR+ cells between 26% and 94.4%. Examination of those cells with PCR did not reveal positivity for KSHV in any of the 34 samples assessed. Amplification of seven samples revealed PCR products of approximately the size of the KS 330(233), which, however, could not be confirmed as KSHV specific after hybridisation. CONCLUSION: We have no evidence that bone marrow derived dendritic cells from patients with MM are infected with KSHV.

Adult↗
PubMed1999

Pentostatin in T-cell malignancies--a phase II trial of the EORTC. Leukemia Cooperative Group.

PURPOSE: Within this phase II EORTC trial, we have investigated the safety and efficacy of pentostatin in lymphoid malignancies. We have previously reported the results in T- and B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia. This report focuses on the outcome in T-cell malignancies: T-CLL, Sézary syndrome (Sézary), mycosis fungoides (MF) and T-zone lymphoma (TZL). PATIENTS AND METHODS: Of the 92 patients with these diagnoses enrolled, 76 were evaluable for response and toxicity, i.e., 25 of 28 with T-CLL, 21 of 26 with Sézary, 22 of 26 with MF, and 8 of 12 with TZL. All patients had progressive and advanced disease. Pentostatin was administered at a dosage of 4 mg/m2 every week for the first 3 weeks, then 4 mg/m2 every 14 days for another 6 weeks, followed by maintenance therapy of 4 mg/m2 monthly for a maximum of 6 months. RESULTS: Response rates (complete and partial responses) in patients with Sézary (n = 22) or MF (n = 21) were 33% and 23%, respectively, and in patients with T-CLL (n = 21) or TZL (n = 8) 8% and 25%, respectively. Sixteen (21%) patients died during the first ten weeks of treatment: twelve of progressive disease, two of infectious complications with progressive disease, one of myocard infarction and one of renal failure related to administration of i.v. contrast. Major toxicity (grade 3-4) included infection in 11% of patients, nausea/vomiting in 4%, diarrhea in 3%. Hematologic toxicity was mild to non-existent. CONCLUSIONS: We conclude that pentostatin is active in Sézary and MF but showed marginal activity in T-CLL or TZL. Toxicities are mild to moderate at the dose schedule administered. Due to its relatively specific lympholytic effect and its favorable toxicity spectrum, pentostatin might be especially useful for the palliative treatment of T-cell malignancies.

Adult↗
PubMed1999

Incidence and clinical significance of 6q deletions in B cell chronic lymphocytic leukemia.

Deletions affecting the long arm of chromosome 6 (6q) are among the most commonly observed chromosomal aberrations in lymphoid malignancies and have been identified as adverse prognostic factor in subsets of tumors. Whereas at least two regions of minimal deletion have been established, one in 6q21-q23 and another in 6q25-q27, no tumor suppressor gene that might be involved in the pathogenesis of lymphoid malignancies has been so far identified from these segments. For B cell chronic lymphocytic leukemia (B-CLL) conflicting data have been reported regarding the incidence and prognostic significance of 6q deletions. In the current study we have used two YAC clones mapping to deletion regions in bands 6q21 and 6q27 as probes for fluorescence in situ hybridization (FISH) in a large series of B-CLL cases to analyze the incidence, localization and clinical significance of 6q aberrations. Among 285 patients with B-CLL studied we identified 21 cases (7%) with 6q deletions. All deletions were found with the probe mapping to 6q21 while the 6q27 region was deleted only in a third of these cases. Analysis of the clinical characteristics and laboratory parameters showed that the patients with 6q deletions had higher white blood cell counts and more extensive lymphadenopathy. However, the overall survival and the treatment-free intervals were similar in the two groups. We conclude that deletions in 6q21 occur in 7% of B-CLL and identify a subgroup of patients characterized by a larger tumor mass but no inferior outcome.

Chromosome Deletion↗
PubMed1999

Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported cytotoxic chemotherapy: an update on 100 patients.

