Molecular characterization of the contribution of autophagy to antigen presentation using quantitative proteomics

L’autophagie est une voie hautement conservée de dégradation lysosomale des constituants cellulaires qui est essentiel à l’homéostasie cellulaire et contribue à l’apprêtement et à la présentation des antigènes. Les rôles relativement récents de l'autophagie dans l'immunité innée et acquise sous-tendent de nouveaux paradigmes immunologiques pouvant faciliter le développement de nouvelles thérapies où la dérégulation de l’autophagie est associée à des maladies auto-immunes. Cependant, l'étude in vivo de la réponse autophagique est difficile en raison du nombre limité de méthodes d'analyse pouvant fournir une définition dynamique des protéines clés impliquées dans cette voie. En conséquence, nous avons développé un programme de recherche en protéomique intégrée afin d’identifier et de quantifier les proteines associées à l'autophagie et de déterminer les mécanismes moléculaires régissant les fonctions de l’autophagosome dans la présentation antigénique en utilisant une approche de biologie des systèmes. Pour étudier comment l'autophagie et la présentation antigénique sont activement régulés dans les macrophages, nous avons d'abord procédé à une étude protéomique à grande échelle sous différentes conditions connues pour stimuler l'autophagie, tels l’activation par les cytokines et l’infection virale. La cytokine tumor necrosis factor-alpha (TNF-alpha) est l'une des principales cytokines pro-inflammatoires qui intervient dans les réactions locales et systémiques afin de développer une réponse immune adaptative. La protéomique quantitative d'extraits membranaires de macrophages contrôles et stimulés avec le TNF-alpha a révélé que l'activation des macrophages a entrainé la dégradation de protéines mitochondriales et des changements d’abondance de plusieurs protéines impliquées dans le trafic vésiculaire et la réponse immunitaire. Nous avons constaté que la dégradation des protéines mitochondriales était sous le contrôle de la voie ATG5, et était spécifique au TNF-alpha. En outre, l’utilisation d’un nouveau système de présentation antigènique, nous a permi de constater que l'induction de la mitophagie par le TNF-alpha a entrainée l’apprêtement et la présentation d’antigènes mitochondriaux par des molécules du CMH de classe I, contribuant ainsi la variation du répertoire immunopeptidomique à la surface cellulaire. Ces résultats mettent en évidence un rôle insoupçonné du TNF-alpha dans la mitophagie et permet une meilleure compréhension des mécanismes responsables de la présentation d’auto-antigènes par les molécules du CMH de classe I. Une interaction complexe existe également entre infection virale et l'autophagie. Récemment, notre laboratoire a fourni une première preuve suggérant que la macroautophagie peut contribuer à la présentation de protéines virales par les molécules du CMH de classe I lors de l’infection virale par l'herpès simplex virus de type 1 (HSV-1). Le virus HSV1 fait parti des virus humains les plus complexes et les plus répandues. Bien que la composition des particules virales a été étudiée précédemment, on connaît moins bien l'expression de l'ensemble du protéome viral lors de l’infection des cellules hôtes. Afin de caractériser les changements dynamiques de l’expression des protéines virales lors de l’infection, nous avons analysé par LC-MS/MS le protéome du HSV1 dans les macrophages infectés. Ces analyses nous ont permis d’identifier un total de 67 protéines virales structurales et non structurales (82% du protéome HSV1) en utilisant le spectromètre de masse LTQ-Orbitrap. Nous avons également identifié 90 nouveaux sites de phosphorylation et de dix nouveaux sites d’ubiquitylation sur différentes protéines virales. Suite à l’ubiquitylation, les protéines virales peuvent se localiser au noyau ou participer à des événements de fusion avec la membrane nucléaire, suggérant ainsi que cette modification pourrait influer le trafic vésiculaire des protéines virales. Le traitement avec des inhibiteurs de la réplication de l'ADN induit des changements sur l'abondance et la modification des protéines virales, mettant en évidence l'interdépendance des protéines virales au cours du cycle de vie du virus. Compte tenu de l'importance de la dynamique d'expression, de l’ubiquitylation et la phosphorylation sur la fonction des proteines virales, ces résultats ouvriront la voie vers de nouvelles études sur la biologie des virus de l'herpès. Fait intéressant, l'infection HSV1 dans les macrophages déclenche une nouvelle forme d'autophagie qui diffère remarquablement de la macroautophagie. Ce processus, appelé autophagie associée à l’enveloppe nucléaire (nuclear envelope derived autophagy, NEDA), conduit à la formation de vésicules membranaires contenant 4 couches lipidiques provenant de l'enveloppe nucléaire où on retrouve une grande proportion de certaines protéines virales, telle la glycoprotéine B. Les mécanismes régissant NEDA et leur importance lors de l’infection virale sont encore méconnus. En utilisant un essai de présentation antigénique, nous avons pu montrer que la voie NEDA est indépendante d’ATG5 et participe à l’apprêtement et la présentation d’antigènes viraux par le CMH de classe I. Pour comprendre l'implication de NEDA dans la présentation des antigènes, il est essentiel de caractériser le protéome des autophagosomes isolés à partir de macrophages infectés par HSV1. Aussi, nous avons développé une nouvelle approche de fractionnement basé sur l’isolation de lysosomes chargés de billes de latex, nous permettant ainsi d’obtenir des extraits cellulaires enrichis en autophagosomes. Le transfert des antigènes HSV1 dans les autophagosomes a été determine par protéomique quantitative. Les protéines provenant de l’enveloppe nucléaire ont été préférentiellement transférées dans les autophagosome lors de l'infection des macrophages par le HSV1. Les analyses protéomiques d’autophagosomes impliquant NEDA ou la macroautophagie ont permis de decouvrir des mécanismes jouant un rôle clé dans l’immunodominance de la glycoprotéine B lors de l'infection HSV1. Ces analyses ont également révélées que diverses voies autophagiques peuvent être induites pour favoriser la capture sélective de protéines virales, façonnant de façon dynamique la nature de la réponse immunitaire lors d'une infection. En conclusion, l'application des méthodes de protéomique quantitative a joué un rôle clé dans l'identification et la quantification des protéines ayant des rôles importants dans la régulation de l'autophagie chez les macrophages, et nous a permis d'identifier les changements qui se produisent lors de la formation des autophagosomes lors de maladies inflammatoires ou d’infection virale. En outre, notre approche de biologie des systèmes, qui combine la protéomique quantitative basée sur la spectrométrie de masse avec des essais fonctionnels tels la présentation antigénique, nous a permis d’acquérir de nouvelles connaissances sur les mécanismes moléculaires régissant les fonctions de l'autophagie lors de la présentation antigénique. Une meilleure compréhension de ces mécanismes permettra de réduire les effets nuisibles de l'immunodominance suite à l'infection virale ou lors du développement du cancer en mettant en place une réponse immunitaire appropriée.

