Tumor cell purging and positive selection of hematopoietic stem cells.
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Antineoplastic Agents↗
Biomedical subjects
A P Gee
Publications and source records attributed to A P Gee.
Design of large-scale separation systems for positive and negative immunomagnetic selection of cells using superparamagnetic microspheres.
The ex vivo selective separation of cells from bone marrow and peripheral blood stem cell preparations is increasingly used as an adjunct to hematopoietic rescue following high-dose therapy for refractory cancer. Immunomagnetic separation, in which the target cells are identified using monoclonal antibodies and separated by attachment to paramagnetic particles and passage through a magnetic field, is widely used for both negative and positive cell selection. In this paper, we discuss the factors that should be considered when developing a magnetic separation device for purging tumor cells and selecting stem cells from bone marrow using superparamagnetic microspheres.
Animals↗
Effects of target antigen density on the efficacy of immunomagnetic cell separation.
Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti-immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly-accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated.
Antibodies, Monoclonal↗
Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion.
The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.
Animals↗
Transporting bone marrow for in vitro purging before autologous reinfusion.
We describe the procedures employed for transporting bone marrow to and from a central facility. Marrow has been harvested from 80 patients with neuroblastoma, at 16 centers which are geographically dispersed throughout North America. Marrow from the outside transplant centers was packed on wet ice or cold packs in insulated containers, and transported by commercial carriers or chartered aircraft to the central processing laboratory. Post processed marrows were frozen in liquid nitrogen and returned by commercial carrier to the referring institution. In comparing transported with non-transported but similarly treated marrows, no differences were found in any of the following parameters: (1) CFU-GM recovery, (2) fraction viable cells at thawing, or (3) time to engraftment in patients. We conclude the transportation of harvested marrows to a central purging facility is safe. Based on this experience, we propose a set of standards, which, if adhered to, will insure the continued safe processing, shipping, and storage of bone marrow in all centers so engaged.
Bone Marrow Transplantation↗
Bone marrow purging and processing--a review of ancillary effects.
This paper differs markedly from the others that are being presented at this symposium, in that it does not describe a particular technique, or have a single unifying theme. Its aim is to draw to the attention of the purging community a list of variables that we all have tended to regard as only secondarily important to our primary goal of target cell depletion, or collection. It is often difficult to remember that ex vivo purging treatments are composed of multiple steps, and that, in turn, purging is a single component of a complex treatment protocol. In order to cope with this diversity, the natural tendency has been to focus on manageable segments. While this can speed development, we need to constantly be aware that there are multiple factors, both within and outside of our own particular segment, that can impact on the final result. Variables that we often regard as unimportant, or techniques that are thought of as routine, may ultimately be influencing clinical outcome. It would be naive to suggest that we can ever have control over all of the variables in any procedure that is part of a clinical treatment. One purpose of this paper is simply to draw the existence of these factors to the attention of investigators, and suggest that their potential impact may not always be fully appreciated. Secondly, the selective separation of cells from bone marrow is sufficiently well established that it should now be possible to evaluate the technology, reagents and variables in a systematic and collaborative fashion. While we may not emerge from such an exercise with unity of option, we may at least lay the foundations for the further development of this type of therapy, and provide the framework for addressing future questions.
Bone Marrow↗
Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres.
A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.
Animals↗
T-cell depletion of human bone marrow.
The myelo-ablative effects of high-dose therapy of refractory cancer can be overcome by the transplantation of bone marrow from an HLA-matched normal donor. Suitable donors are available for only one patient in three, and even minor disparities at HLA loci can produce graft-versus-host disease (GvHD) in transplant recipients. Depletion of T lymphocytes from the marrow in vitro can reduce the incidence and severity of GvHD. In this paper we review the use of immunomagnetic cell separation for the depletion of mature T cells from bone marrow. This procedure uses monoclonal antibodies to identify the target cells. These are then rosetted with anti-immunoglobulin-coated paramagnetic microspheres and collected by exposure of the marrow to a magnetic field. Factors impacting the efficiency of the separation, including choice of anti-immunoglobulin and monoclonal antibodies, incubation conditions and methods for residual cell detection, are outlined. The relative limitations and advantages of the method are discussed in relation to other techniques. It is concluded that the flexibility of the immunomagnetic procedure, in its ability to be used for both positive and negative selection of T-cell subsets, or for pan-T-cell depletion, could make it the method of choice in this application.
