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J M Rojo

Publications and source records attributed to J M Rojo.

At least 19 recordsLinked to original sources
PubMed2002

Dislocation emission around nanoindentations on a (001) fcc metal surface studied by scanning tunneling microscopy and atomistic simulations.

We present a combined study by scanning tunneling microscopy and atomistic simulations of the emission of dissociated dislocation loops by nanoindentation on a (001) fcc surface. The latter consist of two stacking-fault ribbons bounded by Shockley partials and a stair-rod dislocation. These dissociated loops, which intersect the surface, are shown to originate from loops of interstitial character emitted along the <110> directions and are usually located at hundreds of angstroms away from the indentation point. Simulations reproduce the nucleation and glide of these dislocation loops.

Journal Article↗
PubMed2001

A proposal for harmonising laboratory performance assessment criteria in national asbestos fibre counting schemes.

Four European national asbestos fibre counting proficiency testing schemes have been studied in order to compare their criteria for the assessment of laboratory performance. Performance assessment is based on each laboratory's results after counting a certain number of samples. Two methods are currently being applied. To be classified 'satisfactory' laboratories must obtain at least 75% of normalised counts lying within defined performance limits (in three schemes), or the median and coefficient of variation of normalised counts must be within performance limits (in the fourth scheme). Differences in the numbers of test samples mean that the schemes are operating with different selectivity in assessing their laboratories' performances. Differences in the percentage of laboratory results falling within performance limits indicate that the schemes do not operate the same confidence probability in correctly assessing individual counts. It means that some schemes may be more lenient than others. This paper discusses two proposals to move towards harmonisation of the asbestos fibre counting proficiency testing schemes: (i) standardisation of the number of samples used for laboratory assessment and (ii) changes to the criteria to establish the limits of satisfactory performance.

Asbestos↗
PubMed2000

Crry/p65, a membrane complement regulatory protein, has costimulatory properties on mouse T cells.

It is known that certain type I membrane molecules (complement receptors type 1 and 2) belonging to the regulators of complement activation (RCA) family are involved in the regulation of B lymphocyte activation. In contrast, only GPI-anchored RCA molecules (CD55) have been described to be involved in T lymphocyte activation. In this study, we describe a novel function for the mouse RCA type I membrane protein Crry/p65 as a costimulatory molecule in CD4+ T cell activation. This is shown by increased anti-CD3-induced proliferation of CD4+ spleen T lymphocytes in the presence of the Crry/p65-specific mAb P3D2. Furthermore, Ab-induced coligation of Crry/p65 and CD3 favors IL-4 rather than IFN-gamma secretion in these cells. Crry/p65 signaling was also observed regardless of additional Ca2+, protein kinase C, or CD28-mediated costimuli. Analysis of intracellular intermediaries shows that Crry/p65-CD3 coligation enhances certain TCR/CD3-mediated signals, producing increased early tyrosine phosphorylation of many substrates and enhanced activation of the mitogen-activated protein kinase, extracellular signal-related kinase. These data fit well with the association of Crry/p65 with the tyrosine kinase Lck found in T cell lysates. The epitope recognized by the mAb P3D2 interferes with the protective role of Crry/p65 on C3 deposition. The relationship between protective function and costimulation by Crry/p65 is discussed. Our results support a multifunctional role for Crry/p65 in T cells and suggest new links between the natural and adaptive immune responses.

Adjuvants, Immunologic↗
PubMed2000

Variability of invariant mouse CD3epsilon chains detected by anti-CD3 antibodies.

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.

Alleles↗
PubMed1999

Antibody-induced CD3-CD4 coligation inhibits TCR/CD3 activation in the absence of costimulatory signals in normal mouse CD4(+) T lymphocytes.

