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Biomedical subjects

E Ruud

Publications and source records attributed to E Ruud.

At least 19 recordsLinked to original sources
PubMed1989

Heterogeneity of degradation of B-cell endocytosed monoclonal antibodies reacting with different sIgM epitopes.

The degradation of a panel of monoclonal antibodies (MoAb) bound to surface IgM (sIgM) was studied in three human Burkitt's lymphoma cell lines. The panel included MoAb that recognize several distinct epitopes associated with the F(c mu)5 domain, the c mu 2 domain and kappa or lambda light chains. The amount of degraded MoAb and the rate of their degradation varied considerably between the various antibodies. Properties of MoAb such as avidity or ability to cross-link sIgM did not significantly influence their degradation. The most consistent correlation between rate of degradation and MoAb used was the location of the epitope recognized by the individual MoAb. Thus, 7 out of 8 anti-light chain MoAb were degraded at a higher rate than 5 out of 5 anti-F(c mu)5 MoAb. One anti-c mu 2 MoAb was degraded at a rate similar to the majority of anti-light chain MoAb. The intracellular transport of an anti-kappa light chain MoAb and an anti-F(c mu)5 MoAb was studied in detail by subcellular fractionation in sucrose gradients. We found that the anti-kappa light chain MoAb was transported more rapidly to lysosomes than the anti-F(c mu)5 MoAb, showing that they were sorted differently intracellularly.

Animals↗
PubMed1988

Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle.

The surface IgM-mediated endocytosis and intracellular transport of an anti-F(c mu)5 mAb was studied by using subcellular fractionation in sucrose gradients. The results of such experiments showed that antibody was initially endocytosed in vesicles of low density, and later transferred to a presumably lysosomal compartment of higher density. SDS-PAGE analysis of gradient fractions showed that high Mr degradation fragments of the endocytosed antibody were formed in the low density vesicles before terminal degradation could be recorded. The partial degradation of the antibody was not blocked by low temperature or enzyme inhibitors, such as leupeptin and benzyloxycarbonyl-phenylalanylalanine-diazomyethyl-ketone, all of which severely retarded terminal degradation. The data also suggested that the recycling of partially degraded antibody to the cell surface employed a pool of such low density prelysosomal vesicles.

Animals↗
PubMed1988

Cyclic AMP has the ability to influence multiple events during B cell stimulation.

Negative regulators of cellular proliferation are important in maintaining a balanced growth control. In this study we have examined the effects of the diterpene forskolin on various parameters of B cell activation. Forskolin is known to elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and thereby to influence B cell stimulation. We found that forskolin exerted an inhibitory effect on early as well as late events during stimulation of resting normal human B cells. Cells were activated either by antibodies to surface immunoglobulins (anti-mu), by the monoclonal antibody 1F5 reactive with the CD20 antigen or by 12-O-tetradecanoylphorbol 13-acetate. While anti-mu stimulation induces increased phosphatidylinositol (PI) turnover and [Ca2+]i fluxes, the latter two reagents confer an activation of B cells independent of the PI/Ca2+ pathway. We found a clear inhibitory effect of forskolin on the anti-mu-induced PI turnover and [Ca2+]i fluxes as well as on later parameters of cell activation. There was also a clear inhibition of G1 entry and DNA synthesis when PI/Ca2+-independent activation was employed, indicating that cAMP interferes with B lymphocyte stimulation in several ways. Importantly, forskolin maintained its inhibitory effect when added late after anti-mu stimulation, implying an effect also at multiple stages of activation. When examining the inhibitory effect of forskolin on neoplastic B cells, we found essentially no differences from the responses in normal cells.

Antibodies, Monoclonal↗
PubMed1988

Potential multiple functions of the v-myc oncogene within a single cell clone of OK10 retrovirus-transformed quail fibroblasts.

