Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

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Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance Nadia Bakkour1,2,3[, Yea-Lih Lin4,5,6[, Sophie Maire1,2,3, Lilia Ayadi7,8, Florence Mahuteau-Betzer9,10, Chi Hung Nguyen9,10, Clément Mettling4,5,6, Pierre Portales4,5,6, David Grierson9,10, Benoit Chabot11, Philippe Jeanteur1,2,3, Christiane Branlant7,8, Pierre Corbeau4,5,6, Jamal Tazi1,2,3* 1 Université de Montpellier II, Montpellier, France, 2 Institut de Génétique Moléculaire de Montpellier, Montpellier, France, 3 CNRS, UMR 5535, Montpellier, France, 4 Laboratoire d’Immunologie CHU de Montpellier, Montpellier, France, 5 Institut de Genetique Humaine, Montpellier, France, 6 CNRS, UPR1142, Montpellier, France, 7 Université Henri Poincare-Nancy I, Vandoeuvre-les-Nancy, France, 8 CNRS, UMR 7567, Vandoeuvre-les-Nancy, France, 9 Laboratoire de Pharmaco-chimie, Institut Curie, Orsay, France, 10 CNRS-UMR 176, Orsay, France, 11 Département de Microbiologie et d’Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell– tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses. Citation: Bakkour N, Lin YL, Maire S, Ayadi L, Mahuteau-Betzer F, et al. (2007) Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance. PLoS Pathog 3(10): e159. doi:10.1371/journal.ppat.0030159 sequences and non-spliceosomal nuclear RNA-binding proteins (trans-acting factors). These trans-acting factors can be classified as SR proteins (serine-arginine-rich proteins) [18– 21] and hnRNPs (heterogenous nuclear ribonucleoproteins) [22–25]. Binding of the SR proteins to exonic splicing enhancers (ESEs) downstream of the Tat-, Rev-, Vpr-, Env-, and Nef-specific 39 splice sites promotes exon definition by recruiting constitutive factors and preventing the action of nearby splicing silencers [26]. Therefore, members of the SR protein family are thought to play a major role in the regulation of HIV-1 pre-mRNA splicing. By screening a large collection of chemical compounds, our laboratory has discovered several benzopyridoindole and pyridocarbazole derivatives that selectively inhibit the ESEdependent splicing activity of individual SR proteins [27–29]. Selective binding of these compounds to specific SR proteins Introduction The increasing prevalence of drug-resistant human immunodeficiency virus type 1 (HIV-1) has highlighted the challenging issue of the optimal treatment of HIV-1-infected patients [1–3]. Current routine drug regimens, typically consisting of various combinations of compounds targeting the viral proteins reverse transcriptase, protease, and gp120, have revolutionized the treatment of HIV/AIDS [3–5]. However, HIV-1 can acquire resistance to all known inhibitors of these targets, and transmission of multidrugresistant HIV strains is becoming a growing problem [1,2,6– 9]. This, as well as other problems such as viral escape mutants [4,10], persistence of viral reservoirs [11–14], poor patient compliance due to complicated regimens [15,16], and toxic side effects [17], have emphasized the need for the development of new drugs with novel mechanisms of action. In addition to virus-specific enzymes, such as reverse transcriptase and protease, several cellular factors are required for replication of HIV-1 [10]. The identification of these critical host cell factors may provide novel cellular targets for the development of compounds that are potentially capable of inhibiting HIV-1, thereby decreasing the burden of viral replication in cases of transmitted multidrug-resistant HIV-1 infection. To express key viral proteins, HIV-1 uses a combination of several alternative 59 and 39 splice sites to generate more than 40 different mRNAs from its full-length genomic pre-mRNA [10]. The choice of these alternative splice sites strongly depends on specific interactions between HIV pre-mRNA PLoS Pathogens | Editor: Michael H. Malim, King’s College London, United Kingdom Received March 26, 2007; Accepted