Nuevas estrategias terapéuticas frente a la infección por el VIH-1

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-07-2014The present work is composed of two main goals: “Identification and functional analysis of the HIV-1 Nef PGPG motif, responsible for the interaction with the SH2 domain of Lck, as a new potential therapeutic target” and “Study of the in vitro and ex vivo effects of bryostatin-1 in combination with the antiretrovirals maraviroc or Atripla® on viral reactivation and the inhibition of novel infection issues”. SECTION 1: HIV-1 Nef affects pivotal processes in the infected host cell including T cell receptor signalling. Most of these effects involve the physical association with and alteration of subcellular localization of the host cell kinase Lck. Retargeting of Lck depends on a PxxP motif in Nef that binds to SH3 domains. Moreover, the SH2 domain of Lck was reported to contribute to interactions with Nef, however the identity of the HIV-1 Nef motif responsible for this interaction has remained unknown. To search for the elusive SH2 binding site we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified a PGPG motif encompassing residues 121-137 of HIV-1NL4.3 Nef to be required for the interaction with Lck-SH2. The PGPG motif was critical for Lck dependent Nef functions, including CD4 downregulation, optimal infectivity of viruses produced in T cells as well as intracellular recruitment of Lck. Unexpectedly, we found that the PGPG motif also participated in the binding to the Lck-SH3 domain, indicative of the synergy between PxxP-SH3 and PGPG-SH2 interaction. In accordance, the PGPG motif also participated on Nef dependent inhibition of host cell actin dynamics and MHC-I downregulation, which are PxxP dependent activities of Nef that do not involve Lck. In conclusion, the PGPG motif can mediate interactions of Nef with SH2 domains and supports the activity of the Nef PxxP motif. Thus, we propose the conserved PGPG motif in HIV-1 Nef as a potential pharmacological target for interference with Nef function in HIV-1 infection. SECTION 2: Despite antiretroviral therapy successfully controls HIV-1 viremia in most AIDS patients, virus latency establishment early upon infection impedes HIV-1 eradication in HIV-1+ patients. Bryostatin-1 (BRYO) inhibits in vitro HIV-1 infection of CD4 T lymphocytes (as it downregulates CD4 and CXCR4, viral receptor and coreceptor, respectively) and, at the same time, reactivates virus from latency through PKC/NF-kB pathway activation. Prior to design clinical studies with BRYO to assess its real impact on the size of the HIV-1 latent reservoirs, the potential in vitro effect of BRYO in combination with the antiretrovirals (AR) maraviroc (MVC) and Atripla® (ATP) has been determined. Jurkat-LTRGFP-R5 cell line and two latent and reactivatable HIV-1-infected lymphocytic or monocytic clonal cell lines (J89GFP and THP89GFP respectively) were used as latency models. BRYO reactivated latent HIV-1, reaching levels up to 80% in THP89GFP cells, even in combination with MVC or ATP. Moreover, when AR pre-treated reporter TZM-bl cells were co-cultured with BRYO treated THP89GFP, new infections of reactivated HIV-189.6 were inhibited 50% or 80% for MVC or ATP pre-treated cells, respectively. Remarkably, when AR were combined with BRYO, the combinations maintained the antiviral effect of the drugs with the maximum rate of inhibition by its own. In addition, BRYO-mediated downregulation of surface CD4 and CXCR4 in PBMC was not affected when it was used along with other AR and no hiperactivation or high proliferation effects were observed in these cells. Significantly, we found that BRYO strongly stimulated the viral promoter (LTR) transcription by activating the transcription factor NF-κB in human primary astrocytes and in the astrocytoma cell line U87, suggesting that the reactivation effect of BRYO could be especially important in a cellular reservoir such as astrocytes. Moreover, BRYO was also tested ex vivo for HIV-1 induction in CD4 T cells isolated from infected individuals receiving HAART and was found to exhibit potent induction activity. This work is the first to demonstrate that AR combination with BRYO do not interfere with BRYO activity (in terms of reactivation of latently infected cells and partial inhibition of infection) neither with AR antiviral activity. Thus, we propose BRYO as a viral latency reactivator compound appropriate to be combined with actual antiretroviral treatments in order to purge the viral reservoirs

