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Biomedical subjects

M Fresno

Publications and source records attributed to M Fresno.

At least 55 records · Page 3Linked to original sources
PubMed1998

Inhibition of phosphodiesterase type IV suppresses human immunodeficiency virus type 1 replication and cytokine production in primary T cells: involvement of NF-kappaB and NFAT.

Rolipram, a phosphosdiesterase type IV-specific inhibitor, prevented p24 antigen release from anti-CD3-activated human immunodeficiency virus (HIV)-infected T cells and CD4(+)-cell depletion associated with viral replication in a dose-responsive manner but minimally inhibited T-cell proliferation. Moreover, rolipram reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) by HIV-infected T cells. The transcriptional ability of a luciferase reporter gene under control of the HIV long terminal repeat, induced by phorbol myristic acetate plus ionomycin or by TNF-alpha, in primary T and Jurkat cells was also inhibited by rolipram. Rolipram inhibited NF-kappaB and NFAT activation induced by T-cell activation in Jurkat and primary T cells, as measured by transient transfection of reporter genes and electrophoretic mobility shift assays. Exogenous addition of TNF-alpha in the presence of rolipram restored NF-kappaB but not NFAT activation or p24 release. Addition of dibutyryl-cyclic AMP (dBcAMP) mimicked the effects of rolipram on p24 antigen release, NF-kappaB activation, and TNF-alpha secretion, but it did not affect NFAT activation or IL-10 production. The protein kinase A inhibitor KT5720 prevented the inhibition of TNF-alpha secretion but not that of HIV type 1 (HIV-1) replication caused by rolipram. Our data indicate that blockade of phosphodiesterase type IV could be of benefit against HIV-1 disease by modulating cytokine secretion and transcriptional regulation of HIV replication, and they suggest an important role of NFAT in HIV replication in primary T cells. Some of those activities cannot be ascribed solely to its ability to increase cAMP.

Cells, Cultured↗
PubMed1998

Trypanosoma cruzi tubulin eliminated in the urine of the infected host.

In previous studies we have identified and characterized an 80-kDa Trypanosoma cruzi urinary antigen (UAg) eliminated during acute infection. Polyclonal antibodies raised against this antigen revealed by western blotting and immunoprecipitation analyses showed the existence of another antigenic component of 50-55 kDa in the UAg preparation. The antiserum was also used for screening of a T. cruzi expression library. Sequencing of inserts from selected cDNA clones showed high homology with the 3' end of the T.cruzi beta-tubulin gene sequence encoding for the C-terminus of the protein. The presence of T. cruzi tubulin in the UAg was confirmed by immunoprecipitation of a 50-55-kDa protein from 125I-labeled UAg with monoclonal antibodies (MAbs) to human alpha/beta-tubulin. Interestingly, MAbs recognized radiolabeled T. cruzi tubulin eliminated in the urine of infected mice 24 hr postinoculation of [35S]methionine-labeled viable trypomastigotes. Tubulin found in the urine proved to be of T. cruzi origin because this protein could not be identified in urinary specimens from uninfected animals or mice acutely infected with Leishmania infantum or Toxoplasma gondii. We conclude that tubulin is one of the parasite antigens eliminated in the urine of T. cruzi-infected hosts. This finding may be used to develop a noninvasive procedure for early diagnosis of Chagas' disease.

Amino Acid Sequence↗
PubMed1997

Alteration of macrophage function by a Trypanosoma cruzi membrane mucin.

Cytokines secreted by macrophages play important roles in the immune response to Trypanosoma cruzi. Here, we report that a purified glycosylphosphatidylinositol (GPI)-anchored mucin from the T. cruzi membrane, Ag C10, is able to bind to the macrophage cell surface and blocks the subsequent binding of mAb against CD62L/L-selectin, whereas binding of mAbs directed against other monocyte surface molecules is unaffected. In addition, Ag C10 binding to macrophages triggered a CD54/ICAM-1-mediated cell adhesion as well as an increase in intracellular Ca2+, which was further augmented by cross-linking the Ag C10-bound surface receptors by mAb against Ag C10. Interestingly, Ag C10-treated monocytes secreted IL-1beta, but not TNF-alpha or IL-12. Moreover, these cells could secrete IL-1beta, but not TNF-alpha or IL-12, after activation with LPS. T. cruzi-infected macrophages displayed similar alterations in cytokine secretion, with an impaired ability to secrete IL-12 and TNF-alpha, but not IL-1beta, upon LPS activation. These effects were substantially inhibited by neutralizing mAb against Ag C10. These effects appeared to take place at the transcriptional level, since mRNA for TNF-alpha, but not that for IL-1beta, was drastically reduced in LPS-stimulated infected cells treated with Ag C10. Conceivably, inhibition of TNF-alpha and IL-12 by T. cruzi could be involved in the evasion of the immune response by this parasite.