September 14, 2007; Published October 26, 2007 Copyright: Ó 2007 Bakkour 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 credited. Abbreviations: AZT, azidothymidine (39-azido-39-deoxythymidine, zidovudine); ELISA, enzyme-linked immunosorbent assay; ESE, exonic splicing enhancer; HIV-1, human immunodeficiency virus type 1; PBMC, peripheral blood mononuclear cell; SR, serine-arginine-rich * To whom correspondence should be addressed. E-mail: [ These authors contributed equally to this work. 1530 October 2007 | Volume 3 | Issue 10 | e159 Splicing Inhibitor for HIV-1 Therapy prevents spliceosome assembly and splicing in an in vitro system. Given the fact that these proteins play an important role in regulating HIV-1 pre-mRNA splicing, we surmised that specific inhibition of SR proteins by benzopyridoindole and pyridocarbazole derivatives could block HIV-1 replication. Author Summary Over the two decades highly active antiretroviral therapy (HAART) for the treatment of HIV infection has led to a significant decline in morbidity and mortality rates among HIV-infected individuals. HAART uses a combination of molecules that target the virus itself. However, naturally occurring and extensive genetic variation found in the virus allow the emergence of drug-resistant viruses, which rapidly render individuals untreatable. An alternative approach for effective anti-HIV-1 chemotherapy should be targeting cellular factors required for HIV-1 replication. Here, we report the development of a successful therapeutic agent for HIV-1 infection based on inhibition of HIV-1 pre-mRNA splicing, a crucial step of the HIV-1 life cycle that allows production of key viral proteins. The splicing inhibitor IDC16 can efficiently block HIV-1 viral production in primary blood cells infected with different laboratory strains or clinical isolates from patients resistant to anti-HIV multitherapies. These findings may serve as the basis for a new strategy to develop a new class of anti-HIV drugs, the splicing inhibitors, and even of antiviral drugs in general, since any virus needing to splice its RNAs may be targeted. Results Among 220 indole derivatives that were screened for selective inhibition of ESE-dependent splicing events, one selected drug (IDC16) demonstrated a strong inhibitory effect on different substrates harboring SF2/ASF high-affinity binding site (Figure 1A). IDC16 had also no adverse effect on cell growth and viability of different cell lines, including primary cell lines (see below). Given that SF2/ASF plays a major role in HIV-1 pre-mRNA splicing, we decided in the present study to test the effect of IDC16 on HIV replication as well as on specific splicing events of the HIV-1 pre-mRNA. The efficiency of the drug was first assessed by using the pDPSP plasmid containing the HIV-1 proviral genome deleted between nucleotides 1511 and 4550 (Figure 1B), which recapitulates all splicing events of HIV-1 pre-mRNA in Figure 1. Selective Inhibition of HIV-1 RNA Splicing Ex Vivo by IDC16 (A) Drawing and formula of IDC16 compound. (B) Schematic representation of HIV-1 genome. The 59 splice sites (D1–D4) and 39 splice sites (A1–A7) are indicated. The various open reading frames are boxed. (C) HeLa cells transfected with the pDPSP construct were either untreated (lane 7) or treated with 0.05 lM, 0.1 lM, 0.5 lM, 1 lM, 2.5 lM, or 5 lM of compound IDC16 (lanes 1–6, respectively). Multiply spliced products of HIV-1 RNA were amplified by RT-PCR using the oligonucleotide primers BSS and SJ4.7A. The PCR products were analyzed by polyacrylamide gel electrophoresis after normalization with GAPDH (see Materials and Methods). Nomenclature of the RT-PCR products on the left of the panel is according to [30]. Size markers (in bp) are shown on the right of the panel (lane 9). RTPCR from untransfected HeLa cells (lane 8). doi:10.1371/journal.ppat.0030159.g001 PLoS Pathogens | 1531 October 2007 | Volume 3 | Issue 10 | e159 Splicing Inhibitor for HIV-1 Therapy Figure 