The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis

<p>Abstract</p> <p>Background</p> <p>RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight) known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether <it>Leishmania </it>PUF proteins result antigenic during visceral leishmaniasis (VL).</p> <p>Findings</p> <p>Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in <it>Leishmania infantum</it>. Additionally, the 10 genes of the <it>L. infantum </it>PUF family were cloned and used to express the <it>Leishmania </it>PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The <it>Leishmania </it>PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients.</p> <p>Conclusion</p> <p>Our results suggest that <it>Leishmania </it>PUFs are targets of the humoral response during <it>L. infantum </it>infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as <it>Trypanosoma cruzi</it>.</p

Experiencias de investigación feminista : propuestas y reflexiones metodológicas

Aquest text és una presentació del monogràfic "Experiències d'investigació feminista: propostes i reflexions metodològiques ". En ell donem compte de les consideracions i motivacions principals que ens han portat a proposar-ho, així com de la trajectòria i el procés de treball amb metodologies feministes del que formem banda, el seminari SIMReF. D'aquesta manera, exposem algunes dificultats que es mantenen per produir coneixement no androcèntric a l'acadèmia, sostenim l'interès de treballar sobre mètodes amb els quals transversalitzar l'anàlisi de gènere en diferents àrees disciplinàries, plantegem l'oportunitat d'elaborar guies o referents metodològics per aplicar les epistemologies feministes en les nostres investigacions i, especialment, advoquem per la necessitat de donar compte de les decisions pràctiques preses en els processos d'investigació que realitzem, per tal de fer visible així la "cuina" dels mateixos. Per acabar, descrivim amb brevetat el contingut present en el monogràfic.This text means to be an introduction to the monograph named “Feminist research experiences: reflections and proposals on methodologies”. We aim to express the main concerns and motivations that led us to elaborate it, as well as the process of working with feminist research methodologies in the frame of SIMReF. We try and express the difficulties we met when aiming to produce non-androcentric forms of knowledge in academia. At the same time we underline our interest on working with and forward methods to transverse gender analysis in different disciplines. We also draw on the opportunity to elaborate guides or methodological reference texts on the implementation of feminist epistemologies in our research and, specially, advocate the idea of giving account about practical decisions taken during the research processes, revealing the “back room”within them. Finally, we provide a short description of the articles included in the monograph.Este texto es una presentación del monográfico “Experiencias de investigación feminista: propuestas y reflexiones metodológicas”. En él damos cuenta de las consideraciones y motivaciones principales que nos han llevado a proponerlo, así como de la trayectoria y el proceso de trabajo con metodologías feministas del que formamos parte, el seminario SIMReF. De esta manera, exponemos algunas dificultades que se mantienen para producir conocimiento no androcéntrico en la academia, sostenemos el interés de trabajar sobre métodos con los que transversalizar el análisis de género en distintas áreas disciplinarias, planteamos la oportunidad de elaborar guías o referentes metodológicos para aplicar las epistemologías feministas en nuestras investigaciones y, especialmente, abogamos por la necesidad de dar cuenta de las decisiones prácticas tomadas en los procesos de investigación que realizamos, a fin de hacer visible así la “cocina” de los mismos. Para acabar, describimos con brevedad el contenido presente en el monográfico

CD2v interacts with Adaptor Protein AP-1 during African swine fever infection

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.This work was supported by Ministerio de Ciencia e Innovación of Spain, BFU2010-17794 (YR); European Community’s Seventh Framework Programme, KBBE.2012.1.3-02-ASFORCE (YR). Ricardo Madrid was funded by an Amarauto research program and by FIS-641 PS09/01386.Peer Reviewe

A Conserved GPG-Motif in the HIV-1 Nef Core Is required for principal nef-activities

© 2015 Martínez-Bonet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedTo find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121–137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.This work was supported by the Ramon y Cajal research program (MICIIN-RYC-2005-002174; http://www.mineco.gob.es/portal/site/mineco/idi), Fondo de Investigación Sanitaria en España (FIS-PS09/01386; http://www.isciii.es/) to RM, and Comunidad de Madrid (S-2010/BMD-2332; http://www.madrimasd.org/) to MAM-F, and the Deutsche Forschungsgemeinschaft (TRR83, project 15 to OTF). MM-B, CP and VB hold a fellowship from the Fondo de Investigación Sanitaria en España (FIS, http://www.isciii.es/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities.