Animals↗
PubMed1997

Massive glycation of protein HC, a low molecular weight lipocalin, in non-diabetic individuals.

Human protein HC is a member of the lipocalin superfamily with unique properties since it carries a covalently bound fluorescent chromophore mediating the linkage of the major part of protein HC to several plasma proteins, with IgA as the dominating complex partner. Native protein HC displays characteristic absorption and fluorescence spectra similar to those of glycated proteins with advanced glycosylation end products (AGEs). In vitro glycation of protein HC induces the formation of fibril aggregates with a corresponding increase of absorption in the visible region of the spectrum. Boronate-affinity chromatography and a novel galactosyltransferase assay indicate that protein HC is modified with residues of glucose exposed in a terminal non-reducing position which is typical of glycated proteins. The glycation level of several isolated batches of protein HC as measured by both assays was around 35%, which represents the highest level described for human plasma-derived proteins from healthy individuals.

Alpha-Globulins↗
PubMed1997

IL-2 signaling controls actin organization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta.

IL-2 and IL-4 induce proliferation of TS1 alpha beta cells. Activation of the zeta isoform of protein kinase C is an important step in IL-2-, but not IL-4-mediated proliferation. In addition, protein kinase C-zeta is implicated in IL-2-mediated actin organization. Given the established involvement of the Rho family of small guanine nucleotide-binding proteins in organization of actin structures, we analyze the possible relationships between Rho and protein kinase C-zeta. Using the Rho-like protein family-specific toxin B from Clostridium difficile, we report in this work that IL-2, but not IL-4, induces a Rho-dependent activation of protein kinase C-zeta. This signaling event is mediated by the activation of phosphatidylinositol 3-kinase. In contrast, IL-4 induces a Rho-independent, phosphatidylinositol 3-kinase-mediated activation of protein kinase C-zeta, but this pathway has no implications in cytoskeleton organization.

Actins↗
PubMed1997

Replication of human immunodeficiency virus-1 in primary human T cells is dependent on the autocrine secretion of tumor necrosis factor through the control of nuclear factor-kappa B activation.

Tumor necrosis factor (TNF)-alpha controls T-cell activation and is a major inducer of human immunodeficiency virus (HIV)-1 replication in chronically infected cells. Therefore, we have investigated its role in primary cultures of HIV-infected human T lymphocytes by using neutralizing anti-TNF-alpha antibodies or TNF-alpha. Primary resting T lymphocytes produced TNF-alpha and supported HIV replication after T-cell receptor activation. Addition of neutralizing anti-TNF-alpha antibodies drastically reduced p24 antigen release and prevented CD4+ cell depletion associated with infection. Anti-TNF-alpha also prevented nuclear factor-kappa B (NF-kappa B) activation, and a good correlation between this inhibition and inhibition of HIV replication was observed. Moreover, supplementing the cultures with high doses of IL-2 reverted anti-TNF-alpha inhibition of cell proliferation but did not affect the inhibition of HIV p24 antigen release or NF-kappa B activation in the same cultures. Moreover, anti-TNF-alpha inhibited HIV-1 long terminal repeat (LTR)-driven transcription of a reporter gene in primary T cells in response to activation, either in the presence or the absence of HIV-1 Tat. Our results support an important role for autocrine TNF-alpha secretion in controlling HIV replication in primary T cells because of its ability to maintain NF-kappa B elevated in the nucleus of T cells.

Cells, Cultured↗
PubMed1997

p53 expression is of independent predictive value in lymph node-negative breast carcinoma.

The aim of this study was to evaluate p53 expression, determined by immunohistochemistry, in 151 infiltrating ductal breast carcinomas with negative axillary lymph nodes, and to determine whether p53 can be considered as an independent prognostic value for overall and disease-free survival. A monoclonal antibody (DO-7) that reacts with an epitope on the N terminal portion of the human protein p53 was used to detect p53 in paraffin-embedded sections, utilising a standard avidin-biotin-peroxidase complex (ABC) technique with a microwave oven antigen retrieval. Overexpression of p53 (more than 50% of stained cells) was found in 45 cases (30%). Forty-five cases were negative and occasionally or moderately stained cells were present in 61 cases. p53 protein overexpression was significantly associated with high histological grade and tumour necrosis, high MIB-1 value (MIB-1 > 30%) and negative oestrogen receptor status. Univariate analysis (log-rank) showed a shorter overall survival (P = 0.003) in patients with high tumour p53 positivity. This statistical significance was also seen on multivariate analysis (Cox's logistic regression, P = 0.004). p53 protein overexpression is an independent prognostic marker in node-negative breast carcinoma for overall survival and should be used with other prognostic factors.