The effect of CD3-CD4 coligation on CD3-mediated activation of normal mouse CD4(+) T lymphocytes has been analyzed in the absence of exogenous lymphokines. If anti-CD3 and anti-CD4 antibodies are adsorbed to culture wells by means of previously adsorbed anti-Ig antibodies (indirect binding), CD3-CD4 coligation inhibits activation measured as cell proliferation or as secretion of IL-2, IL-4, and IFN-gamma. Addition of IL-2, anti-CD28 antibodies, or phorbol esters, but not IL-1, IL-4, or ionomycin, blocked CD4-mediated inhibition and restored the response to levels equal or higher than those of cultures activated by anti-CD3 alone. In contrast, CD3-CD4 coligation by antibodies directly adsorbed to culture wells potentiated anti-CD3-induced activation, either in the absence or in the presence of exogenous costimuli. Similar results were observed when CD4(+) T cells of naive phenotype (CD44(low), CD45RB(high)) were used in the experiments. The analysis of early tyrosine phosphorylation in CD4(+) T cells shows that phosphorylation of many cell substrates is clearly enhanced upon CD3-CD4 coligation using indirectly or directly bound antibodies, yet certain substrates are mainly phosphorylated under inhibitory conditions. Although CD28 ligation does not produce any clear change in the tyrosine phosphorylation pattern in lysates from cells activated by indirectly bound anti-CD3 plus anti-CD4 antibodies, the analysis of active forms of the MAP kinase ERK suggests that downstream signaling pathways involved in IL-2 gene activation can be differentially activated depending on the direct or indirect CD3-CD4 adsorption and CD28 ligation.

Adsorption↗
PubMed1999

Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle.

CD38 displays lateral association with the HIV-1 receptor CD4. This association is potentiated by the HIV-1 envelope glycoprotein gp120. The aim of this work was to evaluate the CD38 role in T cell susceptibility to HIV-1 infection. Using laboratory X4 HIV-1 strains and X4 and X4/R5 primary isolates, we found that CD38 expression was negatively correlated to cell susceptibility to infection, evaluated as percentage of infected cells, release of HIV p24 in the supernatants, and cytopathogenicity. This correlation was at first suggested by results obtained in a panel of human CD4(+) T cell lines expressing different CD38 levels (MT-4, MT-2, C8166, CEMx174, Supt-1, and H9) and then demonstrated using CD38 transfectants of MT-4 cells (the line with the lowest CD38 expression). To address whether CD38 affected viral binding, we used mouse T cells that are non-permissive for productive infection. Gene transfection in mouse SR.D10.CD4(-).F1 T cells produced four lines expressing human CD4 and/or CD38. Ability of CD4(+)CD38(+)cells to bind HIV-1 or purified recombinant gp120 was significantly lower than that of CD4(+)CD38(-) cells. These data suggest that CD38 expression inhibits lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent viral binding to target cells.-Savarino, A., Bottarel, F., Calosso, L., Feito, M. J., Bensi, T., Bragardo, M., Rojo, J. M., Pugliese, A., Abbate, I., Capobianchi, M. R., Dianzani, F., Malavasi, F., and Dianzani, U. Effects of the human CD38 glycoprotein on the early stages of theHIV-1 replication cycle.

ADP-ribosyl Cyclase↗
PubMed1997

Modulation of lymphocyte interaction with endothelium and homing by HIV-1 gp120.

We have previously shown that HIV-1 glycoprotein 120 (gp120) induces CD4 association with several molecules on the surface of CD4+ lymphocytes. Since one of these molecules was CD38, involved in lymphocyte/endothelium interaction, this article examines the possibility that gp120/CD4 binding alters CD4+ T cell interaction with vascular endothelium in vitro and in vivo. Cocapping experiments showed that gp120 induced CD4 association with CD38, CD29, CD49d, and CD11a in peripheral blood CD4+ T cells. Two in vitro binding assays were used to evaluate the effect of gp120. A static binding assay, performed at 37 degrees C, evaluated stable interactions mediated by integrins, and a dynamic binding assay, performed at 4 degrees C on a rocking shelf, evaluated weak interactions mediated by constitutively active molecules such as selectins and CD38. Gp120 increased dynamic binding and inhibited static binding to the endothelium of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs suggested that the gp120 effect on dynamic binding involved CD38, CD31, and CD49d, whereas the effect on static binding involved CD11a and CD49d. In vivo experiments showed that treatment of 2D4 cells, a CD4- CD8- mouse T cell clone transfected with the human CD4, with gp120 increased their homing into the spleen, intestine, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Alteration of lymphocyte homing may contribute to immune deficiency in HIV-1+ patients by decreasing the probability of an encounter between Ags and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.