To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121-137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.This work was supported by the Ramon y Cajal research program (MICIIN-RYC-2005-002174; http://www.mineco.gob.es/portal/site/mineco/idi), Fondo de Investigación Sanitaria en España (FIS-PS09/01386; http://www.isciii.es/) to RM, and Comunidad de Madrid (S-2010/BMD-2332; http://www.madrimasd.org/) to MAM-F, and the Deutsche Forschungsgemeinschaft (TRR83, project 15 to OTF). MM-B, CP and VB hold a fellowship from the Fondo de Investigación Sanitaria en España (FIS, http://www.isciii.es/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Experiencias de investigación feminista: propuestas y reflexiones metodológicas

Este texto es una presentación del monográfico Experiencias de investigación feminista: propuestas y reflexiones metodológicas. En él damos cuenta de las consideraciones y motivaciones principales que nos han llevado a proponerlo, así como de la trayectoria y el proceso de trabajo con metodologías feministas del que formamos parte, el seminario SIMReF. De esta manera, exponemos algunas dificultades que se mantienen para producir conocimiento no androcéntrico en la academia, sostenemos el interés de trabajar sobre métodos con los que transversalizar el análisis de sexo y género en distintas áreas disciplinarias, planteamos la oportunidad de elaborar guías o referentes metodológicos para aplicar las epistemologías feministas en nuestras investigaciones y, especialmente, abogamos por la necesidad de dar cuenta de las decisiones prácticas tomadas en los procesos de investigación que realizamos, a fin de hacer visible así la “cocina” de los mismos. Para acabar, describimos con brevedad el contenido presente en el monográfico

Early progression to active tuberculosis is a highly heritable trait driven by 3q23 in Peruvians

Abstract: Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5–15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability (hg2) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10−8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function

Increasing Cervical Cancer Screening Coverage: A randomised, Community-Based Clinical Trial

Altres ajuts: The project received a research grant from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain), awarded on the 2010 call under the Health Strategy Action 2013-16, within the National Research Program oriented to Societal Challenges within the Technical, Scientific and Innovation Research National Plan 2013-16 with reference PI10/01275 co-funded with European Union ERDF funds. Additional support was provided by the European Regional Development Fund (ERDF), public grants from the Carlos III Institute of Health (RTIC RD06/0020/0095 RD12/0036/0056 and CIBERESP) and the Agència de Gestió d'Ajuts Universitaris i de Recerca (grants AGAUR 2014SGR1077 and 2014SGR2016) and the Primary Health Care Unit IDIAP Jordi Gol and the Catalan Institute of Health resolved 07/04/2014.Background. Opportunistic cervical cancer screening can lead to suboptimal screening coverage. Coverage could be increased after a personalised invitation to the target population. We present a community randomized intervention study with three strategies aiming to increase screening coverage.Methods. The CRICERVA study is a community-based clinical trial to improve coverage of population based screening in the Cerdanyola SAP area in Barcelona.A total of 32,858 women residing in the study area, aged 30 to 70 years were evaluated. A total of 15,965 women were identified as having no registration of a cervical cytology in the last 3.5 years within the Public Health data base system. Eligible women were assigned to one of four community randomized intervention groups (IGs): (1) (IG1 N = 4197) personalised invitation letter, (2) (IG2 N =3601) personalised invitation letter + informative leaflet, (3) (IG3 N = 6088) personalised invitation letter + informative leaflet + personalised phone call and (4) (Control N = 2079) based on spontaneous demand of cervical cancer screening as officially recommended. To evaluate screening coverage, we used heterogeneity tests to compare impact of the interventions and mixed logistic regression models to assess the age effect. We refer a ªrescueº visit as the screening visit resulting from the study invitation. Results. Among the 13,886 women in the IGs, 2,862 were evaluated as having an adequate screening history after the initial contact; 4,263 were lost to follow-up and 5,341 were identified as having insufficient screening and thus being eligible for a rescue visit. All intervention strategies significantly increased participation to screening compared to the control group. Coverage after the intervention reached 84.1% while the control group reached 64.8%. The final impact of our study was an increase of 20% in the three IGs and of 9% in the control group (p<0.001). Within the intervention arms, age was an important determinant of rescue visits showing a statistical interaction with the coverage attained in the IGs. Within the intervention groups, final screening coverage was significantly higher in IG3 (84.4%) (p<0.001). However, the differences were more substantial in the age groups 50±59 and those 60+. The highest impact of the IG3 intervention was observed among women 60+ y.o with 32.0% of them being rescued for screening. The lowest impact of the interventions was in younger women.Conclusions. The study confirms that using individual contact methods and assigning a fixed screening date notably increases participation in screening. The response to the invitation is strongly dependent on age