[Sugar molecules, cell adhesion and cancer etiology].
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Carbohydrate Conformation↗
Biomedical subjects
S Funderud
Publications and source records attributed to S Funderud.
A new method for detachment of Dynabeads from positively selected B lymphocytes.
This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.
Antigens, CD↗
Cell cycle-dependent regulation of CDw75 (beta-galactoside alpha-2,6-sialyltransferase) on human B lymphocytes.
Within the hematopoietic system, CDw75 is primarily expressed on cells of the B cell lineage. Cloning and sequencing of the gene has shown CDw75 to be a beta-galactoside alpha-2,6-sialyltransferase. This enzyme plays an important role in the intracellular terminal glycosylation pathways in various cell types. In this article, we demonstrate that COS cells transfected with the CDw75 cDNA clone displayed sialyltransferase activity, in contrast to mock-transfected cells. We also found that activated B cells displayed an increased enzyme activity compared to resting cells, in accordance with the staining data. Moreover, CDw75 expression was found to be up-regulated approximately 7-9-fold from early G1 to the G2/M phases of the cell cycle in peripheral blood leukocyte B cells. This was shown by staining of in vitro activated B cells with the anti-CDw75 monoclonal antibody HH2, using cell fractions corresponding to different stages of the cell cycle. Using a combination of Hoechst 33258 and propidium iodide after bromodeoxyuridine incorporation, it is possible to distinguish between different phases of the first and second cell cycle. By combining this with HH2 immunofluorescence staining, using a multistation multiparameter flow cytometry program, we confirmed the cell cycle-dependent expression of CDw75. Immunocytochemical stainings of cytospin specimens of elutriated B cells showed that the antigen was up-regulated in late G1 before the appearance of the nuclear activation antigen Ki67. Finally, we showed that activated B cells secreted soluble CDw75 into the medium, as demonstrated by a specific blocking of HH2 staining of B cells using suboptimal concentrations of HH2. In accordance with this, we observed small, but detectable levels of soluble sialyltransferase activity in supernatants of activated B cells.
Antibodies, Monoclonal↗
Human Golgi beta-galactoside alpha-2,6-sialyltransferase generates a group of sialylated B lymphocyte differentiation antigens.
The role of the human beta-galactoside alpha-2,6-sialyltransferase (hu alpha-2,6-ST) in the generation of B cell surface antigens was investigated by selecting subclones of COS cells (monkey kidney epithelial cells) constitutively expressing a transfected cDNA which encodes the hu alpha-2,6-ST (COS alpha-2,6-ST cells). Expression of hu alpha-2,6-ST in COS cells was sufficient to generate sialylated cell surface epitopes on different glycosylated antigens recognized by monoclonal antibodies to CDw75, CD76, and the unclustered monoclonal antibodies HB-4 and EBU-65. These epitopes were sensitive to sialidase treatment and are likely to contain terminal alpha-2,6-linked sialic acid residues. A novel antiserum raised against bacterially expressed hu alpha-2,6-ST fusion protein was used to localize the sialyltransferase in two cell lines with high expression of either endogenous (B cell line JOK-1) or recombinant (COS alpha-2,6-ST cells) hu alpha-2,6-ST. In both cell lines, the enzyme was detected only intracellularly in the juxtanuclear region and not on the cell surface. In contrast, CDw75, formerly proposed to be identical with an alpha-2,6-ectosialyltransferase, was strongly expressed on the cell surface. The different expression patterns show that neither the CDw75 antigen nor any of the other sialylated antigens analyzed is identical with the hu alpha-2,6-ST. Furthermore, the presence of a surface-expressed alpha-2,6-ST appears unlikely in these cell lines. We propose that CDw75, CD76, HB-4, and EBU-65 represent a unique group of B cell differentiation antigens the production of which requires the enzymatic activity of alpha-2,6-ST.
Animals↗
Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.
Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.
Antigens, CD↗
Interleukin-7 differentiates a subgroup of acute lymphoblastic leukemias.
The bone marrow stromal cell-derived growth factor interleukin-7 (IL-7) is known to stimulate growth of normal human B-cell precursors. In the present report, we have examined the effect of IL-7 on neoplastic B-cell precursors. Leukemic cells from 20 patients with common acute lymphoblastic leukemia (ALL) were highly purified by removing contaminating T cells and monocytes by rosetting with immunomagnetic beads. IL-7 markedly reduced the DNA synthesis in leukemic cells from three patients. This inhibition of DNA synthesis was accompanied by maturation of the cells, as demonstrated by the induced expression of the differentiation antigens CD19, CD20, CDw75, and surface mu-chain, and a decreased expression of terminal deoxynucleotidyl transferase. By examining G1 parameters, such as MYC, 4F2, and transferrin-receptor levels analyzed by flow cytometry as well as RNA and the cell cycle regulated antigen Ki67, it appeared that the cells were inhibited late in G1. Leukemic cells from the majority of the cases (12 of the 20 patients) responded to IL-7 with enhanced DNA synthesis without detectable maturation, as has been reported for their normal counterparts. Low molecular weight B-cell growth factor greatly potentiated the IL-7-induced growth stimulation of these cells. Thus, we have shown that IL-7 is capable of inhibiting proliferation of leukemic cells isolated from a subgroup of ALLs, and that this growth inhibition is accompanied by maturation of the cells.