Peripheral blood progenitor cells (PBPC) can be mobilized using chemotherapy and granulocyte colony-stimulating factor (G-CSF). We and others previously reported a correlation of steady-state PBPC counts and the PBPC yield during mobilization in a small group of patients. Here we present data on 100 patients (patients: 25 non-Hodgkin's lymphoma (NHL), five Hodgkin's disease, 35 multiple myeloma (MM), 35 solid tumour) which enabled a detailed analysis of determinants of steady-state PBPC levels and of mobilization efficiency in patient subgroups. Previous irradiation (P = 0.0034) or previous chemotherapy in patients with haematological malignancies (P = 0.0062) led to a depletion of steady-state PB CD34+ cells. A correlation analysis showed steady-state PB CD34+ cells (all patients: r = 0.52, P < 0.0001; NHL patients, r = 0.69, P = 0.0003; MM patients: r = 0.66, P = 0.0001) and PB colony-forming cells can reliably assess the CD34+ cell yield in mobilized PB. In patients with solid tumour a similar trend was observed in mobilization after the first chemotherapy cycle (r = 0.51, P = 0.05) but not if mobilization occurred after the second or further cycle of a sequential dose-intensified G-CSF-supported chemotherapy regimen, when premobilization CD34+ counts were 18-fold elevated (P = 0.004). When the patients with MM (r = 0.63, P = 0.0008) or with NHL (r = 0.65, P = 0.006) were analysed separately, a highly significant correlation of the steady-state PB CD34+ cell count to the mean leukapheresis CD34+ cell yield was found, whereas no correlation was observed for patients with a solid tumour. For patients with haematological malignancies estimates could be calculated which, at a specific steady-state PB CD34+ cell count, could predict with a 95% probability a defined minimum progenitor cell yield. These results enable recognition of patients who mobilize PBPC poorly and may assist selection of patients for novel mobilization regimens.

Adult↗
PubMed1999

Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation.

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.

Adult↗
PubMed1999

Mobilization of blood-derived stem and progenitor cells in normal subjects by granulocyte-macrophage- and granulocyte-colony-stimulating factors.

BACKGROUND: It was previously reported that the combination of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) for 4 days mobilized more primitive CD34+ subsets than did either G-CSF or GM-CSF alone. STUDY DESIGN AND METHODS: The studies determine the optimal number of days of growth factor dosing for mobilization and collection of peripheral blood progenitor cells, by increasing the days of administration of GM-CSF and/or G-CSF or employing the sequential administration of GM-CSF followed by G-CSF. Sixty normal subjects were given injections of G-CSF or GM-CSF alone; GM-CSF and G-CSF concurrently for 4, 5, or 6 days; or a sequential regimen of GM-CSF for 3 or 4 days followed by G-CSF for 2 or 3 days. A 10-L apheresis was performed 24 hours after the last dose. RESULTS: The three most efficacious mobilization regimens consisted of sequential GM-CSF for 3 days followed by G-CSF for either 2 or 3 days and G-CSF alone for 5 days. Each of these regimens resulted in the collection of significantly greater numbers of CD34+ cells by apheresis than any of the 4-day dosing regimens with G-CSF and/or GM-CSF (sequential GM-CSF/G-CSF: 3 days/2 days = 3.58 +/- 0.53 x 106 CD34+ cells/kg; GM-CSF/G-CSF: 3 days/3 days = 4.45 +/- 1.08 x 10(6) CD34+ cells/kg; G-CSF: 5 days = 3.58 +/- 0.97 x 10(6) CD34+ cells/kg; all p<0.05 vs. G-CSF and/or GM-CSF for 4 days). Clonogenic assays generally paralleled the level of CD34+ cells. Regimens containing GM-CSF resulted in a higher percentage of the cells from primitive CD34+/CD38-/HLA-DR+ subset than G-CSF alone. CONCLUSION: Compared with 4-day dosing regimens with G-CSF and/or GM-CSF, mobilization of CD34+ cells in normal subjects using sequential GM-CSF for 3 days followed by G-CSF for 2 or 3 days or using G-CSF alone for 5 days increased the number CD34+ cells that can be collected by a single 10-L apheresis 24 hours after the last dose of cytokine.

Antigens, CD34↗
PubMed1998

Gene therapy targeting cord blood-derived CD34+ cells from HIV-exposed infants: preclinical studies.

Hematopoietic CD34+ cells from placental and umbilical cord blood (PUCB) can be valuable vehicles for gene therapy of immunodeficiencies and genetic disorders. We have conducted preclinical studies towards the treatment of HIV-1-infected infants with genetically 'immunized' CD34+ cells derived from PUCB using anti-HIV-1 hairpin ribozyme genes. PUCB was collected from 10 newborns of HIV-1-positive mothers. CD34+ cells were enriched with a modified procedure using Dynal immunomagnetic beads and chymopapain, stimulated with stem cell factor (SCF), IL-3 and IL-6, and transduced using cell-free recombinant retroviral vector (MJT) expressing a ribozyme against the U5 region of HIV-1. No significant differences were observed in the growth pattern of CD34+ cells from normal infants, HIV-1 exposed infants or infants confirmed to be infected by HIV-1. The transduction efficiency of the CD34+ cells from all the infants was also comparable. MJT-transduced CD34+ cells from an HIV-1-infected infant were maintained in a liquid culture system for 4 weeks, and the progeny macrophage cells were challenged with the monocyte-tropic laboratory strain, HIV-Bal, or the HIV-1 isolate from the infant's mother. Significant inhibition of virus replication was observed in ribozyme-transduced cells. Thus, we have demonstrated efficient and stable gene transfer into progenitor cells from the cord blood of HIV-1-exposed or -infected infants and shown that protection from HIV-1 infection was conferred to the progeny cells produced by CD34+ cells transduced with the anti-HIV ribozyme gene construct.

