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T E Thomas

Publications and source records attributed to T E Thomas.

17 recordsLinked to original sources
PubMed1994

Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.

The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.

Adolescent↗
PubMed1994

Selective separation of cells using magnetic colloids.

We have developed a reliable, purely immunological means of quantitatively labeling cells with magnetic colloidal dextran iron. Labeled cells can be efficiently separated with a high gradient magnetic filter inside a magnetic field. Separation conditions and filter design can be changed to accommodate large and small scale positive or negative selection. This separation technique has been applied to tumor cell depletions, stem cell enrichment, and large scale preclinical T-cell depletions.

Animals↗
PubMed1994

Age-related decline in proliferative potential of purified stem cell candidates.

Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here. Extensive qualitative changes in hematopoietic cells from various stages of development should be taken into account in the design of novel therapeutic strategies.

Adult↗
PubMed1993

Positive selection of human blood cells using improved high gradient magnetic separation filters.

High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool. Wire elements arranged regularly in a filter should allow more efficient separation of cells. Filters were constructed containing ordered wire arrays composed of 430 series stainless steel wire mesh with wire diameters of 50, 100, or 150 microns. The ability of these filters to separate T cells from peripheral blood mononuclear cell suspensions was tested and found superior to random arrays of 302 series stainless steel wire (Thomas et al, 1992). Target cells recognized by OKT5 monoclonal antibody were cross-linked to dextran-iron particles of approximately 20 nm in diameter. Separation conditions were optimized and after one passage through the filter 88% of the OKT5+ cells were recovered in the enriched fraction with 85% purity (%OKT5+). Multiple passages (3 times) could achieve 99% purity with 68% recovery. Variations in separation flow rate had a large effect on the balance between purity and recovery. Optimum separation efficiencies were achieved only when > 10(8) cells were processed. The primarily cause of nonspecific entrapment of CD8- cells was not nonspecific magnetic labeling of cells but the physical (nonmagnetic) characteristics of the filter/filter chamber.

Adult↗
PubMed1992

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens.

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (less than 0.2 microns) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.

Antibodies, Monoclonal↗
PubMed1992

Use of lectins for characterization and purification of human bone marrow cells that express CD34.

The binding of lectins to nucleated cells from human bone marrow was studied in a search for markers that can be used to subdivide further immature hemopoietic cells that are characterized by their expression of CD34. Low-density bone marrow cells were indirectly labeled with biotinylated lectins and streptavidin-R-phycoerythrin (SA-RPE) together with FITC-labeled monoclonal anti-CD34. Four-parameter flow cytometric analysis was then performed and list mode data analyzed. Of the 21 lectins tested, only a few showed differential staining of CD34+ versus CD34- cells. These include soybean agglutinin (SBA) and Ulex europaeus agglutinin I (UE). Lycopersicon esculentum (LE) and Erythrina cristigalli (EC) reacted preferentially with, respectively, CD34+ and CD34- cells, suggesting their usefulness in some method to enrich for CD34+ cells. This possibility was tested by passing cells labeled with biotinylated lectins over a column containing streptavidin-coated beads. CD34+ cells could be enriched > 10-fold by competitive (sugar) elution of LE-labeled cells from the column. Similarly, depletion of biotinylated EC-labeled cells by passage through the streptavidin column enriched CD34+ cells several fold. The results of these studies document the reactivity of a large panel of lectins with subpopulations of nucleated bone marrow cells and indicate that certain lectins could possibly be used for development of cell separation procedures aimed at the selective enrichment of cells that express CD34.

Antigens, CD↗
PubMed1992

Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures.

Clinical uses of gene transfer to bone marrow transplants require the establishment of a reproducible method for infecting large numbers of very primitive hematopoietic cells at high efficiency using cell-free retrovirus-containing media. In this study we report the results of experiments with preparations of a high-titer (2-5 x 10(7)/ml) helper-free recombinant neo(r) retrovirus that indicate this goal can now be achieved based on measurements of gene transfer efficiencies to cells referred to as long-term culture initiating cells (LTC-IC) because they give rise to clonogenic cells after greater than or equal to 5 wk in long-term culture (LTC). Intermittent, repeated exposure of normal human marrow mononuclear cells to virus-containing supernatant over a 3-d period of cell maintenance on an IL-3/granulocyte colony-stimulating factor (G-CSF) producing stromal layer resulted in gene transfer efficiencies to LTC-IC of 41%; a level previously obtainable only using co-cultivation infection techniques. Marrow cells enriched greater than or equal to 500-fold for LTC-IC (1-2% pure) by flow cytometry showed gene transfer efficiencies of 27% when infected in a similar fashion over a shorter period (24 h), but in the presence of added soluble IL-3 and G-CSF without stromal feeders, and this increased to 61% when Steel factor was also present during the infection period. By using a less highly enriched population of LTC-IC obtained by a bulk immunoselection technique applicable to large-scale clinical marrow harvests, gene transfer efficiencies to LTC-IC of 40% were achieved and this was increased to 60% by short-term preselection in G418. Southern analysis of DNA from the nonadherent cells produced by these LTC over a 6-wk period provided evidence of clonal evolution of LTC-IC in vitro. Leukemic chronic myelogenous leukemia LTC-IC were also infected at high efficiency using the same supernatant infection strategy with growth factor supplementation. These data demonstrate the feasibility of using cell-free virus preparations for infecting clinical marrow samples suitable for transplantation, as well as for further analysis of human marrow stem cell dynamics in vitro.

