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Biomedical subjects

A Bank

Publications and source records attributed to A Bank.

At least 19 recordsLinked to original sources
PubMed2001

Arterial distensibility and physical activity in the ARIC study.

PURPOSE: Arterial distensibility decreases with age. This decrease may be associated with the initiation and/or progression of hypertension and atherosclerosis and may be attenuated by positive lifestyle habits, including habitual physical activity. We tested the hypothesis that self-reported sport, leisure, and work physical activity is associated with greater arterial distensibility (i.e., carotid artery pulsatile diameter changes). METHODS: The Atherosclerosis Risk in Communities (ARIC) study assessed left common carotid arterial diameters and intimal-medial wall thickness (IMT) using B-mode ultrasound techniques, in 10,644 African-American and white men and women aged 45-64 yr and free of cardiovascular disease. RESULTS: Work activity, but not sports or leisure activity, was weakly associated with greater arterial distensibility in an ANCOVA model adjusted for blood pressure and other covariates (diastolic arterial diameter, pulse pressure, pulse pressure squared, age, race, sex, smoking, dietary fat intake, height, education, and clinical center) (P for linear trend = 0.03). Vigorous sports activity was weakly positively associated with arterial distensibility (arterial diameter change (mean +/- SE in mm) 0.42 +/- 0.004 vs 0.41 +/- 0.002 for the 12.7% of participants reporting any vs no vigorous activity, P = 0.02), and this association was not attenuated by adjustment for IMT, body mass index, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, or diabetes. Repeated analyses with traditional arterial stiffness indices showed similar findings for vigorous but not work activity. CONCLUSION: In contrast to several smaller studies, these findings do not support the hypothesis that habitual physical activity has a strong, consistent positive effect on arterial distensibility.

Arteries↗
PubMed2000

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Adolescent↗
PubMed2000

Transcatheter closure of post-infarction ventricular septal defect with the Amplatzer Septal Occluder device.

There is an 80-90% mortality rate within the first 2 months of the occurrence of a post-infarction ventricular septal defect (VSD) with medical treatment alone. The muscular VSD presents a technical problem for the surgeon. Surgical treatment was unsuccessful in two patients. They were treated successfully using the Amplatzer Septal Occluder, with improvement in their condition.

Aged↗
PubMed2000

An ikaros-containing chromatin-remodeling complex in adult-type erythroid cells.

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching.

Adenosine Triphosphatases↗
PubMed1999

HemT-3, an alternative transcript of mouse gene HemT specific to male germ cells.

A number of genes are known to be expressed primarily in hematopoietic cells and testis and are thought to function in the control of both blood cell and male germ cell differentiation. We have recently identified a mouse gene, HemT, that encodes two alternatively spliced transcripts specific to hematopoietic cells (HemT-1 and HemT-2) and kidney (HemT-2). We have now isolated a third HemT transcript, HemT-3, that is found only in testis by Northern blot analysis and RT-PCR. HemT-3 is alternatively spliced and may be initiated differently from HemT-1 and HemT-2. RNA in-situ hybridization of testis from wild-type and germ-cell-deficient adult mice, as well as from mice at different developmental stages, indicates that HemT-3 is expressed only in early spermatocytes. HemT-3 cDNA has a major open reading frame related to a human glycosylphosphatidylinositol (GPI)-anchored protein, GML. Using an antibody generated against a peptide derived from the HemT-3 open reading frame, we have detected a testis-specific 22kDa protein by Western blot analysis.

Alternative Splicing↗
PubMed1999

A novel mouse gene, HemT, encoding an hematopoietic cell-specific transcript.

We performed differential display to identify hematopoietic cell-specific genes that are expressed differentially before and after mouse erythroleukemia cell (MELC) induction. In this study we report the identification, cloning, and characterization of one such mouse gene, HemT. Using Northern blot, RT-PCR and in situ hybridization, we show that HemT encodes three different transcripts. One of them, HemT-1, is hematopoietic-cell specific and may be erythroid cell-specific. Another, HemT-2, is expressed mainly in hematopoietic cells. HemT-1 and HemT-2 are alternative splicing products. A third type of HemT transcript is expressed only in testis. The erythroid transcript is expressed preferentially in uninduced MELC as compared to induced MELC, and is likely to be expressed in early erythroid cell populations.