Antigens, CD34↗
PubMed1998

Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.

PURPOSE: The CD20 antigen is expressed on more than 90% of B-cell lymphomas. It is appealing for targeted therapy, because it does not shed or modulate. A chimeric monoclonal antibody more effectively mediates host effector functions and is itself less immunogenic than are murine antibodies. PATIENTS AND METHODS: This was a multiinstitutional trial of the chimeric anti-CD20 antibody, IDEC-C2B8. Patients with relapsed low grade or follicular lymphoma received an outpatient treatment course of IDEC-C2B8 375 mg/m2 intravenously weekly for four doses. RESULTS: From 31 centers, 166 patients were entered. Of this intent-to-treat group, 48% responded. With a median follow-up duration of 11.8 months, the projected median time to progression for responders is 13.0 months. Serum antibody levels were sustained longer after the fourth infusion than after the first, and were higher in responders and in patients with lower tumor burden. The majority of adverse events occurred during the first infusion and were grade 1 or 2; fever and chills were the most common events. Only 12% of patients had grade 3 and 3% grade 4 toxicities. A human antichimeric antibody was detected in only one patient. CONCLUSION: The response rate of 48% with IDEC-C2B8 is comparable to results with single-agent cytotoxic chemotherapy. Toxicity was mild. Attention needs to be paid to the rate of antibody infusion, with titration according to toxicity. Further investigation of this agent is warranted, including its use in conjunction with standard chemotherapy.

Adult↗
PubMed1998

Candidate hematopoietic stem cells from fetal tissues, umbilical cord blood vs. adult bone marrow and mobilized peripheral blood.

As part of our ongoing effort to identify a rich source of pluripotent progenitor cells for transplantation and gene therapy, we cultured single-sorted CD34+ subpopulations from different human hematopoietic tissues to assess the relationship between immunophenotype expression and functional characteristics. In combination with index sorting, single cell culture permits precise assessment of the colony efficiency (CE), growth characteristics, and replating potential (RP) of each individual phenotype without interference from other cell types. CD34+ cells from fetal liver (FL), fetal bone marrow (FBM), umbilical cord blood (UCB), adult BM (ABM), and mobilized peripheral blood (MPB) were sorted and cultured as single cells according to the coexpression of CD38. With the exception of FL, higher CEs were found among CD34+/CD38+ cells vs. CD34+/CD38- cells from the same cell source. However, colonies with dispersed growth pattern (DGP) and mixed growth pattern (MGP) were found predominantly in the CD34+/CD38- subsets. We next examined the functional characteristics of CD34+ subsets defined by three-color analysis and coexpression of CD38 and HLA-DR or CD38 and CDw90 (Thy-1). Using the combination CD34, CD38, and HLA-DR, the highest CEs, the highest percentages of colonies with DGP/MGP and the maximum RP were found in the CD34+/CD38-/HLA-DR+ subset among samples from FL, FBM, and UCB. The CEs and percentages of colonies with DGP/MGP of single-sorted CD34+/CD38-/HLA-DR+ cells were 72.7+/-11.8% (mean +/- standard deviation) and 20.1+/-10.4%, respectively, in FL; 60.9+/-11.1% and 11.5+/-5.4% in FBM; 57.0+/-16.5% and 24.1+/-7.3% in UCB; 27.2+/-12.8% and 9.0+/-4.9% in MPB, and 9.6+/-7.8% and 4.6+/-3.2% in ABM. Using the combination CD34, CD38, and CDw90(Thy-1), the subset with the highest CEs and highest percentages of colonies with DGP/MGP was found to be CD34+/CD38-/CDw90- in FL and FBM, but colonies with high RP were distributed evenly among CDw90+ and CDw90- subsets derived from FL, FBM, UCB, or MPB. We conclude that the CD34+/CD38-/HLA-DR+ subset contained the highest number of candidate stem cells among the various immunophenotypes, and that FL contained the highest concentration of CD34+ cells (11.4+/-7.5%) and the highest level of CD34+/CD38-/HLA-DR+ subsets (34.7+/-8.2%) among cells from various ontogenic age. Our estimate of candidate stem cells using single cell suspension culture correlated with that obtained by single cell long-term culture-initiating cells. CD34+/CD38-/HLA-DR+ cells from FL appear to represent the best targets for ex vivo stem cell expansion and genetic manipulation.

