Terry E. Thomas

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, an 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.

Rapid ammonium uptake and nitrogen interactions in five intertidal seaweeds grown under field conditions

Time courses of ammonium and nitrate uptake rates were determined for five intertidal macrophytes collected directly from the field. There were two perennials, Pelvetiopsis limitata Gardner and Fucus gardneri Silva, and three annuals, Mastocarpus papillatus (C. Ag. Kutzing, Gracilaria pacifica Abbott and Enteromorpha intestinalis (L.) Grev. When a 15-μM pulse of ammonium was added to the incubation medium, all three annuals and one of the two perennials, Fucus gardneri, showed enhanced ammonium uptake rates for the first 15 min of a 30-min exposure. This is the first report of non-linear nutrient depletion by plants grown in the field. There was no enhancement in the initial nitrate uptake rate. In fact, nitrate uptake had to be induced in the low intertidal Gracilaria pacifica by incubating it in 30 μM nitrate for 20 min. All species took up both ammonium and nitrate simultaneously. Enteromorpha intestinalis and the high intertidal Gracilaria pacifica exhibited a 50% decrease in nitrate uptake in the presence of ammonium. Ammonium concentrations of > 5 μM were required to inhibit nitrate uptake in the former.

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.

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.

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.

Cell separation improves the sensitivity of detecting rare human normal and leukemic hematopoietic cells in vivo in NOD/SCID mice

BACKGROUNDnThis report describes a novel cell-separation procedure developed to improve detection and analysis of rare human hematopoietic populations, obtained from NOD/SCID mice engrafted with normal and/or leukemic stem cells.nnnMETHODSnIn preliminary experiments, artificial mixtures of murine and human BM cells were labeled with a combination of Abs specific for murine hematopoietic cells, prior to immunomagnetic negative selection using StemSep. In subsequent experiments, BM was harvested from individual NOD/SCID mice transplanted 6-12 weeks earlier with either human cord blood or primary CML cells and a similar immunomagnetic selection procedure was applied to enrich human cells present.nnnRESULTSnApplication of this selection procedure to mixtures of murine and human hematopoietic cells using anti-mouse CD45 and Ter-119 allowed a > 1000-fold depletion of murine cells with > 50% recovery of human cells, including progenitors. This level of depletion and recovery were found to be reproducible for NOD/SCID mice transplanted and engrafted with human cord blood stem cells, thus facilitating detection of human progenitors, including colony-forming cells (CFC) and LTCIC. For NOD/SCID mice previously transplanted with CML cells, this procedure increased the sensitivity of detecting rare human cell subsets by up to > 100-fold. This, in turn, improved the sensitivity of RT-PCR for BCR-ABL and made possible the identification by FACS of various minor subsets of human cells, including CD34(-)CD19/20(+) B-lineage cells, CD34(+) progenitors, mature CD15(+) myeloid cells and CD3(+) T cells present in the mice.nnnDISCUSSIONnThis simple cell-depletion procedure should facilitate future investigations of normal and CML stem cell populations in vitro and in NOD/SCID mice.

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.