PubMed Health⌕ Search

Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 19 recordsLinked to original sources
PubMed2006

Role of mitochondria in apoptosis induced by the 2-5A system and mechanisms involved.

The 2-5A system (2-5OAS/RNaseL) is composed of the 2',5'oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (delta psi m), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of delta psi m induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.

Adenine Nucleotides↗
PubMed2006

Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action.

The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5' untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-kappaB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IkappaB and NF-kappaB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results.

Animals↗
PubMed2006

Severe hypernatremic dehydration secondary to undetected lactation failure: usefulness of sodium levels in breast milk.

There is a concern that lactation failure and neonatal morbidity might be more common than previously thought. Maybe the early discharge, the lack of timely follow-up, or poor information during pregnancy or after delivery play important roles. A case of severe hypernatremic dehydration secondary to lactation failure is reported and we analyze recent recommendations.

Breast Feeding↗
PubMed2003

Tissue distribution of the Ankara strain of vaccinia virus (MVA) after mucosal or systemic administration.

MVA is a candidate vector for vaccination against pathogens and tumors. Little is known about its behaviour in mucosal tissues. We have investigated the fate and biosafety of MVA, when inoculated by different routes in C57BL/6 mice. Intranasal inoculation targeted the virus to the nasal associated lymphoid tissue and the lungs, whereas systemic inoculation led to distribution of MVA in almost all lymphoid organs, lungs and ovaries. Intravaginal, intrarectal and intragastric inoculations failed to induce efficient infection. After 48 h no virus was detectable any more in the organs analyzed. Upon intranasal inoculation, no inflammatory reactions were detected in the central nervous system as well as the upper and lower airways. These results show the tropism of MVA and indicate that high doses of recombinant MVA are safe when nasally administered, a vaccination route known to elicit strong cellular and humoral immune responses in the female genital tract.

Administration, Intranasal↗
PubMed2003

The latency protein LANA2 from Kaposi's sarcoma-associated herpesvirus inhibits apoptosis induced by dsRNA-activated protein kinase but not RNase L activation.

Kaposi's sarcoma-associated herpesvirus (KSHV) uses several strategies to counteract the interferon (IFN) system. In this study, the relationship of the protein LANA2 from KSHV to the IFN-activated protein kinase (PKR) and 2-5A system was analysed. It was found that LANA2 could not abrogate apoptosis or RNA degradation mediated by the 2-5A system. However, expression of LANA2 inhibited apoptosis triggered by PKR. LANA2 also counteracted the PKR-mediated inhibition of protein synthesis and partially blocked PKR-induced phosphorylation of eIF-2alpha. Analysis of PKR-induced activation of caspases 3 and 9 revealed that LANA2 abrogated activation of caspase 3 but not of caspase 9. These findings show that LANA2 is able to interfere with downstream events triggered by PKR. Hence, LANA2 should be considered as a KSHV defence protein against IFN.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice.

Immunization protocols based on priming with plasmid DNA and boosting with recombinants of vaccinia virus (rVV) encoding the same antigen offer great promise for the prevention and treatment of many parasitic and viral infections for which conventional vaccination has little or no effect. To overcome some of the potential problems associated to the use of plasmids, we have developed minimalistic, immunogenically defined, gene expression (MIDGE((R))) vectors. These linear vectors contain only the minimum sequence required for gene expression and can be chemically modified to increase the immune response. Here, we demonstrate that MIDGE vectors coding for the LACK antigen confer a highly effective protection against Leishmania infection in susceptible Balb/c mice. Protection is achieved at lower doses of vector compared to conventional plasmids. This efficacy could be greatly improved by the addition of a nuclear localization signal (NLS) peptide to the end of the MIDGE vector. In fact, immunization with two doses of NLS-modified MIDGE conferred similar or even better protection than that achieved by priming with plasmid DNA followed by boosting with rVV. These results demonstrate that MIDGE vectors are a good alternative to plasmid and rVV for immunization.

Animals↗
PubMed2002

Visna/maedi virus Env protein expressed by a vaccinia virus recombinant induces cell-to-cell fusion in cells of different origins in the apparent absence of Env cleavage: role of glycosylation and of proteoglycans.

The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV- env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV- env-MV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV- env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, beta-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA.

3T3 Cells↗
PubMed2001

Enhanced CD8+ T cell immune response against a V3 loop multi-epitope polypeptide (TAB13) of HIV-1 Env after priming with purified fusion protein and booster with modified vaccinia virus Ankara (MVA-TAB) recombinant: a comparison of humoral and cellular immune responses with the vaccinia virus Western Reserve (WR) vector.

