Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

From diabetes to heart disease : studies on dyslipidemia-induced B-cell dysfunction, immunomodulation in heart transplantation, and cardiac stem cell therapy

Diabetes is a growing epidemic with devastating human, social and economic impact. It is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent LDL and HDL particles in insulin-secreting β-cells. Purified human VLDL and LDL particles reduced insulin mRNA levels and β-cell proliferation, and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of β-cells involved caspase-3 cleavage and reduction in levels of the c-Jun N-terminal (JNK) Interacting Protein-1 (IB1/JIP-1). In contrast, the pro-apoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of JNK. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of the protein kinase Akt/PKB. Heart disease is a major cause of morbidity and mortality among patients with diabetes. When heart failure is refractory to medical therapy and cannot be improved by electrical resynchronization, percutaneous angioplasty or coronary graft bypass surgery, heart transplantation remains a "last resort" therapy. Nevertheless, it is limited by the side effects of immunosuppressive drugs and chronic rejection. Localized expression of immunomodulatory genes in the donor organ can create a state of immune privilege within the graft, and was performed in rodent hearts by infecting cells with an adenovirus encoding indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophane. Other strategies are based on genetic manipulation of dendritic cells (DCs) with immunosuppressive genes and in vitro exposure of DCs to agents that prevent their maturation by inflammatory cytokines. Finally, we used 5-bromo-2'-deoxyuridine, which is incorporated into DNA and diluted with cell division, to identify long-term label retaining cells in the adult rodent heart. The majority of these cells were positive for the stem cell antigen-1 (Sca-1) and negative for the endothelial precursor marker CD31. They formed cardiospheres in vitro and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. Taken together, these data provide evidence of slow-cycling stem cells in the rodent heart. Chronic shortage of donor organs opens the way to cardiac stem cell therapy in humans, although the long way from animal experimentation to routine therapy in patients may still take several years. - Du diabète de type 2 à la maladie coronarienne : trois études sur les dysfonctions de la cellule sécrétrice d'insuline induites par les dyslipidémies, l'immunomodulation dans la transplantation cardiaque, et la thérapie par des cellules souches myocardiques. Le diabète de type 2 a pris les dimensions d'une épidémie, avec des conséquences sociales et économiques dont nous n'avons pas encore pris toute la mesure. La maladie s'accompagne souvent d'une dyslipidémie caractérisée par une hypertriglycéridémie, des taux abaissés de cholestérol HDL, et des concentrations de cholestérol LDL à la limite supérieure de ce qui est considéré comme acceptable. L'hypothèse à la base de cette étude est qu'une modification des taux plasmatiques de lipoprotéines pourrait avoir une influence directe sur la cellule β sécrétrice d'insuline en modifiant sa fonction, sa durée de vie et son taux de régénération. Dans un premier temps, nous avons mis en évidence, sur la cellule β, la présence de plusieurs récepteurs impliqués dans la captation des lipoprotéines. Nous avons confirmé la fonctionnalité de ces récepteurs en suivant l'internalisation de LDL et de HDL marqués. En présence de VLDL ou de LDL humains, nous avons observé une diminution de la transcription du gène de l'insuline, une prolifération cellulaire réduite, et une augmentation de l'apoptose, toutes fonctions de la dose et du temps d'exposition. L'apoptose induite par les VLDL passe par une activation de la caspase-3 et une réduction du taux de la protéine IB1/JIP-1 (Islet Brain1/JNK Interacting Protein 1), dont une mutation est associée à une forme monogénique de diabète de type 2. Par opposition, les HDL, ainsi que des peptides inhibiteurs de JNK, sont capables de contrer la cascade pro-apoptotique déclenchée, respectivement, par les LDL et les VLDL. Ces effets protecteurs comprennent l'inhibition du clivage de la caspase-3 et l'activation de la protéine kinase Akt/PKB. En conclusion, les lipoprotéines sont des éléments clés de la survie de la cellule β, et pourraient contribuer au dysfonctionnement observé dans le pancréas endocrine au cours du développement du diabète. La maladie cardiaque, et plus particulièrement la maladie coronarienne, est une cause majeure de morbidité et de mortalité chez les patients atteints de diabète. Plusieurs stratégies sont utilisées quotidiennement pour pallier les atteintes cardiaques: traitements médicamenteux, électromécaniques par resynchronisation électrique, ou communément appelés « interventionnels » lorsqu'ils font appel à l'angioplastie percutanée. La revascularisation du myocarde par des pontages coronariens donne également de très bons résultats dans certaines situations. Il existe toutefois des cas où plus aucune de ces approches n'est suffisante. La transplantation cardiaque est alors la thérapie de choix pour un nombre restreint de patients. La thérapie génique, en permettant l'expression locale de gènes immunomodulateurs dans l'organe greffé, permet de diminuer les réactions de rejet inhérentes à toute transplantation (à l'exception de celles réalisées entre deux jumeaux homozygotes). Nous avons appliqué chez des rongeurs cette stratégie en infectant le coeur greffé avec un adénovirus codant pour l'enzyme indoleamine 2,3-dioxygénase (IDO), une enzyme clé dans le catabolisme du tryptophane. Nous avons procédé de manière identique in vitro en surexprimant IDO dans les cellules dendritiques, dont le rôle est de présenter les antigènes aux lymphocytes Τ du receveur. Des expériences similaires ont été réalisées en traitant les cellules dendritiques avec des substances capables de prévenir, en partie du moins, leur maturation par des agents pro-inflammatoires. Finalement, nous avons exploré une stratégie utilisée couramment en hématologie, mais qui n'en est encore qu'à ses débuts au niveau cardiaque : la thérapie par des cellules souches. En traitant des rongeurs avec un marqueur qui s'incorpore dans l'ADN nucléaire, le 5-bromo- 2'-deoxyuridine, nous avons identifié une population cellulaire se divisant rarement, positive en grande partie pour l'antigène embryonnaire Sca-1 et négative pour le marqueur endothélial CD31. En culture, ces cellules forment des cardiosphères et sont capables de se différencier dans les principaux types tissulaires mésenchymateux. Dans un milieu de differentiation adéquat, ces cellules expriment des gènes cardiomyocytaires. En résumé, ces données confirment la présence chez le rongeur d'une population résidente de précurseurs myocardiques. En addenda, on trouvera deux publications relatives à la cellule β productrice d'insuline. Le premier article démontre le rôle essentiel joué par la complexine dans l'insulino-sécrétion, tandis que le second souligne l'importance de la protéine IB1/JIP-1 dans la protection contre l'apoptose de la cellule β induite par certaines cytokines.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Vitellogenin Underwent Subfunctionalization to Acquire Caste and Behavioral Specific Expression in the Harvester Ant Pogonomyrmex barbatus.

