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  • 1
    Publication Date: 1993-05-01
    Description: Self-renewal and differentiation of B-cell precursors is dependent on interactions with bone marrow (BM) stromal cells and associated extracellular matrix. We have recently developed an interleukin (IL)-7- dependent, BM-derived stromal cell culture that supports the growth of normal human B-cell precursors. In the current study, we have characterized the constitutive expression, cytokine-regulated expression, and function of adhesion molecules on BM stromal cells that are critical for adhesion of B-cell precursors. Flow cytometric analysis showed that cultured adult BM stromal cells expressed higher constitutive levels of vascular cell adhesion molecule (VCAM)-1 than intercellular adhesion molecule (ICAM)-1 (CD54). IL-1 beta upregulated VCAM-1 and CD54 in a dose-dependent manner, whereas IL-4 upregulated VCAM-1, but had no effect on CD54. In contrast, transforming growth factor (TGF)-beta decreased the level of BM stromal cell VCAM-1. Using an assay to measure the adhesion of 51Cr-labeled B-cell precursors to BM stromal cells, we observed a direct correlation between cytokine- regulated levels of VCAM-1 and the capacity of stromal cells to support the adhesion of B-cell precursors. Blocking studies using a panel of monoclonal antibodies (MoAb) showed that adhesion of B-cell precursors to untreated and cytokine-treated (IL-1 beta, IL-4) BM stromal cells was mediated by very late antigen (VLA)-4 (CD49d/CD29) and VCAM-1. Adhesion of B-cell precursors could also be enhanced by direct stimulation with MoAb to the CD29 subunit. Our collective results indicate that B-cell precursor/BM stromal cell adhesion is mediated by a VLA-4-VCAM-1 interaction, which in turn can be regulated at the level of the BM stromal cell by cytokines that specifically increase or decrease cell surface VCAM-1.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 2
    Publication Date: 1985-04-01
    Description: We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.
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    Electronic ISSN: 1528-0020
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  • 3
    Publication Date: 1995-10-01
    Description: A common sequela to allogeneic or autologous bone marrow transplantation (BMT) is a delay in the reconstitution of a functional B-cell immune response. Therefore, we examined whether the posttransplant BM microenvironment is deficient in supporting the proliferation and/or differentiation of B-cell precursors. BM stromal cell cultures were established from patients who received allogeneic or autologous BMT for acute lymphoblastic leukemia, Hodgkin's disease, or non-Hodgkin's lymphoma. These cultures were then compared with normal donor BM stromal cell cultures for expression of adhesion molecules and the capacity to support the adhesion and interleukin-7 (IL-7)-dependent growth of normal B-cell precursors. Analysis of BM stromal cell cultures established from 28 BMT recipients showed a significantly reduced expression of cell surface vascular cell adhesion molecule-1 (VCAM-1/CD106), compared with normal donor BM stromal cells. Transplant BM stromal cell CD106 expression was responsive to regulatory cytokines in a manner qualitatively comparable with normal donor BM stromal cells. The level of B-cell precursor adhesion to transplant BM stromal cells correlated with the level of CD106 expression. Of 19 evaluable transplant BM stromal cell cultures, eight exhibited a reduced capacity to support the growth of CD19+/light chain-normal B-cell precursors. The capacity of transplant BM stromal cells to support B-cell precursor growth correlated with the level of CD106 expression, and the level of B-cell precursor adhesion. Our collective results may provide new mechanistic insight into why B-cell recovery is delayed post-BMT and underscore the importance of VCAM-1/CD106 in regulating B lymphopoiesis.
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  • 4
    Publication Date: 1982-05-01
    Description: Monoclonal antibody BA-1 binds to B lymphocytes, to cells from most cases of non-T acute lymphoblastic leukemia (ALL), and weakly to neutrophils. To determine whether BA-1 also reacts with hematopoietic progenitor cells (HPC), we studied the effect of removal of BA-1+ cells from human bone marrow on the proliferation in vitro of the trilineage precursor cell CFU-GEMM, and on the committed progenitor cells of granulopoiesis (CFU-C) and erythropoiesis (BFU-E/CFU-E). Complement- mediated cytotoxicity using BA-1 at concentrations far beyond those required to lyse BA-1+ bone marrow cells and ALL cells did not result in inhibition of colony formation in any of the assays. A rosette separation method, using ox red blood cells coated with BA-1, resulted in enrichment of HPC in the BA-1-depleted interface, whereas very few HPC were found in the BA-1-enriched pellet. Both methods indicate that BA-1 does not bind to HPC, although binding of the antibody to the lymphohematopoietic stem cell cannot be excluded yet. The high cytotoxic capacity of the IgM antibody BA-1, and the lack of reactivity with HPC, make the antibody particularly suitable for use in autologous bone marrow transplantation for patients with ALL.
