Biomedical subjects

B Alarcón

Publications and source records attributed to B Alarcón.

At least 19 recordsLinked to original sources

PubMed2006

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food.

AIMS: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. METHODS AND RESULTS: Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). CONCLUSIONS: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.

Animals↗

PubMed2003

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity.

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.

Animals↗

PubMed2002

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers.

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.

DNA Primers↗

PubMed2000

Intracellular redistribution of nucleolin upon interaction with the CD3epsilon chain of the T cell receptor complex.

T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3gamma, CD3delta, CD3epsilon, and CD3zeta. Whereas the biological significance of the cytoplasmic tails of these molecules is suggested, in part, by their evolutionarily conserved sequences, their interactions with signal transduction molecules are not completely understood. We used affinity chromatography columns of glutathione S-transferase fused to the CD3epsilon cytoplasmic tail to isolate proteins that specifically interact with this subunit. In this way, we identified the shuttling protein nucleolin as a specific CD3epsilon-interacting molecule. Using competition studies and affinity chromatography on peptide columns, we were able to identify a central proline-rich sequence as the nucleolin-interacting sequence in CD3epsilon. Transfection in COS cells of wild type CD3epsilon, but not of nonbinding mutants of CD3epsilon, resulted in redistribution of nucleolin from the nucleus and nucleoli to the cytoplasm. This property was transferred to a CD8 protein chimera by appending the cytoplasmic tail of CD3epsilon. We also found that nucleolin associated with the TCR complex. This association was increased upon TCR engagement, suggesting that the CD3epsilon/nucleolin interaction may have a role in T cell activation.

Amino Acid Sequence↗

PubMed2000

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection.

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Rho regulates T cell receptor ITAM-induced lymphocyte spreading in an integrin-independent manner.

T cell receptor (TCR) engagement increases integrin-mediated adhesion to APC, resulting in the stabilization of the T cell : APC interaction and the close apposition of the two cell membranes. Here we show that engagement of either the TCR or CD3 chimeras with immobilized antibodies causes the rapid spreading of T cells in an integrin-independent fashion. This effect concurs with the polymerization of the actin cytoskeleton and is dependent on the integrity of the immunoreceptor tyrosine-based activation motifs of the CD3 subunits. Expression of a dominant negative mutant of RhoA, as well as the Rho-specific inhibitor C3 toxin, abolished TCR-induced spreading. In contrast, constitutively active or dominant negative forms of Rac and Cdc42 did not affect cell spreading. We conclude that signals emanating from the TCR can directly induce T cell spreading, independently of integrins, and via a Rho-dependent reorganization of the actin cytoskeleton.

Actins↗

PubMed2000

Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism.

Downregulation of the TCR complex is believed to be intimately tied to T cell activation, allowing serial triggering of receptors and desensitization of stimulated cells. We studied transfected and transgenic T cells expressing CD3zeta chimeras to demonstrate that ligand engagement of the TCR or chimeras causes comodulation of nonengaged receptors. Comodulation required protein tyrosine kinase activity but not trans-phosphorylation of nonengaged receptors. The TCR appears to be downregulated by at least two mechanisms. One mechanism requires direct engagement, independent of signaling. The second requires signaling and downregulates nontriggered receptors. These results shed new light on the process of TCR downregulation and indicate that the number of downregulated TCRs cannot be assumed to equal the number of engaged receptors.

Animals↗

PubMed2000

Internalization and intracellular fate of TCR-CD3 complexes.

The number of surface TCR-CD3 complexes is maintained by an equilibrium between the synthesis and secretion of new polypeptides, their internalization, recycling, and degradation. The different subunits of the TCR-CD3 complex do not display the same intracellular trafficking dynamics. Thus, in the absence of stimuli, TCR and zeta chains may be degraded at a higher rate than CD3 subunits, which are mostly recycled. T-cell activation by antigen, anti-TCR-CD3 antibodies, or pharmacological activators of protein kinase C, results in increased TCR-CD3 internalization, followed by the downmodulation of TCR-CD3 surface levels. Once internalized, TCR-CD3 complexes may either enter a recycling pathway or be sorted to lysosomes and degraded. Protein serine kinases and protein tyrosine kinases may influence the internalization and intracellular sorting of TCR-CD3 complexes. In line with these results TCR-CD3 ligands stimulate both TCR-CD3 internalization and degradation, whereas protein kinase C activators stimulate internalization only. Depending on the stimulus applied, internalization motifs from one or several TCR-CD3 subunits mediate endocytic routing of the complex. The involvement of signaling molecules in the intracellular fate of TCR-CD3, the nature and location of sequences for internalization and intracellular sorting, and the role of downregulation in T-cell activation are still the main open questions.

Animals↗

PubMed1999

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma.

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

Blotting, Northern↗

PubMed1999

Receptor engagement transiently diverts the T cell receptor heterodimer from a constitutive degradation pathway.

In the absence of ligand, the T cell receptor (TCR)/CD3 complex is continuously internalized and recycled to the cell surface, whereas receptor engagement results in its down-regulation. The present study shows that the TCR and CD3 components follow different fates accompanying their constitutive internalization. Although the CD3 moiety is recycled to the cell surface, the TCR heterodimer is degraded and replaced by newly synthesized chains. Since the TCR heterodimer cannot reach the cell membrane on its own, we propose a model in which recycling CD3 is transported along a retrograde pathway to the endoplasmic reticulum, where it associates with newly made TCR. Interestingly, engagement of the TCR.CD3 complex by superantigen resulted not only in the down-regulation of the TCR and CD3 components but also caused a transient stabilization of the TCR heterodimer. This suggests that TCR engagement diverts the TCR heterodimer from a degradation to a recycling pathway. Contrary to CD3, the intracellular fate of the TCR heterodimer is thus regulated, providing a mechanism for rapidly replacing nonfunctional TCR during intrathymic development of T cells.

CD3 Complex↗

PubMed1999

The CD3 epsilon subunit of the TCR contains endocytosis signals.

Ligand binding to TCR induces its internalization and cell surface down-modulation. These phenomena contribute to the extinction of activation signals. Due to the multicomponent nature of the TCR-CD3 complex, its internalization may be mediated by one or several of its subunits. Although it has been reported that CD3 gamma and CD3 delta contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3 epsilon and CD zeta display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3 epsilon bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3 epsilon that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3 epsilon displays endocytosis determinants. These data indicate that CD3 epsilon could contribute to the internalization and cell surface down-regulation of TCR-CD3 complexes. Moreover, the existence of endocytosis signals in this polypeptide could serve to retrieve unassembled CD3 epsilon subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.

Amino Acid Sequence↗

PubMed1999

Multivalent structure of an alphabetaT cell receptor.

Whether there is one or multiple alphabetaT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRbeta chains incorporate at least two alphabetaTCRs in a single TCR/CD3 complex. Evidence for bispecific alphabetaTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRbeta-variable regions. Such (alphabeta)2TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (alphabeta)2TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.

Animals↗

PubMed1999

Apoptosis-resistant T cells have a deficiency in NF-kappaB-mediated induction of Fas ligand transcription.

Apoptosis induced through the TCR in CD4+ T cells is mostly mediated by the inducible expression of Fas ligand (FasL) as a primary event leading to the commitment to death. To gain a better understanding of the transcriptional events that regulate this expression, we took advantage of our previously described mutant Jurkat cells. These cells are deficient in FasL expression and apoptosis induced upon TCR triggering, although their cytokine (IL-2 and IFN-gamma) production is normal. Here we show that both a FasL- and a consensus NF-kappaB-reporter construct are inefficiently induced in these cells compared to wild-type cells. In addition, we demonstrate that the inducible transcriptional activity of the FasL reporter is abolished by specific inhibitors of NF-kappaB activation. Thus, we could trace the deficit of the mutant cells to an inefficient NF-kappaB activation, evidencing a relevant role for NF-kappaB in the regulation of FasL expression in activated T cells. Furthermore, our results suggest that the induction of FasL versus cytokine gene expression is differentially sensitive to NF-kappaB deprivation.

Apoptosis↗

PubMed1998

Retroviral vector-mediated expression in primary human T cells of an endoplasmic reticulum-retained CD4 chimera inhibits human immunodeficiency virus type-1 replication.

Intracellular expression of genes that inhibit key steps in the human immunodeficiency virus (HIV-1) replicative cycle could offer an alternative therapy for AIDS treatment. One of these approaches involves the inhibition of env protein maturation through the expression of CD4 molecules with added exogenous sequences that promote their retention in the endoplasmic reticulum (ER). We have tested this strategy using a CD4 chimera (CD4epsilon10) containing an ER retention sequence derived from the TCR CD3-epsilon chain. Transfection of CD4epsilon10 in the human T cell line Jurkat made it resistant to infection with two different HIV-1 isolates, which was evaluated by measuring p24 antigen production, induction of apoptosis, and syncytia formation. Furthermore, polymerase chain reaction (PCR) analysis of genomic DNA showed no traces of the proviral HIV-1 genome in CD4epsilon10-transfected cells, suggesting it was not maintained latently in these cells. To facilitate the delivery of the CD4epsilon10 chimera to primary cells from AIDS patients, a Moloney-based retroviral vector was constructed that expresses CD4epsilon10 under the transcriptional control of the HIV-1 long terminal repeat (LTR) promoter. Transduction of the MT-2 human T cell line with this vector rendered it resistant to infection with HIV-1 by a process that involved the inhibition of gp160 proteolytic processing. Finally, transduction of the CD4epsilon10 chimera into T lymphoblasts derived from asymptomatic HIV-infected individuals demonstrated a protective effect, resulting in both an increased cellular proliferation rate and an increased percentage of CD4+ cells. These results suggest that it is feasible to use retroviral transduction of CD4epsilon10 as a gene therapy approach for AIDS treatment.

CD4 Antigens↗

PubMed1998

Characterization of the region involved in CD3 pairwise interactions within the T cell receptor complex.

Assembly of the six-chain T cell antigen receptor-CD3 complex takes place by pairwise interactions. Thus, CD3-epsilon interacts with either CD3-gamma or CD3-delta, and these dimers then associate with the TCR heterodimer (alpha.beta or gamma.delta) and the CD3-zeta homodimer to constitute a full complex. We have now mapped the site in CD3-epsilon responsible for the interaction with CD3-gamma and CD3-delta by analysis of a series of deletional mutants encompassing the most conserved regions. We found that the highly conserved juxtamembrane domain is mainly responsible for the interaction. Thus, deletion of this 16-amino acid extracellular sequence resulted in the inhibition of up to 95% of the CD3-epsilon/gamma interaction. A highly conserved sequence is also present in both CD3-gamma and CD3-delta, suggesting that the domain in these two chains may reciprocally be involved in the interaction with CD3-epsilon. Indeed, an immobilized synthetic peptide corresponding to the CD3-gamma sequence specifically associated to a bacterially expressed CD3-epsilon protein, suggesting the 16-amino acid domain is sufficient to promote CD3-epsilon/CD3-gamma assembly. The conservation of the motif in the CD3 chains suggest that, in addition to CD3-epsilon/CD3-gamma and CD3-epsilon/CD3-delta interactions, it may also mediate homotypic interactions. Indeed, it is shown that it mediates the formation of disulfide-linked homodimers and that the formation of homo- and heterodimers are mutually excluded. Finally, this domain contains a Cys-X-X-Cys sequence that resembles that of p56(lck), which is responsible for the interaction with the cytoplasmic tails of CD4 and CD8. Since the replacement of the two cysteines (Cys97 and Cys100) in CD3-epsilon by alanines strongly inhibited pair formation, the existence of a Cys-X-X-Cys motif involved in protein-protein interactions is suggested.

Amino Acid Sequence↗

PubMed1998

T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2)-producing, T cells results in impaired ZAP70/CD3-zeta association.

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.

Apoptosis↗

PubMed1998

Assembly of the TCR/CD3 complex: CD3 epsilon/delta and CD3 epsilon/gamma dimers associate indistinctly with both TCR alpha and TCR beta chains. Evidence for a double TCR heterodimer model.

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3 epsilon with either CD3 gamma or CD3 delta. These dimers assemble with TCR alpha and TCR beta chains, and finally, the CD3 zeta homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3 epsilon/CD3 gamma dimer interacts exclusively with TCR beta and the CD3 epsilon/CD3 delta dimer with TCR alpha to form a complex with a single TCR alpha/beta heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3 epsilon/CD3 gamma and CD3 epsilon/CD3 delta dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCR alpha-negative T cell line MOLT-4 in which TCR beta is found separately associated with CD3 epsilon/CD3 gamma and with CD3 epsilon/CD3 delta dimers. Indirectly, our results support a model of stoichiometry in which two TCR alpha/beta heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3 gamma and anti-CD3 delta antibodies from 1% NP40 and 1% Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3 zeta homodimer. This suggests that CD3 zeta mediates the interaction between both TCR alpha/beta heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCR beta that impairs the association of CD3 zeta. In this mutant cell line, unlike a control line with wild-type TCR beta, the CD3 gamma- and CD3 delta-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-alpha/beta heterodimers form two hemicomplexes containing either CD3 epsilon/gamma or CD3 epsilon/delta dimers which become associated via the CD3 zeta homodimer.

Dimerization↗

PubMed1998

Antiparasitic effects of the intra-Golgi transport inhibitor megalomicin.

The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC50) of 0.2 microg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 microg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC50, 2 microg/ml) and Leishmania donovani and Leishmania major promastigotes (IC50, 3 and 8 microg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 microg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.

Animals↗