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Biomedical subjects

T E Thomas

Publications and source records attributed to T E Thomas.

At least 19 recordsLinked to original sources
PubMed2001

A negative-selection strategy for depleting myeloma cells from patients' BM and/or leukapheresis blood.

BACKGROUND: Autologous transplantation improves survival in multiple myeloma patients, however, most eventually relapse. As an attempt towards improving relapse-free survival, we designed a negative-selection purging strategy, to remove myeloma cells from leukapheresis harvests using MAbs specific for Ags on myeloma cells. METHODS: CD38 is highly expressed on myeloma plasma cells, but expressed at lower levels on normal progenitors and absent on in vivo repopulating cells. We evaluated depletion of CD38-expressing cells, with or without depletion of B-cell Ag-expressing cells. Using myeloma BM or blood cells diluted into allogeneic G-CSF primed leukapheresis cells, bispecific tetrameric Ab complexes that bind dextran iron particles were used to label and retain cells in a magnetic column, StemSep. Depletion efficacy was measured by semi-quantitative allele-specific oligonucleotide (ASO)-PCR amplification of patients' clonotypic IgH gene. RESULTS: Low (0.2 microg/mL) concentrations of anti-CD38 with CD19 and CD20 complexes depleted approximately 3-5 logs of clonotypic cells, with recovery of approximately 19% of colony-forming cells, approximately 50% primitive progenitors measured by LTCIC and retention of non-obese diabetic /SCID engrafting ability. Scale-up experiments using leukapheresis harvests and 0.5-1 x 10(10) cell capacity columns demonstrated no loss of log depletion of highly positive cells, or recovery of unlabelled cells. DISCUSSION: These results compare favorably with other purging techniques and allow the retention of most normal BM cells, including T cells, which may be important for immunity. These results support the development of a clinical trial using this strategy for purging myeloma cells.

Animals↗
PubMed1999

Purification of hematopoietic stem cells for further biological study.

For many years, the hematopoietic system has provided a convenient and fascinating model for studies of the molecular processes regulating cell growth and differentiation. However, this system also poses considerable challenges because the most primitive "stem" cells as well as their initial differentiating progeny are normally present in hematopoietic tissues at extremely low frequencies and no unique, stable phenotype has yet been identified to allow hematopoietic cells with specific stem and progenitor functions to be measured directly. Rather, this requires the use of functional assays that detect their developmental properties and take several weeks to complete. Accordingly, many investigations of primitive hematopoietic cell behavior and their responses to molecular cues in the environment have relied on the development of cell separation techniques specifically designed for obtaining highly enriched populations of primitive hematopoietic cells. Key to these procedures is the use of a preenrichment step(s) in which differences in cell density, size, or sensitivity to pharmacological agents or surface phenotype are exploited to first "debulk" the sample. This step can then be followed by a more selective antibody-mediated procedure to generate useful numbers of highly purified cells. Batchwise immunoadsorption techniques offer many advantages for obtaining enriched populations of hematopoietic progenitors because they avoid the nonspecific toxicity seen with antibody-mediated cell killing and are suitable for rapidly processing large samples. For any cell separation procedure, a balance must be struck between the purity and the recovery of the desired cells because steps to increase cell purity usually reduce yields. Both the negative and the positive selection techniques are useful strategies but negative selection usually requires one less manipulation step and circumvents potential effects incurred by the presence of antibody on the surface of the cell being isolated. Specific details for the use and results obtained with an immunomagnetic negative column selection technique are then presented.

Animals↗
PubMed1999

Retroviral marking of acute myelogenous leukemia progenitors that initiate long-term culture and growth in immunodeficient mice.