The reproductive ground plan hypothesis (RGPH) proposes that the physiological pathways regulating reproduction were co-opted to regulate worker division of labor. Support for this hypothesis in honeybees is provided by studies demonstrating that the reproductive potential of workers, assessed by the levels of vitellogenin (Vg), is linked to task performance. Interestingly, contrary to honeybees that have a single Vg ortholog and potentially fertile nurses, the genome of the harvester ant Pogonomyrmex barbatus harbors two Vg genes (Pb_Vg1 and Pb_Vg2) and nurses produce infertile trophic eggs. P. barbatus, thus, provides a unique model to investigate whether Vg duplication in ants was followed by subfunctionalization to acquire reproductive and non-reproductive functions and whether Vg reproductive function was co-opted to regulate behavior in sterile workers. To investigate these questions, we compared the expression patterns of P. barbatus Vg genes and analyzed the phylogenetic relationships and molecular evolution of Vg genes in ants. qRT-PCRs revealed that Pb_Vg1 is more highly expressed in queens compared to workers and in nurses compared to foragers. By contrast, the level of expression of Pb_Vg2 was higher in foragers than in nurses and queens. Phylogenetic analyses show that a first duplication of the ancestral Vg gene occurred after the divergence between the poneroid and formicoid clades and subsequent duplications occurred in the lineages leading to Solenopsis invicta, Linepithema humile and Acromyrmex echinatior. The initial duplication resulted in two Vg gene subfamilies preferentially expressed in queens and nurses (subfamily A) or in foraging workers (subfamily B). Finally, molecular evolution analyses show that the subfamily A experienced positive selection, while the subfamily B showed overall relaxation of purifying selection. Our results suggest that in P. barbatus the Vg gene underwent subfunctionalization after duplication to acquire caste- and behavior- specific expression associated with reproductive and non-reproductive functions, supporting the validity of the RGPH in ants.

