Covadonga López Alonso

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.

Naïve sea bass juveniles (38.4 ± 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-β, TCRβ, CD4, CD8α, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable “in vitro” increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-β and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.

Autophagy is a relevant cellular defense mechanism that directly eliminates intracellular pathogens and has a crucial role for innate and adaptive immune responses. Some viruses have developed tools to counteract this cellular response. A179L, the viral Bcl2 homolog of African swine fever virus, interacts with proapoptotic Bcl2 family proteins to inhibit apoptosis. Here we report that this gene manipulates autophagy by interacting with Beclin 1 through its BH3 homology domain. At subcellular level, A179L colocalized with Beclin 1 at mitochondria and the endoplasmic reticulum. Virus infection inhibited autophagosome formation in cells; however, when autophagy was induced prior to or at the time of infection the number of infected cells was severely decreased. Keywords: African swine fever virus, ASFV, autophagy, Beclin 1, BH3-only proteins, ER stress, viral Bcl2, viral Bcl2 homolog, autophagosomes, stress, starvation, homeostasis, hemorrhagic disease, pathogenesis, lymphoid hyperplasia.

Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens.Protamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406).

We have analyzed the production of tumor necrosis factor alpha (TNF-alpha) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-alpha mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-alpha protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-alpha expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-alpha were detected in serum, corresponding to the onset of clinical signs. TNF-alpha has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-alpha containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-alpha specific antibody. These results suggest a relevant role for TNF-alpha in the pathogenesis of ASF.

Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.

It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in protein p54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the p54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against p54 protein. The sequence of this recombinant phage allowed the location of the p54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a protein of 183 amino acids with a calculated molecular weight of 19,861. This protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for protein processing of several ASFV structural proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the p54 gene is translated from a late mRNA and the protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the p54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when p54 genes from viral clones generated by cell culture passage and coding for p54 proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in p54, resulting in stepwise modifications in the molecular weight of the protein. These duplications and deletions of a tandem repeat sequence array within a protein coding region constitute a novel mechanism of genetic diversification in ASFV.

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.

