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H K Blomhoff

Publications and source records attributed to H K Blomhoff.

At least 55 records · Page 3Linked to original sources
PubMed1993

Constitutive expression of c-myc does not relieve cAMP-mediated growth arrest in human lymphoid Reh cells.

The Reh cell system is suitable for evaluating events important for control of proliferation independently of mechanisms involved in differentiation, as Reh cells are unable to differentiate. In the human pre-B cell line Reh, activation of adenylate cyclase by forskolin induces a five to tenfold rapid, transient down-regulation of steady-state c-myc RNA within 4 hours. Concurrently, the cells are strongly growth arrested in the G1 phase of the cell cycle. To clarify if the observed growth arrest could be relieved by constitutive expression of c-myc, an exogenous c-myc gene under constitutive promoter control was introduced into Reh cells by electroporation. The c-myc-expressing construct pDMmycHyg contained human c-myc exons 2 and 3 driven by the Mo-MLV LTR and conferred hygromycin resistance. Exogenous c-myc RNA transcripts and protein were constitutively expressed in the transfected clones at levels roughly twice as high as the level in nontransfected cells. Total c-myc protein levels were unchanged upon treatment of transfected clones with forskolin. Yet, the transfected cells were not released from growth arrest. Furthermore, the transfected Reh cells did not differentiate upon forskolin treatment. Constitutive overexpression of c-myc is therefore not sufficient for relieving forskolin-mediated effects on growth arrest in Reh cells.

B-Lymphocytes↗
PubMed1992

Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells.

In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.

B-Lymphocytes↗
PubMed1992

Direct mobilization of retinol from hepatic perisinusoidal stellate cells to plasma.

We have studied the mechanism for mobilization of retinol from stellate cells. Our data show that perisinusoidal stellate cells isolated from liver contained retinol-binding protein (RBP) mRNA. By Western blot analysis we found that cultivated liver stellate cells secreted RBP into the medium. Cultivated stellate cells loaded in vitro with [3H]retinyl ester mobilized radioactive retinol as a complex with RBP. Furthermore, exogenous RBP added to the medium of cultured stellate cells increased the secretion of retinol to the medium. These data suggest that liver stellate cells in vivo mobilize retinol directly to the blood and that a transfer to parenchymal cells for secretion as holo-RBP is not required. The direct mobilization of retinol from liver stellate cells as retinol-RBP to blood is indirectly supported by the demonstration of RBP mRNA production and RBP secretion by lung stellate cells. The data suggest that the same mechanism for retinol mobilization may exist in hepatic and extrahepatic stellate cells. This is, vitamin A-storing stellate cells in liver, lungs, and probably also in other organs may synthesize their own RBP (or alternatively use exogenous RBP) and mobilize holo-RBP directly to the blood.

Animals↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

Retinyl esters in chylomicron remnants inhibit growth of myeloid and lymphoid leukaemic cells.

We have studied the effects of retinyl esters in chylomicron remnants on cell growth and differentiation of myeloid and lymphoid leukaemic cells. Ten mumol l-1 retinyl ester in chylomicron remnants effectively reduced proliferation of the myeloid leukaemic cell lines HL60, U937 and KG-1, and induced differentiation of 68% and 53% of the HL60 and U937 cells, respectively, in 5 days. While no effect on cell growth of the lymphoid cell lines Daudi, Raji and SOS was observed, 10 mumol 1-1 retinyl esters in chylomicron remnants reduced the growth of the B lymphoid cell line Reh by more than 50%. Primary cell cultures from six patients with acute leukaemia (four non-lymphocytic and two lymphocytic) were incubated with chylomicron remnant retinyl esters and proliferation was measured by means of thymidine incorporation. Among the myeloid leukaemic cells, the monomyelocytic, the two promyelocytic and the monoblastic leukaemic cells were growth inhibited. Chylomicron remnants had no effect on the growth of the c-ALL primary culture, but reduced proliferation of the T-ALL primary culture by approximately 20% after 48 h. These data suggest that high doses of retinol may be used in the treatment of some forms of acute leukaemia.

Cell Differentiation↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells.

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.

Animals↗
PubMed1990

Effect of retinoids and 1,25(OH)2 vitamin D3 bound to their plasma transport proteins on growth and differentiation of HL-60 cells.

We have compared the effect of physiological and pharmacological concentrations of retinoids and 1,25(OH)2 vitamin D3 bound to their plasma transport proteins upon the proliferation and differentiation of HL-60 cells. Concentrations of chylomicron remnant retinyl ester similar to that obtained in plasma after a vitamin A-rich meal reduced the proliferation in more than 50% of HL-60 cells. Pharmacological concentrations of chylomicron remnant retinyl ester completely blocked the proliferation of the cells, and induced differentiation in 60% of the cells after 5 days. Physiological and pharmacological concentrations of retinoic acid bound to albumin had comparable effects. In contrast to earlier published data, which have been obtained with retinoids dissolved in ethanol, our results suggest that physiological and pharmacological concentrations of retinol (i.e. retinyl esters in chylomicron remnants) are as active as retinoic acid in reduction of proliferation and induction of differentiation of HL-60 cells. Physiological concentrations of 1,25(OH)2 vitamin D3 bound to vitamin D-binding protein (DBP) and retinol bound to retinol-binding protein had only a small effect on differentiation and proliferation of HL-60 cells.

Calcitriol↗
PubMed1989

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↗
PubMed1989

Levels of myc protein, as analyzed by flow cytometry, correlate with cell growth potential in malignant B-cell lymphomas.

We have analyzed c-myc protein expression during the cell cycle in malignant B-cell lymphomas by dual flow cytometric detection of a fluoresceinated polyclonal anti-myc antibody and propidium iodide which binds stochiometrically to DNA. The data obtained were correlated to other parameters of cell activation such as histopathological grading, expression of the activation antigen 4F2, light scatter (proportional to cellular volume), DNA synthesis and percentage of S-phase cells. The c-myc protein level was strongly correlated to parameters of DNA synthesis/content. In addition, the oncoprotein level was largely unvarying from the late G1 phase through the rest of the cell cycle in both malignant cells and normal purified B cells stimulated to proliferate in vitro.

B-Lymphocytes↗
PubMed1989

Intracellular events associated with inhibition of B cell activation by monoclonal antibodies to HLA class II antigens.

We have investigated several aspects of the inhibitory effects of monoclonal antibodies (mAb) directed against MHC class II antigens in B cell activation/proliferation, using a panel of mAb specifically reactive with antigens encoded by HLA class II loci (DP, DQ, DR). All mAb except the anti-DP mAb inhibited significantly anti-mu plus B cell growth factor-induced DNA synthesis. Only one mAb, however, which was reactive with gene products of all three class II loci (DP, DQ, DR) inhibited anti-mu-induced DNA synthesis as well as c-myc mRNA expression. In addition, the same mAb inhibited the early events induced by anti-mu stimulation alone, including phosphatidylinositol turnover and elevation of [Ca2+]i. In contrast to previous findings in the murine system, none of the anti-MHC class II mAb used in this study increased the cAMP levels.

Antibodies, Monoclonal↗