Evolution and expression of vitellogenin genes.

A major event during the growth period of oocytes in nonmammalian animals is the accumulation of yolk. The genes coding for the yolk protein precursor, known as vitallogenin, are well characterized in a few vertebrate and invertebrate species. Studies on the evolution of these genes and on the regulatory mechanisms involved in their time, tissue- and hormone-specific expression are presented and discussed in this review.

Familial evaluation in catecholaminergic polymorphic ventricular tachycardia: disease penetrance and expression in cardiac ryanodine receptor mutation-carrying relatives.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene (Ryr2) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease penetrance, expression, genotype-phenotype correlations, and arrhythmic event rates in relatives carrying the Ryr2 mutation is limited. METHODS AND RESULTS: One-hundred sixteen relatives carrying the Ryr2 mutation from 15 families who were identified by cascade screening of the Ryr2 mutation causing CPVT in the proband were clinically characterized, including 61 relatives from 1 family. Fifty-four of 108 antiarrhythmic drug-free relatives (50%) had a CPVT phenotype at the first cardiological examination, including 27 (25%) with nonsustained ventricular tachycardia. Relatives carrying a Ryr2 mutation in the C-terminal channel-forming domain showed an increased odds of nonsustained ventricular tachycardia (odds ratio, 4.1; 95% CI, 1.5-11.5; P=0.007, compared with N-terminal domain) compared with N-terminal domain. Sinus bradycardia was observed in 19% of relatives, whereas other supraventricular dysrhythmias were present in 16%. Ninety-eight (most actively treated) relatives (84%) were followed up for a median of 4.7 years (range, 0.3-19.0 years). During follow-up, 2 asymptomatic relatives experienced exercise-induced syncope. One relative was not being treated, whereas the other was noncompliant. None of the 116 relatives died of CPVT during a 6.7-year follow-up (range, 1.4-20.9 years). CONCLUSIONS: Relatives carrying an Ryr2 mutation show a marked phenotypic diversity. The vast majority do not have signs of supraventricular disease manifestations. Mutation location may be associated with severity of the phenotype. The arrhythmic event rate during follow-up was low.

MicroRNA-122 modulates the rhythmic expression profile of the circadian deadenylase Nocturnin in mouse liver.

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3'-untranslated region (3'-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3'-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control.

Interferon-Induced Gene Expression Is a Stronger Predictor of Treatment Response Than IL28B Genotype in Patients With Hepatitis C.

BACKGROUND & AIMS: The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS: We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS: IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

Role of ATH5 in the specification of retinal ganglion cells and expression and cell compartmentalization of EFEMP1, a protein associated with Malattia Leventinese