Apoptosis is implicated in the progressive cell loss and fibrosis both at glomerular and tubulointerstitial level. In this study, we examined the potential mechanisms by which persistent proteinuria (protein-overload model) could induce apoptosis. After uninephrectomy (UNX), Wistar rats received daily injections of 0.5 g of bovine serum albumin (BSA)/100 g body weight or saline. Both at day 8 and day 28, rats receiving BSA had proteinuria and renal lesions characterized by tubular atrophy and/or dilation and mononuclear cell infiltration. In relation to control-UNX rats, renal cortex of nephritic rats showed an increment in AT2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) expression. In both groups, AT2 receptor immunostaining was mainly localized in proximal tubular cells. Rats with persistent proteinuria showed a significantly increased number of terminal dUTP nick-end labeling positive apoptotic cells compared with UNX-controls, both in glomeruli and tubulointerstitium. Double staining for apoptosis and AT2 receptor showed that most terminal dUTP nick-end labeling positive cells were found in tubules expressing AT2 receptor. Using an antibody that recognizes the active form caspase-3, we observed an increment in caspase-3 activation in rats receiving BSA with respect to those receiving saline. Rats with persistent proteinuria showed a diminution in the phosphorylation of Bcl-2 with respect to UNX-controls both at day 8 and day 28. By contrast, no changes were observed either in the Bax or in the Bcl-2 protein levels. The administration of BSA to UNX rats induced a diminution in the phosphorylation of ERK with respect to UNX-control at all times studied. The changes observed in ERK activities took place without alterations of ERK1/2 protein levels. In summary, our data suggest that persistent proteinuria causes apoptosis in tubular cells through the activation of AT2 receptor, which can, in turn, inhibit MAP kinase (ERK1/2) activation and Bcl-2 phosphorylation. Apoptosis is implicated in the progressive cell loss and fibrosis both at glomerular and tubulointerstitial level. In this study, we examined the potential mechanisms by which persistent proteinuria (protein-overload model) could induce apoptosis. After uninephrectomy (UNX), Wistar rats received daily injections of 0.5 g of bovine serum albumin (BSA)/100 g body weight or saline. Both at day 8 and day 28, rats receiving BSA had proteinuria and renal lesions characterized by tubular atrophy and/or dilation and mononuclear cell infiltration. In relation to control-UNX rats, renal cortex of nephritic rats showed an increment in AT2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) expression. In both groups, AT2 receptor immunostaining was mainly localized in proximal tubular cells. Rats with persistent proteinuria showed a significantly increased number of terminal dUTP nick-end labeling positive apoptotic cells compared with UNX-controls, both in glomeruli and tubulointerstitium. Double staining for apoptosis and AT2 receptor showed that most terminal dUTP nick-end labeling positive cells were found in tubules expressing AT2 receptor. Using an antibody that recognizes the active form caspase-3, we observed an increment in caspase-3 activation in rats receiving BSA with respect to those receiving saline. Rats with persistent proteinuria showed a diminution in the phosphorylation of Bcl-2 with respect to UNX-controls both at day 8 and day 28. By contrast, no changes were observed either in the Bax or in the Bcl-2 protein levels. The administration of BSA to UNX rats induced a diminution in the phosphorylation of ERK with respect to UNX-control at all times studied. The changes observed in ERK activities took place without alterations of ERK1/2 protein levels. In summary, our data suggest that persistent proteinuria causes apoptosis in tubular cells through the activation of AT2 receptor, which can, in turn, inhibit MAP kinase (ERK1/2) activation and Bcl-2 phosphorylation. Tubulointerstitial damage, regardless of its etiology, has been shown to be an important predictor of progressive renal failure.1Remuzzi G Ruggenenti P Benigni A Understanding the nature of renal disease progression.Kidney Int. 1997; 51: 2-15Crossref PubMed Scopus (609) Google Scholar Although the mechanisms leading to end-stage renal failure are not completely elucidated, persistent proteinuria is considered an aggravating factor.1Remuzzi G Ruggenenti P Benigni A Understanding the nature of renal disease progression.Kidney Int. 1997; 51: 2-15Crossref PubMed Scopus (609) Google Scholar Apoptosis (programmed cell death) is a gene-regulated process that is now recognized to play an important role in maintaining cell number homeostasis both in health and disease.2Ortiz A Lorz C Catalán MP Justo P Egido J Role and regulation of apoptotic cell death in the kidney. Y2K update.Front Biosci. 2000; 5: D735-D749Crossref PubMed Google Scholar, 3Ortiz A Lorz C Justo P Catalán MP Egido J Contribution of apoptotic cell death to renal injury.J Cell Mol Med. 2001; 5: 18-32Crossref PubMed Scopus (62) Google Scholar Cell deletion by apoptosis has been implicated in the repairing process of several renal diseases, such as anti-Thy-1 antibody-induced nephritis and crescentic glomerulonephritis. However, unregulated excessive apoptosis can contribute to renal damage by depletion of glomerular and tubulointerstitial cells characteristic of progressive chronic nephropathies, including protein-overload nephropathy.4Thomas ME Brunskill NJ Harris KPG Bailey E Pringle JH Furness PN Walls J Proteinuria induces tubular cell turnover: a potential mechanism for tubular atrophy.Kidney Int. 1999; 55: 890-898Crossref PubMed Scopus (112) Google Scholar Apoptosis is controlled in part by the Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, and others).5Cory S Adams JM The Bcl2 family: regulators of the cellular life-or-death switch.Nat Rev Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3258) Google Scholar Apoptotic and anti-apoptotic members of the family can interact, and the overall effect on cell survival might depend on the balance between the activity of apoptotic (such as Bax and Bcl-xS) and anti-apoptotic (such as Bcl-2 and Bcl-xL) proteins. The biochemical basis for most of the morphological changes associated with apoptosis can be traced directly or indirectly to the actions of caspases, a family of intracellular cysteine proteases that function as effectors of programmed cell death.6Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar Initiator caspases (−2, −8, −9, −10), situated at the upstream of the caspase cascade, can activate the downstream executioner caspases (−3, −6, −7). Executioner caspases can, in turn, activate initiator caspases, thus propagating the apoptotic process.6Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar Caspase cellular targets include inactivation of protective proteins, such as Bcl-xL and Bcl-2, which can even yield proapoptotic fragments.6Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar In addition, the balance between the pro- and anti-apoptotic Bcl-2 protein family members has been found to control caspase-3 activity.7Cosulich SC Savory PJ Clarke PR Bcl-2 regulates amplification of caspase activation by cytochrome c.Curr Biol. 1999; 9: 147-150Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The renin-angiotensin system (RAS) has been implicated in the development of renal damage. In humans and experimental models, RAS blockade with angiotensin-converting enzyme inhibitors or angiotensin type 1 receptor (AT1) antagonists has been demonstrated to ameliorate organ damage, even in situations of normal blood pressure.8Egido J Vasoactive hormones and renal.Kidney Int. 1996; 49: 578-597Crossref PubMed Scopus (207) Google Scholar, 9Burnier M Angiotensin II type 1 receptor blockers.Circulation. 