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Biomedical subjects

M Fresno

Publications and source records attributed to M Fresno.

At least 73 records · Page 4Linked to original sources
PubMed1995

Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR zeta-chain.

In addition to being an iron transporter, the transferrin receptor (TfR) has been shown to play a role in T cell activation. Stimulation of the TfR with specific Abs results in T cell proliferation, IL-2 secretion, and protein kinase C activation. In this paper we have analyzed early events caused by activation of the TfR. We have found several protein substrates to be tyrosine phosphorylated upon TfR stimulation in the human Jurkat T cell line. Interestingly, the TfR induced tyrosine phosphorylation in cell lines expressing TCR but not in TCR-negative mutants. Restoration of the TCR surface expression in these mutants reestablished the ability of the TfR to induce tyrosine phosphorylation. This result suggests that activation through the TfR is functionally dependent upon the expression of the TCR. Moreover, the functional relationship of the TfR with the TCR complex is also supported by data showing that TfR stimulation resulted in the tyrosine phosphorylation of the TCR zeta-chain; conversely, stimulation of the TCR complex resulted in an increased tyrosine phosphorylation of the TfR. More importantly, the TfR is shown to associate physically with the TCR zeta-chain as well as with the zeta-binding ZAP70 tyrosine kinase. The TfR/zeta complex is expressed on the cell surface independent of the expression of the other subunits of the TCR complex. We suggest that the TfR/zeta complex is responsible for transducing the TfR-induced signals, and that it could serve to amplify signals delivered by Ag binding to the TCR.

Animals↗
PubMed1995

Biphasic control of nuclear factor-kappa B activation by the T cell receptor complex: role of tumor necrosis factor alpha.

The regulation of nuclear factor (NF)-kappa B activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-kappa B protein bound the kappa B element of interleukin-2 receptor (IL-2R) alpha chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7-16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-alpha prevented all inhibition. These results indicate that the regulation of NF-kappa B activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-alpha is present. TNF-alpha, therefore, emerges as the main factor responsible for a second phase of NF-kappa B regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50.

Antibodies, Monoclonal↗
PubMed1995

Physical association and functional relationship between protein kinase C zeta and the actin cytoskeleton.

Protein kinase C (PKC) was initially identified as a serine/threonine protein kinase dependent on calcium and phospholipids and shown to be involved in intracellular signaling pathways. PKC isoforms have been classified into four groups: Ca(2+)-dependent conventional PKC alpha, beta I, beta II, gamma; Ca(2+)-independent, novel PKC delta, epsilon, eta, phi; atypical PKC zeta, lambda, iota which are not activated by Ca2+ or diacylglycerol, and the recently discovered PKCmu. We reported that activation of the zeta PKC isoform is an important step in interleukin-2 (IL-2)-mediated proliferation (Gómez, J., Pitton, C., García, A., Martínez, A., Silva, A. and Rebollo, A., Exp. Cell Res. 1995. 218: 105.). zeta PKC is also required for mitogenic activation of fibroblasts and for the maturation pathway activated by insulin and Ras. Contradictory results have been reported regarding the subcellular redistribution of zeta PKC upon activation. We report here, using confocal microscopy, that IL-2 induces expression, translocation and association of zeta PKC to a structure coincident with the actin cytoskeleton. Furthermore, we show that zeta PKC has a role in maintaining the integrity of the actin cytoskeletal structure in IL-2-stimulated cells. On the contrary, zeta PKC is not involved in the actin cytoskeleton organization when cells are maintained in IL-4, confirming our previous results showing that IL-4-induced signal transduction is PKC independent.

Actins↗
PubMed1994

Regulation of interleukin-2 receptor alpha chain expression and nuclear factor.kappa B activation by protein kinase C in T lymphocytes. Autocrine role of tumor necrosis factor alpha.

The regulation of interleukin-2 receptor alpha chain (IL-2R alpha) expression and nuclear factor (NF) activation by protein kinase C (PKC) in resting T cells, has been studied. Treatment of human resting T cells with phorbol esters strongly induced the expression of IL-2R alpha and the activation of NF.kappa B. This activation was due to the translocation of p65 and c-Rel NF.kappa B proteins from cytoplasmic stores to the nucleus, where they bound the kappa B sequence of the IL-2R alpha promoter either as p50.p65 or as p50.c-Rel heterodimers. Interestingly, all of those events were largely indirect and mediated by endogenously secreted tumor necrosis factor alpha (TNF alpha), as they were strongly inhibited by a neutralizing anti-TNF alpha monoclonal antibody. Furthermore, cyclosporin A, which blocked TNF alpha production induced by PKC, strongly inhibited IL-2R alpha and NF.kappa B activation. The addition of either TNF alpha or IL-2 partially recovered cyclosporin A-induced IL-2R alpha inhibition, but only TNF alpha completely recovered NF.kappa B activation. Those results indicate that, in resting T cells, PKC activation has only a triggering role, whereas the endogenously secreted TNF alpha plays an essential role in the quantitative control of the expression of IL-2R alpha chain or NF.kappa B activation.