2. In Vitro Inhibition of HIV1-D1-A2 pre-mRNA Splicing by IDC16 and Complementation with SR Proteins SC35 and SF2/ASF (A) Schematic representation of HIV1-D1-A2 transcript is shown. Numbering of the HIV-1/BRU RNA sequences is according to Ratner et al. [49]. (B) Polyacrylamide gel electrophoresis of the in vitro splicing products of HIV1-D1-A2 transcript. Uniformly labeled RNA was prepared as described under Materials and Methods and was incubated alone (lane 1) in a HeLa cell nuclear extract under splicing conditions in the absence (lane 2), in the presence of 10% DMSO (lane 3), or 5 lM, 10 lM, 20 lM, and 40 lM of compound IDC16 (lanes 4–7, respectively). (C) The same reactions as in (B) lanes 3–7 were complemented with 0.21 lM of recombinant SF2/ASF (lanes 1–5, respectively). (D) The same reactions as in (B) lanes 3–7 were complemented with 0.19 lM of recombinant SC35 (lanes 1–5, respectively). (E) Splicing products of the Minx transcript analyzed by polyacrylamide gel electrophoresis. Uniformly labeled RNA was incubated alone (lane 1), in HeLa nuclear extract in the absence (lane 2) or presence of 5 lM, 10 lM, 20 lM, 30 lM, or 50 lM of compound IDC16 (lanes 3–7), respectively. doi:10.1371/journal.ppat.0030159.g002 transfected HeLa cells [30]. The mRNAs produced by splicing were then analyzed by reverse transcriptase (RT)-PCR using forward and reverse primers that amplify several splicing isoforms encoding the viral proteins Nef, Rev, and Tat. Compared to untreated cells, the synthesis of all splicing products was less efficient (compare lane 7 and lanes 1–6). The effect of IDC16 was dose dependent, especially for the synthesis of larger splicing isoforms, with a complete block at 2.5 lM. At this concentration of the drug neither global nor HIV-1 RNA synthesis were affected (unpublished data, see below), implying that IDC16 has a specific action on HIV-1 pre-mRNA splicing. The dose-dependent profile of splicing inhibition indicated that IDC16 inhibits the use of several weak 39 splice sites whose utilization is required for the production of key viral regulatory proteins. Utilization of these 39 splice sites is known to critically depend upon the binding of SR proteins [31]. To examine the specificity of IDC16, we tested the effect of this drug in an in vitro splicing assay using a pre-mRNA substrate (HIV1-D1-A2) harboring the D1 and A2 HIV donor and acceptor splice sites, respectively (see Figure 2A, [32]). Splicing of this substrate is activated by the SR protein ASF/ SF2, both in vitro and ex vivo, and therefore it constitutes an ideal target for IDC16, which inhibits most of SF2/ASF ESEdependent splicing tested ([26] and unpublished data). Treatment with increasing concentrations of IDC16 inhibits splicing of HIV1-D1-A2 in a dose-dependent manner (Figure 2B). Complementation of the extract with recombinant SR PLoS Pathogens | protein ASF/SF2 strongly limited the IDC16 splicing inhibition (Figure 2C), whereas the addition of similar amounts of another SR protein, SC35, fails to do so (Figure 2D), demonstrating that IDC16 specifically impedes SF2/ASFdependent splicing. Consistently, concentration up to 50 lM of IDC16 did not alter splicing of synthetic mRNA precursors derived from the adenovirus major late-transcription unit (Minx, Figure 2E), which is a single intron premRNA not requiring ESE sequences in the second exon for efficient splicing. Given the key role played by ASF/SF2 in the activation of several HIV-1 acceptor sites, it is expected that the treatment of infected cells with IDC16 might block HIV-1 replication. Initial experiments were designed to determine the concentration of IDC16 with minimal side effects on cell viability and cell cycle progression. Treatment of stimulated peripheral blood mononuclear cells (PBMCs) with 1 or 2.5 lM IDC16 next showed no effect on cell proliferation, as measured by tritiated thymidine incorporation into cellular DNA (Figure 3A). Since no adverse cellular effect was observed up