Alternative Splicing↗
PubMed1999

Retrovirus-mediated gene transfer of the human multidrug resistance-associated protein into hematopoietic cells protects mice from chemotherapy-induced leukopenia.

Utilization of chemotherapy for the treatment of tumors is mainly limited by its hematological toxicity. Because of the low-level expression of drug resistance genes, transduction of hematopoietic progenitors with multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes should provide protection from chemotherapeutic agent toxicity. Successful transfer of drug resistance genes into hematopoietic cells may allow the administration of higher doses of chemotherapy and, thus, increase regression of chemosensitive tumors. The interest in the use of MRP as an alternative to MDR1 for bone marrow protection lies in its different modulation. This would allow, in the same patient, the use of MDR1 reversal agents to decrease MDR1 tumor resistance without reversing bone marrow (BM) protection of the MRP-transduced hematopoietic cells, since MRP expression is not reversed by these agents. We have constructed MRP-containing retroviral vectors using the phosphoglycerate kinase promoter and generated ecotropic producer cells. Lethally irradiated mice were engrafted with BM cells transduced by coculture with MRP producer cells. Evidence of long-term (9 months) gene transfer was provided by PCR of peripheral blood from MRP-transduced mice. Southern blot analysis confirmed the integrity of the provirus in the MRP-transduced mice. Long-term MRP expression (>5 months) was detected by RT-PCR and fluorescence-activated cell sorting of blood from living mice. High-level expression of MRP in murine hematopoietic cells reduces doxorubicin-induced leukopenia and mortality. Furthermore, we show in vivo selection of MRP-transduced cells following doxorubicin administration, with better and more significant chemoprotection after the second chemotherapy cycle. These data indicate that MRP retroviral gene transfer may be useful for chemoprotection and selection.

ATP-Binding Cassette Transporters↗
PubMed1999

Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching.

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine beta-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult beta-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of beta-globin transcriptional activators.

Animals↗
PubMed1999

Up-regulation of amphotrophic retroviral receptor expression in human peripheral blood CD34+ cells.

Retroviral-mediated gene transfer into hematopoietic stem cells provides the only means of stable transduction of these cells and their progeny for use with a variety of potentially therapeutic genes. Expression of the Moloney amphotropic retroviral receptor-pit-2 or GLVR-2-is critical to the recognition and entry of Moloney leukemia virus-derived viruses into human target cells such as CD34+ hematopoietic cells. GLVR-2 functions as a sodium-dependent phosphate transporter as well as a receptor. We have previously shown that the expression of the murine homologue of the amphotropic receptor Ram 1, also a phosphate transporter, is developmentally regulated in murine hematopoietic fetal liver cells. We also demonstrated that culture of murine fetal liver cells in phosphate-free (PO(4)-free) medium increases levels of receptor mRNA and makes murine fetal liver cells susceptible to Moloney amphotropic viral gene transfer. We now examine the effect of culture conditions on the expression of GLVR-2 in human CD34+ cells. In this report, we demonstrate that there is a 2-3 fold increase in GLVR-2 mRNA levels in CD34+ cells after 3 days in culture with interleukin 3, interleukin 6, and stem-cell factor. In addition, the use of PO(4)-free medium increases expression of GLVR-2 an additional 2-fold in these cells during this time. These results indicate that GLVR-2 expression can be up-regulated on these cells, and may permit improved retroviral gene transfer efficiencies.

Animals↗
PubMed1999

Competitive repopulation of retrovirally transduced haemopoietic stem cells.

Gene transfer into haemopoietic stem cells (HSC) may be useful in gene therapy for a variety of inherited and acquired human diseases. Cell division is required for retroviral transduction, and cytokine stimulation is often used to increase mitosis of quiescent HSC. Exposure to cytokines has been shown to have an unfavourable effect on the engraftment of these cells when competed with unmanipulated HSC. We now show that a similar engraftment defect is present when HSC are manipulated and transduced with the human multiple drug resistance (MDR) gene. The extent of the unfavourable competition depended on the relative numbers of cytokine-treated and fresh cells when the two populations of cells were administered simultaneously into marrow-ablated isogenic mice. When the manipulated transduced cells were given 2 or 4 d before the unmanipulated cells there was a much greater engraftment of the manipulated cells. The data suggested that the manipulated cells were at a relative disadvantage for marrow engraftment as compared to fresh cells, presumably due to the more efficient homing and engraftment properties of these latter unmanipulated cells. However, the manipulated cells had no intrinsic inability to engraft when they were the predominant donor cell population. In all cases the percent of MDR transduced cells in the engrafting manipulated cells remained relatively constant at about 25-30%. These results have implications for the use of manipulated transduced stem cells in gene therapy, suggesting that administering them before adding fresh cells can overcome their engraftment defect.

Animals↗
PubMed1998

Retroviral vectors aimed at the gene therapy of human beta-globin gene disorders.

We are focusing on the development of complex retroviral vectors containing human beta-globin gene and beta-LCR for the gene therapy of sickle cell disease and beta-thalassemias. First generation vectors containing mutated splice-sites to insure stability of proviral transfer enabled long-term reconstitution in 10/12 transplanted mice for a least 8 months with high expression levels in 2 out of 3 mice analyzed (5% and 20% murine beta). Transfer and expression were also achieved in secondary recipients (range: 3-11% murine beta). Position independent expression was not observed. In an effort to increase the efficiency of gene transfer and obtain complete reconstitution of recipient mice with exclusively transduced cells while enriching for proviral integration into active chromatin regions, we have incorporated a cassette expressing CD24 or the green fluorescent protein (GFP). Stable transfer to murine bone marrow cells allowed efficient FACS-sorting of pure populations of transduced cells. A family of vectors based on these principles and containing segments of gamma- or delta-globin genes were also designed for systematic analysis of their anti-sickling properties.

Anemia, Sickle Cell↗
PubMed1998

High-level transfer and long-term expression of the human beta-globin gene in a mouse transplant model.

Insertion of a normally functioning human beta-globin gene into the hematopoietic stem cells (HSC) of patients with beta-thalassemia may be an effective approach to the therapy of this disorder. Safe, efficient gene transfer and long-term, high-level expression of the transferred human beta-globin gene in animal models are prerequisites for HSC somatic gene therapy. We have recently shown for the first time that, using a modified beta-globin retroviral vector in a mouse transplant model, long-term, high-level expression of a transferred human beta-globin gene is possible. The human beta-globin gene continues to be detected up to eight months post-transplantation of beta-globin-transduced hematopoietic cells into lethally irradiated mice. The transferred human beta-globin gene is detected in three of five mice surviving long-term (> 4 months) transplanted with bone marrow cells transduced with high-titer virus. The unrearranged 5.1 kb human beta-globin gene-containing provirus is seen by Southern blotting in two of these mice. More importantly, long-term expression of the transferred gene is seen in two mice at levels of 5% and 20% that of endogenous murine beta-globin. We document stem cell transduction by showing continued high-level expression of the human beta-globin gene in secondarily transplanted recipient mice. These results provide evidence of HSC transduction with a human beta-globin gene in animals and demonstrate that retroviral-mediated unrearranged human beta-globin gene transfer leads to a high level of human beta-globin gene expression in the long term for the first time. A gene therapy strategy may be a feasible therapeutic approach to the beta-thalassemias if consistent human beta-globin gene transfer and expression into HSC can be achieved.

Animals↗
PubMed1998

Feasibility of multidrug resistance (MDR-1) gene transfer in patients undergoing high-dose therapy and peripheral blood stem cell transplantation for lymphoma.

We have performed a pilot study of MDR-1 gene transfer in patients receiving CD34-selected peripheral blood stem cell (PBSC) transplant for lymphoma. To ensure minimum engraftment thresholds and facilitate CD34 purification, mobilisation of > 2 x 10(6) CD34 cells/kg was a condition for recruitment. Of 11 patients counselled for study entry, only five achieved this target in a single apheresis. In three consenting patients, purified CD34 cells were exposed to A12M1 MDR-1 retroviral supernatant for 6 h, cryopreserved then thawed and readministered following ablative chemotherapy. No delay in engraftment was observed, although one patient received additional back-up cells. Gene transfer was demonstrated by polymerase chain reaction (PCR) for vector-derived MDR-1 cDNA sequence in all cases. Analysis of peripheral blood and bone marrow cells after transplant has, however, shown no evidence of in vivo gene transfer with a follow-up of 12, 15 and 18 months. The effect of MDR-1 substrate drugs has not yet been tested as all patients remain in clinical and radiological remission of their lymphoma. These results confirm the difficulty of achieving in vivo gene transfer in human haemopoietic cells and indicate major logistical constraints in PBSC mobilisation in patients with relapsed and resistant disease in whom initial studies are appropriate.

Adolescent↗
PubMed1998

Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation.

PURPOSE: Normal bone marrow cells have little or no expression of the MDR p-glycoprotein product and, therefore, are particularly susceptible to killing by MDR-sensitive drugs, such as vinca alkaloids, anthracyclines, podophyllins, and paclitaxel and its congeners. Here we report the results of a phase I clinical trial that tested the safety and efficacy of transfer of the human multiple drug resistance (MDR1, MDR) gene into hematopoietic stem cells and progenitors in bone marrow as a means of providing resistance of these cells to the toxic effects of cancer chemotherapy. PATIENTS AND METHODS: Up to one third of the harvested cells of patients who were undergoing autologous bone marrow transplantation as part of a high-dose chemotherapy treatment for advanced cancer were transduced with an MDR cDNA-containing retrovirus; these transduced cells were reinfused together with unmanipulated cells after chemotherapy. RESULTS: High-level MDR transduction of erythroid burst-forming unit (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) derived from transduced CD34+ cells was shown posttransduction and prereinfusion. However, only two of the five patients showed evidence of MDR transduction of their marrow at a low level at 10 weeks and 3 weeks, respectively, posttransplantation. The cytokine-stimulated transduced cells may be out-competed in repopulation by unmanipulated normal cells that are reinfused concomitantly. The MDR retroviral supernatant that was used was shown to be free of replication-competent retrovirus (RCR) before use, and all tests of patients' samples posttransplantation were negative for RCR. In addition, no adverse events with respect to marrow engraftment or other problems related to marrow transplantation were encountered. CONCLUSION: These results indicate the feasibility and safety of bone marrow gene therapy with a potentially therapeutic gene, the MDR gene.

Adult↗
PubMed1997

Long-term transfer and expression of the human beta-globin gene in a mouse transplant model.

Somatic gene therapy of hemoglobinopathies depends initially on the demonstration of safe, efficient gene transfer and long-term, high-level expression of the transferred human beta-globin gene in animal models. We have used a beta-globin gene/beta-locus control region retroviral vector containing several modifications to optimize gene transfer and expression in a mouse transplant model. In this report we show that transplantation of beta-globin-transduced hematopoietic cells into lethally irradiated mice leads to the continued presence of the gene up to 8 months posttransplantation. The transferred human beta-globin gene is detected in 3 of 5 mice surviving long term (>4 months) transplanted with bone marrow cells transduced with high-titer virus. Southern blotting confirms the presence of the unrearranged 5.1-kb human beta-globin gene-containing provirus in 2 of these mice. In addition, long-term expression of the transferred gene is seen in 2 mice at levels of 5% and 20% that of endogenous murine beta-globin at 6 and 8 months posttransplantation. We further document stem cell transduction by the successful transfer and high-level expression of the human beta-globin gene from mice transduced 9 months earlier into irradiated secondary recipient mice. These results demonstrate high-level, long-term somatic human beta-globin gene transfer into the hematopoietic stem cells of an animal for the first time, and suggest the potential feasibility of a retroviral gene therapy approach to sickle cell disease and the beta thalassemias.

Animals↗
PubMed1997

Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents.

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.

3T3 Cells↗
PubMed1997

Efficient retroviral gene transfer of a Tat-regulated herpes simplex virus thymidine kinase gene for HIV gene therapy.

We have previously reported that lymphoid and monocytic CD4+ cells transfected with and expressing a herpes simplex virus thymidine kinase (HSV-TK) gene, under the transcriptional control of the HIV long terminal repeat, are protected from HIV spread in the presence of 10 microM acyclovir. Furthermore, the expression of HSV-TK was able to enhance the antiviral effect of AZT. We now investigate the ability of retroviral vectors containing the HSV-TK gene under the transcriptional control of HIV regulatory sequences to transduce target cells. Bone marrow cells or lymphocytes might be genetically modified by these vectors in an HIV gene therapy strategy. In this study, we describe high-titer retroviral producer clones expressing the HSV-TK gene from an HIV LTR. Lymphoid cells transduced with one of these retroviruses express HSV-TK at a relatively low basal level. When the same cells express the HIV regulatory protein Tat, they are 10-fold more sensitive to killing by ganciclovir. Thus, this vector has potential application for gene therapy of HIV infection.

3T3 Cells↗