Antibodies, Monoclonal↗
Immunomagnetic isolation of NK and LAK cells.
The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity.
Antigens, Differentiation, T-Lymphocyte↗
Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry.
7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.
Antigens, Surface↗
Independent regulation of 55-kDa and 75-kDa tumor necrosis factor receptors during activation of human peripheral blood B lymphocytes.
We have studied the expression of two different tumor necrosis factor receptors (TNFR; 55 kDa and 75 kDa) on resting and activated human peripheral blood B lymphocytes using specific monoclonal antibodies (mAb). Flow cytometric analysis revealed that most resting B cells expressed small amounts of the 75-kDa TNFR, and that the 75-kDa TNFR was markedly up-regulated upon stimulation with anti-mu or Staphylococcus aureus Cowan strain I (SAC). In contrast, the expression of the 55-kDa TNFR was low on resting as well as on activated cells. B cell activation was accompanied by an increased binding of biotinylated TNF-alpha, and this binding could be blocked by preincubation by utr-1 (anti-75-kDa TNRF), but not the htr (anti-55-kDa TNFR) antibodies. Notably, a number of cytokines tested (interleukin 1 to 8, interferon-gamma, TNF-alpha and -beta) did not influence the expression of either the 75-kDa or the 55-kDa TNFR when given to resting B cells. Moreover, phorbol 12-myristate 13-acetate led to an early, marked down-regulation of the 75-kDa TNFR expression, followed by a later modest increase after greater than 24 h. In contrast to other cell systems where htr mAb have been found either to mimic or to inhibit TNF action, htr mAb had insignificant effects in assays for restimulation of preactivated B cells. However, utr-1 markedly inhibited the TNF-beta but only partly inhibited the TNF-alpha-induced proliferation. Taken together, our data suggest that changes in 75-kDa protein expression is responsible for the increased TNFR expression on activated vs. resting peripheral blood B cells and that this protein also may play an important functional role.
Antibodies, Monoclonal↗
Differential effects of interferon-gamma and low molecular weight BCGF on growth of human B lymphocytes; interferon-gamma prolongs the increased c-MYC mRNA levels after activation.
We have characterized the growth-stimulating effect of Interferon-gamma (IFN-gamma) on various parameters of B cell growth, and compared the effects with those of low molecular weight B cell growth factor (lmw BCGF). We have found that IFN-gamma did not affect early changes induced by anti-mu, like initial calcium-flux and rise in mRNA-and protein levels of the proto-oncogene c-myc measured at 3 h. On the other hand, IFN-gamma enhanced the effect of anti-mu on parameters measured later in the G1 phase of the cell cycle, such as expression of the transferrin receptor and general transcriptional activity, measured as an increase in 7-aminoactinomycin D binding. In particular, whereas the c-myc levels in anti-mu-treated cells peaked at 3 h and then gradually declined, IFN-gamma together with anti-mu maintained the c-myc levels at 24 h at approximately the same levels as seen at 3 h. Overall, lmw BCGF had a more potent effect on the parameters affected by IFN-gamma, correlating with stronger enhancement of DNA synthesis. However, in contrast to IFN-gamma lmw BCGF did not affect anti-mu-induced c-myc mRNA levels. Thus this study has revealed differences between two B cell growth factors in effects on B cell cycle parameters.
Antibodies, Anti-Idiotypic↗
Use of size fractionation of in vitro-activated human B lymphocytes for studies of cell cycle-dependent growth regulation.
Cell cycle progression of in vitro-stimulated human B lymphocytes occurs asynchronously. In order to allow detailed studies of growth control in G1, B cells were stimulated with anti-mu and low molecular weight B-cell growth factor (LMW BCGF) for 50 h and subsequently separated into nine fractions of cells by means of centrifugal elutriation. As judged by volume profiles, activation antigen expression and DNA content, the cells in fractions 1-4 were in early to mid-G1, while fractions 5-7 mainly contained cells in late G1, and fractions 8-9 contained cells mainly in S and G2. Cells in fractions 5-7 had passed the commitment point, as demonstrated by a high spontaneous incorporation of [3H]thymidine when recultured in medium alone. Moreover, S-phase entry of these cells was largely unaffected by exogenous growth-promoting or growth-inhibitory signals. Cells in early (fractions 1-2) and intermediate fractions (fractions 3-4) showed a negligible spontaneous [3H]thymidine incorporation, but a significant proportion of these cells progressed to S phase upon restimulation. Moreover, while IL-4 or the anti-CD40 MoAb G28-5 potently stimulated cells in early and intermediate fractions, the responsiveness of LMW BCGF alone was obtained just prior to the commitment point.
B-Lymphocytes↗
Cell cycle-specific expression and nuclear binding of DNA polymerase alpha.