The humoral and cytotoxic T-lymphocyte (CTL) responses have been shown to be determinant in the clearance of many viral infections and because of those characteristics, vaccine candidates against AIDS are designed to enhance both arms of the immune system. While a protocol of immunization able to confer protection in humans against HIV will have to await the results of current clinical trials, it remains important to identify protocols of immunization in animals that achieve significant levels of humoral and cellular immune responses to HIV. In this study we have carried out a comparative analysis of the immune responses elicited in mice immunized with recombinants based on the modified vaccinia virus Ankara strain (rMVA) versus the Western Reserve strain (WR) of vaccinia virus (rVV), both expressing a V3 loop multi-epitopic protein from eight different HIV isolates (TAB13). We found that during priming, rMVA elicited a two- to three-fold higher specific CD8+ T cell response than rVV. Similar enhancement was observed during priming with purified protein TAB13 followed by a booster with rMVA. The epitopes LR150, MN and IIIB, located at the ends and in the middle of the chimeric protein, were able to induce a specific CD8+ T cell response, both after priming or prime/booster with the recombinant viruses but not after prime/booster with TAB13. By examining the cytokine pattern, the immune response triggered by these vectors was of Th-1 type. Humoral immune responses were higher in animals immunized with TAB13/TAB13 or TAB13/rVV than in animals immunized with TAB13/rMVA. These findings demonstrate that during priming or in a prime/booster immunizations, rMVA is superior to rVV in the ability to enhance specific cellular responses to an HIV-1 protein, and that both humoral and cellular immune responses to theV3 loop epitope of HIV-1 Env can be obtained by priming with TAB13 followed by a booster with viral vectors.

AIDS Vaccines↗
PubMed2001

The A17L gene product of vaccinia virus is exposed on the surface of IMV.

The p21 membrane protein of vaccinia virus (VV), encoded by the A17L gene, has been reported to localize on the inner of the two membranes of the intracellular mature virus (IMV). It has also been shown that p21 acts as a membrane anchor for the externally located fusion protein p14 (A27L gene). Since p14 is located on the surface of IMVs, it is hard to envision that p21 should be located only on the inner membrane. Our results from (i) immunoelectron microscopy, (ii) biotinylation, and (iii) protease treatment of purified IMVs showed that the N-terminus of p21 is exposed on the surface of virus particles, while the C-terminus is embedded in the membrane. Mono-specific antibodies to the N-terminus of p21 neutralize infection of VV while antibodies to the C-terminal domain do not. We suggest that p21 molecules are located both in the inner and in the outer membrane of IMV.

Animals↗
PubMed2001

Voltammetric analysis of heterogeneity in metal ion binding by humics.

The complexation of Cd, Pb, and Cu by fulvic acids at a fixed pH and ionic strength is studied by means of different voltammetric techniques at any metal-to-ligand ratio. When using Reverse Pulse Polarography (RPP) the complex species are electrochemically labile and not subject to significant electrodic adsorption. RPP titrations of fulvic acid with metal ions are interpreted on the basis of a recently proposed analytical expression for limiting currents valid for fully labile heterogeneous complexation. The voltammetric data are transformed into the corresponding binding curve, i.e., the fraction of occupied sites vs free metal concentration. Finally, the competition between metal ions and protons in their interaction with the fulvic binding sites as well as the concomitant polyelectrolytic effects are analyzed in terms of the NICCA-Donnan model. The results show that voltammetric techniques can be applied to the studies of heterogeneous complex systems in a broad range of metal-to-ligand ratios.

Benzopyrans↗
PubMed2001

The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-kappaB activation.

The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-kappaB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-kappaB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-kappaB induction. In PKR(0/0) cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-kappaB. An inactive PKR mutant (K296R), was unable to induce NF-kappaB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-kappaB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2alpha phosphorylation and NF-kappaB activation. However, PKR mutants unable to activate NF-kappaB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-kappaB.

Animals↗
PubMed2001

Growth of moderately halophilic bacteria isolated from sea water using phenol as the sole carbon source.

Moderately halophilic bacteria utilizing phenol as the sole carbon source were isolated by selective enrichment from sea water. The isolate (Gram-negative motile rods) was identified as Deleya venusta. It grew well in the presence of up to 1600 mg/L of phenol and 8% NaCl under aerobic conditions. When the cells were treated with chloramphenicol prior to the addition of phenol they did not utilize added phenol, even after prolonged incubation. Thus, the enzymes necessary for phenol metabolism appeared to be inducible.

Arthrobacter↗
PubMed2001

Recombinant proteins produced by vaccinia virus vectors can be incorporated within the virion (IMV form) into different compartments.