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  • 5
    Publication Date: 1984-05-01
    Description: We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.
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  • 6
    Publication Date: 1982-12-01
    Description: Analysis of surface antigenic determinants of hematopoietic progenitor cells has relevance both to basic biologic study of cell differentiation and to potential clinical application in the diagnosis and treatment of hematologic neoplasia. The production and characterization of monoclonal antibody BA-2 by immunization with a pre- B-ALL cell line has been reported previously. In this study we utilized complement-dependent cytotoxicity and rosette-separation with antibody indirectly coupled to ox RBC to determine if the antigen (p24) recognized by the antibody BA-2 is represented on human pluripotential (CFU-GEMM) or committed hematopoietic progenitors (CFU-GM, BFU-E, CFU- E). BA-2 showed no reactivity with normal hematopoietic progenitors by either method. In contrast, BA-2 exhibited potent complement-mediated cytotoxicity for selected ALL-derived cell lines. These results show that normal human hematopoietic progenitors do no express antigenic sites represented on ALL cells that are recognized by BA-2 and suggest that this monoclonal antibody may serve as a potent and specific agent for treatment of lymphocytic leukemia, perhaps most useful in ex vivo marrow conditioning for autologous bone marrow transplantation.
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  • 7
    Publication Date: 1981-12-01
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  • 8
    Publication Date: 1988-05-01
    Description: The purpose of this study was to examine the expression and structure of CD22 in B cell precursor acute lymphoblastic leukemia (BCP-ALL), acute myeloid leukemia (AML), and T cell acute lymphoblastic leukemia (T-ALL). By using immunofluorescence microscopy and flow cytometry we observed that CD22 is expressed not only in the cytoplasm (as previously reported) but also on the cell surface of virtually all (15/16) BCP-ALL examined. CD22 that was biosynthetically labeled with 35S-cysteine and immunoprecipitated from the uncommon cytoplasmic CD22- positive/surface CD22-negative BCP-ALL cells was analyzed by single- dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our results indicated that the cytoplasmic form of CD22 comigrated with 125I/lactoperoxidase-labeled surface CD22. Therefore, cytoplasmic CD22 is probably a pool of fully processed glycoprotein. We also observed unusual cases of AML (approximately 20%) that expressed cytoplasmic CD22 based on immunofluorescent staining; however, biosynthetic labeling and immunoprecipitation revealed an apparently cross-reactive protein(s) of approximately 250 to 300 kd in AML cells. No T-ALL cell lines examined expressed either cytoplasmic or surface CD22. Thus, cytoplasmic and surface expression of bona fide CD22 appears restricted to B cells, which suggests that this molecule subserves a function unique to B cells.
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  • 9
    Publication Date: 1985-04-01
    Description: We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
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  • 10
    Publication Date: 1983-05-01
    Description: CLL B cells may be induced to form B-cell colonies in vitro. Colonies formed are monoclonal and appear to reflect the circulating malignant B- cell clone in vitro. Using hybridoma-produced monoclonal antibodies (MAB) and an in vitro B-cell colony assay, we have provided a characterization of the antigenic phenotype of the clonogenic CLL B cell. B-cell colony growth in both patients and normals was not altered by prior incubation with either MAB or complement (C') alone. CLL B- cell colony formation was markedly reduced after treatment with T101 and C', while normal colonies were unaffected (8 +/- 2 versus 107 +/- 10). None of the residual CLL B-cell colonies after T-101 and C' treatment reacted with T-101. However, BA-1 and la reactivity were still seen in residual CLL B-cell colonies following T-101 treatment. In contrast, a similar percentage reduction of B-cell colony growth was seen for both normals and CLL patients following treatment with BA-1 (76% versus 81%) and Q5/13 (89% versus 92%). These studies suggest that the CLL progenitor cell is characterized by the phenotype la+, BA-1+, T- 101+. Better definition of the CLL progenitor cell has potential implications with regards to clinical utilization of MAB in the treatment of CLL.
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