ADP-ribosyl Cyclase↗
PubMed1997

Gene therapy targeting peripheral blood CD34+ hematopoietic stem cells of HIV-infected individuals.

Gene therapy is a promising treatment modality for acquired immunodeficiency syndrome (AIDS). Autologous transplantation with genetically altered pluripotent hematopoietic stem cells encoding anti-human immunodeficiency virus (HIV) genes could in theory completely and permanently reconstitute all blood lineages and immune functions with cells resistant to HIV. Recent studies showed that CD34+ stem cell can be mobilized in HIV-infected individuals after granulocyte colony-stimulating factor (G-CSF) administration without major side effects or increase of viral load. In this study, peripheral blood CD34+ cells of five HIV-infected individuals were mobilized with G-CSF and after leukapheresis and enrichment, subjected to retroviral transduction with genes encoding anti-HIV ribozyme-decoy fusion molecules. These cells were tested for the ability to give rise to progeny cells, for retroviral transduction efficiency, and for expression of the transgene. CD34+-derived macrophage-like cells were also challenged with HIV. Results showed that CD34+ cells from HIV-infected individuals gave rise to similar numbers of progeny colonies as cells from healthy donors. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. CD34+-derived macrophages expressed anti-HIV genes and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. Furthermore, we showed that after thawing, cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. Thus, CD34+ cells from HIV-infected patients can be stored for further genetic manipulations with improved vectors or anti-HIV genes as they become available.

Animals↗
PubMed1997

Recombinant methionyl human stem cell factor and filgrastim for peripheral blood progenitor cell mobilization and transplantation in non-Hodgkin's lymphoma patients--results of a phase I/II trial.

To examine the safety and efficacy of recombinant-methionyl human stem cell factor (r-metHuSCF), 38 patients with intermediate-grade or immunoblastic high-grade non-Hodgkin's lymphoma who were eligible for autologous transplantation were randomized to receive r-metHuSCF (5, 10, 15, or 20 microg/kg/d) plus Filgrastim (10 microg/kg/d) or Filgrastim (10 microg/kg/d) alone to mobilize peripheral blood progenitor cells. Subcutaneous administration of r-metHuSCF was well tolerated in conjunction with a multi-agent pre-medication regimen; local injection site reactions were the most commonly seen adverse event. The total mononuclear cell count, CD34+ cell content, granulocyte-macrophage colony-forming cells (GM-CFC), and burst-forming units-erythroid (BFU-E) per kilogram in the apheresis product was similar when all patients were analyzed by treatment cohort and mobilization regimen (Filgrastim or r-metHuSCF in combination with Filgrastim); however, when prior chemotherapy was taken into account in a supplementary analysis, clinically important differences were observed. Extensive prior therapy was defined as the amount of exposure to specific stem cell toxic chemotherapeutic agents that patients received. These agents include procarbazine, nitrogen mustard, melphalan, nitrosoureas (> or = 2 cycles of any of these drugs) or greater than 7.5 g of cytosine arabinoside. In these patients, there was an increased number of CD34+ cells (1.76 v 0.28 x 10(6)/kg), GM-CFC (20.5 v 5.0 x 10(4)/kg), and BFU-E (36.9 v 8.9 x 10(4)/kg) in patients receiving r-metHuSCF and Filgrastim (N = 18) compared with Filgrastim alone (N = 5). These patients also had a decreased time to an untransfused platelet count of 20 x 10(9)/L that was 10.5 days shorter in the patients who received r-metHuSCF and Filgrastim (12.5 v 23 days). These differences were not found to be statistically significant, possibly because of small size, but are clinically important.

Adult↗
PubMed1997

Interaction of vesicular stomatitis virus-G pseudotyped retrovirus with CD34+ and CD34+ CD38- hematopoietic progenitor cells.