Antibodies, Monoclonal↗
Low antigen density leukemia cells: selection and comparative resistance to antibody-mediated marrow purging.
Low tumor-associated antigen (TAA) expressing tumor cells present an obstacle to effective antibody directed purging of tumor cells from bone marrow. In this study, a comparison was made of the efficiency with which low TAA expressing leukemia cells could be depleted using two monoclonal antibody (MoAb) directed purging techniques: 1) complement (C)-mediated cytolysis, and 2) physical separation using magnetic microspheres. Low TAA sublines were selected from a cultured human leukemia cell line by growing out cells remaining after treatment with anti-TAA and C, or after immunomagnetic (IM) purging. IM-selected sublines showed lower TAA expression than did C-selected sublines, and sublines resulting from multiple selections expressed less TAA than those that had only been through one selection. These sublines were then examined for sensitivity to C or IM purging. The highly selected, lowest TAA expressing sublines were markedly resistant to both IM and C. Less selected sublines were resistant to C, but not to IM. In both techniques, addition of MoAbs against a second TAA restored the efficiency of purging to that observed with the parental line. When low TAA subline cells were seeded into simulated bone marrow and subjected to purging, C-mediated lysis removed less than 40% of leukemia cells, whereas IM purging removed 85% of the cells. These results indicate that there are low antigen density cells that are resistant to C-mediated purging, but which retain sensitivity to IM removal.
Antibodies, Monoclonal↗
Bone marrow transplantation (BMT) for advanced neuroblastoma (NBL): a multicenter POG pilot study.
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Adolescent↗
Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo.
The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.
Animals↗
T lymphocyte depletion of human peripheral blood and bone marrow using monoclonal antibodies and magnetic microspheres.
It has previously been demonstrated that graft-versus-host disease can be overcome in patients receiving HLA-mismatched bone marrow transplants by prior in vitro depletion of T lymphocytes from the marrow. In this report we describe the use of monoclonal antibodies and magnetic microspheres for the depletion of T cells from peripheral blood and bone marrow. The target cells are sensitized with antibodies directed against the CD2, CD3, CD4 and/or CD8 cell surface antigens, captured by magnetic beads coated with sheep anti-mouse IgG antibody and collected by placing the cell suspension in a magnetic field. This simple, rapid procedure results in the efficient removal of T cells from peripheral blood and from bone marrow without affecting the colony-forming potential of normal hematopoietic stem cells. The procedure is capable of being scaled up for the treatment of larger volumes of marrow that are required for clinical transplantation.
Antibodies, Monoclonal↗
Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.
The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.
Anthracenes↗
Studies on the terminal stages of immune hemolysis. VI. Osmotic blockers of differing Stokes' radii detect complement-induced transmembrane channels of differing size.
We have previously shown that 0.1 M EDTA could be used to distinguish functionally different transmembrane channels produced during complement-(C) mediated hemolysis of E. In this paper we have studied the ability of sugars of varying Stokes' radii to prevent hemoglobin release from E intermediates whose lysis was inhibitable or not inhibitable by EDTA. On the basis of these experiments we propose that the inhibition of E transformation by high molarity EDTA occurs by virtue of the size of the EDTA molecule in solution. Studies on the effect of EDTA on red cell lysis induced by polyene antibiotics that form transmembrane channels of a defined size support this conclusion. The results of these experiments were interpreted to mean: 1) The EDTA inhibitable lesion of E has a smaller effective radius than the noninhibitable lesion; 2) the effective radius of the smallest lesion that yields a lytic site was less than 3.6 A; 3) the lesions produced in the red cell membrane by C are not uniform but vary in size depending on the C9 to SACl-8 ratio used to produce E.
Animals↗