Alkaloids↗
PubMed1994

Control of T lymphocyte activation and IL-2 receptor expression by endogenously secreted lymphokines.

The relative contribution of endogenously secreted lymphokines, TNF-alpha and IL-2, to T cell activation has been studied. Interestingly, a neutralizing anti-TNF-alpha mAb strongly inhibited the proliferation, IL-2R alpha expression, and activation of the nuclear factor (NF)-kappa B induced on human purified T lymphocytes by an immobilized anti-CD3 mAb (imm.anti-CD3). Furthermore, the addition of exogenous TNF-alpha to T cells activated by imm.anti-CD3 or phorbol esters strongly stimulated all those activities. Similarly, neutralizing anti-IL-2R alpha Abs inhibited the cell proliferation and the IL-2R alpha expression induced by imm.anti-CD3, whereas exogenous addition of IL-2 enhanced both activities. However, exogenous addition of IL-2 did not affect NF-kappa B activation. Cyclosporin A, which prevented lymphokine mRNA transcription, inhibited more than 90% of the IL-2R alpha and NF-kappa B levels induced by TCR/CD3-mediated activation. Exogenous TNF-alpha and IL-2 were equally important to the partial recovery of this IL-2R alpha inhibition, and high doses of both lymphokines together completely abolished the inhibitory effect of cyclosporin A. However, only TNF-alpha (but not IL-2) efficiently recovered the nuclear NF-kappa B levels. Those results indicate that the autocrine secretion of TNF-alpha and IL-2, and not the primary stimulus itself, contribute mostly to the regulation of IL-2R; thereby playing a very important role in quantitative control of T cell proliferation and leaving to the specifically TCR/CD3-derived signals the triggering role in T cell activation.

Antibodies, Monoclonal↗
PubMed1994

Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, and interleukin-6 but not TNF-beta induce differentiation of neuroblastoma cells: the role of nitric oxide.

Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), but not TNF-beta, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-alpha and IFN-gamma can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, L-NG-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by L-NG-monomethylarginine. These results indicate that TNF-alpha and IFN-gamma, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture supernatants of N103 cells induced by TNF-alpha and IFN-gamma, but not that by IL-6, contained high levels of NO2-, the production of which was inhibited by L-NG-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-alpha and IFN-gamma and that neuronal cells, in addition to the macrophage-like brain cells, can be induced by immunological stimuli to produce large quantities of NO.

Amino Acid Oxidoreductases↗
PubMed1994

Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a GPI anchor.

The biochemical and functional properties of T. cruzi GP50/55, a novel glycosylphosphatidylinositol (GPI)-anchored membrane antigen have been investigated. A 50-52-kDa thiol proteinase activity could be immunoprecipitated with monoclonal antibodies (mAb) directed against GP50/55 (mAb C10), different from the one reactive with mAbs against lysosomal cysteine proteinase GP57/51. Furthermore, the mAb C10-reactive proteinase corresponded to the GPI-anchored surface antigen since the proteolytic and antigenic activity partitioned to the aqueous phase after Triton X114 phase separation of phosphatidylinositol specific phospholipase C (PI-PLC)-treated parasites. Of several proteins immunoprecipitated by a polyclonal anti-lysosomal cysteine proteinase, an mAb to GP57/51 recognized a 60-kDa protein, whereas mAb C10 recognized antigens ranging between 52 and 50 kDa. The GP50/55 antigen detected by mAb C10 is expressed on the parasite surface whereas the GP57/51 antigen is mainly intracellular. The internal peptide sequence obtained from purified GP50/55 showed that it is more homologous to the prototype of the cysteine proteinases superfamily, papain, than to the two T. cruzi lysosomal cysteine proteinases so far described. Our data indicate that the T. cruzi GP50/55 is a novel GPI-anchored cysteine proteinase and may represent another isoform of this heterogeneous group of proteinases.

Animals↗
PubMed1993

Involvement of nitric oxide on the cytokine induced growth of glial cell.