Propagation of in vitro transformed OK10 QDP 9c cells, cloned in soft agar, results in selective amplification of the OK10 pro-viral genome to yield a 12-fold increase in v-myc oncogene expression. In addition, increased v-myc oncogene dosage correlates with an increase of the proliferative potential of already transformed cells and relieves dependence on high serum concentrations for optimal cell growth. The increased rate of cell proliferation is reflected by a much more rapid progression through all parts of the cell cycle. These results suggest that the attainment of transformed status on one hand and progressive increase in growth factor independence for optimal cell proliferation on the other hand, may be initiated by different myc oncogene dosages within the OK10 QDP 9c cell clone.

Animals↗
PubMed1988

Attention deficit disorders: a study of peptide-containing urinary complexes.

In several behavioral disorders, we have observed that abnormal amounts of peptides and protein-associated peptide complexes are excreted in the urine. The gel filtration patterns of these excreted substances have some specificity for the different disorders. The urinary excretion of peptide-containing complexes was studied in 91 boys and 13 girls (mean age 9.4 years, range 1-23) with the clinical diagnosis of attention deficit disorder (ADD), with or without hyperactivity. The gel filtration of urine precipitate showed patterns in all patients that were different from those seen in 36 normal controls. Sixty-four patients had increased benzoic acid-glycoprotein-peptide complexes in the late peaks. The symptoms of all these patients fit the criteria for diagnosis of attention deficit disorder with hyperactivity (ADDH). Thirty-five patients showed reduced amounts of uric acid complexes in the late peaks. Clinically, this group, with the exception of three patients, fit the criteria for diagnosis of attention deficit disorder without hyperactivity. Five patients showed reduced amounts of all urinary complexes; four of these were hyperactive. Moderate exercise in control children did not change the urinary pattern. One urinary peptide fraction from hyperactive patients, purified to homogeneity, increased the uptake of 14C[5-HT] in platelets. Strict clinical, neuropsychological, and psychophysiological selection of the patients reduced the heterogeneity of the patterns. Although more studies are needed, the findings seem promising for the possibility of developing biochemical tests that may be helpful diagnostically.

Adolescent↗
PubMed1987

Activation of human B cells: alternate options for initial triggering and effects of nonmitogenic concentrations of anti-IgM antibodies on resting and activated cells.

The monoclonal antibody 1F5, which is reactive with the CD20 (Bp 35) pan-B cell antigen, was shown to activate resting human peripheral blood B cells into the middle to late G1 phase of the cell cycle. However, in contrast to F(ab')2 fragments of polyclonal anti-mu, 1F5 synergized only weakly with B cell growth factor (BCGF) for DNA synthesis in these cells. We provide evidence that the CD20 molecule and surface immunoglobulin represent two alternative activation pathways in resting B cells. We also show that anti-immunoglobulins, during co-stimulation with BCGF, may play an important role in G1 as well as for the initial cell triggering. Thus, anti-mu in nonmitogenic concentrations was shown to provoke distinct effects on 1F5-pretreated G1 cells, as monitored by increases in cellular volumes as well as in cytoplasmic Ca2+ levels. Moreover, anti-mu could increase c-myc mRNA levels in 1F5-primed cells, implying that c-myc expression can be regulated in G1 as well as during the initial G0 to G1 transition. Partially purified human BCGF neither induced G1 entry in resting peripheral blood cells nor primed the cells for DNA synthesis. The finding that BCGF did not influence c-myc mRNA levels in resting or in activated B cells suggests that its mitogenic action does not involve the c-myc function.

Antibodies, Anti-Idiotypic↗
PubMed1987

Cyclic AMP-mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha-ras protooncogenes.