The expression and distribution of DNA polymerase alpha was measured by cytometry and confocal laser scanning microscopy. Expression was proportional to DNA content in proliferating cells, while only S-phase cells retained DNA polymerase alpha after detergent extraction. Nuclear DNA polymerase alpha binding may be one of the key events of S-phase entry.
Animals↗
Autologous transplantation of an immunomagnetic bead purged marrow in patients with relapsed acute lymphoblastic leukemia.
In acute lymphoblastic leukemia (ALL) the expected duration of a second complete remission (CR) following conventional aggressive multi-drug treatment is rather short, and long-term survival is rarely seen. Myeloablative consolidation treatment with ABMT in second CR is thought to benefit "high risk" patients thus allowing the escalation of cytoreductive treatment into a lethal range. In three patients with relapsed ALL bone marrow was collected in the very early second remission phase. The autografts were incubated with a panel of monoclonal antibodies bound to magnetobeads (DYNABEADS M450). The pretransplant regimen consisted of total body irradiation (TBI) (14.4 Gy) and cyclophosphamide (CY) (50 mg/kg b.w. x4). Following autologous transplantation of those immunomagnetobead purged marrow grafts, complete hematologic reconstitution was seen in all three patients. They are alive and disease-free for +26, +24 and +17 months post transplant, which is also proven by the absence of detectable gene rearrangements after transplantation. In two of them disease-free survival exceeds the duration of the preceding remission y 20 and 12 months respectively, which is a definitive parameter for therapy effectiveness. Therefore, "high dose" consolidation treatment in very early second CR followed by autologous transplantation of purged bone marrow seems to allow long-term disease-free survival. The technique of ex vivo immunomagnetic bead purging, i.e. the "direct" and "indirect" method of binding monoclonal antibodies to magnetobeads is described.
Antibodies, Monoclonal↗
The B cell antigen CD75 is a cell surface sialytransferase.
In this work we have isolated a cDNA clone encoding the B cell antigen CD75. The amino acid sequence of CD75 is shown to be identical to that of human alpha 2,6 sialyltransferase, believed to be primarily associated with the Golgi complex. This is the first demonstration of cell surface expression of sialytransferase which, in B cells, may play an important role in intercellular adhesion and antigen presentation events.
Amino Acid Sequence↗
Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation.
Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-mu plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.
Antibodies, Anti-Idiotypic↗
Immunological typing of acute leukaemias by rosetting with immunomagnetic beads: comparison with immunofluorescence staining.
Immunological phenotyping of acute leukaemias is important for a more precise diagnosis with respect to both cell lineage and maturation level. We have developed a rapid and reliable method for immunophenotyping, based on the use of magnetic monodisperse beads coated with monoclonal antibodies. After only a 10-min incubation of immunomagnetic beads (IMB) with mononuclear cells isolated from bone marrow or peripheral blood, the percentage of rosetting cells can be counted in the microscope. A panel of 16 monoclonal antibodies against haematopoietic cell-surface antigens was applied on 29 cases of acute myelogenic (AML) or lymphocytic (ALL) leukaemias, in order to compare immunological typing by immunomagnetic beads with immunofluorescence staining (IF). In all the cases tested, the two methods showed a virtually identical antigen distribution. The procedure described offers the advantages of being fast and simple to perform. Moreover, it has a high specificity and is easy to interpret in cases with low antigen expression.
Antibodies, Monoclonal↗
Inhibition of polyphosphoinositide breakdown and c-myc induction accompanying inhibition of human B-cell activation by two monoclonal antibodies against the leucocyte common antigen (CD45).
In this report we show that the two monoclonal anti-CD45 antibodies, EO-1 and FN-126, potently inhibit G0 to G1 transition and S phase entry in human B cells stimulated with anti-mu and low molecular weight B-cell growth factor. Both antibodies were found to inhibit anti-mu-induced inositol phospholipid breakdown and c-myc mRNA induction. In contrast, EO-1 and FN-126 only partially inhibited the early anti-mu-induced increase in cytoplasmic Ca2+ levels, both in normal and in Ca2(+)-depleted medium. B-cell activation provoked by 12-O-tetradecanoylphorbol 13-acetate (TPA) was not inhibited by these antibodies, except when using high concentrations of EO-1. In addition, both antibodies were found to inhibit G1 entry induced by the anti-CD20 antibody 1F5, which confers an activation of B cells without any detectable increase in [Ca2+]i or in phospholipid metabolism. This indicates that alternative mechanisms in addition to the inhibition of polyphosphoinositide (PI) breakdown are involved in the inhibitory action of these antibodies.
Antibodies, Monoclonal↗
Interleukin 4 induces selective production of interleukin 6 from normal human B lymphocytes.
In this paper we have shown that extensively purified human B lymphocytes respond to IL-4 treatment with a marked production of IL-6. Addition of anti-mu potentiated the effect of IL-4 on IL-6 production. Other cytokines tested like TNF-alpha and-beta, IFN-gamma, IL-1, IL-2, and IL-5 did not induce IL-6 secretion when given to resting B cells. Although B cells generally also produced TNF-alpha and TNF-beta upon stimulation, IL-4 did not induce TNF secretion and seemingly had a specific effect on IL-6 production.
B-Lymphocytes↗