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Visceral leishmaniasis is not endemic in West Africa, but prevalence of canine leishmaniasis and seroprevalence of Leishmania infantum infection in humans are high in the Mont Rolland community (Thiès region, Senegal). Previous studies in this area showed that Sergentomyia schwetzi could be the potential vector of Le. infantum. To precisely describe the biology and population structure of this potential vector, we identified eight novel microsatellite loci to characterize Se. schwetzi populations. We tested these loci in Se. schwetzi populations from five locations at Mont Rolland (Thiès, Senegal). All the loci were polymorphic, with a mean of 17.25 alleles (observed heterozygosity: 0.455). We did not detect any evidence of scoring errors due to stuttering and large allele dropout. Moreover, several of these loci were also amplified in six other sand fly species (Sergentomyia magna, Sergentomyia dubia, Sergentomyia minuta, Phlebotomus duboscqi, Phlebotomus perniciosus, and Phlebotomus ariasi). These preliminary results demonstrate the utility of these microsatellite markers for Se. schwetzi (and for the other sand fly species) population genetic studies.

Differences in codon frequency between genomes, genes, or positions along a gene, modulate transcription and translation efficiency, leading to phenotypic and functional differences. Here, we present a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells, quantifying the phenotypic consequences of codon usage bias at different molecular and cellular levels, with an emphasis on translation elongation.Six synonymous versions of an antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was analysed by means of: quantitative transcriptome and proteome assessment, as proxies for gene expression; cellular fluorescence, as a proxy for single-cell level expression; and real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness.We show that differences in codon usage bias strongly impact the molecular and cellular phenotype: (i) they result in large differences in mRNA levels and in protein levels, leading to differences of over fifteen times in translation efficiency; (ii) they introduce unpredicted splicing events; (iii) they lead to reproducible phenotypic heterogeneity; and (iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression.In human cells in culture, codon usage bias modulates gene expression by modifying mRNA availability and suitability for translation, leading to differences in protein levels and eventually eliciting functional phenotypic changes.IMPORTANCESynonymous codons encode for the same amino acid, but they are not neutral regarding gene expression or protein synthesis. Bias between synonymous codons have evolved naturally and are also applied in biotechnology protein production. We have studied the multilevel impact of codon usage on a human cell system. We show that differences in codon usage lead to transcriptomic, proteomic and functional changes, modulating gene expression and cellular phenotype.

International audience; The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including β-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.

Despite their role in Leishmania transmission, little is known about the organization of sand fly populations in their environment. Here, we used 11 previously described microsatellite markers to investigate the population genetic structure of Phlebotomus ariasi, the main vector of Leishmania infantum in the region of Montpellier (South of France). From May to October 2011, we captured 1,253 Ph. ariasi specimens using sticky traps in 17 sites in the North of Montpellier along a 14-km transect, and recorded the relevant environmental data (e.g., altitude and hillside). Among the selected microsatellite markers, we removed five loci because of stutter artifacts, absence of polymorphism, or non-neutral evolution. Multiple regression analyses showed the influence of altitude and hillside (51% and 15%, respectively), and the absence of influence of geographic distance on the genetic data. The observed significant isolation by elevation suggested a population structure of Ph. ariasi organized in altitudinal ecotypes with substantial rates of migration and positive assortative mating. This organization has implications on sand fly ecology and pathogen transmission. Indeed, this structure might favor the global temporal and spatial stability of sand fly populations and the spread and increase of L. infantum cases in France. Our results highlight the necessity to consider sand fly populations at small scales to study their ecology and their impact on pathogens they transmit.