Le CpG et le poly(I:C) agissent en synergie avec le trastuzumab contre le cancer du sein HER2+

Chez la souris, la thérapie anti-HER2 est dépendante de la présence de cellules T CD8+IFN-γ+ et des réponses IFN de type I. Ces IFN sont induits par les TLRs suite à la reconnaissance de signaux de danger, appelés PAMPs et DAMPs. Les TLR-3 et TLR-9 sont tous deux de bons inducteurs d’IFN de type I et sont également capable d’agir en synergie afin d’augmenter les niveaux d’IFN-γ, de TNF-α et d’IL-12. Notre hypothèse fut que la stimulation de ces deux TLRs mènerait à l’amélioration de l’activité anti-tumorale du trastuzumab via le recrutement et l’activation des cellules immunitaires. Nos buts furent de confirmer le potentiel thérapeutique de la combinaison de l’anticorps anti-HER2, de l’agoniste de TLR-3, le poly(I:C), et de l’agoniste de TLR-9, le CpG ODN. Des études in vivo et in vitro nous ont permis de découvrir une synergie entre ces agents qui résulte en une cytotoxicité ciblée plus efficace. De plus, cette thérapie s’avéra efficace chez des modèles CD8-dépendants et CD8-indépendents. Les souris purent rejeter leur tumeur et demeurer sains plusieurs semaines après l’arrêt des injections. Ces souris étaient également protégées lors d’un challenge, soulignant ainsi la présence d’une immunité mémoire. Nous avons aussi découvert que l’administration combine de trastuzumab des deux agonistes de TLRs mène à des réponses systémiques. Des études de déplétion confirmèrent que les cellules T CD8+ sont cruciales pour la protection à long terme des animaux, mais que les pDC sont moins impliquées que ce que l’on pourrait croire. Leur absence n’a que modestement affecté les effets de notre thérapie. À l’opposé, les cellules NK sont d’importants médiateurs des effets thérapeutiques. Des expériences d’ADCC ont révélé que le CpG ODN et poly(I:C) ont tous deux la capacité d’améliorer les fonctions des cellules NK, mais que la stimulation simultanée des TLR-3 et TLR-9 permet de maximiser les effets bénéfiques du trastuzumab. De la même manière, l’addition de CpG ODN et de poly(I:C) aux anticorps anti-HER2 a permis d’augmenter les réponses pro-inflammatoires, plus spécifiquement l’IFN-γ, le TNF-α, l’IP-10 et l’IL-12.

