PubMed Health⌕ Search

Biomedical subjects

T J Farley

Publications and source records attributed to T J Farley.

10 recordsLinked to original sources
PubMed1999

Human CD34+ hematopoietic cells transduced by retrovirus-mediated interferon alpha gene maintains regeneration capacity and engraftment in NOD/SCID mice.

To achieve long-term expression of human interferon alpha-5 (IFNalpha) gene in the bone marrow (BM) hematopoietic microenvironment, replication-deficient retroviral vector LSN-IFNalpha was used to deliver the IFNalpha gene into human BM CD34+ cells. After fibronectin-facilitated transduction, a fraction of CD34+ cells was plated in methylcellulose medium with or without G418 to assess transduction efficiency and the effect of IFNalpha gene transfer on colony formation. Colony-forming assay in the presence of G418 (400 microg/mL) revealed that 41% CFU-GM colonies are G418 resistant after infection with LSN-IFNalpha retrovirus. There was no significant difference in CFU-GM/BFU-E colony formation among IFNalpha gene-transduced CD34+ cells, control vector (LXSN) transduced-CD34+ cells and nontransduced CD34+ cells. Another portion of CD34+ cells was grown in liquid medium to measure IFNalpha production. RIA revealed that IFNalpha gene-transduced CD34+ cells produced 72.2 +/- 15.4 U/mL (10(6) cells/24 hours) of IFNalpha compared with 8.3 +/- 2.1 U/mL and 4.3 +/- 1.2 U/mL in LXSN-transduced or nontransduced CD34+ cells, respectively. The remaining portion of transduced CD34+ cells was transplanted into immunodeficient (NOD/SCID) mice to allow analysis of long-term expression of IFNalpha. Transplantation of 1x10(6) CD34+ cells into sublethally irradiated NOD/SCID mice showed that IFNalpha and neo(r) mRNA were detectable in engrafted mouse BM cells for up to 6 months. We conclude that continual local expression of IFNalpha in transduced CD34+ cells does not impair either CD34+ cell growth and differentiation or engraftment and long-term survival in NOD/SCID mice.

Animals↗
PubMed1999

Distinct effect of retroviral-mediated IFN-alpha gene transfer on human erythroleukemic and CD34+ cell growth and differentiation.

Human interferon-alpha (IFN-alpha) has been used in the management of leukemia, but its diverse adverse effects may influence the ability of IFN-alpha to treat this disease. We constructed two retroviral vectors, LSN-IFN-alpha and LNC-IFN-alpha, in which IFN-alpha cDNA was driven by viral LTR and CMV promoters, respectively. After transduction into the PA317 and PG13 retroviral packaging cells, high titers of retrovirus were produced and were used to infect K562 and human BM CD34+ hematopoietic cells. The IFN-alpha gene expression in transduced K562 cells was confirmed by Northern blot, RT-PCR, RIA, and biologic assay. Cell proliferation and cell viability in IFN-alpha-transduced K562 cells were significantly suppressed as compared with control K562 cells. Although the IFN-alpha expression in K562 cells did not affect BCR/ABL expression, it apparently upregulated the production of adhesion molecules (VLA-4 and Mac-1). We evaluated the effect of IFN-alpha gene transfer on human CD34+ cells infected with LSN-IFN-alpha retrovirus with the aid of fibronectin (FN) fragment CH-296 and growth factors. RIA showed that IFN-alpha-transduced CD34+ cells produced 72.2+/-15 U/ml of IFN-alpha compared with 4.3+/-1.2 U/ml in control CD34+ cells. Methylcellulose clonogenic assay indicated that IFN-alpha-transduced CD34+ cells produced similar numbers of burst-forming units-erythrocytes (BFU-E)/colony-forming units-GM (CFU-GM) colonies as compared with control CD34+ cells. Selected colonies expressed IFN-alpha and neo(r) mRNA, as measured by RT-PCR. These studies indicate that retrovirus-mediated IFN-alpha gene transfer may provide a useful tool for studying the effect of IFN-alpha gene transfer on leukemic cells and long-lived CD34+ cells.

Antigens, CD↗
PubMed1999

Adenovirus and retrovirus mediated interferon alpha gene transfer into CD34+ cells maintains regeneration capacity and enhances adhesion molecules in K562 cells.

BACKGROUND: Systemic administration of interferon-alpha (IFN-alpha) results in cytogenetic remissions and enhanced survival in a significant percentage of patients with chronic mylogenous leukemia (CML) and lymphoma. However, this treatment is associated with deleterious toxic effects. Gene transfer of the IFN-alpha gene into hematopoietic progenitors represents a novel strategy to deliver high concentrations of IFN-alpha to a local area. METHODS: We compared the effect of the transfer of the IFN-alpha gene on the cell growth and differentiation of several CD34+ cells in culture and in a NOD/SCID animal model, using adenovirus and retrovirus constructs. RESULTS: Transient local expression of the IFN-alpha gene using an adenovirus vector was associated with normal proliferation of CD34+ progenitors as measured by a colony forming unit of granulocyte-macrophage (CFU-GM) growth. Flow cytometric determination revealed that there was no significant difference in viability of these cells for 24-hour transduction periods. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of RNA from CD34+ harvested CFU-GM progenitors demonstrated expression of IFN-alpha mRNA; radioimmunoassay (RIA) revealed that transduced cells secreted substantial levels of IFN-alpha protein. Furthermore, we constructed a retroviral vector in which IFN-alpha cDNA was driven by a viral LTR promoter to evaluate the effect of permanent IFN-alpha gene expression on cell growth. Retroviral packaging cells PA317 with high titers of retrovirus were produced and used to infect CD34+ and K562 cells. RIA showed that IFN-alpha-transduced CD34+ cells (with the aid of fibronectin fragment CH-296) produced approximately 400 units of IFN-alpha protein compared to CD34+ cells, or cells transduced with empty vector. IFN-alpha transduced CD34+ generated similar numbers of CFU-GM colonies as compared to control CD34+ cells. Engraftment of CD34+ cells transduced with IFN-alpha gene in NOD/SCID mice was successful for the first 30 days. Additionally, we studied the effect of local IFN-alpha expression on the cellular adhesion molecules, VLA-4, Mac-1, ICAM-1, and L-selectin in K562 cells, and human umbilical endothelial vein cells. K562 cells transduced with the IFN-alpha gene expressed a significantly elevated level of VLA-4, Mac-1, and ICAM-1. CONCLUSIONS: We conclude that expression of the IFN-alpha gene using retrovirus vectors results in an adequate localized expression of IFN-alpha mRNA and protein.

Adenoviridae↗
PubMed1997

An intralaboratory quality control program for quantitation of CD34+ cells by flow cytometry.

This study was undertaken to develop a quality control protocol to monitor instrument, operator, and CD34 assay performance. A dual level control system was established by cryopreserving aliquots of cells from peripheral blood progenitor cell (PBPC) collections exhibiting different percentages of CD34+ cells. Twenty-five samples from each control specimen were assayed to establish a control range (mean +/- 2 SD). Levey-Jennings graphs were prepared for each control specimen to plot multiple measurements of CD34%. No significant differences were observed between fresh or cryopreserved PBPC aliquots in terms of light scatter properties or CD34 antigen density within the gated cell population. Cryopreserved PBPC samples are ideal for serving as a positive methodology control for daily CD34 analysis. Furthermore, such a system can help identify problems with assay reagents, sample preparation technique, or incorrect data analysis.

Antigens, CD34↗
PubMed1997

Optimization of CD34+ cell selection using immunomagnetic beads: implications for use in cryopreserved peripheral blood stem cell collections.

Isolation of CD34+ cells from bone marrow, umbilical cord blood, and mobilized peripheral blood stem cell (PBSC) collections has many potential clinical benefits. The aim of this study was to evaluate the use of the ISOLEX 300 system to select hematopoietic precursors and determine the effectiveness at depleting contaminating tumor cells from cryopreserved/thawed PBSC. Median recovery of CD34+ cells and CFU-GM colonies was 71% and 51.5%, respectively, using a protocol optimized for our laboratory. A mean 2.9 log10 decrease in contaminating breast carcinoma cells was seen after the selection process. Selected CD34+ cells underwent a second round of cryopreservation/thawing while retaining 85.6% viability and 72.3% recovery of CFU-GM colonies.

Antibodies, Monoclonal↗
PubMed1994

The combined use of soybean agglutinin and immunomagnetic beads for T lymphocyte subset depletion of bone marrow allografts: a laboratory analysis.

The prevention of graft-versus-host disease by T-lymphocyte depletion of allografts prior to bone marrow transplantation has resulted in an increase in graft failure/rejection and relapse of disease. Evidence for the selective roles of specific T-lymphocyte subsets in each of the engraftment, graft-versus-host disease, and disease relapse processes has been presented, but sufficient clinical verification to support any hypothesis in this regard is lacking. In this paper we describe a convenient and highly flexible clinical laboratory method for depletion of specific T-lymphocytes in controllable quantities by the use of select monoclonal antibodies. Almost 3 log10 removal of CD2+ and CD8+ cells without significant loss of hematopoietic progenitors (CFU-GM, BFU-E, and CD34+ cells) can be reproducibly achieved. This method, employing soybean agglutination and immunomagnetic beads, is potentially adaptable to depletion of any cell subset or any tumor cell for which unique cell-surface antigen characteristics have been defined. In addition, our protocol is equally suited to the positive selection of stem cells and hematopoietic progenitors bearing the CD34 surface antigen.

Bone Marrow Transplantation↗
PubMedDate not supplied

Serial MRI and CT findings in infantile Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive childhood disorder characterized by severe motor and mental deterioration. The disease has been divided into 3 main types and further subdivided into several subtypes based on age of onset and symptoms. Initial clinical findings and magnetic resonance imaging (MRI) with several follow-up scans are presented to identify the order and extent of white matter involvement and developing brain atrophy in a child with the floppy infant variant of Krabbe disease. When the patient's clinical condition proceeded to stage 2, MRI disclosed severe involvement of the deep white matter around the atria and posterior limbs of the internal capsules. At the same time there was progression of normal myelination around the frontal horns. At age 32 months, the patient's clinical condition proceeded to stage 3; she did not interact with her environment. MRI revealed a significant decrease of white matter volume, generalized atrophy, and abnormal high signal in all white matter areas except the anterior limbs of the internal capsules. At the same time the volume of the central gray nuclei was decreased and also demonstrated abnormal high signal. Despite its sensitivity, MRI could not differentiate the findings of this variant of Krabbe disease from the classic form; therefore, subclassifications of Krabbe disease should be made on clinical grounds because they cannot be distinguished by biochemical or radiologic (MRI) criteria.

Brain↗