2001; 103: 904-912Crossref PubMed Scopus (418) Google Scholar, 10Gómez-Garre D Largo R Tejera N Fortés J Manzarbeitia F Egido J Activation of NF-κB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (138) Google Scholar, 11Suzuki Y Lopez-Franco O Gómez-Garre D Tejera N Gómez-Guerrero C Sugaya T Bernal R Blanco J Ortega L Egido J Renal tubulointerstitial damage caused by persistent proteinuria is attenuated in AT1-deficient mice: role of endothelin-1.Am J Pathol. 2001; 159: 1895-1904Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Angiotensin II (Ang II), the main peptide of the RAS, exerts its biological actions via the activation of two major classes of specific cellular receptors, designated AT1 and AT2. Most effects of Ang II, such as cell proliferation, fibrosis, and the inflammatory response are mediated by AT1 receptors.9Burnier M Angiotensin II type 1 receptor blockers.Circulation. 2001; 103: 904-912Crossref PubMed Scopus (418) Google Scholar, 12Ruiz-Ortega M Lorenzo O Suzuki Y Rupérez M Egido J Proinflammatory actions of angiotensins.Curr Opin Nephrol Hypertens. 2001; 10: 321-329Crossref PubMed Scopus (341) Google Scholar Much less is known about the physiological role of AT2 receptors. Recent evidence suggests involvement of AT2 receptors in development, cell differentiation, apoptosis, and inflammation.12Ruiz-Ortega M Lorenzo O Suzuki Y Rupérez M Egido J Proinflammatory actions of angiotensins.Curr Opin Nephrol Hypertens. 2001; 10: 321-329Crossref PubMed Scopus (341) Google Scholar, 13de Gasparo M Siragy HM The AT2 receptor: fact, fancy and fantasy.Regul Pept. 1999; 81: 11-24Crossref PubMed Scopus (135) Google Scholar Throughout the last years, several studies have been focused on AT2 receptor-mediated apoptosis.13de Gasparo M Siragy HM The AT2 receptor: fact, fancy and fantasy.Regul Pept. 1999; 81: 11-24Crossref PubMed Scopus (135) Google Scholar Proapoptotic effects of AT2 receptor have been noted in different cell types, including fibroblasts (R3T3), vascular smooth muscle cells, and proximal tubular cells (LLC-PK1). It has been demonstrated that the AT2 receptor activates a tyrosine phosphatase that induces the inactivation of extracellular signal-regulated kinases (ERK1 and ERK2),14Ito T Deng X Carr B May WS Bcl-2 phosphorylation required for anti-apoptosis function.J Biol Chem. 1994; 269: 26865-26870PubMed Google Scholar resulting in Bcl-2 dephosphorylation, which may contribute to apoptosis.14Ito T Deng X Carr B May WS Bcl-2 phosphorylation required for anti-apoptosis function.J Biol Chem. 1994; 269: 26865-26870PubMed Google Scholar, 15Horiuchi M Hayashida W Kambe T Yamada T Dzau VJ Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating mitogen-activated protein kinase phosphatase-1 and induces apoptosis.J Biol Chem. 1997; 272: 19022-19026Crossref PubMed Scopus (262) Google Scholar Little is known, however, about the AT2 receptor-signaling pathway in vivo. Protein-overload proteinuria is an experimental model characterized by early and intense proteinuria associated to tubulointerstitial lesions, which has proven to be a valuable model to investigate the relationship between proteinuria and renal damage.10Gómez-Garre D Largo R Tejera N Fortés J Manzarbeitia F Egido J Activation of NF-κB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (138) Google Scholar, 16Eddy AA Interstitial nephritis induced by protein-overload proteinuria.Am J Pathol. 1989; 135: 719-733PubMed Google Scholar We have previously demonstrated that bovine serum albumin (BSA)-overloaded animals showed important morphological renal lesions, characterized by tubular atrophy and/or vacuolization and protein casts within proximal and distal tubules, coinciding with an up-regulation of the RAS proteins located in the proximal renal tubules.17Largo R Gómez-Garre D Soto K Marrón B Blanco J Gazapo MR Plaza JJ Egido J Angiotensin converting enzyme is upregulated in the proximal tubules of rats with intense proteinuria.Hypertension. 1999; 33: 732-739Crossref PubMed Scopus (92) Google Scholar However, in that study the expression of the AT2 receptor was not investigated. Since Thomas and colleagues4Thomas ME Brunskill NJ Harris KPG Bailey E Pringle JH Furness PN Walls J Proteinuria induces tubular cell turnover: a potential mechanism for tubular atrophy.Kidney Int. 1999; 55: 890-898Crossref PubMed Scopus (112) Google Scholar at have recently reported that protein overload proteinuria in rats induces tubular cell apoptosis, in the current study we extend these findings by investigating the expression of pro- and anti-apoptotic proteins and their relationship with the activation of the AT2 receptor. In addition, to elucidate the potential intracellular mechanisms controlling the AT2-mediated apoptosis, we examined the expression of caspase-3 as well as that of the ERK family during tubulointerstitial damage progression. Female Wistar rats (150 to 200 g) were fed standard rat chow ad libitum and given free access to water. The animals underwent unilateral left nephrectomy 5 days before the initiation of the study. Since day 0, uninephrectomized (UNX) rats received intraperitoneal and daily injections of BSA (Sigma, St. Louis, MO) (0.5 g BSA/100 g body weight) or saline.10Gómez-Garre D Largo R Tejera N Fortés J Manzarbeitia F Egido J Activation of NF-κB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (138) Google Scholar, 17Largo R Gómez-Garre D Soto K Marrón B Blanco J Gazapo MR Plaza JJ Egido J Angiotensin converting enzyme is upregulated in the proximal tubules of rats with intense proteinuria.Hypertension. 