Supervivencia cáncer específica posterior a nefrectomía radical frente a nefrectomía parcial en carcinoma renal t2: revisión sistemática

Introducción: El tratamiento estándar para los tumores renales localizados es la nefrectomía radical, sin embargo debido a la variación el tamaño del tumor renal en el momento del diagnóstico, se ha reemplazado en algunos casos por la nefrectomía parcial. Objetivo: Este estudio busca comparar el resultado oncológico de la nefrectomía parcial en términos de supervivencia cáncer específica, respecto a la nefrectomía radical, en pacientes mayores de 50 años con carcinoma renal estadio II (T2N0M0) Métodos: Se realizó una revisión sistemática de la literatura, con inclusión de estudios de casos y controles, cohortes y experimentos clínicos aleatorizados incluidos en las bases de datos de MEDLINE , EMBASE y CENTRAL Resultados: La búsqueda inicial emitió un total de 101 resultados, 11 artículos fueron preseleccionados y sólo un artículo cumplió con los criterios de selección; éste se clasificó como nivel de evidencia II. Conclusión: No fue posible concluir su equivalencia oncológica de la nefrectomía radical con la nefrectomía parcial, dado que no hay diseños de estudios que permitan llegar a esta conclusión.

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10-8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10-7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10-7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10-4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Nanotecnologia e a sua aplicação à terapêutica oncológica

A nanotecnologia, e a sua aplicação na área da biomedicina, em particular na terapêutica e diagnóstico oncológico, tem sido alvo de um desenvolvimento exponencial, com impacto profundo no que respeita a cuidados de saúde. O cancro é uma das doenças que afecta mais pessoas a nível mundial, com elevados índices de mortalidade, níveis de sofrimento físico e emocional e, encargos para o doente, família e sociedade. É uma doença complexa, e a prática clínica convencional constitui um paradigma, na medida em que se revela insuficiente e extremamente agressiva, expondo o doente a medicamentos citotóxicos, não específicos, com elevada toxicidade sistémica e efeitos adversos. A Nanomedicina, e a exploração das propriedades únicas das nanopartículas, apresenta a potencialidade de melhorar a capacidade de detecção e diagnóstico do cancro e, aumentar a especificidade e efectividade no tratamento das células tumorais. No entanto, o recurso a nanotecnologias continua a ser alvo de alguma controvérsia e cepticismo por parte de alguns elementos das comunidades científicas e académica. Com nanoprodutos já aprovados e utilizados em prática clínica e muitos outros em desenvolvimento e investigação em ensaios clínicos, a realização deste trabalho tem como objectivo compreender os conceitos de nanotecnologia e Nanomedicina, perceber o estado da arte, ponderando as vantagens e desvantagens da abordagem “nano” e a sua real aplicação à terapêutica oncológica.

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultraperformance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats.Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0±10.4%) andheart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influencedbymicrobial activitiesormodulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Prebiotic effects: metabolic and health benefits