ABSTRACT : The development of the retina is a very complex process, occurring through the progressive restriction of cell fates, from pluripotent cell populations to complex tissues and organs. In all vertebrate species analyzed so far, retinal differentiation starts with the generation of retinal ganglion cells (RGC)s. One of the documented key essential events in the specification of RGCs is the expression of ATHS, an atonal homolog encoding a bHLH transcription factor. Despite the putative role of master regulator of RGC differentiation, the mechanism of integrating its functions into a coherent program underlying the production of this subclass of retinal neurons has not yet been elucidated. By using chromatin immunoprecipitation combined with microarray (ChIP-on-chip) we have screened for ATH5 direct targets in the developing chick retina at two consecutive periods: E3.5 (stage HH22) and E6 (stage HH30), covering the stages of progenitor proliferation, neuroepithelium patterning, RGC specification, cell cycle exit and early neuronal differentiation. In parallel, complementary analysis with Affymetrix expression microarrays was conducted. We compared RGCs versus retina to see if the targets correspond to genes preferentially expressed in RGCs. We also precociously overexpressed ATH5 in the retina of individual embryo, and contralateral retina vas used as a control. Our integrated approach allowed us to establish a compendium of ATH5-targets and enabled us to position ATH5 in the transcription network underlying neurogenesis in the retina. Malattia Leventinese (ML) is an autosomal, dominant retinal dystrophy characterized by extracellular, amorphous deposits known as drusen, between the retinal pigment epithelium (RPE) and Bruch's membrane. On the genetic level, it has been associated with a single missense mutation (R345W) in a widely expressed gene with unknown function called EFEMP1. We determined expression patterns of the EFEMP1 gene in normal and ML human retinas. Our data shown that the upregulation of EFEMP1 is not specific to ML eye, except for the region of the ciliary body. We also analyzed the cell compartmentalization of different versions of the protein (both wild type and mutant). Our studies indicate that both abnormal expression of the EFEMP1 gene and mutation and accumulation of EFEMP 1 protein (inside or outside the cells) might contribute to the ML pathology. Résumé : 1er partie : L'ontogenèse de la rétine est un processus complexe au cours duquel des cellules progénitrices sont engagée, par vagues successives, dans des lignées où elles vont d'abord être déterminées puis vont se différencier pour finalement construire un tissu rétinien composé de cinq classes de neurones (les photorécepteurs, les cellules horizontales, bipolaires, amacrines et ganglionnaires) et d'une seule de cellules gliales (les cellules de Muller). Chez tous les vertébrés, la neurogenèse rétinienne est d'abord marquée par la production des cellules ganglionnaires (RGCs). La production de cette classe de neurone est liée à l'expression du gène ATH5 qui est un homologue du gène atonal chez la Drosophile et qui code pour un facteur de transcription de la famille des protéines basic Helix-Loop-Helix (bHLH). Malgré le rôle central que joue ATH5 dans la production des RGCs, le mécanisme qui intègre la fonction de cette protéine dans le programme de détermination neuronale et ceci en relation avec le développement de la rétine n'est pas encore élucidé. Grâce à une technologie qui permet de combiner la sélection de fragments de chromatine liant ATH5 et la recherche de séquences grâce à des puces d'ADN non-codants (ChIP-on-chip), nous avons recherché des cibles potentielles de la protéine ATH5 dans la rétine en développement. Nous avons conduit cette recherche à deux stades de développement de manière à englober la phase de prolifération cellulaire, la détermination des RGCs, la sortie du cycle cellulaire ainsi que les premières étapes de la différentiation de ces neurones. Des expériences complémentaires nous ont permis de définir les patrons d'expression des gènes sélectionnés ainsi que l'activité promotrice des éléments de régulation identifiés lors de notre criblage. Ces approches expérimentales diverses et complémentaires nous ont permis de répertorier des gènes cibles de la protéine ATH5 et d'établir ainsi des liens fonctionnels entre des voies métaboliques dont nous ne soupçonnions pas jusqu'alors qu'elles puissent être associées à la production d'une classe de neurones centraux. 2ème partie : Malattia Leventinese (ML) est une maladie génétique qui engendre une dystrophie de la rétine. Elle se caractérise par l'accumulation de dépôt amorphe entre l'épithélium pigmentaire et la membrane de Bruch et connu sous le nom de drusen. Cette maladie est liée à une simple mutation non-sens (R345W) dans un gène dénommé EFEMP1 qui est exprimé dans de nombreux tissus mais dont la fonction reste mal définie. Une étude détaillée de l'expression de ce gène dans des rétines humaines a révélé une expression à un niveau élevé du gène EFEMP1 dans divers tissus de l'oeil ML mais également dans des yeux contrôles. Alors que l'accumulation d'ARN messager EFEMP1 dans les cellules de l'épithélium pigmentaire n'est pas spécifique à ML, l'expression de ce gène dans le corps cilié n'a été observée que dans l'oeil ML. Nous avons également comparé la sécrétion de la protéine sauvage avec celle porteuse de la mutation. En résumé, notre étude révèle que le niveau élevé d'expression du gène EFEMP1 ainsi que l'accumulation de la protéine dans certains compartiments cellulaires pourraient contribuer au développement de pathologies rétiniennes liées à ML.

Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

On the significance of CD8 alpha alpha expression for T cell memory.

Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8alphaalpha homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8alphaalpha is in the generation of memory CD8 T cells. The involvement of the high affinity CD8alphaalpha ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8alphaalpha in this process and call for additional research focus into these issues.

Pancreatic-specific expression of the glucose transporter type 2 gene: identification of cis-elements and islet-specific trans-acting factors.

A defect in glucose sensing of the pancreatic beta-cells has been observed in several animal models of type II diabetes and has been correlated with a reduced gene expression of the glucose transporter type 2 (Glut2). In a transgenic mouse model, expression of Glut2 antisense RNA in pancreatic beta-cells has recently been shown to be associated with an impaired glucose-induced insulin secretion and the development of diabetes. To identify factors that may be involved in the specific decrease of Glut2 in the beta-cells of the diabetic animal, an attempt was made to localize the cis-elements and trans-acting factors involved in the control of Glut2 expression in the endocrine pancreas. It was demonstrated by transient transfection studies that only 338 base pairs (bp) of the murine Glut2 proximal promoter are needed for reporter gene expression in pancreatic islet-derived cell lines, whereas no activity was detected in nonpancreatic cells. Three cis-elements, GTI, GTII, and GTIII, have been identified by DNAse I footprinting and gel retardation experiments within these 338 bp. GTI and GTIII bind distinct but ubiquitously expressed trans-acting factors. On the other hand, nuclear proteins specifically expressed in pancreatic cell lines interact with GTII, and their relative abundance correlates with endogenous Glut2 expression. These GTII-binding factors correspond to nuclear proteins of 180 and 90 kilodaltons as defined by Southwestern analysis. The 180-kilodalton factor is present in pancreatic beta-cell lines but not in an alpha-cell line. Mutation of the GTI or GTIII cis-elements decreases transcriptional activity directed by the 338-bp promoter, whereas mutation of GTII increases gene transcription. Thus negative and positive regulatory sequences are identified within the proximal 338 bp of the GLUT2 promoter and may participate in the islet-specific expression of the gene by binding beta-cell specific trans-acting factors.

Genome-wide analysis of cancer/testis gene expression.

Cancer/Testis (CT) genes, normally expressed in germ line cells but also activated in a wide range of cancer types, often encode antigens that are immunogenic in cancer patients, and present potential for use as biomarkers and targets for immunotherapy. Using multiple in silico gene expression analysis technologies, including twice the number of expressed sequence tags used in previous studies, we have performed a comprehensive genome-wide survey of expression for a set of 153 previously described CT genes in normal and cancer expression libraries. We find that although they are generally highly expressed in testis, these genes exhibit heterogeneous gene expression profiles, allowing their classification into testis-restricted (39), testis/brain-restricted (14), and a testis-selective (85) group of genes that show additional expression in somatic tissues. The chromosomal distribution of these genes confirmed the previously observed dominance of X chromosome location, with CT-X genes being significantly more testis-restricted than non-X CT. Applying this core classification in a genome-wide survey we identified >30 CT candidate genes; 3 of them, PEPP-2, OTOA, and AKAP4, were confirmed as testis-restricted or testis-selective using RT-PCR, with variable expression frequencies observed in a panel of cancer cell lines. Our classification provides an objective ranking for potential CT genes, which is useful in guiding further identification and characterization of these potentially important diagnostic and therapeutic targets.

Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes') in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.