Cyclic AMP functions as a negative regulator of cell proliferation in a variety of cell systems. We show here that the proliferation of normal and neoplastic B cells can be inhibited by high intracellular levels of cAMP. Thus forskolin treatment of the neoplastic B precursor cell line Reh induced a rapid increase in the cAMP level, which was followed by an accumulation of cells in the G0/G1 phase of the cell cycle over a period of 2-3 days. Similar inhibition of Reh cell proliferation after 3 days was observed whether forskolin was present continuously or only during the first 5 hr. Both c-myc and c-Ha-ras protein levels were transiently down-regulated at 4 hr of forskolin treatment, suggesting that these protooncogenes play a role in the process leading to cAMP-mediated growth cessation. Northern-blot analysis showed that the steady-state levels of c-myc RNA rapidly declined in all phases of the cell cycle, to return to control levels within a time period of 24 hr. In contrast, the c-Ha-ras mRNA level was steadily maintained. Thus the expression of c-myc and c-Ha-ras protein was regulated at different metabolic levels. The reduced proliferative capacity of the B precursor cell line in the presence of forskolin was not linked to induced differentiation. This was judged from the lack of appearance of three different B cell differentiation markers; cytoplasmic immunoglobulin heavy chain and two antigens recognized by the monoclonal antibodies B1 (CD20) and HH1 (CD37). We also showed that forskolin partially inhibited the proliferation of normal B lymphocytes stimulated by anti-immunoglobulins (anti-mu) and B cell growth factor (BCGF). The burst of c-myc mRNA during activation of normal B cells was also reduced by forskolin.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Downregulation of c-myc RNA is not a prerequisite for reduced cell proliferation, but is associated with G1 arrest in B-lymphoid cell lines.

We related the effects of c-myc expression on the ability of growth inhibitors to block the cells in the G0/G1 phase of the cell cycle. In two different B-cell lines, there was an association between the accumulation of cells in the middle to late G1 phase of the cell cycle and a rapid transient downregulation of c-myc mRNA levels. The phorbol ester TPA and the adenylate cyclase activator forskolin reduced the c-myc RNA, levels and after 3 days of treatment a proportion of the cells accumulated in G1. In contrast, neither interferon-gamma, tumor necrosis factor-alpha nor the monoclonal antibody 33-1 against DQ major histocompatibility antigens changed the cell-cycle distribution or regulated the c-myc RNA levels. Yet, all five growth inhibitors reduced the proliferation to approximately the same extent. The growth reduction was not accompanied by definite differentiation, as judged by the absence of the B-cell differentiation marker B1 (CD20).

B-Lymphocytes↗
PubMed1987

Transforming growth factor type beta (TGF beta) inhibits G1 to S transition, but not activation of human B lymphocytes.

Type beta transforming growth factor (TGF beta) is a polypeptide that may influence the growth of a variety of cell types in a positive or negative fashion. In this study we show that TGF beta markedly inhibits DNA synthesis in normal and neoplastic human B lymphocytes stimulated to proliferate with anti-immunoglobulins and B-cell growth factor (BCGF). Although TGF beta was needed during the initial 12 h of the culture to promote optimal inhibition, we found that it had little or no effect on several early to intermediate parameters of cell activation [( Ca2+]i increase, c-myc mRNA increase, cellular enlargement, RNA increase, and the increase in the expression of the 4F2 activation antigen). In contrast, TGF beta almost completely blocked the induction of transferrin receptor expression, which normally occurs in the late G1 phase of the cell cycle. Therefore, we conclude that TGF beta treatment leads to arrest of the cells in the middle to late G1 phase, prior to transferrin receptor expression.

Antibodies, Anti-Idiotypic↗
PubMed1986

Distinct effect of forskolin and interferon-gamma on cell proliferation and regulation of histocompatibility antigen expression in hematopoietic cells.

The adenylate cyclase activator, forskolin, was found to induce expression of class I and class II major histocompatibility complex antigens in a B precursor cell line, Reh, as well as in a B lymphoid cell line, Raji. No such effect was, however, observed when the promyelocytic cells line HL-60 was treated with either forskolin or the cAMP analogue 8-bromoadenosine cyclic monophosphate. As expected, all three cell lines showed reduced proliferation upon forskolin treatment. Forskolin induced expression of class I and class II major histocompatibility complex antigens in cell lines not affected by interferon-gamma and vice versa, indicating that cAMP is not involved in the regulation of histocompatibility antigens by interferon-gamma. We also compared the effect of interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate on major histocompatibility complex class I and class II expression, and despite differences in the response on the tested cell lines, we can not at this point exclude the possibility that protein kinase C is involved in the action of interferon-gamma.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Internalization and processing of antibodies to surface antigens on human B cells. Monoclonal anti-IgM antibodies are processed differently than monoclonal antibodies towards non-Ig surface receptors.