Animals↗
PubMed1997

CD4 dependence of activation threshold and TCR signalling in mouse T lymphocytes.

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.

Animals↗
PubMed1996

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes.

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56lck, p59fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolés, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4+ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4+ T lymphocytes. Our results show that, in normal CD4+ T lymphocytes, as in CD4+ T cell lines, there is a stable and readily detectable association between CD4:p56lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4+ lymphocytes have detectable amounts of p56lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4+ T cells and CD4+ T cell lines in kinase association to the TcR/CD3 complex.

Animals↗
PubMed1996

Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor.

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single-positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide-linked dimer, precipitating as a broad band at about 50-65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N-glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross-blocking, co-capping, co-modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co-stimulatory molecules such as CD69, CD2, Ly-6, CD25, OX-40, Mac-1 and LFA-1. By in vitro kinase assay, H4 was found to co-precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co-modulation and co-capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell-specific co-stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.

Animals↗
PubMed1995

A hyperreactive variant of a CD4+ T cell line is activated by syngeneic antigen presenting cells in the absence of antigen.

A variant of the murine CD4+ T helper cell clone D10.G4.1 (D10) has been isolated and cloned. This line, which we have named "syngeneic-reactive D10", or SR.D10, maintains the I-Ak-restricted specificity for Conalbumin and the allogeneic specificities characteristic of D10 cells. However, it is hyperreactive to TCR-dependent and-independent stimuli, indicating a lower activation threshold than the original D10.G4.1 clone. The hyperreactivity of SR.D10 runs in parallel with the acquisition of a reactive phenotype against syngeneic antigen presenting cells (APCs). As in antigen activation, reactivity to syngeneic APCs can be inhibited by anti-TCR, anti-CD4, or anti-class II monoclonal antibodies. The role of CD4 in this phenomenon is highlighted as "syngeneic reactivity" disappears in CD4- mutants of SR.D10 and is recovered in CD4 transfectants. The expression of several cell surface molecules involved in T cell activation show qualitative and/or quantitative differences between SR.D10 and the original D10. No significant differences in quantity and activity of p56lck and p59fyn were detected between the hyperreactive and the original clone. Our results suggest that high sensitivity to activation, concomitant with expression of CD4, might allow the acquisition of an autoreactive phenotype and confirm the important contribution of coreceptors to determine the activation threshold of the cells. The characteristics of SR.D10 and the possibility of growing them in the presence of interleukins make this cell line a experimental model of great interest for analyzing activation mechanisms in T cells.

Animals↗
PubMed1995

Suppression of immune parameters in animal models of morphine dependence.

Implantation of pellets containing 75 mg of morphine induced short term (4 day) morphine dependence and markedly reduced total number of spleen cells of BALB/c mice, without affecting total body or liver weight. Polyclonal responses induced by anti-CD3 antibodies, Concanavalin A or Escherichia coli lipopolysaccharide in the remaining spleen cells of morphine-treated mice were also inhibited. Cytofluorimetric analysis indicated that the proportion of major functional lymphocyte populations (Ig+, CD3+, CD4+ and CD8+ lymphocytes) were not significantly changed in the spleen from morphine-dependent mice. Furthermore, expression levels of surface Ig, CD3, CD4, and CD8, were similar in spleen cells from control or morphine-treated mice. So, morphine dependence in BALB/c mice under these controlled conditions results in a specific defect in lymphoid cell number and function, with no incidence on body weight or particular lymphocyte subsets.

Animals↗
PubMed1994

Genetic and immunochemical evidence for CD4-dependent association of p56lck with the alpha beta T-cell receptor (TCR): regulation of TCR-induced activation.

Recent observations suggest that the tyrosine kinase p56lck is involved in the transduction of transmembrane signals through the antigen specific T cell receptor (TCR) in CD4+ T cells. By means of in vitro kinase assays, we have found that p56lck coprecipitated with the TCR from lysates of a murine CD4+ T cell line in the absence of TCR-mediated stimuli. Analysis of CD4- mutants and CD4-transfected cells shows that p56lck-TCR association occurred only when CD4 was present. The functional importance of CD4:p56lck-TCR association was demonstrated by low activating potential of rare clonotypic antibodies which did not coprecipitate CD4:p56lck, as well as by total or partial loss of anti-TCR or antigen induced stimulation in CD4- cells, which could be recovered by CD4 transfection. Complementation assays using different anti-TCR antibodies suggest that cross linking of TCR-p56lck:CD4 plus structural changes in the complex are needed for efficient transduction of activating signals through the TCR in these cells.

