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M Patarroyo

Publications and source records attributed to M Patarroyo.

At least 91 records · Page 5Linked to original sources
PubMed1986

Identification of a cell-surface glycoprotein mediating cell adhesion in EBV-immortalized normal B cells.

Phorbol ester treatment markedly enhanced aggregation of Epstein-Barr virus (EBV)-immortalized normal B cells. Monoclonal antibody (MAb) 60.3, recognizing a leukocyte common antigen, completely inhibited intracellular adhesion, whereas MAbs reacting with leukocyte common antigen T200, C3b receptor, T-cell-associated antigen TA-I, C3d (EBV) receptor, brain-granulocyte/T-lymphocyte antigen, transferrin receptor, surface immunoglobulin, Class-I or Class-II transplantation antigens or a B-cell-specific antigen (BI) showed no inhibitory effect. Both the spontaneous and phorbol-ester-enhanced cell aggregations were similarly inhibited by Fab fragments made from antibody 60.3. Phorbol esters also enhance binding between EBV-immortalized normal B cells and autologous or allogeneic blood mononuclear cells depleted of B lymphocytes. This process, which is independent of immunity to EBV, was similarly blocked by the antibody fragments. Antibody 60.3 precipitated 3 non-covalently associated surface glycopolypeptides with apparent MW of 90, 130 and 160/kDa from EBV-infected B cells. Although dissociation of a similar protein complex from granulocytes by sodium dodecyl sulfate treatment clearly indicated the presence of the epitope on the smallest component, the same treatment did not dissociate the protein complex from EBV-immortalized normal B cells. It is thus concluded that the 90-kDa glycopolypeptide, either alone or associated with the larger glycopolypeptides, mediates cell adhesion of the B cells.

Animals↗
PubMed1986

Genetic assignment of GP90, leukocyte adhesion glycoprotein to human chromosome 21.

Intercellular adhesion among human leukocytes involves a cell-surface glycoprotein with an apparent molecular weight of 90,000 which forms complexes with higher-molecular-weight glycoproteins. A monoclonal antibody (60.3) against this glycoprotein blocks induced adhesion. Here we have shown that the antibody reacts with cell clones carrying human chromosome 21 in lymphocyte hybrids between an AKR mouse thymoma (BW5147) and human concanavalin A-activated peripheral blood lymphocytes. Cell sorting by FACS of a hybrid clone heterogeneous in the expression of the antigen identified by the 60.3 antibody yielded a positive fraction expressing the antigen and carrying human chromosome 21, and a negative fraction lacking both the antigen and chromosome 21. The gene coding for the cell adhesion glycoprotein is thus provisionally assigned to chromosome 21.

Antibodies, Monoclonal↗
PubMed1986

Phorbol ester regulation of Ca2+ flux during natural, lectin and antibody-dependent killing.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, suppresses natural, lectin and antibody-dependent killing by normal human lymphocytes in short-term radioisotope release assays. Fifty percent inhibition of killing of lymphoid target cells was seen at approximately 5 ng/ml TPA and inhibition was further potentiated by the presence of monocytic cells. In contrast, TPA increased killing of K-562 erythroleukaemic cells by non-adherent NK cells with optimal activity around 1 ng/ml. Two anti-estrogenic drugs, tamoxifen and clomiphene, known to inhibit protein kinase C, gave near to complete inhibition of NK killing at concentrations 12 microM and 30 microM, respectively. Retinal, another protein kinase C inhibitor, inhibited both antibody-dependent killing and lectin-dependent killing. An influx of 45Ca2+ into the effector population was found during effector-target cell conjugation and this flux was suppressed at TPA concentrations similar to those that suppressed killing. The results suggest that killing depends on a co-ordinated activation of protein kinase C together with a channel-dependent calcium influx. TPA may suppress killing by a negative feedback effect of protein kinase C on the hydrolysis of inositol phospholipids, as demonstrated in many other systems, or through the down-regulation of cell surface receptors required for triggering of lysis.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1985

Phorbol ester-induced adhesion (binding) among human mononuclear leukocytes requires extracellular Mg++ and is sensitive to protein kinase C, lipoxygenase, and ATPase inhibitors.