1999; 33: 732-739Crossref PubMed Scopus (92) Google Scholar Periodically, animals were housed in metabolic cages and 24-hour urine was collected for protein measurement by the sulfosalicylic acid method.18Gyure WL Comparison of several methods for semiquantitative determination of urinary protein.Clin Chem. 1977; 23: 876-879PubMed Google Scholar Animals were killed in groups (n = 7 per group) at days 8 and 28 after the initiation of saline or BSA injections based on previous experiments in which proteinuria peaked on day 8 and persisted up to day 28. Rats were anesthetized with sodium pentobarbital (5 mg/100 g body wt), and kidneys were perfused with cold sodium saline and removed. For light microscopy, paraffin-embedded sections (4 μm thick) were prepared and stained with hematoxylin and eosin and Masson's trichrome. For each animal, glomerular damage (mesangial cell proliferation and matrix expansion) and tubulointerstitial injury (tubular dilation and/or atrophy, interstitial fibrosis, and inflammatory cell infiltrate) were graded from 0 to 3 by a semiquantitative score: 0, no changes; 1, focal changes that involve 33% of the sample; 2, changes affecting >33 to 66%; 3, lesions affecting >66%. Two independent observers performed the semiquantification of morphological lesions in a blinded manner. Pieces of renal cortex were homogenized and total RNA was obtained by the acid guanidinium-phenol-chloroform method.19Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar Isolated RNA was reverse-transcribed and then amplified with a commercial kit (Access RT-PCR System; Promega, Madison, WI), using specific primers of rat AT2 (anti-sense: 5′-TAATACGACTCACTATAGGGG-3′, sense: 5′-AATTAGGTCACACTATAGGAT-3′; fragment 498-bp length). We performed reverse transcriptase (RT)-PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. The optimum number of amplification cycles used for semiquantitative reverse transcriptase-PCR (37 and 20 cycles, respectively) was chosen based on previous experiments that establish the exponential range of the reaction (data not shown). Then, samples were size-fractionated with 4% acrylamide-bisacrylamide gels. The gels were dried and exposed to X-OMAT AR films (Eastman Kodak, Rochester, NY). Autoradiograms were analyzed using scanning densitometry (Molecular Dynamics, Sunnyvale, CA). Results were expressed as arbitrary densitometric units. Renal cortex samples were homogenized in 1 ml of lysis buffer (50 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mmol/L phenylmethyl sulfonyl fluoride, 0.1 mmol/L sodium orthovanadate) at 4°C. Soluble lysates (60 to 80 μg) were loaded in each lane and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/0.5% Tween-20 (v/v) for 1 hour, washed with Tris-buffered saline/Tween-20, and incubated with the following antibodies: rabbit polyclonal anti-AT2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-caspase-3 (1:200, Santa Cruz Biotechnology), rabbit polyclonal cleaved caspase-3 (Asp175) (1:1000; New England Biolabs Inc., Beverly, MA), rabbit polyclonal anti-Bax, (1:200, Santa Cruz Biotechnology), rabbit polyclonal anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-phospho ERK1/2 (Thr/Tyr) (1:500, New England Biolabs Inc.), and goat polyclonal anti-ERK2 (1:500, Santa Cruz Biotechnology). Blots were washed with Tris-buffered saline/Tween-20 and subsequently incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:1500; Amersham, Aylesbury, UK). After washing with Tris-buffered saline/Tween-20 the blots were developed with the enhanced chemiluminescence method (Amersham). The intensity of the identified bands was quantified by densitometry (Molecular Dynamics). Results were expressed as arbitrary densitometric units. For immunoprecipitations, 200 μg of homogenized renal cortex were incubated overnight at 4°C with either goat polyclonal anti-ERK2 (Santa Cruz Biotechnology) or rabbit polyclonal anti-Bcl-2 (Santa Cruz Biotechnology). Immune complexes were absorbed to protein A-Sepharose and washed twice with lysis buffer. Western blot analysis was subsequently performed with mouse monoclonal anti-phosphotyrosine antibody (1:500, Transduction Laboratories, Newington, NH) as first antibody. Incubation with the secondary antibody and detection were performed as described above. Immunolocalization of AT2 receptor was performed with a polyclonal anti-rat AT2 (Santa Cruz Biotechnology) in paraffin-embedded renal tissues. After dewaxed and hydrated, endogenous alkaline phosphatase was blocked in 5 mmol/L levamisole and sections were subsequently incubated with blocking serum for 30 minutes at room temperature to reduce nonspecific background staining and with a polyclonal anti-rat AT2 (Santa Cruz Biotechnology) overnight at 4°C. After being washed, the sections were incubated with biotinylated donkey anti-rabbit IgG (Amersham) and processed using alkaline phosphatase-streptavidin-biotin immunoperoxidase method (ABComplex/AP; DAKO A/S, Glostrup, Denmark) and developed with Fast Red TR/naphthol AS-MX solution (DAKO A/S). The tissue sections were counterstained with Mayer's hematoxylin (Sigma, Madrid, Spain). Negative controls for specific labeling were performed in parallel by replacing the primary antibody with a nonimmune normal rabbit serum. Renal cortex sections were quantitatively examined using the computer-assisted image analysis software (Optimas 6.5; Media Cybernetics, Silver Spring, MD) and digitized images. For each renal cortex section, AT2 receptor expression was analyzed in four nonoverlapping random fields viewed at ×200 magnification and expressed as the percentage of area occupied by an AT2 staining threshold that was arbitrarily considered as positive. An independent observer evaluated the immunostaining results in a blinded manner. Apoptosis was detected by terminal dUTP nick-end labeling (TUNEL) using a commercial kit (Frag EL DNA Fragmentation Detection kit; Oncogene Research Products, Cambridge, MA). Biotinylated-dUTP-labeled DNA was detected and visualized using peroxidase-streptavidin conjugated with diaminobenzidine (Sigma) as the chromogen. The number of TUNEL-positive cells was quantified in a blinded manner with computer-assisted image analysis software (Optimas 6.5, Media Cybernetics) and digitized images. The specificity of the technique was evaluated with positive and negative controls. In positive controls, generated by pretreating slides with 1 μg/μl of DNase I before labeling reaction, nearly all of the cells stained, although they showed normal shape without cytoplasmic condensation. No staining was observed in negative controls that were incubated in labeling buffer without TdT. For each renal cortex section, glomerular cells undergoing apoptosis were calculated by counting at least 10 glomeruli per biopsy and expressed as the mean number ± SEM per glomeruli. In tubules, apoptotic cells were calculated by counting all positive apoptotic tubular cells in four to six nonoverlapping random fields viewed at ×200 magnification and expressed as the mean number ± SEM per field. Staining of a size between 5 and 25 pixels (that is, the size of an apoptotic nucleus) was counted as a single particle. Only intense brown-stained nuclei, including pyknotic nuclei with apoptotic bodies that stained positive, were considered as TUNEL-positive staining. Double-immunohistochemical staining was performed in paraffin-embedded renal tissue. Sections were dewaxed, rehydrated by descending ethanol concentrations, and processed with the FragEl DNA Fragmentation Detection kit (Oncogene Research Products). Afterward, sections were