The growth of C6 glioma cells was stimulated by TNF-alpha, IFN-gamma and IL-6 but not by TNF-beta. However, TNF-alpha and IFN-gamma but not IL-6 induced the synthesis of NO in C6 cells. Moreover, N-monomethyl-L-arginine a competitive inhibitor of NO synthase blocked TNF-alpha and IFN-gamma-dependent proliferation and NO induction on C6 cells, but had no effect on IL-6-dependent proliferation. In addition, C6 proliferation induced by TNF-alpha and IFN-gamma was specifically blocked by inhibitors of cyclic nucleotide dependent protein kinases such us H-9. Those results suggest that TNF-alpha and IFN-gamma but not IL-6 induce the growth of glia cells through the generation of NO which in turns activate a cyclic nucleotide dependent kinase.

Animals↗
PubMed1993

Involvement of beta 1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages.

We have investigated the role of integrin molecules in the binding and entry of Trypanosoma cruzi into human macrophages, with the help of monoclonal antibodies (mAb). Addition of Lia 1/2 mAb and in lesser extent of Lia 1/5 mAb, which are both specific for the beta 1 subunit of the VLA integrin family, to human macrophages blocked T. cruzi uptake and subsequent replication inside the macrophages. This inhibition correlated with their respective ability to block Fibronectin (Fn) binding to macrophages. Furthermore, another anti-beta 1 mAb, Alex 1/4, which binds to a different epitope on the beta 1 molecule and was unable to block Fn binding, did not affect T. cruzi invasion. The inhibition by Lia 1/2 and Lia 1/5 was dose dependent and clearly observable with doses as low as 1 microgram/ml. Moreover, this inhibition was T. cruzi specific since the Lia 1/2 and Lia 1/5 were unable to block uptake of Leishmania pifanoi or Escherichia coli by human macrophages. In contrast, the TS 1/18 mAb, which blocks ligand binding to beta 2 integrin, inhibited entry of L. pifanoi but not of T. cruzi. Finally, mAb specific for the alpha 4 and alpha 5 subunits of the two major Fn binding molecules of macrophages (VLA-4 and VLA-5, respectively), either alone or in combination, were poor inhibitors of T. cruzi uptake, suggesting that several members of the VLA family, including VLA-4 and VLA-5, are involved in binding and entry of T. cruzi into macrophages.

Animals↗
PubMed1993

Localization of metallothionein in breast carcinomas. An immunohistochemical study.

Metallothionein (MT) is a cysteine-rich, low molecular weight protein that binds zinc, copper, and cadmium. It is present in a number of normal cells including hepatocytes particularly during fetal and early postnatal life. It has been suggested that developmental profile of MT is similar to other oncofetal gene products and hence, it could be used as a marker for aggressive tumour behaviour. In order to test that hypothesis, we used a monoclonal antibody to MT and immunohistochemically evaluated formalin-fixed, paraffin-embedded tissues from 79 breast carcinomas. In non-neoplastic breast tissue, a strong nuclear and cytoplasmic staining was observed in myoepithelial cells. Positive staining for MT was present in 35 (44%) of breast carcinomas. In most positive cases, nuclear, or both nuclear and cytoplasmic staining was seen. All positive tumours were invasive ductal carcinomas, including a medullary and a metaplastic carcinoma. None of the mucinous, lobular, or intraductal papillary carcinomas reacted for MT. A statistically significant association was found between MT immunostaining and histological grade (P < 0.01) as well as with nuclear grade (P < 0.01). We also observed an inverse relationship between MT staining and oestrogen receptor content of tumours (P < 0.01). Similarly, a statistically significant association was found between moderate and strong MT immunostaining and decreased overall survival and shorter disease-free survival (P < 0.01). MT immunostaining was also predictive of a worse prognosis in the subgroup of lymph node negative (P < 0.001) and oestrogen receptor negative patients (P < 0.01). No statistically significant association was found between MT staining and size of tumour or the presence of lymph node metastasis. We conclude that MT staining may be a useful marker of less differentiated and more aggressive carcinomas of the breast.

Breast Neoplasms↗
PubMed1993

GP 50/55, a membrane antigen of Trypanosoma cruzi involved in autoimmunity and immunosuppression.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and the induction of autoantibodies crossreacting with host cells and tissues. We described here a GPI-linked 50/55 kDa antigen (GP50/55) present on the T. cruzi membrane, but not in the membrane of other parasites of the family Trypanosomatidae. We have obtained several monoclonal antibodies which specifically recognize this molecule. One of these GP50/55-specific mAbs (C10) crossreacts with a 28 kDa antigen expressed on the membrane of activated mouse and human T and B lymphocytes, after "in vitro" activation with mitogens, phorbol esters, or antigen, and on several murine T and B lymphocyte cell lines. Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from T. cruzi-infected mice or Chagasic patients but not from uninfected mice or control patients contain antibodies which recognize a similar p28 antigen and also suppress the proliferation of human T lymphocytes. These results suggest a possible role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Animals↗
PubMed1993

[Epidemiological data on chronic myeloid leukemia in Asturias, 1972-1986].