Rare primitive progenitors among the malignant cells from most patients with AML include AML long-term culture-initiating cells (AML LTC-IC) and NOD/SCID mouse leukemia-initiating cells (NOD/SL-IC). To evaluate the feasibility of genetic modification of these progenitors for gene marking and/ or gene therapy strategies, cells from patients with newly-diagnosed AML were cocultured with retroviral producer cells and then placed in colony (AML-CFC) assays, LTC, and injected intravenously into NOD/SCID mice. Southern blotting demonstrated transfer of the neo(r) gene to 30% to 80% of leukemic blasts when cells were cultured for 48 hours in the presence of IL-3 and steel factor (SF) prior to 48-hour coculture with viral producers. Three of six retrovirally-infected AML samples showed both engraftment in NOD/SCID mice and the presence of the neo(r) transgene in mouse tissues 8-15 weeks after injection of transduced cells. Thirteen weeks after injection of one of these samples, >80% of cells from mouse bone marrow were the progeny of two retrovirally-transduced AML progenitors. Four of the remaining five samples showed markedly reduced ability to engraft in mice after retroviral infection. Subsequent experiments demonstrated that the loss of engraftment potential took place within 24 hours of culture initiation in the absence of retroviral producers and regardless of the cytokines present. Interestingly, the majority of AML-CFC or AML LTC-IC survived the 24-hour culture period. A retroviral vector containing the murine cell surface marker heat stable antigen (HSA), which allows purification of transduced cells on immunomagnetic columns, was used to obtain an enriched population of gene-modified AML cells following an infection protocol that eliminated the 48 hours of prestimulation in IL-3 and SF and reduced coculture with viral producers to 10-36 hours. These modifications failed to improve engraftment of the infected cells. In addition, in these experiments more than 10 hours of cocultivation with viral producer cells was necessary to achieve gene transfer and expression in AML LTC-IC. These data demonstrate that although retroviral-mediated gene transfer can be achieved to AML progenitors, including NOD/SL-IC, improved culture conditions will be required before substantial numbers of such transduced primitive progenitors can be obtained. In addition, the difference in the ability of AML LTC-IC and NOD/SL-IC to survive ex vivo suggests that these assays may detect different populations of cells or that changes are induced in vitro in primitive cells which can only be detected in the mouse assay.

Acute Disease↗
PubMed1996

Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells.

In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.

Adenoviruses, Human↗
PubMed1996

Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow.

The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, and enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction-based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34-cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.

Adolescent↗
PubMed1995

Nonlinear effects of radiation dose on donor-cell reconstitution by limited numbers of purified stem cells.

Despite the increasing use of bone marrow transplantation (BMT) as a treatment for a wide variety of diseases, the numbers and types of cells required for both rapid and sustained recovery of hematopoiesis are not well defined. To investigate further the potential of transplants consisting of highly enriched populations of long-term repopulating cells, we transplanted a series of Ly-5.2 mice given various doses (750, 800, 850, 900, or 950 cGy) of total-body irradiation (TBI) with 30 or 90 Sca-1+Lin-WGA+ marrow cells isolated from congenic Ly-5.1 donors. As expected, mature progeny derived from these cells, belonging to both myeloid and lymphoid compartments, could be detected with increasing case in recipients given radiation doses from 750 to 900 cGy TBI. Surprisingly, expression of this potential was significantly reduced in mice that had received 950 cGy TBI. This contrasts with the capacity of the same number of purified Sca-1+Lin-WGA+ cells to generate readily detectable progeny in 950 cGy treated mice given a simultaneous transplant of 10(5) normal marrow cells or 2 x 10(5) serially passaged marrow cells. We suggest that this variable behavior of purified stem cells in differently treated recipients may reflect radiation dose-dependent differences in the types or levels of expression of factors that regulate transplanted stem cell proliferation and differentiation in vivo and that above a certain threshold radiation dose, this may result in an irreversible loss of long-term reconstituting potential. Regardless of the nature of the underlying mechanism, this study shows that the extent of donor repopulation after BMT can be a function not only of the number of stem cells transplanted but also of the conditioning of the recipient and whether other cell types are also injected.

Animals↗
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↗
PubMed1992

Designing the next generation of American hospitals.

Change has always been a part of the hospital's operating environment, but the changes predicted for American hospitals during the next two decades pose considerable challenge to those who manage health care services and those who design medical facilities. The next generation of American Hospitals are responding to changes in technology, availability of human resources and demand of the public, private and government interest groups. Hospitals and architects that understand the predictable changes and anticipate the unknown, will be able to plan a facility that responds to the needs of future health care delivery.

Forecasting↗
PubMed1992

Designing the next generation of American hospitals.

Change has always been a part of the hospital's operating environment, but the changes predicted for American Hospitals during the next two decades pose considerable challenge to those who manage health care services and those who design medical facilities. The next generation of American Hospitals are responding to changes in technology, availability of human resources and demand of the public, private and government interest groups. Hospitals and architects that understand the predictable changes and anticipate the unknown, will be able to plan a facility that responds to the needs of future health care delivery.

Aged↗