Under gentle shaking, the phorbol 12,13-dibutyrate (P(Bu)2)-induced adhesion among human blood mononuclear leukocytes started within a few minutes, increased with time and was almost complete after 12 h. During this moment and thereafter more than 60% of the cells were in aggregates. Induction of the cell aggregation by 20 min treatment with P(Bu)2 did not occur in Ca++/Mg++-free medium but was almost complete with Mg++ alone and reached its maximal manifestation with both divalent cations present. The intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate had a minimal inhibitory effect, and simultaneous treatment of the cells with Ca++ ionophore A23187 and P(Bu)2 did not increase the intercellular binding, whereas A23187 alone induced some cell aggregation. Retinal, a protein kinase C inhibitor, inhibited the intercellular adhesion by more than 50%, and treatment of intact cells with 1-oleoyl-2-acetyl-glycerol, an activator of the enzyme, induced some cell aggregation that was slightly increased when A23187 was added simultaneously. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid and 5,8,11,14-eicosatetraynoic acid, which inhibit lipoxygenation of arachidonic acid, reduced the cell aggregation in contrast to indomethacin and acetylsalicylic acid that had no effect. Cellular ATPases, inhibited by quercetin, but not the ouabain-sensitive Na+, K+-ATPase, appeared to participate, whereas the amiloride-sensitive plasma membrane Na+/H+ exchanger and the intracellular levels of cGMP did not seem to influence the system.

Adenosine Triphosphatases↗
PubMed1985

Identification of a cell surface protein complex mediating phorbol ester-induced adhesion (binding) among human mononuclear leukocytes.

Phorbol esters rapidly induce aggregation of human mononuclear leukocytes in vitro. Previous studies have indicated that cell surface proteins are involved. We report now that the monoclonal antibody 60.3, either as purified IgG or as Fab' fragments, to an antigen common to leukocytes completely inhibited the phorbol ester-induced intercellular adhesion (binding). No inhibition of cell aggregation was observed with monoclonal antibodies to common leukocyte antigen T 200, T-cell-associated antigen, monocyte-granulocyte antigen, brain granulocyte-T-lymphocyte antigen, transferrin receptor, mature T-cell antigens (mol.wt either 67,000 or 19,000/29,000), T helper/inducer cell antigen, sheep erythrocyte receptor, class I or class II antigens, or T cytotoxic/suppressor cell antigen. The antibody 60.3 did not inhibit stimulation of the cells since the characteristic phorbol ester-induced morphological changes and phorbol ester-enhanced cap formation of membrane glycoproteins were readily observed. Two major cell surface polypeptides with apparent molecular weights of 90,000 and 160,000 were immunoprecipitated. We conclude that this protein complex, or at least one of its components, mediates adhesion among mononuclear leukocytes.

Antibodies, Monoclonal↗
PubMed1985

Identification of a cell-surface glycoprotein mediating adhesion in human granulocytes.

Previous studies have shown that monoclonal antibody 60.3 reacting with a surface antigen common to human leukocytes inhibits phorbol ester-induced adhesion among blood mononuclear cells and precipitates from these cells three surface polypeptides with apparent molecular weights of 90,000, 130,000 and 160,000. Now we report that the same antibody, either as purified IgG or Fab fragments, also inhibits the extensive adhesion among granulocytes induced by phorbol ester. Inhibition of cell aggregation was not observed with monoclonal antibodies to C3b receptor, common leukocyte antigen T200, C3bi receptor, brain granulocyte-T lymphocyte antigen, IgG Fc receptor, class I transplantation antigen, or a granulocyte-specific antigen. Intercellular adhesion induced by either the chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (FMLP) or the ionophore A23187 was also inhibited by antibody 60.3. However, this antibody did not affect phorbol ester-induced superoxide (O2-) generation or lysozyme release. Two major surface glycopolypeptides with apparent molecular weights of 92,000 and 155,000 were immunoprecipitated from granulocytes. Dissociation of the protein complexes obtained from blood mononuclear cells and granulocytes indicated the presence of the epitope on the 90,000-92,000 molecular-weight components. It is thus concluded that the smallest glycopolypeptides mediate adhesion in human granulocytes and mononuclear leukocytes.

Antibodies, Monoclonal↗
PubMed1984

Phorbol 12,13-dibutyrate enhances lateral redistribution of membrane glycoproteins in human blood lymphocytes.