Animals↗
PubMed1994

Polyerga, a biological response modifier enhancing T-lymphocyte-dependent responses.

Cancer patients are often treated with biological response modifiers to enhance immunological functions. However, little is known about the actual mechanism of action of many of these substances. Therefore, we were interested in the effect of i.p. treatment with porcine low-molecular-weight spleen peptides, which are used during supportive cancer therapy, on lymphoid cell populations and function in mice. After treatment with 0.5 microgram peptides/kg body weight for 14 consecutive days, lymphokine secretion and the generation of cytotoxic T-cells were significantly enhanced as compared with controls. However, there was no effect on the number of cells or the percentage of cells expressing functional surface markers in secondary lymphoid organs.

Animals↗
PubMed1990

A simple method for the radioactive iodination of CD4 molecules.

A method for the vectorial radioiodination of CD4 and other membrane proteins having few or no tyrosine residues in the extracytoplasmic domains is described. Incubation of the cells with sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP), a water-soluble derivative of the Bolton-Hunter reagent results in the coupling of hydroxyphenyl groups to free amino groups of cell surface proteins and these groups are then vectorially radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The method is highly efficient, gentle, fast, simple, and does not require the previous radiolabelling of the Bolton-Hunter reagent.

Animals↗
PubMed1989

Antibodies against a peptide representative of a conserved region of human IFN-alpha. Differential effects on the antiviral and antiproliferative effects of IFN.

mAb generated from mice immunized with a synthetic peptide representative of a conserved region (amino acids 133 to 147) of human IFN-alpha are described. Antisera from mice immunized with the peptide coupled to keyhole limpet hemocyanin were able to specifically bind to the peptide and also to bind and precipitate human IFN-alpha. Binding of the antibodies to IFN-alpha was inhibited by the immunizing peptide, but not by an unrelated peptide. Immunized mice were used to obtain three hybridomas producing mAb able to recognize both the immunizing peptide and human IFN-alpha, as determined by RIA and immunoprecipitation. These antibodies also neutralized the antiviral effect of human leukocyte IFN. In contrast, none of the mAb significantly affected the inhibition of Daudi cell proliferation induced by IFN-alpha.

Amino Acid Sequence↗
PubMed1989

Selective induction of growth factor production and growth factor receptor expression by different signals to a single T cell.

Stimulation of lymphokine production and the expression of receptors for growth factors can be dissociated in AK-8, a line of CD4+, I-A-restricted, conalbumin-specific mouse T cells. When activated by antibodies specific for the T cell receptor (TcR; F23.1) or the CD3 complex (145-2C11) adsorbed to plastic culture wells, AK-8 cells produce lymphokines but are unable to proliferate. Proliferation takes place using the same stimuli upon addition of interleukin 1 (IL 1). Autocrine growth induced by anti-TcR, anti-CD3 or by antigen is dependent on IL 4 and not on IL 2 in this cell line, as shown by the effect of antibodies against IL 4 or the IL 2 receptor. Similarly to plastic-adsorbed antibodies, phorbol myristic acetate (PMA) or a combination of PMA and the calcium ionophore Ionomycin also induces secretion of growth factors without inducing proliferation, but in this case addition of IL 1 is ineffective in inducing AK-8 proliferation. When incubated with anti-TcR or anti-CD3 antibodies in soluble form these cells neither proliferate nor produce IL 4 even in the presence of IL 1. However, soluble antibodies in the presence of IL 1 induce enhanced expression of IL 2 receptors, as measured both by induction of responsiveness to exogenous IL 2 or flow cytometry analysis using anti-IL 2 receptor antibodies. These results show that the pathways for the activation of growth factor receptor expression and the induction of lymphokine secretion can be differentiated in this cell line using anti-TcR or anti-CD3 reagents in different physical forms. The transmembrane signals delivered by these different forms of anti-receptor antibody may allow an understanding of these distinct requirements for T cell growth.

Animals↗