PURPOSE: To evaluate the incidence of CML in Asturias during the period 1972-1986, studying the variations in relation to age, sex and geographical zone. MATERIAL AND METHODS: Information relating to the date of diagnosis, name, age, sex and home address of the 108 cases (65 men and 43 women) diagnosed CML during the period 1972-1986 according to conventional criteria was collected from the hospital records of the region. For the calculations, SADEI demographic data were taken as a reference, comparing the incidence between sexes and geographical zones by means of the CHI2. RESULTS: A rate of incidence of 0.64/100,000 inhabitants/year was obtained, this being 0.8 in men and 0.5 in women (p < 0.05). The rate of incidence increased progressively with age until the 45-59 age group when it was maximum. There were no differences between the three main centres of population Gijón (0.53), Oviedo (0.55) and Avilés (0.69) nor the latter with the rest of the zones in Asturias; nor was three either any difference between coastal towns (0.56) and those of the interior (0.71) (p = NS). CONCLUSIONS: Limited incidence of CML in Asturias without significant differences in its geographical distribution within the region. The incidence, which was higher among males, increased progressively with age until reaching maximum importance in the 45-59 age group.

Adolescent↗
PubMed1993

Sino-nasal adenocarcinoma: epidemiological and clinico-pathological study of 34 cases.

Thirty-four cases of sino-nasal adenocarcinoma treated between 1976 and 1992 are presented. The purpose of this work is the epidemiological analysis of this disease and to put forward a classification of the lesions according to their degree of differentiation, based on the histopathological examination of 29 tumors. The risk increase of having a sino-nasal neoplasia was significant in the population exposed to wood dust in their working environment--896 times greater (540 in the male population) for adenocarcinoma and 20 times greater for squamous-cell adenocarcinoma. The proposed histopathological classification divides these lesions according to their degree of differentiation. In this series 23 well-differentiated cases were found, three moderately differentiated and three colloid-type. The five-year determinate survival of this series was 30%. The tumor stage (especially intracranial involvement) was the only significant clinico-pathological parameter that impaired the survival.

Adenocarcinoma↗
PubMed1992

CD4 gene transcription is transiently repressed during differentiation of myeloid cells to macrophage-like cells.

The CD4 glycoprotein, which serves as receptor for human immunodeficiency virus (HIV), is expressed in several types of cells of hematopoietic origin, including T lymphocytes and monocytes. Triggering differentiation of peripheral blood monocytes, monocytic U-937 or promyelocytic HL-60 precursor cells to macrophage-like cells by phorbol ester treatment transiently induced both a rapid reduction in surface CD4, demonstrated by flow-cytometry analysis, and a gradual loss of CD4 mRNA, revealed by Northern-blot analysis. Experiments in HL-60 cells to determine the cause of the observed decay in CD4 mRNA levels suggested that the half-life of CD4 transcripts did not diminish but increased after phorbol ester stimulation. Direct measurement of CD4 gene transcription by run-on analysis indicated that the rate of synthesis of new CD4 mRNA molecules was reduced approximately 10-fold after phorbol ester stimulation, whereas the rate of synthesis of c-fos mRNA resulted in a 2.5-fold increase. These data suggest that phorbol ester treatment specifically reduces CD4 mRNA levels by repressing CD4 gene transcription. These findings may be relevant to understand the regulation of CD4 gene expression during differentiation.

Blotting, Northern↗
PubMed1992

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.

Amino Acid Sequence↗
PubMed1992

A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Animals↗
PubMed1992

Synergism between tumor necrosis factor-alpha and interferon-gamma on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism.

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi-infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell-free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage-like cell line. when the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin 2, whereas the A28 line did not secrete IFN-gamma upon stimulation. Furthermore, the trypanocidal-inducing ability of A10 supernatant was completely abrogated by neutralizing anti-IFN-gamma antibodies and partially abrogated by neutralizing anti-TNF-alpha antibodies. When recombinant cytokines were added to J774 cells, IFN-gamma was able to induce significant trypanocidal activity whereas TNF-alpha was almost ineffective. However, TNF-alpha or lipopolysaccharide (LPS) showed a synergistic effect with IFN-gamma on macrophage activation. IFN-gamma triggered nitric oxide (NO) synthesis by J774 cells whereas TNF-alpha was almost ineffective. TNF-alpha and LPS were also synergistic with IFN-gamma in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N-monomethyl-L-arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN-gamma and TNF-alpha, secreted by T. cruzi-immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO-dependent mechanism.

Animals↗