to the 2.5 lM IDC16 concentration range, we then asked whether IDC16 could block HIV-1 replication in this potential therapeutic window. Stimulated PBMCs were infected with either NL4.3 (unpublished data) or Ada-M R5 (Figure 3B) HIV-1 strains and cultured for 14 d in the presence or absence of 0.1 lM, 0.5 lM, or 1 lM IDC16. At the indicated days, virus replication was determined by p24 antigen enzyme-linked immunosorbent assay (ELISA). Replication of 1532 October 2007 | Volume 3 | Issue 10 | e159 Splicing Inhibitor for HIV-1 Therapy Figure 3. Inhibition of HIV-1 Production in PBMC-Infected Cells by IDC16 (A) PBMCs from healthy donors were cultured in the presence of labeled 3H-thymidine and incorporated radioactivity was measured after 72 h stimulation with phytohemagglutinin A (PHA) in the absence (-) or presence of 1 lM or 2.5 lM of IDC16. (B) 100 TCID50 of Ada-M was used to infect triplicate of 106 activated PBMCs (stimulated for 2 d with PHA and IL2) in the absence or presence of 0.1 lM, 0.5 lM, or 1 lM of IDC16. Supernatant was harvested at the indicated days and viral capsid protein p24 antigen was quantitated using standard ELISA protocol. (C) Concurrently, cell viability was measured by trypan blue exclusion and indicated as percentage as compared with untreated cells. doi:10.1371/journal.ppat.0030159.g003 human cells infected with either macrophage-tropic (R5) HIV-1, Ada-M, or T-cell-line-tropic (X4), NL4–3 HIV-1 laboratory strains, suggesting that IDC16 could be effective on a variety of viral strains detected in vivo. We next used an in vitro system that may be more relevant to the clinical situation, since it involves infecting primary cells with HIV-1 isolates from patients resistant to conventional antiretroviral therapies. We chose five extreme cases in which viruses harbored mutations in different regions of the viral genome, including those encoding the reverse transcriptase and protease domains. Using two strains that previously demonstrated robust resistance to different therapeutic agents in vitro, we show that IDC16 also very efficiently inhibits the replication of these clinical isolates, since no viral particles were detected 14 d post-infection (Figure 4D). In order to provide further evidence that the anti-HIV activity we observed with IDC16 is actually the consequence of its inhibitory effect on viral RNA splicing, we examined various steps of the viral cycle in cells treated with the drug and submitted to one-round infection. For this purpose, we both NL4.3 (unpublished data) and Ada-M R5 strains (Figure 3B) is very efficiently blocked in these primary cells following treatment with 1 lM of IDC16. Meanwhile, cell viability was not affected by the drug throughout the assay as shown in Figure 3C. To generalize the effect of IDC16 on HIV-1 replication in other primary cells, the same protocol was repeated using infected macrophages, which act as viral reservoirs. Cells were treated with 0.1 lM, 0.5 lM, or 1 lM of IDC16 and p24 antigen levels were monitored both in culture supernatant (Figure 4A) and in cell lysates (Figure 4B). Again, IDC16 blocked virus replication efficiently and in a dose-dependent manner, reaching inhibition levels up to 90% in primary macrophages at 1 lM. However, cell viability was not decreased under IDC16 treatment (Figure 4C). It is important to note that macrophages survival was rather increased in the presence of IDC16 at day 7 and day 14 post-infection (Figure 4C), suggesting that IDC16 may protect these cells from deleterious effects induced by viral infection. The previous experiments were all performed with primary PLoS Pathogens | 1533 October 2007 | Volume 3 | Issue 10 | e159 Splicing Inhibitor for HIV-1 Therapy Figure 4. Inhibition of HIV-1 Production in Macrophage-Infected Cells by IDC16 Macrophages were infected with 100 TCID50 of Ada-M for 18 h in the absence or presence of 0.1 lM, 0.5 lM, or 1 lM concentrations of IDC16 and then washed intensively with PBS. (A and