The different compartments of the gastrointestinal tract are inhabited by populations of micro-organisms. By far the most important predominant populations are in the colon where a true symbiosis with the host exists that is a key for well-being and health. For such a microbiota, 'normobiosis' characterises a composition of the gut 'ecosystem' in which micro-organisms with potential health benefits predominate in number over potentially harmful ones, in contrast to 'dysbiosis', in which one or a few potentially harmful micro-organisms are dominant, thus creating a disease-prone situation. The present document has been written by a group of both academic and industry experts (in the ILSI Europe Prebiotic Expert Group and Prebiotic Task Force, respectively). It does not aim to propose a new definition of a prebiotic nor to identify which food products are classified as prebiotic but rather to validate and expand the original idea of the prebiotic concept (that can be translated in 'prebiotic effects'), defined as: 'The selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host.' Thanks to the methodological and fundamental research of microbiologists, immense progress has very recently been made in our understanding of the gut microbiota. A large number of human intervention studies have been performed that have demonstrated that dietary consumption of certain food products can result in statistically significant changes in the composition of the gut microbiota in line with the prebiotic concept. Thus the prebiotic effect is now a well-established scientific fact. The more data are accumulating, the more it will be recognised that such changes in the microbiota's composition, especially increase in bifidobacteria, can be regarded as a marker of intestinal health. The review is divided in chapters that cover the major areas of nutrition research where a prebiotic effect has tentatively been investigated for potential health benefits. The prebiotic effect has been shown to associate with modulation of biomarkers and activity(ies) of the immune system. Confirming the studies in adults, it has been demonstrated that, in infant nutrition, the prebiotic effect includes a significant change of gut microbiota composition, especially an increase of faecal concentrations of bifidobacteria. This concomitantly improves stool quality (pH, SCFA, frequency and consistency), reduces the risk of gastroenteritis and infections, improves general well-being and reduces the incidence of allergic symptoms such as atopic eczema. Changes in the gut microbiota composition are classically considered as one of the many factors involved in the pathogenesis of either inflammatory bowel disease or irritable bowel syndrome. The use of particular food products with a prebiotic effect has thus been tested in clinical trials with the objective to improve the clinical activity and well-being of patients with such disorders. Promising beneficial effects have been demonstrated in some preliminary studies, including changes in gut microbiota composition (especially increase in bifidobacteria concentration). Often associated with toxic load and/or miscellaneous risk factors, colon cancer is another pathology for which a possible role of gut microbiota composition has been hypothesised. Numerous experimental studies have reported reduction in incidence of tumours and cancers after feeding specific food products with a prebiotic effect. Some of these studies (including one human trial) have also reported that, in such conditions, gut microbiota composition was modified (especially due to increased concentration of bifidobacteria). Dietary intake of particular food products with a prebiotic effect has been shown, especially in adolescents, but also tentatively in postmenopausal women, to increase Ca absorption as well as bone Ca accretion and bone mineral density. Recent data, both from experimental models and from human studies, support the beneficial effects of particular food products with prebiotic properties on energy homaeostasis, satiety regulation and body weight gain. Together, with data in obese animals and patients, these studies support the hypothesis that gut microbiota composition (especially the number of bifidobacteria) may contribute to modulate metabolic processes associated with syndrome X, especially obesity and diabetes type 2. It is plausible, even though not exclusive, that these effects are linked to the microbiota-induced changes and it is feasible to conclude that their mechanisms fit into the prebiotic effect. However, the role of such changes in these health benefits remains to be definitively proven. As a result of the research activity that followed the publication of the prebiotic concept 15 years ago, it has become clear that products that cause a selective modification in the gut microbiota's composition and/or activity(ies) and thus strengthens normobiosis could either induce beneficial physiological effects in the colon and also in extra-intestinal compartments or contribute towards reducing the risk of dysbiosis and associated intestinal and systemic pathologies.

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKQ, which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

Effects of consumption of probiotics and prebiotics on serum lipid levels in humans

The objective of this article is to review existing studies concerning the effects of probiotics and prebiotics on serum cholesterol concentrations, with particular attention on the possible mechanisms of their action. Although not without exception, results from animal and human studies suggest a moderate cholesterol-lowering action of dairy products fermented with appropriate strain(s) of lactic acid bacteria and bifidobacteria. Mechanistically, probiotic bacteria ferment food-derived indigestible carbohydrates to produce short-chain fatty acids in the gut, which can then cause a decrease in the systemic levels of blood lipids by inhibiting hepatic cholesterol synthesis and/or redistributing cholesterol from plasma to the liver. Furthermore, some bacteria may interfere with cholesterol absorption from the gut by deconjugating bile salts and therefore affecting the metabolism of cholesterol, or by directly assimilating cholesterol. For prebiotic substances, the majority of studies have been done with the fructooligosaccharides inulin and oligofructose, and although convincing lipid-lowering effects have been observed in animals, high dose levels had to be used. Reports in humans are few in number. In studies conducted in normal-lipidemic subjects, two reported no effect of inulin or oligofructose on serum lipids, whereas two others reported a significant reduction in serum triglycerides (19 and 27%, respectively) with more modest changes in serum total and LDL cholesterol. At present, data suggest that in hyperlipidemic subjects, any effects that do occur result primarily in reductions in cholesterol, whereas in normal lipidemic subjects, effects on serum triglycerides are the dominant feature.