processed for AT2 immunostaining as described above. The tissue sections were counterstained with Mayer's hematoxylin (Sigma). Negative control sections were performed in parallel for specific labeling by replacing the primary antibody with nonimmune normal rabbit serum and with the omission of TdT enzyme. Results are expressed as mean ± SEM. Comparisons between groups were made using the unpaired Student's t-test or the Kruskal-Wallis nonparametric analysis of variance test when appropriate. Differences were taken as significant when P < 0.05. In BSA-overloaded rats, proteinuria was significantly increased at day 8, reaching a plateau between days 8 and 28 (day 8: UNX-control, 3.0 ± 0.4 and BSA-overloaded, 544 ± 105; day 28: UNX-control, 7.0 ± 0.3 and BSA-overloaded, 460 ± 65 mg/24 hours; n = 7, P < 0.05). In UNX-control rats, no clear evidence of tubulointerstitial injury was seen, although mild glomerular proliferative changes could be detected both at days 8 and 28 (Figure 1, A and B). However, animals with persistent proteinuria showed interstitial infiltration of mononuclear cells, tubular atrophy and/or vacuolization, and protein casts within distal and proximal tubules both on days 8 and 28 (Figure 1, C and D). BSA-overloaded rats also showed mesangial expansion (Figure 1; C to E). The semiquantitation of the morphological lesions demonstrated a significant increase in renal injury in BSA-overloaded rats with respect to UNX-control animals (day 8: UNX-control, 0.2 ± 0.1 and BSA-overloaded, 1.3 ± 0.4; day 28: UNX-control, 0.3.0 ± 0.2 and BSA-overloaded, 2.57 ± 0.75; n = 7, P < 0.05 versus UNX-control at the same time). By reverse transcriptase-PCR, we could not detect the mRNA for AT2 receptor in renal cortex from Wistar-Kyoto rats with two kidneys, in agreement with previous data.20Shanmugam S Llorens-Cortes C Clauser E Corvol P Gasc JM Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland.Am J Physiol. 1995; 268: F922-F930PubMed Google Scholar, 21Wehbi GJ Zimpelmann J Carey RM Levine DZ Burns KD Early streptozotocin-diabetes mellitus downregulates rat kidney AT2 receptors.Am J Physiol. 2001; 280: F254-F265PubMed Google Scholar However, a band of the predicted size (498 bp) of the AT2 receptor mRNA was found in renal cortex from UNX-control and BSA-overloaded rats both at days 8 and 28 (Figure 2). Figure 2A shows representative examples of AT2 receptor PCR products from the different groups. Densitometric analysis of the bands demonstrated a significant increment in the AT2 receptor expression in BSA-overloaded rats compared to UNX-control at all times studied (Figure 2B). By Western blot analysis, a significant increase in AT2 receptor protein expression was observed in the renal cortex from BSA-overloaded rats compared to UNX-control rats (Figure 2, C and D). Adrenal gland, which expresses abundant AT2 receptor protein,20Shanmugam S Llorens-Cortes C Clauser E Corvol P Gasc JM Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland.Am J Physiol. 1995; 268: F922-F930PubMed Google Scholar was used as positive control. To localize the AT2 receptor protein in the rat kidney, immunohistochemistry was performed on paraffin-embedded kidney sections from UNX-control and BSA-overloaded animals. As shown in Figure 3, in both groups of rats, AT2 receptor protein was localized in proximal and distal tubules, mainly in the basolateral membranes of the tubules. No appreciable immunostaining of AT2 receptor was observed in glomeruli. Negative controls of the immunohistochemistry showing the specificity of the AT2 receptor antibody is shown in Figure 3D. Quantitation of AT2 receptor staining by computer-based image analysis revealed a significant increase in expression of AT2 receptor in BSA-overloaded rats with respect to UNX-control rats (Figure 4). This AT2 expression was higher, but not significantly, at day 8 than at day 28 (Figure 4), corroborating the results obtained by Western blot.Figure 4Percentage of renal cortex occupied by AT2 receptor immunostaining. AT2 receptor immunostaining was quantified as described in Materials and Methods. Results as mean ± SEM, n = 7 animals per group. *, P < 0.05 with respect to UNX-control at same time.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Measured by TUNEL technique, UNX-control rats showed occasional TUNEL-positive staining cells (Figure 5A). However, the administration of BSA to UNX rats induced an increment of cells showing an intense brown TUNEL-stained nuclei mainly in tubules but also in the glomeruli (Figure 5; B to G). In our experimental conditions, no staining is detected either in normal Wistar rats or in the negative control sections using buffer lacking TdT as previously reported.22Soto K Gómez-Garre D Largo R Gallego J Tejera N Catalán MP Ortiz A Plaza JJ Alonso C Egido J Tight blood pressure control decreases apoptosis during renal damage.Kidney Int. 2004; 65: 811-822Crossref PubMed Scopus (14) Google Scholar Quantitation of TUNEL-stained apoptotic cells demonstrated a significantly increased number of apoptotic cells mainly in tubules and, to a lesser extent, in glomeruli from BSA-overloaded animals with respect to UNX-control rats at all times studied (Figure 5H). Double staining for apoptosis and AT2 receptor showed that most of TUNEL-positive cells were found in tubules expressing AT2 receptor (Figure 6). No staining was observed in renal sections incubated with a nonimmune normal rabbit serum and with the omission of TdT enzyme, demonstrating the specificity of labelings (Figure 6C). Among executioner caspases, caspase-3 is the best studied and represents the central molecule situated at the crossroad of all known apoptotic pathways.6Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar Western blot analysis and quantification of procaspase-3 (32 kd) showed a transient increase of the caspase-3 precursor in the renal cortex from BSA-overloaded rats as compared with UNX-controls at day 8 (Figure 7). At day 28, the procaspase-3 expression was slightly, but not significantly, higher in BSA-overloaded rats than in UNX-controls. All known caspases require proteolytical cleavage to liberate one large and one small subunit that heterodimerize to form the active enzyme.23Fernandes-Alnemri T Litwack G Alnemri ES CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme.J Biol Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar Western blots using an antibody to the large fragment of activated caspase-3 (17 to 20 kd) identified two proteins of 17 and 19 kd in BSA-overloaded rats, corresponding to large fragment and prodomain + large fragment, respectively (Figure 8). By Western blot, no changes were observed either in the Bax or in the Bcl-2 protein levels in BSA-overloaded rats with respect to UNX-controls at all time studied (Figure 9). Because phosphorylation of Bcl-2 is essential for its physiological role,14Ito T Deng X Carr B May WS Bcl-2 phosphorylation required for anti-apoptosis function.J Biol Chem. 1994; 269: 26865-26870PubMed Google Schola