Adult↗
PubMed1997

Induction of adhesion/differentiation of human neuroblastoma cells by tumour necrosis factor-alpha requires the expression of an inducible nitric oxide synthase.

Neuroblastoma cell lines (SK-N-SH and SK-N-MC) were induced to differentiate, as detected by the expression of neurofilament proteins of 68 and 200 kDa, and to express adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule) after stimulation with tumour necrosis factor-alpha (TNF-alpha). This induction was accompanied by the arrest of cell growth. The induction of neuroblastoma adhesion by TNF-alpha could be inhibited by the nitric oxide synthase inhibitors, L-N-monomethyl arginine (L-NMMA) and L-N6-(1-imidoethyl)-lysine (highly specific for the inducible enzyme), but not by the inactive enantiomer D-NMMA. These results indicate that TNF-alpha induces the adhesion of neuroblastoma cells via nitric oxide. This was confirmed by the finding that the adhesion/differentiation of SK-N-SH and SK-N-MC cells can be directly induced by the addition of nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine, into the culture medium. The isoform of the nitric oxide synthase induced in human neuroblastoma cells by TNF-alpha treatment was identified enzymatically as isoform II by Western blotting and by the polymerase chain reaction. Thus TNF-alpha induces the in vitro adhesion/differentiation of human neuroblastoma cells through nitric oxide synthesized by a calcium-independent inducible form of nitric oxide synthase, clearly indicating that isoform II of nitric oxide synthase can be expressed in human neuronal cell types.

Animals↗
PubMed1997

Megalomicin disrupts lysosomal functions.

Megalomicin (MGM) has been shown to cause a dilation of the most distal cisternae of the Golgi complex. The effects of MGM on Golgi morphology correlated with an inhibition of protein transport to the trans-Golgi resulting in an accumulation of poorly sialylated glycoproteins. Here we show that the addition of 50 microM MGM caused a rapid swelling of lysosomes in cultured cells and inhibited the degradation of the newly synthesized T cell antigen receptor CD36 subunit. Although MGM did not affect the uptake of fluid phase markers, it prevented their degradation. Interestingly, endocytosed ovalbumin did not colocalize with lysosomes in MGM-treated cells, suggesting an MGM-induced impairment in the delivery to lysosomes. This was confirmed by Percoll density gradients, where the fluid phase marker remained in endosomal fractions, even after long chase times, whereas in control cells the endocytosed marker was located in lysosomes. The effect of MGM was not confined to soluble proteins since it did also inhibit the delivery of the membrane-bound epidermal growth factor receptor to lysosomes. Finally, MGM strongly inhibited the ATP-dependent acidification of lysosomes in vitro, suggesting a possible mechanism for its in vivo activity.

Animals↗
PubMed1996

Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation.

A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimer's disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

Amyloid beta-Protein Precursor↗
PubMed1996

Intra-Golgi transport inhibition by megalomicin.

Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.

Animals↗
PubMed1996

Cathepsin D in host stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative breast cancer.

One hundred fifty-four axillary lymph node-negative invasive ductal carcinomas of the breast were immunohistochemically evaluated for the expression of cathepsin D. Formalin-fixed paraffin sections of each tumor were stained using a polyclonal antibody raised against recombinant procathepsin D. Cathepsin D content of tumor cells and host histiocytes and fibroblasts within or immediately at the invasive border of tumors were assessed separately and correlated with histomorphology, estrogen-receptor content, and patients' survival data. Positive cathepsin D staining of tumor cells was associated with a lower nuclear grade and well-differentiated histology, whereas moderate to strong staining of host cells correlated with larger tumor size, higher nuclear grade, poorly differentiated histomorphology, and lack of estrogen-receptor (ER) protein. No statistically significant correlation was found between cathepsin D in tumor cells and survival. There was, however, a statistically significant correlation between moderate to strong cathepsin D staining of host cells and shorter disease-free and overall survivals. Expression of cathepsin D by host cells, however, did not have an independent influence on survival. The authors conclude that cathepsin D in stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative invasive ductal carcinomas of breast. Furthermore, determination of cathepsin D in cytosolic extracts of tumors is of no practical value because it may represent cathepsin D content of tumor cells, intratumoral host cells, or both.