B) The culture medium and cells were collected at days 4, 7, and 14, and extracellular (A) or intracellular (B) viral production was measured by the quantification of viral capsid protein p24 using ELISA. (C) Concurrently, cell viability was measured by trypan blue exclusion and indicated as percentage as compared with untreated cells. (D) Activated PBMCs from healthy donors were infected with two HIV-1 clinical isolates (GAR and CLO) in the absence (Ctl) or presence of 1 lM of IDC16 (1DC16). Supernatants were harvested after 14 d of infection days, and viral capsid protein p24 antigen was quantitated enfocar la importancia de la producción mediática de los niños en su descubrimiento del mundo, sobre todo utilizando el periódico escolar y la imprenta. Asimismo las asociaciones de profesores trabajaron en esta línea e incluso la enseñanza católica se comprometió desde los años sesenta realizando trabajos originales en el marco de la corriente del Lenguaje Total. Páginas 43-48 45 Comunicar, 28, 2007 En el ámbito de los medios, también desde el principio del siglo XX hay ciertas corrientes de conexión. Pero es a lo largo de los años sesenta cuando se constituyeron asociaciones de periodistas apasionados por sus funciones de mediadores, que fomentaron la importancia ciudadana de los medios como algo cercano a los jóvenes, a los profesores y a las familias. Así se crearon la APIJ (Asociación de Prensa Información para la Juventud), la ARPEJ (Asociación Regional de Prensa y Enseñanza para la Juventud), el CIPE (Comité Interprofesional para la Prensa en la Escuela) o la APE (Asociación de Prensa y Enseñanza), todas ellas para la prensa escrita Estas asociaciones fueron precedidas por movimientos surgidos en mayo de 1968, como el CREPAC que, utilizando películas realizadas por periodistas conocidos, aclaraba temas que habían sido manipulados por una televisión demasiado próxima al poder político y realizaba encuentros con grupos de telespectadores. cipio del siglo XX, y nos han legado textos fundadores muy preciados, importantes trabajos de campo y muchos logros educativos y pedagógicos. La educación en medios ha tenido carácter de oficialidad de múltiples maneras, aunque nunca como una enseñanza global. Así la campaña «Operación Joven Telespectador Activo» (JTA), lanzada al final de los años setenta y financiada de manera interministerial para hacer reflexionar sobre las prácticas televisuales de los jóvenes, la creación del CLEMI (Centro de Educación y Medios de Comunicación) en el seno del Ministerio de Educación Nacional en 1983, la creación de la optativa «Cine-audiovisual» en los bachilleratos de humanidades de los institutos en 1984 (primer bachillerato en 1989) y múltiples referencias a la educación de la imagen, de la prensa, de Internet. La forma más visible y rápida de evaluar el lugar de la educación en medios es valorar el lugar que se le ha reservado en los libros de texto del sistema educa- 2. Construir la educación en los medios sin nombrarla El lugar que ocupa la edu- La denominación «educación en medios», que debería cación en los medios es muy ambiguo, aunque las cosas están cambiando recientemente. entenderse como un concepto integrador que reagrupase todos los medios presentes y futuros, es a menudo percibida En principio, en Francia, co- por los «tradicionalistas de la cultura» como una tendencia mo en muchos otros países, la educación en los medios no es hacia la masificación y la pérdida de la calidad. una disciplina escolar a tiempo completo, sino que se ha ido conformado progresivamente a través de experiencias y reflexiones teóricas que han tivo en Francia. Una inmersión sistemática nos permi- permitido implantar interesantes actividades de carác- te constatar que los textos oficiales acogen numerosos ter puntual. Se ha ganado poco a poco el reconoci- ejemplos, citas, sin delimitarla con precisión. miento de la institución educativa y la comunidad es- colar. Podemos decir que ha conquistado un «lugar», 3. ¿Por qué la escuela ha necesitado casi un siglo en el ámbito de la enseñanza transversal entre las dis- para oficilializar lo que cotidianamente se hacía en ciplinas existentes. ella? Sin embargo, la escuela no está sola en esta aspi- Primero, porque las prácticas de educación en me- ración, porque el trabajo en medios es valorado igual- dios han existido antes de ser nombradas así. Recor- mente por el Ministerio de Cultura (campañas de foto- demos que no fue hasta 1973 cuando aparece este grafía, la llamada «Operación Escuelas», presencia de término y que su definición se debe a los expertos del colegios e institutos en el cine ), así como el Minis- Consejo Internacional del Cine y de la Televisión, que terio de la Juventud y Deportes que ha emprendido en el seno de la UNESCO, definen de esta forma: numerosas iniciativas. «Por educación en medios conviene entender el estu- Así, esta presencia de la educación en los medios dio, la enseñanza, el aprendizaje de los medios moder- no ha sido oficial. ¡La educación de los medios no apa- nos de comunicación y de expresión que forman parte rece oficialmente como tal en los textos de la escuela de un dominio específico y autónomo de conocimien- francesa hasta 2006! tos en la teoría y la práctica pedagógicas, a diferencia Este hecho no nos puede dejar de sorprender ya de su utilización como auxiliar para la enseñanza y el que las experiencias se han multiplicado desde el prin- aprendizaje en otros dominios de conocimientos tales Páginas 43-48 46 Comunicar, 28, 2007 como los de matemáticas, ciencias y geografía». A pe- mente en todas las asignaturas. Incluso los nuevos cu- sar de que esta definición ha servido para otorgarle un rrículos de materias científicas en 2006 para los alum- reconocimiento real, los debates sobre lo que abarca y nos de 11 a 18 años hacen referencia a la necesidad no, no están totalmente extinguidos. de trabajar sobre la información científica y técnica y En segundo lugar, porque si bien a la escuela fran- el uso de las imágenes que nacen de ella. cesa le gusta la innovación, después duda mucho en Desde junio de 2006, aparece oficialmente el tér- reflejar y sancionar estas prácticas innovadoras en sus mino «educación en medios» al publicar el Ministerio textos oficiales. Nos encontramos con una tradición de Educación los nuevos contenidos mínimos y las sólidamente fundada sobre una transmisión de conoci- competencias que deben adquirir los jóvenes al salir mientos muy estructurados, organizados en disciplinas del sistema educativo. escolares que se dedican la mayor parte a transmitir Este documento pretende averiguar cuáles son los conocimientos teóricos. La pedagogía es a menudo se- conocimientos y las competencias indispensables que cundaria, aunque los profesores disfrutan de una ver- deben dominar para terminar con éxito su escolaridad, dadera libertad pedagógica en sus clases. El trabajo seguir su formación y construir su futuro personal y crítico sobre los medios que estaba aún en elaboración profesional. Siete competencias diferentes han sido te- necesitaba este empuje para hacerse oficial. nidas en cuenta y en cada una de ellas, el trabajo con Aunque el trabajo de educación en los medios no los medios es reconocido frecuentemente. Para citar esté reconocido como disciplina, no está ausente de un ejemplo, la competencia sobre el dominio de la len- gua francesa definen las capa- cidades para expresarse oral- La metodología elaborada en el marco de la educación en mente que pueden adquirirse con la utilización de la radio e, medios parece incluso permitir la inclinación de la sociedad incluso, se propone fomentar de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir el interés por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. Ruth Rousing, Hanne Willumsen, Carsten Nielsen and Flemming Jessen are thanked for excellent technical help. The Danish HIV-Cohort is thanked for providing us HIV-related data. 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