Nanomolar concentrations of 4 beta-phorbol 12,13-dibutyrate markedly enhanced the redistribution of concanavalin A receptors, the common leukocyte antigen and the Lyt-3 antigen in human blood lymphocytes, as measured by cap formation. The effect on the lateral mobility of these cell surface molecules was dose dependent and occurred within a few minutes of treatment. 12-O-Tetradecanoyl phorbol 13-acetate, another tumor promoter, was similarly active. 4 alpha-Phorbol 12,13-didecanoate, which does not have tumor-promoting activity, did not enhance cap formation. The effect of various drugs and treatments indicated that the phorbol ester-enhanced cap formation was energy and temperature dependent and required functional microfilaments. Retinoic acid, an antitumor-promoting agent, was inhibitory and trifluoperazine, an inhibitor of calmodulin-dependent processes, had a minor inhibitory effect. Protein secretion and synthesis, extracellular Ca2+/Mg2+ and functional microtubules did not seem to be involved. The enhanced capping was inhibited by the alkylating agents tosyl phenylalanyl chloromethyl ketone and tosyl lysyl chloromethyl ketone but not by other protease inhibitors. The effect of various amino acid derivatives suggested the participation of an esterase. A comparative study of dose response, kinetics and sensitivity to drugs indicated a direct correlation between the phorbol 12,13-dibutyrate-enhanced redistribution of membrane glycoconjugates and the phorbol ester-induced binding (adhesion) between human blood lymphocytes, a phenomenon previously described.

Glycoproteins↗
PubMed1983

Characterization of the phorbol 12,13-dibutyrate (P(Bu)2) induced binding between human lymphocytes.

The mechanisms, cell surface structures, and cell types involved in the phorbol 12,13-dibutyrate (P(Bu)2)-induced binding between human lymphocytes were studied. Induction of cell aggregation by 20 min treatment with P(Bu)2 required Ca2+, an intact membrane, functional microfilaments, and the possible participation of an esterase or, less likely, a protease. Trypsin-sensitive cell surface structures were needed and neuraminidase (NANase) treatment slightly increased the intercellular binding. Retinoic acid, an anti-tumor promoting agent, was inhibitory. Calmodulin-dependent processes, microtubules, phospholipid methylation, intracellular levels of cyclic adenosine monophosphate, and cellular secretion did not seem to be involved. Cell conjugation between 24 hr P(Bu)2-treated and untreated cells required participation of trypsin-sensitive cell surface structures in each of the interacting cells and NANase treatment of one partner slightly increased the intercellular binding. Thymocytes, T cells, mature B and Epstein-Barr virus-transformed B cells aggregated while pre-B, early B, and intermediate B lymphocytes derived from representative malignancies did not. The lack of aggregation was not due to the absence of phorbol ester receptors. It is concluded that the P(Bu2)-induced intercellular binding is mediated by cell surface proteins, depends on certain enzymatic activities and metabolic events and involves certain cell types.

Amino Acids↗
PubMed1983

Activation of the EBV-cycle and aggregation of human blood lymphocytes by the tumor promoters teleocidin, lyngbyatoxin A, aplysiatoxin and debromoaplysiatoxin.

A variety of tumor promoters such as the phorbol esters were found to be capable of inducing the viral cycle in cell lines latently infected with Epstein-Barr virus (EBV). We tested two classes of new tumor promoters; indole alkaloids and polyacetates, for their ability to induce the synthesis of the Epstein-Barr virus determined early antigen (EA) complex. Teleocidin and lyngbyatoxin A are indole alkaloids. Aplysiatoxin and debromoaplysiatoxin are polyacetates. Of these four tumor promoters all but debromoaplysiatoxin induced the synthesis of the EA complex. However, in combination with 3 mM n-butyrate, all four induced EA synthesis. The potent tumor promoters teleocidin, lyngbyatoxin A and aplysiatoxin induced maximal synthesis of EA at the concentration of 5 to 10 ng/ml, whereas the weak tumor promoter debromoaplysiatoxin required a concentration of 250 ng/ml to achieve maximal induction. Phorbol esters induce quick morphological changes and aggregation of human blood lymphocytes. The latter phenomenon has been interpreted as the expression of a "cell binding phenotype" (Patarroyo et al., in press). We showed that all four promoters induced aggregation of human lymphocytes at similar concentrations. The induction seemed to be a common effect which could be induced by both strong and weak tumor promoters.

Alkaloids↗
PubMed1983

Phorbol 12,13-dibutyrate (P(Bu)2)-treated human blood mononuclear cells bind to each other.

Treatment of human blood mononuclear cells with nanomolar concentrations of phorbol 12,13-dibutyrate (P(Bu)2) induced their aggregation. The phenomenon was seen within a few minutes and reached its maximum manifestation after 20 min. At this time, 20-30% of unfractionated and nylon wool-passed mononuclear leukocytes were in the aggregates. The influence of pretreatment with 2-deoxyglucose, NaN3, EDTA, cyclohexamide, or incubation at 4 degrees C on the phenomenon indicated that it is energy and temperature dependent, requires the presence of extracellular divalent cations, and is independent of protein synthesis. Nonaggregating 2-deoxyglucose-and NaN3-pretreated cells could still bind [3H]P(Bu)2 which rules out the possibility that the phorbol ester molecule acts as a bridge between the aggregated cells. Seventeen percent of the P(Bu)2-treated T-cell population bound untreated autologous and allogeneic cells. The binding property has a certain species specificity because only 4% of the cells interacted with mouse lymphocytes. At the ultrastructural level, the intercellular binding showed broad areas of surface contact (both between lymphocytes and lymphocyte-monocyte) and "trapping" by surface processes was not seen. Aggregated cells did not show cytopathogenic changes.

Animals↗
PubMed1983

Lymphocyte-mediated lysis of autologous and allogeneic B-cell lines in man.

The lytic potential of human blood lymphocytes was assayed against autologous and allogeneic EBV-transformed B-cell lines (LCL). The effects--if present--were very weak. Short-term interferon (IFN) treatment of the lymphocytes induced cytotoxic potential which could be manifested against autologous and allogeneic LCL. The sensitivity of the targets increased after superinfection with the P3HR-1 strain of EBV. Thus the strongest lytic effects were obtained with IFN-treated lymphocytes acting on EBV superinfected targets. Individuals with and without previous EBV encounter reacted against autologous LCL, indicating that the lysis did not represent an EBV-specific cellular memory. No evidence for alloantigen recognition emerged from the tests.

B-Lymphocytes↗
PubMed1980

Induction of the EBV cycle in B-lymphocyte-derived lines is accompanied by increased natural killer (NK) sensitivity and the expression of EBV-related antigen(s) detected by the ADCC reaction.

Human B-lymphocyte-derived lines were forced to enter the EBV-cycle by superinfection with the P3HR-1 substrain of EBV or sodium butyrate treatment. The induced cells were used as targets for natural killing (NK) and EBV-specific, antibody-dependent cellular cytotoxicity (ADCC). Two Burkitt lymphoma lines, Raji and Daudi, and one normal adult derived lymphoblastoid cell line, NAD-7, were comparable in their ADCC-sensitivity after induction, but only the Burkitt lymphoma-derived lines showed a major increase in NK-sensitivity. The superinfection-induced membrane change, responsible for both NK and ADCC sensitivity, is an early function of the viral cycle, correlated with the appearance of early antigens (EA). Indirect evidence indicates that the NK and ADCC target sites are different but this problem requires further investigation. Sodium butyrate induced an increased NK sensitivity and EBV-related ADCC sensitivity in the Burkitt lymphoma-derived P3HR-1 line. Lymphocyte effectors from different donors showed great differences in their NK and ADCC activity. Optimal ADCC could be demonstrated with effectors that were intermediate in their NK-activity.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1980

Increased sensitivity of human lymphoid lines to natural killer cells after induction of the Epstein-Barr viral cycle by superinfection or sodium butyrate.

Superinfection of latently Epstein-Barr virus (EBV)-carrying Raji cells with the P3HR-1 substrain EBV, known to induce the entry of a substantial fraction of cells into an abortively lytic cycle, increased the susceptibility of the cells to natural killer (NK) effect of human blood lymphocytes. Reciprocal cold-target competition tests with known NK-cell sensitive and -resistant lymphoid cell ines showed that the increased susceptibility is a result of the appearance of an NK-sensitive target, rather than to a general increase in membrane fragility. Lymphocytes of EBV-seropositive and -negative donors were equally effective killers against P3HR-1 virus-superinfected targets. EBV-induced NK sensitivity increased with time. It was a result of some event associated with the intracellular viral cycle, and not to the adherence of viral particles to the cell surface. Induction of EBV-carrying P3HR-1 cells to entry into the viral cycle with n-butyrate also increased their NK sensitivity. A transforming, noncytopathic prototype strain of EBV, B95-8, failed to increase the susceptibility of theRaji cells to NK-lysis, although it had some effect on the Daudi line. Because NK cells can kill virus-producing cells at an early stage of the cycle, before the virus particles are assembled, they may restrict, in vivo, the spread of the virus from latently infected cells.

Antigens, Viral↗