The internalization and intracellular processing of monoclonal antibody to immunoglobulin mu heavy chain (Mamu) have been investigated in two human Burkitt lymphoma cell lines (Ramos and Raji), in a human B cell lymphoma and in normal human peripheral blood B cells. In addition to the degradation of 125I-labeled Mamu to trichloroacetic acid (TCA) soluble material, a distinct pattern of larger 125I-Mamu fragments was detected in all sources of B cells tested. The particular fragmentation pattern, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, involved the cleavage of both peptide bonds and disulfide bridges. This type of antibody fragmentation appeared to be a selective mechanism associated with sIgM, as no other degradation than that leading to TCA-soluble material could be detected after the internalization and degradation of radiolabeled monoclonal antibodies towards a variety of non-Ig B cell surface receptors. Three fragments of 125I-Mamu degradation were also detected in the supernatant of Ramos cells, implying that the recycling and exocytosis of certain 125I-Mamu fragments also took place.

Antibodies, Monoclonal↗
PubMed1985

The specific induction of myc protooncogene expression in normal human B cells is not a sufficient event for acquisition of competence to proliferate.

Resting human B cells can be activated to proliferate in the presence of both polyclonal antibodies to immunoglobulin mu heavy chains and B-cell growth factor (BCGF). This process appears to be temporally controlled in that the initial activation of the B cells and their responsiveness to BCGF is carried out by polyclonal anti-mu-chain antibodies alone. We have used this system to investigate the role of the c-myc gene in the cell cycle of normal human peripheral blood B cells. Our results show that the polyclonal anti-mu-chain antibody-induced B-cell activation is accompanied by a specific induction of c-myc gene expression without promoting subsequent entry into the S phase unless BCGF is added. Monoclonal antibodies to either mu chain or the pan-B-cell antigen Bp35 also revealed a similar G0-to-G1 transition and activation of c-myc gene expression. However, unlike activation with polyclonal anti-mu-chain antibodies, cells stimulated with these monoclonal antibodies do not acquire responsiveness to BCGF. The results imply that additional inducible functions must be present to potentiate the myc-specific function in order for the B cells to acquire the capacity to proliferate in response to BCGF. These findings are discussed in relation to the origin of B-cell malignancies.

Antibodies, Anti-Idiotypic↗
PubMed1985

Characterization of two murine monoclonal antibodies reactive with human B cells. Their use in a high-yield, high-purity method for isolation of B cells and utilization of such cells in an assay for B-cell stimulating factor.

We describe two monoclonal antibodies, HH1 and HH2. Both reacted selectively with surface immunoglobulin (sIg)-positive human B cells. Both antibodies stained on average 7-8% of peripheral blood mononuclear cells. They have not been found to react with cells or cell lines of other haematopoietic cell lineages, except that HH2 was positive on a small percentage of cells of the erythroid cell line K562. The molecular weight of the HH1 antigen was 95 kD, as established by Western blotting. Neither of these two antibodies reacted with Ig determinants, Fc receptors, complement receptors, or known class-I or class-II molecules. A combination of these antibodies was used in a direct panning technique for high-yield enrichment of normal B lymphocytes from peripheral blood. The enriched B cells could be further purified by lysis of T cells (final yield, on average 72 +/- 8% of initial B cells) or by a second panning (yield, 35 +/- 11%). The purified B cells contained less than 1% contaminating T cells and less than 0.5% monocytes and were used in an assay for B-cell-stimulating factor which they showed a normal and very reproducible proliferative response.

Animals↗