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.

To delineate the interactions between rabbit hemorrhagic disease virus (RHDV) and host cells, organ and cellular targets of infection were identified in vivo. Viral specific antigens were detected by immunohistochemistry in liver, lung, spleen and lymph nodes cells. Also, intravascular infected cells were detected in most organs including kidneys, myocardium, thymus and central nervous system. To further characterize infected target cells, viral proteins and cell-specific surface antigens were identified simultaneously in double labeling experiments. Numerous lymphoid organ macrophages, from the splenic red pulp, circulating monocytes, alveolar macrophages and Kupffer cells were double labeled, demonstrating that cells of the mononuclear phagocyte lineage are major hosts for RHDV. Double labeling for other specific cell markers were negative. The distribution of viral antigens in these tissues coincided with those areas where cells presented morphology of apoptosis. Association of intravascular monocyte infection and apoptosis, could represent a possible mechanism to develop disseminated intravascular coagulation.

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.

The adenovirus (Ad) E1A gene exerts an antitumor effect and can induce sensitivity to treatment with DNA-damaging agents. In contrast, the Ad 19-kDa E1B protein inhibits E1A-mediated apoptosis and the 55-kDa E1B inactivates the p53 protein. In this paper, we study the in vitro and in vivo effects of a 19-kDa and 55-kDa E1B-defective Ad in several malignant human tumor cell lines. Materials and Methods. Nontumorigenic human fibroblasts (CCD-45SK and Hs67), peripheral blood lymphocytes, and several human tumor cell lines derived from cervix, colon, and breast carcinomas, epidermoid carcinoma, and osteosarcoma (HeLa, HT29, MCF7, Saos-2, and A431 cell lines) were studied. Wild-type (wt) Ad type 5 and H5 dL118 Ad, a mutant with the deleted E1B region, were employed. The cells were infected at 20 plaque-forming units, and cell viability was evaluated by the crystal violet method. In the in vivo experiments, 2 × 106 cells from the carcinoma cell lines HeLa, A431. and HT29 were injected into nude mice. The tumorigenicity of previously infected cells and after an intratumoral injection of Ad was analyzed. The mice received whole-body γ-irradiation. Results. The H5 dL118 mutant produced a marked cytopathic effect in all of the malignant cells, surpassing that of the wt Ad; viability at 72 hours ranged from 11% to 20% for H5 dL118 Ad and from 70% to 93% for the wt Ad with respect to uninfected controls. In the in vivo experiments, a total inhibition of tumorigenicity was detected when cells were infected prior to injection and a partial and transitory decrease in tumorigenicity was detected when the mutant H5 dL118 was injected intratumorally. γ-irradiation enhanced the in vivo antitumor effects. Conclusions. These results indicate that infection with completely E1B-deficient Ads induced a marked cytopathic effect on malignant cells that was higher than that seen for wt Ads; in addition, infection with such Ads exerts a tumor suppressor effect in vivo.

Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.

It has previously been reported that expression of the adenoviral gene E1A dramatically increases sensitivity to DNA damaging agents and decreases the tumorigenicity of murine and human malignant cells by both p53 dependent and independent pathways (1–3, for a review see reference 4). Given the fact that the E1a protein is expressed at high levels during adenovirus 5 infection and due to its highly cytotoxic potential 5, we decided to study the effects of an adenovirus lacking the E1B gene. 19K E1B has been reported to overcome the cytotoxicity of E1A by blocking apoptosis, and the 55K-E1B inactivates the p53 protein (5, 6). Consequently, we hypothesized that deletions in both E1B ORFs could result in enhanced antitumor cytotoxicity and induction of sensitivity to DNA-damaging agents.

Infection with African swine fever virus (ASFV) leads to a short haemorrhagic course of disease that, depending on the virus isolate, results in up to 100% lethality in domestic and Eurasian wild pigs. Consequently, ASFV infection in swine is of considerable economic significance. This chapter explains the basics of antiviral immunity in swine, focusing on the 'knowns' and 'unknowns' of the response against ASFV. In particular, monocytes and macrophages play an essential role as the main targets of infection and are crucial in viral persistence and dissemination. Furthermore, ASFV has developed several mechanisms to influence the antiviral and cell biological activity of infected monocytes, including down-regulation of cell surface receptors (e.g. CD14 and MHC-I) and modulation of interferon and cytokine/chemokine responses. ASFV infected pigs also develop virus-specific antibodies that can be used diagnostically, and while the neutralising effect of these antibodies has led to their involvement in protective immunity being controversially discussed, they may still exhibit protective functions through complement-mediated lysis and/or antibody dependent cell-mediated cytotoxicity. Indeed, T cells (presumably CD8+) also play a central role in the elimination of the virus, as can be seen in experiments where, after depletion of these cells, pigs previously primed with an avirulent ASFV become ill, while non-depleted animals are protected from highly virulent challenge. Nonetheless, despite these advances in our knowledge, much remains unknown about antiviral immunity generated during the course of a natural ASFV infection, or in response to attenuated virus strains or immunisation. Although such studies would undoubtedly be technically challenging, a deeper understanding of the immunity developed by the natural hosts (i.e. bushpigs and warthogs) against ASFV infection would teach us a lot about an effective protection from ASFV infection, and the involvement of both the innate and adaptive immune systems in this process.

En esta comunicacion se describe y analiza la experimentacion de un trabajo colaborativo realizado en la asignatura de Analisis del Discurso en el Campus Virtual. Los resultados obtenidos nos permiten avanzar en este entorno didactico que se adecua a una pedagogia neoconstructivista en la que se valoran las capacidades de los sujetos que interactuan con objetivos y finalidades comunes.

Esta obra se enmarca dentro de la ciberpragmatica y la pragmatica cognitiva y pretende ser una contribucion a los estudios de los discursos y textos electronicos; en efecto, el ordenador ha transformado los discursos sociales creando nuevos generos que implican diferentes estrategias de intercambio de informacion, produccion, comprension y lectura de textos. La comunicacion por Internet, ultima forma de comunicacion humana, se ha desarrollado en todos los esferas sociales y, por una parte, ha sustituido, en gran medida, a generos tradicionales como la carta, el dialogo, la conversacion o el debate y, por otra, ha modificado, a su vez, los generos del discurso de transmision de conocimientos como los diccionarios y los metodos de ensenanza.

The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca 2+ -dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.

African swine fever virus (ASFV) is a double-stranded DNA virus that infects domestic and wild swine, warthogs and bushpigs, causing a devastating disease in affected geographical regions (Dixon et al. 2000). Soft ticks of the genus Ornithodoros are also infected, with no disease signs, acting as a vector. African swine fever (ASF) is a very high mortality hemorrhagic syndrome characterized clinically by anorexia, fever, leukopenia and disseminated intravascular coagulation (Tulman and Rock 2001). Widespread cell death caused by apoptosis occurs in lymphoid tissues affecting macrophages, T and B lymphocytes, and endothelial cells (Ramiro-Ibanez et al. 1996, 1997; Oura et al. 1998a).

En este trabajo se propone la incorporacion de LAMS a un LMS para mejorar el control del espacio virtual de aprendizaje de caracter socio-constructivista E-Ling. Este nuevo espacio tiene como objetivo desarrollar la competencia de ‘aprender a investigar’ como base para otros aprendizajes en educacion superior. Esta propuesta de integracion de distintos tipos de Entornos Virtuales de Aprendizaje (VLE) se explica dentro de un marco conceptual de referencia que admite analizar y definir las aportaciones de otros modelos y herramientas diferentes. En este trabajo planteamos la hipotesis de que con LAMS se podra definir, construir y utilizar con mas precision escenarios de aprendizaje de tipo instruccional. Los resultados obtenidos pueden ser contrastados con trabajos anteriores que miden la rentabilidad de un diseno de aprendizaje aplicado a dos escenarios diferentes: 1) presencial, 2) virtual sobre un LMS. En consecuencia, planteamos crear, utilizar y evaluar la rentabilidad de un nuevo espacio de aprendizaje que integre las funciones de gestion de cursos de caracter general de un LMS, con funciones mas especificas de creacion de escenarios de aprendizaje en linea, su ejecucion con profesores y alumnos, y el control de la evolucion del aprendizaje en un sistema gestor de actividades.

La comprension de los textos, tal como la hemos venido definiendo en nuestros trabajos, no exige unicamente una reflexion de tipo linguistico sino que debe situarse en un campo multidisciphinar en donde la semantica de los prototipos y ha teoria de los modelos mentales jueguen y ocupen un lugar especifico y basico. En efecto, estas dos disciplinas nos permiten definir esta actividad a partir de la interaccion de tres factores de naturaleza muy diferente: cognoscitivos, linguisticos y pragmaticos.

El objetivo principal del proyecto Quizzes es mejorar el proceso de aprendizaje tanto mediante un modelo dinamico que implique activamente a los estudiantes, a la vez que se generan materiales didacticos que sirvan para la consolidacion del conocimiento y la autoevaluacion. Con el desarrollo de este proyecto buscamos implicar al alumno en su propio aprendizaje y lograr que su trabajo, tanto fuera como dentro del aula, sirva para genere materiales didacticos adaptados a sus necesidades educativas en distintos momentos del proceso de aprendizaje. Nuestro proyecto es una novedosa aplicacion de los quiz a la rama de Ciencias Sociales y Humanidades, de modo que este modelo de evaluacion formativa se convierta a su vez en un metodo que permita la generacion de conocimiento y su consolidacion.

El presente proyecto de innovacion docente y educativa esta basado en la metodologia de la simulacion aplicada a uno de los modelos de reunion cientifica, el simposio, con la finalidad de lograr aunar formacion academica y comunicacion profesional en un desarrollo metodologico todavia poco experimentado en asignaturas presenciales de los estudios de Grado. La actividad propuesta requiere poner en juego las habilidades comunicativas de los estudiantes en distintas lenguas, de cara a su futuro desempeno profesional. Si bien en los estudios de posgrado enfocados a la investigacion se fomenta la participacion de los estudiantes en simposios especializados adaptados a su estatus, los alumnos de grado no tienen apenas oportunidades de probar sus capacidades y competencias comunicativas fuera del aula. Asi pues, el objetivo principal del proyecto es mejorar la competencia comunicativa de los estudiantes dentro del marco formativo que proporcionan algunas asignaturas (de ultimo curso del Grado) que se prestan especialmente a la aplicacion de esta metodologia. Con el desarrollo del proyecto Encuentro buscamos implicar al alumno en su propio aprendizaje y lograr que, desde el inicio, todo el trabajo que realiza en las actividades propuestas contribuya a mejorar las destrezas relacionadas con su competencia comunicativa y, asimismo, que este pueda afrontar actividades similares en entornos profesionales.

El cambio metodologico que estan abordando las universidades en Europa, como consecuencia de la introduccion de nuevos enfoques educativos y la implantacion de las nuevas tecnologias en el ambito docente, obliga a apoyar y asumir nuevas formas de entender la construccion y comunicacion del conocimiento. En particular, la implantacion de tecnologias especificas del ambito docente, como son los campus virtuales, hace necesaria la compatibilidad de metodos y tecnologias. Uno de los desarrollos que se han mostrado mas eficaces son las plataformas b-learning, que permite a profesores y alumnos combinar ensenanza presencial con una ensenanza flexible. La especificidad de las ensenanzas universitarias lleva a la creacion de espacios metodologicos apropiados a las necesidades de alumnos y profesores. En este marco es en el que hemos desarrollado el espacio E-Ling, que presentamos en este articulo, modelo mixto que compagina acertadamente presencia fisica con ensenanza a distancia en linea. Se trata de un entorno plural de comunicacion que resulta innovador, ya que se han disenado estrategias especificas b-learning y metodologias dinamicas con el fin de lograr un aprendizaje efectivo de la Linguistica, tanto en la ensenanza general como en la iniciacion a la investigacion. Esta metodologia significa un cambio de paradigma en la Ensenanza Superior.

Within the framework of the Project E-Ling, at Universidad Complutense de Madrid (Spain), our research team is working on the creation of educational materials and defining online pedagogies in the area of Linguistics. We have been using LAMS, for some years now, to design activity sequences in higher education. Our learning sequences have fundamentally focussed on collaborative learning environments, however, we have realised that LAMS can be employed in many different online pedagogies. One of our main interests in LAMS is to point out its adaptability, as some of the designers/ teachers working with us can only see the use of sequences as an instructor-led type of learning. We have transformed some of our tested sequences into pedagogical planners, using the activity planner tool provided in LAMS. In the second stage of our work, we are surveying teachers about the use of these planners, in order to determine f planners help them not only to save time but open new possibilities of using different pedagogies in LAMS. In this presentation, we will show some examples of planners, sequences obtained through these planners and some of the analysed opinions. [RESUMEN] Desde el proyecto E-Ling, en la Universidad Complutense de Madrid, estamos trabajando en la creacion de pedagogias y materiales online para la ensenanza de la Linguistica. Desde hace varios anos estamos utilizando LAMS para crear secuencias de aprendizaje a nivel universitario. Hemos trabajado con esta herramienta en entornos de ensenanza/ aprendizaje colaborativos, sin embargo, hemos notado que su uso puede adaptarse a numerosas pedagogias online. Uno de nuestros intereses en LAMS es mostrar esta adaptabilidad, ya que algunos de los disenadores de cursos/ profesores con los que hemos trabajado unicamente utilizan LAMS en contextos de aprendizaje colaborativo y liderado por el instructor. Una parte de nuestro trabajo ha sido trasformar secuencias de aprendizaje basadas en diferentes pedagogias utilizadas en entornos en linea ya experimentadas en planes pedagogicos utilizando la herramienta de activity planner disponible en LAMS. En estos momentos, estamos recogiendo la opinion de los profesores que trabajan con nosotros sobre el uso de estos planners. En esta presentacion mostraremos varios ejemplos de planners, secuencias creadas con estos planners y las primeras opiniones recogidas.

It has previously been reported that expression of the adenoviral gene E1A dramatically increases sensitivity to DNA damaging agents and decreases the tumorigenicity of murine and human malignant cells by both p53 dependent and independent pathways (1–3, for a review see reference 4). Given the fact that the E1a protein is expressed at high levels during adenovirus 5 infection and due to its highly cytotoxic potential 5, we decided to study the effects of an adenovirus lacking the E1B gene. 19K E1B has been reported to overcome the cytotoxicity of E1A by blocking apoptosis, and the 55K-E1B inactivates the p53 protein (5, 6). Consequently, we hypothesized that deletions in both E1B ORFs could result in enhanced antitumor cytotoxicity and induction of sensitivity to DNA-damaging agents.

El contexto ocupa un papel privilegiado en cualquier acto comunicativo porque la intención y el sentido del hablar no pueden construirse si no están situados y su comprensión e interpretación dependen siempre del contexto y de sus circunstancias. En este capítulo respondemos a las preguntas de qué es el contexto y cómo podemos observarlo y analizarlo. Presentamos, en primer lugar, cuatro acercamientos de carácter interdisciplinar: las dimensiones lingüística, pragmática, interaccional y cognitiva del contexto. En segundo lugar, ofrecemos una propuesta metodológica como posible guía de análisis, partiendo de géneros discursivos y tipos de textos en los que se van integrando 1) los participantes y sus relaciones interpersonales, 2) el sentido e intencionalidad del texto, y 3) los canales y medios de producción.

Desde la metodologia de investigacion-accion, el proyecto incentiva a los estudiantes universitarios a ser agentes activos en la difusion de conocimientos cientificos y academicos en internet utilizando mecanismos discursivos de divulgacion en la red.

La divulgacion del conocimiento cientifico es una responsabilidad social que la Universidad debe abordar desde la perspectiva de la investigacion y la innovacion. El proyecto incentiva a usar la comunicacion multimodal en la red para divulgar ciencia.