Retroviral vectors have had limited success in mediating gene transfer to hematopoietic stem cells, particularly in primates, due in part to low or absent expression of the amphotropic receptor (RAM-1). We have been interested in determining whether retrovirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) would allow more efficient gene delivery to hematopoietic stem cells as the VSV-G receptors appear to be ubiquitously present phospholipids. However, we previously found that completion of retroviral vector reverse transcription does not occur in CD34+ CD38- hematopoietic stem cells that were exposed to VSV-G pseudotyped retrovirus. To determine at which stage the block to infection of CD34+ CD38- cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped viral particles could bind to CD34+ CD38- cells. Virus binding to CD34+ cells was saturable at 4 degrees C but nonsaturable at 37 degrees C, up to a multiplicity of infection of 1080. This suggests that surface levels of phospholipid receptors available for viral binding are limiting on CD34+ cells. Cytokine stimulation increased virus binding to CD34+ cells. However, no increase in the level of surface phosphatidylserine (PS), a strong candidate for the VSV-G receptor, was seen as detected by the PS-specific reagent, annexin V. This suggests that another molecule is serving as the VSV-G receptor on CD34+ cells. Here, we show that once virus binding to cytokine-stimulated CD34+ CD38- cells has occurred, virus fusion proceeds efficiently as determined by octadecyl rhodamine (R18) fusion assays. Taken together with our previous observation that reverse transcription does not occur in CD34+ CD38- cells, we suggest that there are intracellular mechanisms leading to blockage of complete reverse transcription of the retrovirus in CD34+ CD38- cells. This has important implications for retrovirus-mediated gene transfer to quiescent stem cells.

ADP-ribosyl Cyclase↗
PubMed1997

Decrease in tumor cell contamination and progenitor cell yield in leukapheresis products after consecutive cycles of chemotherapy for breast cancer treatment.

In this retrospective study, we assessed the impact of each of three consecutive cycles of conventional-dose chemotherapy on CD34+ cells, colony-forming units granulocyte-macrophage (CFU-GM), and contaminating breast cancer cells collected in the leukapheresis products of patients with metastatic breast cancer. The patients subsequently underwent high-dose chemotherapy followed by autologous blood progenitor cell transplantation. We analyzed 172 leukapheresis products from 17 patients and have correlated the long-term clinical outcome with tumor cell contamination. The induction chemotherapy regimen consisted of three cycles of cyclophosphamide 750 mg/m2 i.v., epirubicin 100 mg/m2, and 5-fluorouracil (5-FU) 750 mg/m2 i.v., followed by 5 microg/kg body weight of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) daily until leukapheresis was completed. An average of 10 leukapheresis products (three to four collections after each cycle of chemotherapy) were obtained from each patient. Numbers of CD34+ cells, CFU-GM, and mononuclear cells (MNCs) in the leukapheresis products were determined at the time of collection. Aliquots from the same products were frozen and breast cancer cells were detected by immunocytochemistry with a cocktail of anti-cytokeratin antibodies (AE-1, AE-3, CAM 5.2, Keratin 8+18+19) using a standardized immunoalkaline phosphatase method. A minimum of 10(6) cells were examined by light microscopy and by at least two blinded observers. Cells were considered positive when immunostaining was detected in the cytoplasm and on the cell membrane, and cellular morphology was consistent with a malignant phenotype. Of the 172 samples analyzed, 13 of 57 (23%) leukapheresis products collected after cycle I were positive for tumor cells; 3 of 60 (5%) after cycle II; and 4 of 55 (7%) after cycle III. The likelihood of contamination by breast cancer cells after cycle I was significantly higher than after subsequent cycles of chemotherapy (p = 0.0052). Simultaneously, there was a significant decrease in quantity of CD34+ cells and CFU-GM (p < 0.0001 for both comparisons). Our study indicated that leukapheresis products collected after the second or third cycles of induction chemotherapy carry a significantly lower likelihood of tumor cell contamination, albeit the quantity of CD34+ cells or CFU-GM collected was also significantly reduced.

Adult↗
PubMed1996

Retrovirus-mediated gene expression in hematopoietic cells correlates inversely with growth factor stimulation.

Cells of the hematopoetic system, especially hematopoietic progenitor and stem cells, are perceived as ideal targets for human gene therapy. In this report, the stability of retrovirus-mediated gene expression driven by three different potent promoters has been examined in purified human CD34+ cells. The promoters, murine stem cell virus (MSCV) long terminal repeat (LTR) and pgk, show gene expression in 10 times more hematopoietic colonies derived from CD34+ cells than the commonly used Moloney murine leukemia virus (Mo-MLV) LTR. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, however, demonstrates that the levels of gene expression in retrovirus-transduced cells decrease with time in long-term bone marrow cultures and in suspension cultures containing hematopoietic growth factors. Removal of hematopoietic growth factors from the suspension culture medium was associated with a decrease in cell proliferation and differentiation, but with stable gene expression. Retrovirus-mediated gene expression is, therefore, inversely related to proliferation and differentiation of the transduced CD34+ cells. These observations may have implications in future design and implementation of human gene therapy protocols.

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