The cannabinoid CB1 receptor antagonist SR141716 blocks the orexigenic effects of intrahypothalamic ghrelin

The paraventricular nucleus (PVN) of the hypothalamus plays a key role in the control of appetite and energy balance. Both ghrelin and cannabinoid receptor agonists increase food intake when administered into this nucleus: this study investigated possible interactions between the two systems in relation to eating. The orexigenic effect of ghrelin (100 pmol) when infused in to the PVN was reversed by a small, systemic dose of the CB1 cannabinoid receptor antagonist SR141716 (1 mg kg(-1)). This is the first demonstration of a functional relationship between brain ghrelin and endocannabinoid systems, and, although it needs to be further investigated, the effect of ghrelin on food intake when injected into the PVN seems to be mediated by stimulation of cannabinoid release.

Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli.

Normative, systemic and procedural aspects: a review of indicator‐based sustainability assessments in agriculture

Several methods for assessing the sustainability of agricultural systems have been developed. These methods do not fully: (i) take into account the multi‐functionality of agriculture; (ii) include multidimensionality; (iii) utilize and implement the assessment knowledge; and (iv) identify conflicting goals and trade‐offs. This paper reviews seven recently developed multidisciplinary indicator‐based assessment methods with respect to their contribution to these shortcomings. All approaches include (1) normative aspects such as goal setting, (2) systemic aspects such as a specification of scale of analysis, (3) a reproducible structure of the approach. The approaches can be categorized into three typologies. The top‐down farm assessments focus on field or farm assessment. They have a clear procedure for measuring the indicators and assessing the sustainability of the system, which allows for benchmarking across farms. The degree of participation is low, potentially affecting the implementation of the results negatively. The top‐down regional assessment assesses the on‐farm and the regional effects. They include some participation to increase acceptance of the results. However, they miss the analysis of potential trade‐offs. The bottom‐up, integrated participatory or transdisciplinary approaches focus on a regional scale. Stakeholders are included throughout the whole process assuring the acceptance of the results and increasing the probability of implementation of developed measures. As they include the interaction between the indicators in their system representation, they allow for performing a trade‐off analysis. The bottom‐up, integrated participatory or transdisciplinary approaches seem to better overcome the four shortcomings mentioned above.

Effects of fermentation products of pro- and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon

Evidence from in vivo and in vitro studies suggests that the consumption of pro- and prebiotics may inhibit colon carcinogenesis; however, the mechanisms involved have, thus far, proved elusive. There are some indications from animal studies that the effects are being exerted during the promotion stage of carcinogenesis. One feature of the promotion stage of colorectal cancer is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. We have used the Caco-2 human adenocarcinoma cell line as a model for the intestinal epithelia. Trans-epithelial electrical resistance measurements indicate Caco-2 monolayer integrity, and we recorded changes to this integrity following exposure to the fermentation products of selected probiotics and prebiotics, in the form of nondigestible oligosaccharides (NDOs). Our results indicate that NDOs themselves exert varying, but generally minor, effects upon the strength of the tight junctions, whereas the fermentation products of probiotics and NDOs tend to raise tight junction integrity above that of the controls. This effect was bacterial species and oligosaccharide specific. Bifidobacterium Bb 12 was particularly effective, as were the fermentation products of Raftiline and Raftilose. We further investigated the ability of Raftilose fermentations to protect against the negative effects of deoxycholic acid (DCA) upon tight junction integrity. We found protection to be species dependent and dependent upon the presence of the fermentation products in the media at the same time as or after exposure to the DCA. Results suggest that the Raftilose fermentation products may prevent disruption of the intestinal epithelial barrier function during damage by tumor promoters.

Adeno-associated virus-8-Mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle

Myostatin is a member of the transformating growth factor-_ (TGF-_) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a ‘double muscling’ phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects.