Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study.

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs.

Evidence for non-axial A/P patterning in the nonneural ectoderm of Xenopus and zebrafish pregastrula embryos.

Recent studies in early Xenopus and zebrafish embryos have demonstrated that posteriorizing, non-axial signals arising from outside the organizer (or shield) contribute to A/P patterning of the neural axis, in contradiction to the classical Spemann model in which such signals were proposed to be solely organizer derived. Our studies on the early expression of the transcription factors GATA-2 and 3 in both Xenopus and zebrafish nonneural ectoderm lend support to the existence of such non-axial signaling in the A/P axis. Thus we find that the earliest expression of GATA-2 and 3 is located in nonneural ectoderm and is strongly patterned in a graded manner along the A/P axis, being high anteriorly and absent from the most posterior regions.

T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect.

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 x 10(6) cells/kg on day 30 and 5 x 10(7) cells/kg on day 45 (schedule 1), the overall probability of grade > or = II aGVHD developing was 31.5%, with a 15.

T cell-depleted granulocyte colony-stimulating factor (G-CSF) modified allogenic bone marrow transplantation for hematological malignancy improves graft CD34+ cell content but is associated with delayed pancytopenia.

To increase the stem cell content of T cell-depleted bone marrow transplants (BMT), we treated 12 patients with hematological malignancies with BMT from HLA-identical sibling donors given G-CSF 10 microg/kg/day for 5 days before marrow harvest. After CD34+ cell selection, patients received a median of 1.7 (range, 0.

Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients.

Seroepidemiology of Trypanosoma cruzi, etiologic agent of Chagas' disease, in US blood donors.

A comprehensive seroepidemiologic study was conducted in two Red Cross regions (Los Angeles and Miami) to determine the prevalence of Trypanosoma cruzi antibodies in at-risk blood donors, to identify additional risk factors, and to assess the likelihood of transmitting T. cruzi by transfusion. At-risk and control donors were stratified by a broad risk question, tested for T.

Enumeration of CD34+ hematopoietic progenitor cells in peripheral blood and leukapheresis products by microvolume fluorimetry: a comparison with flow cytometry.

There is increasing interest in both standardization and simplification of methods for enumeration of CD34+ hematopoietic progenitor cells (HPC) to facilitate cellular therapies and to improve interinstitutional comparison of clinical and laboratory results. We evaluated a novel method for CD34+ cell enumeration based on microvolume fluorimetry (MVF) compared with our laboratory's routine flow cytometric method on samples of peripheral blood and leukapheresis products. The MVF method is semiautomated and uses a 633-nm light from a helium-neon laser to scan fluorochrome-labeled cells held in stasis in a capillary known volume.

CD34+ cell dose predicts survival, posttransplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies.

After autologous or allogeneic transplants of peripheral blood stem cells (PBSC), an adequate dose of CD34+ cells is necessary to ensure early and sustained hematopoietic engraftment and favorable clinical outcome. There are no comparable data on the relationship between CD34+ cell dose and recovery after allogeneic bone marrow transplants (BMT). Twenty-eight patients with hematologic malignancies received a BMT from an HLA-identical sibling, using T-cell depletion and cyclosporin for graft-versus-host disease prophylaxis and delayed donor lymphocyte transfusions in an attempt to prevent leukemia relapse.

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP.

Developmental effects of trichloroacetonitrile administered in corn oil to pregnant Long-Evans rats.

Trichloroacetonitrile (TCAN) is a by-product of the chlorine disinfection of water containing natural organic material. When administered by gavage to pregnant Long-Evans rats in a medium-chain triglyceride vehicle, tricaprylin oil (Tricap), at a volume of 10 ml/kg, TCAN induced fetal cardiovascular anomalies at doses as low as 1 mg/kg/d (Smith et al., 1988).

Lack of sensitivity of indium-111 mixed leukocyte scans for active disease in Takayasu's arteritis.

Objective: Currently, a reliable, noninvasive means of detecting the active vasculitic lesions of Takayasu's arteritis does not exist. Based on the cellular infiltrates seen in active lesions, we postulated that indium-111 labelled mixed leukocytes could be used to scintigraphically detect sites of active vasculitis.

Methods: Fifteen In-111 mixed leukocyte scans were performed in 10 patients with Takayasu's arteritis and correlated with clinical, laboratory, and radiographic findings.

Cardiopathic effects of dichloroacetate in the fetal Long-Evans rat.

Dichloroacetic acid (DCA) is a by-product of the chlorine disinfection of water and may occur in treated water at levels exceeding 100 micrograms/L. Previous studies revealed teratogenic effects, particularly heart malformations, at high doses (900-2,400 mg/kg given on days 6-15 of pregnancy). In a series of three studies, groups of 7-10 Long-Evans rats were dosed with 1,900 mg/kg of DCA on days 6-8, 9-11, or 12-15; with 2,400 mg/kg on days 10, 11, 12, or 13; and with 3,500 mg/kg on days 9, 10, 11, 12, or 13, in an attempt to determine the most sensitive period and further characterize the heart defect.

Developmental toxicity of dichloroacetate in the rat.

Dichloroacetic acid (DCA) is a principal by-product of the chlorine disinfection of water containing humic and fulvic acids, and is also a drug of interest in the therapeutic management of metabolic disorders. The developmental effects of DCA were evaluated in the pregnant Long-Evans rat. In two separate studies, animals were dosed by oral intubation on gestation days 6-15 (plug = 0) with 0, 900, 1,400, 1,900 or 2,400 mg/kg/day and 0, 14, 140, or 400 mg/kg/day.

The telomere-associated MAL3 locus of Saccharomyces is a tandem array of repeated genes.

Saccharomyces strains capable of fermenting maltose contain any one of five telomere-associated MAL loci. Each MAL locus is a complex of three genes encoding the three functions required to ferment maltose: maltose permease (GENE 1), maltase (GENE 2) and the MAL trans-activator (GENE 3). All five loci have been cloned and all are highly sequence homologous over at least a 9.

Adverse male reproductive effects following subchronic exposure of rats to sodium dichloroacetate.

Dichloroacetate (DCA) activates the pyruvate dehydrogenase complex enhancing carbohydrate and lactate utilization in animals. As a result it is used clinically in the treatment of acute lactic acidosis and has therapeutic potential in the treatment of stroke. Adverse effects of chronic DCA treatment include polyneuropathy and testicular degeneration.

Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation.

Several conditional-lethal mutant alleles of the single-copy Saccharomyces cerevisiae beta-tubulin and actin genes were used to evaluate the roles of microtubules and actin filaments in the pheromone-induced extension of mating projections. Mutants defective in tubulin assembly form projections indistinguishable in appearance from those formed by wild-type cells. However, the tubulin mutants are unable to move their nuclei into the projections and to orient the spindle pole body associated with each nucleus toward the projection tip.

Enumeration of cells in bone marrow and peripheral blood stem cell collections: technical issues and prospects for standardization.

Enumeration of total nucleated cells and mononuclear cells is a fundamental part of the laboratory evaluation and quality control program for bone marrow and peripheral blood stem cell collections intended for transplantation. Measurement of the total nucleated cell content is especially useful for providing rapid feedback about the bone marrow product during or immediately after the harvest. However, the mononuclear cell content may be more informative because the nucleated cell population contains a variable number of mature granulocytes and nucleated red cells, which do not contribute to hematopoietic engraftment.

Hematologic improvement in a patient with Gaucher disease on long-term enzyme replacement therapy: evidence for decreased splenic sequestration and improved red blood cell survival.

We describe here an investigation of the hematologic response of a child with Gaucher disease to a six-year therapeutic trial of human placental mannose-terminated glucocerebrosidase. While on enzyme replacement therapy, the patient's hemoglobin and platelet count significantly increased (6.9 g/dl to 12.

Flow cytometric detection of human red cells labeled with a fluorescent membrane label: potential application to in vivo survival studies.

In vivo survival studies of human red cells (RBCs) are commonly carried out by using chromium-51 (51Cr), a gamma-emitting radionuclide, as the cell label. The effects of labeling human RBCs with PKH-2, a nonradioactive lipophilic fluorescent dye that binds to the cell membrane, and the feasibility of detecting the labeled cells by flow cytometric analysis were investigated. Optimal labeling, defined as maximum mean fluorescence intensity with minimal cell-to-cell variability in fluorescence intensity and minimal cell loss, was achieved with the use of 15.