Base Sequence↗
PubMed1991

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.

Animals↗
PubMed1990

Purification and analysis of bispecific tetrameric antibody complexes.

In order to study the type and yield of immune complexes obtained by the mixing of purified F(ab')2 fragments of rat monoclonal antibodies specific for mouse IgG1 with equimolar amounts of purified mouse IgG1 size exclusion HPLC of the reaction mixture was performed. Immune complexes eluted as a single peak at a position compatible with a tetrameric antibody complex configuration. The yield of tetramers could be increased by incubation of the antibody mixture for several hours at 37 degrees C, indicating a preference of the tetrameric composition over other immune complex compositions. Size exclusion HPLC also showed that greater than 80% of purified tetramers retained their original dimensions after storage for 1 year at 4 degrees C, thus indicating the long-term stability of tetrameric antibody complexes. When complexes were prepared with a mixture of two different mouse IgG1 antibodies, bispecific tetramers were obtained that could be separated from monospecific tetramers using DEAE-HPLC. Purified bispecific antibody complexes of mouse IgG1 anti-CD34 (My10) cross-linked to mouse IgG1 anti-desferal with F(ab')2 rat anti-mouse IgG1 were useful for the purification of cells expressing CD34 from human bone marrow. For this purpose cells were labelled with the antibody complexes, selectively adsorbed onto columns containing desferal coated glass beads and then selectively eluted by treatment with dithiothreitol resulting in reductive cleavage of the disulfide bonds of the F(ab')2 fragments. This relatively simple cell fractionation technique illustrates the unique cross-linking properties of bispecific tetrameric antibody complexes. The procedure appears useful for further studies of hemopoietic cells and bone marrow transplantation.

Antibodies↗
PubMed1989

Specific binding and release of cells from beads using cleavable tetrameric antibody complexes.

A two-step separation procedure is described for the positive selection of cells based on their reactivity with mouse monoclonal antibodies. In the first step cells are specifically cross-linked to hapten-modified glass beads using tetrameric monoclonal antibody complexes. In the second step bound cells are selectively eluted by reductive cleavage of the tetrameric antibody complexes. The latter are comprised of two mouse IgG1 monoclonal antibodies (one recognizing a cell surface antigen on target cells and the other a hapten coupled to the glass beads) bound together by two F(ab')2 fragments of rat anti-mouse IgG1 monoclonal antibody. The complexes provide a specific cleavable cross-link between cell and bead because the disulfide bonds between the two Fab' arms of the F(ab')2 fragments can be broken under relatively mild conditions using dithiothreitol. This specific cleavage of the cross-linker allows elution of the specifically absorbed cells without co-elution of non-specifically bound cells. This is shown in the purification of CD3+ T cells from human peripheral blood, where the removed fractions were over 90% pure and approximately 50% of the positive cells were recovered. Separation of cells labelled with limiting amounts of tetrameric antibody complexes demonstrated that this separation technique was also effective for the purification of cells expressing low amounts of antigens. This was confirmed by the purification of CD34-positive cells from human bone marrow. With this approach, colony-forming cells were enriched 15-24-fold over density separated marrow.

Antibodies, Monoclonal↗
PubMed1988

Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies.

Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin. Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclonal antibodies and monoclonal rat anti-mouse IgG1. Optimum conditions for depletion of CD8 positive cells from peripheral blood mononuclear cell suspensions were determined with 1 ml columns. 90-99% of the CD8 positive cells could be removed with 0-17% non-specific adsorption of CD8 negative cells. The weakest link in this system was the bond between hapten-modified albumin and the glass beads. These results indicate that tetramolecular antibody complexes are useful for the specific immunoadsorption of cells to a defined affinity matrix.

Antibodies, Anti-Idiotypic↗
PubMed1987

Use of tetrameric antibody complexes to stain cells for flow cytometry.

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.

Animals↗
PubMed1977

NMR in cancer: XVII. dewar for a 53-inch superconducting NMR magnet.

A giant nitrogen-jacketed liquid-helium metal dewar built in this laboratory is described for housing 53-inch superconducting magnet used in the human FONAR experiments. This dewar is 10 feet tall, 6 feet wide, 18 inches deep, and weighs 1 1/2 tons. It consists of the main magnet hoop connected through a demountable gooseneck to a liquid helium reservoir tank.

Electromagnetic Phenomena↗