Axilla↗
PubMed1996

Pentoxifylline inhibits acute HIV-1 replication in human T cells by a mechanism not involving inhibition of tumour necrosis factor synthesis or nuclear factor-kappa B activation.

OBJECTIVE AND DESIGN: To study the in vitro activity of pentoxifylline (PTX), which may be of benefit in AIDS, on cell proliferation, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma (a type 1 cytokine) and interleukin (IL)-10 (a type 2 cytokine) production, viral replication and CD4+ depletion in acutely HIV-1-infected human T cells. METHODS: T cells were stimulated with anti-CD3 antibody or phytohaemagglutinin (PHA) and infected with HIV-1 in presence or absence of PTX. Cell proliferation, CD4+ cell number, nuclear factor (NF)-kappa B activation, p24 antigen release or lymphokine content of the supernatants were evaluated by [3H]-thymidine incorporation, cytofluorimetry, electrophoretic mobility shift assays and specific enzyme-linked immunosorbent assay, respectively. RESULTS: In HIV-1-infected T cells, PTX inhibited cell proliferation and p24 release and prevented CD4+ depletion associated with HIV replication. Moreover, PTX reduced TNF-alpha, IFN-gamma and IL-10 production and NF-kappa B activation. PTX inhibited with similar potency IFN-gamma, TNF-alpha and cell proliferation. However, the inhibition of p24 release and specially of IL-10 production required significantly lower doses of PTX. Exogenous addition of IL-2 or TNF-alpha in presence of PTX restore T-cell proliferation and NF-kappa B activation respectively, but did not affect p24 inhibition. CONCLUSIONS: Our data suggest that the inhibitory effect of PTX on HIV replication cannot be satisfactorily explained by the inhibition of NF-kappa B or TNF-alpha. Moreover, PTX cannot be primarily considered as a TNF-alpha inhibitor and has several immunomodulatory and antiviral properties which could be of benefit against HIV-1 at various levels.

Antiviral Agents↗
PubMed1996

[Leydig cell tumor in the adult].

Presentation of 3 cases of Leydig's cells tumour in the adult seen in our hospital over the last 10 years. They account for 3% of all testicular tumours, 20 to 25% of them appearing during the pre-puberty period. Although an isolated testicular nodule is the most frequent symptom, gynaecomastia can be found in about 30% of adult patients, as it happened in 2 of our patients. Even though no malignant case has been described in children, up to 10% of cases in adults have metastasis, this being the only demonstration of malignant behaviour in these tumours, so radical orchiectomy is the choice therapy. Certain histological and hormonal parameters may be suggestive of the aggressive behaviour of this neoplasia which, however, we have not found in our series of all whom presented a good evolution after surgery. The most common diagnostic and therapeutic approaches, based on the different characteristic of the tumour, are reviewed.

Adult↗
PubMed1996

[Castleman's disease: a histological concept for a wide clinical spectrum].

A literature review about Castleman's disease after a clinical case with different manifestations of this entity was done. Special emphasis on the histological and clinical aspects of the sickness was given. The different pathological patterns and their broader spread do not match necessarily with the different clinical presentations described. Further conclusions regarding their evolution, prognosis and treatment response can not be easily obtained from these data.

Castleman Disease↗
PubMed1995

Characterization of carbohydrate binding proteins in Trypanosoma cruzi.

Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans and invades a great variety of mammalian cells. The nature of the ligand(s) and receptor components in both T. cruzi and target cells remains controversial, although it seems to involve an interaction with oligosaccharides. In an attempt to identify possible ligands on the parasite, we have searched for the presence of carbohydrate binding proteins (CBPs) in T. cruzi. By fluorescence-activated cell sorter analysis using a panel of fluoresceinated glyco- and neoglycopeptides with well characterized glycans, the presence of at least two different CBPs was identified on the surface of T. cruzi epimastigotes and trypomastigotes. The specificity of binding of the two CBPs seems to be mediated by galactose and mannose residues. The mannose- and galactose-mediated CBPs from epimastigotes and trypomastigotes were purified to homogeneity by affinity chromatography on immobilized thyroglobulin and identified as 60-70-kDa glycoproteins. Purified CBPs were able to specifically bind with high affinity to murine and human macrophages as well as other cell types susceptible to infection by T. cruzi but not to fat or neuronal cells. This binding was inhibited by the corresponding ligands. Moreover, the mannose-mediated CBP binding was completely abolished by alpha-mannosidase treatment of the cells. These results suggest a possible role for the CBPs in the recognition events between the parasite and target cells and/or in the interaction of the epimastigotes with the insect gut cells.

Animals↗