Vaccinia virus (VV) is one of the largest and most complex of animal viruses, with a virion that contains about 100 different polypeptides. Assembly of the viral proteins occurs in discrete cytoplasmic sites leading to formation of two infectious forms, an abundant (>90%) intracellular mature virus (IMV) with an envelope, and a minor extracellular enveloped virus (EEV) with an extra membrane acquired from the trans-Golgi network. It has been shown that while EEV contains in the outer membrane cellular proteins probably acquired during virus release from cells, however, IMV exclude host proteins during assembly. Since VV recombinants (VVr) expressing genes of interest are candidates as potential vaccines against pathogens and cancer, it becomes important to know if VVr can acquire foreign proteins during morphogenesis. In this investigation we show that purified virions (IMVs) from VVr can incorporate foreign proteins into different sites in the virus particle. By sequential fractionation of virion compartments with detergents, we found foreign proteins in the lipid envelope (cytokine IL-12 and CS antigen of Plasmodium yoelii), as part of a protein matrix-like membrane (HIV-1 gp41 of env), or more closely associated with the core containing the DNA complexes (HIV-1 gp160; a multiepitopic protein with the V3 loop of HIV-1 Env from different isolates, and beta-galactosidase). Similar findings were observed with purified virions derived from the WR strain as well as from the highly attenuated modified vaccinia virus Ankara (MVA) strain. These observations should be taken into consideration when VVr are used in clinical trials or in other vaccination approaches.

Animals↗
PubMed2001

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36.

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.

Animals↗
PubMed2001

Implementation of a chemical equilibrium constraint in the multivariate curve resolution of voltammograms from systems with successive metal complexes.

A multivariate curve resolution (MCR) method, using a constrained alternating least squares (ALS) procedure with a new chemical equilibrium constraint, was applied to differential-pulse polarograms of successive metal complexes. This new restriction imposes the fulfillment of a chemical model defined by a set of stability constants that are optimised along the iterative ALS procedure. The reliability of the method was tested with simulated data and with polarograms measured for the systems Zn(II) + glutathione and Cd(II) + 1,10-phenanthroline. These systems respectively yield two and three successive and electroactive complexes, which are inert from the electrochemical point of view, that is, the complexes virtually do not dissociate during the measurement. Although the presence of electrode adsorption could induce overestimation of some concentrations and losses of linearity between concentrations and signals, the results showed that the proposed method can yield satisfactory estimations of the stability constants in this kind of system. The performance of the new method is compared with the performances obtained using MCR-ALS without the equilibrium constraint and using traditional curve fitting least-squares approaches.

Journal Article↗
PubMed2001

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Animals↗
PubMed2001

Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens.

A main goal of the industrialized world is the development of effective vaccines to control infectious diseases with major health and socio-economic impact. Current understanding of the immune response triggered during infection with pathogens causing malaria, hepatitis C and AIDS emphasizes the importance of cytotoxic T lymphocytes (CTLs) in combating these infections. This has led to the development of new vaccination strategies, some of which are in phase I/II clinical trials. Promising strategies of vaccination are based on highly attenuated viral vectors, such as Vaccinia virus (VV) in combination with heterologous like vectors naked DNA, referred to as priming/booster vaccination. While these immunization schedules increased the production of specific CTLs, there is a need to further expand the CD8+T cell population to control an infection. Among molecules that play a significant role in the modulation of the CTL response is the cytokine IL-12. Immunoregulation by IL-12 is of central importance in cell-mediated immunity (CMI) against those pathogens and tumors that are controlled by cell-mediated mechanisms, supported by Thl cells. The use of this cytokine in combination with highly immunogenic VV-derived vectors is a promising system for development of future vaccination schedules. In this review, we summarize recent data on the use of IL-12 in vaccination procedures, as well as undesired side-effects of the cytokine that can be overcome by accurate use of dose, route and time-window administration of IL-12 encoding vectors. Results described here indicate that VV IL-12-mediated enhancement of the specific CMI response against a model antigen HIV-1 env was time- and dose-dependent and that the antigen and the cytokine could be expresed from two different rVVs modulating the doses of the vectors and allowing for enhancement of a specific CMI response. Moreover, the use of IL-12 during DNA prime/VV boost regimens enhanced the specific anti-HIV-1 env cellular response 20 times compared to that generated after a single rVVenv inoculation. Variables such as: a) dose of the cytokine applied, b) time of its administration and c) routes of inoculation play a critical role in the final outcome of the response. The findings presented here can be extended to other antigens, suggesting that immunomodulatory cytokines can be useful in the development of the future vaccines against numerous infectious diseases and tumors.

Adjuvants, Immunologic↗