Introduction In Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of extrapulmonary tuberculosis (EPTB) are zoonotic TB, caused by M. bovis. Although several studies have analyzed bovine TB in cattle in Tunisia, no study has evaluated the risk of transmission to humans in such an endemic country. We aimed to study the genetic diversity of M. bovis human isolates, to ascertain the causes of human EPTB infection by M. bovis and to investigate the distribution and population structure of this species in Tunisia. Materials and methods A total of 110 M. bovis isolates taken from patients with confirmed EPTB were characterized by spoligotyping and MIRU-VNTR typing methods. Results Among the 15 spoligotypes detected in our study, 6 (SB0120, SB0121, SB2025, SB1200, SB1003 and SB0134) were the most prevalent (83.5%) of which SB0120, SB0121 and SB2025 were the most prevailing. MIRU-VNTR typing method showed a high genotypic and genetic diversity. The genetic differentiation based on MIRU-VNTR was significant between populations from South East (Tataouine, Medenine) and Central West (Gafsa, Sidi Bouzid, Kasserine) regions. Of note, 13/15 (86.7%) spoligotypes detected in our study were previously identified in cattle in Tunisia with different frequencies suggesting a peculiar ability of some genotypes to infect humans. Using combined spoligotyping and MIRU-VNTR method, a high clustering rate of 43.9% was obtained. Our results underlined that human EPTB due to M. bovis was more commonly found in female gender and in young patients. Most of our patients, 66.4% (73/110) were raw milk or derivatives consumers, whereas 30.9% (34/110) patients would have contracted EPTB through contact with livestock. The findings suggest that the transmission of Zoonotic TB caused by M. bovis to humans mainly occurred by oral route through raw milk or derivatives. Conclusion Our study showed the urgent need of a better veterinary control with the implementation of effective and comprehensive strategies in order to reach a good protection of animals as well as human health., Author summary In South Tunisia, the prevalence of bovine TB is high with Mycobacterium bovis as causative agent and cattle as reservoir of the bacteria. However as previously mentioned in several studies, M. bovis is also responsible for human extrapulmonary tuberculosis (EPTB) cases in South Tunisia. Despite the veterinary and medical problems, M. bovis is still little studied. In this context, this work aimed to study the molecular epidemiology of M. bovis in EPTB patients in south Tunisia in order to determine the main risk factors of transmission. Our results underlined that SB0120, SB0121 and SB2025, previously described in cattle in Tunisia, represent the predominant genotypes. The findings highlighted that human EPTB caused by M. bovis mainly occurred through the consumption of raw milk or derivatives. These data demonstrate the urgent need to implement strategies for preventing and controlling zoonotic TB.

The study aims to make an update on the distribution and ecology of sand flies in the Quang Ninh province, Northern Vietnam, where Leishmania cases were reported in 2001. Seventeen sites were chosen in three districts of the province: Ha Long, Cam Pha, and Hoanh Bo. Phlebotomine sand flies were collected using 68 CDC light traps from May 30 to 3 June 2016. Captured specimens were transferred individually into Eppendorf tubes with 90% ethanol. The sand fly heads and genitalia were removed and were mounted in Euparal after successive different baths. Specimen identification was determined based on the morphology of the cibarium, pharynx, and/or male genitalia or female spermathecae. A total of 416 sand flies (125 females, 283 males) belonging to four genera were collected and 10 sand fly species were identified: Sergentomyia silvatica, Se. barraudi, Se. hivernus, Se. bailyi, Phlebotomus mascomai, Ph. stantoni, Ph. yunshengensis, Ph. betisi, Chinius junlianensis, Idiophlebotomus longiforceps. The Sergentomyia genus prevailed (79.7% of the collected sand flies), followed by the Phlebotomus genus (13.7%), the Chinius genus (6.1%), and the Idiophlebotomus genus (0.8%). Besides these well-defined taxa, five specimens, named sp1, showed unknown morphological characteristics, requiring further study. The majority of sand flies were collected in rock caves suggesting the cavernicolous character of the species in the Quang Ninh province. However, specimens were also collected in intra and peridomiciliary sites in which Ph. stantoni and Se. hivernus were found as the main species. It is worth noting that two Ph. stantoni were found in the house of a patient affected by Leishmania.

International audience; One of the major insulin resistance instigators is excessive adiposity and visceral fat depots. Individually, exercise training and polyphenol intake are known to exert health benefits as improving insulin sensitivity. However, their combined curative effects on established obesity and insulin resistance need further investigation particularly on white adipose tissue alterations. Therefore, we compared the effects on different white adipose tissue depot alterations of a combination of exercise and grape polyphenol supplementation in obese insulin-resistant rats fed a high-fat diet to the effects of a high-fat diet alone or a nutritional supplementation of grape polyphenols (50 mg/kg/day) or exercise training (1 hr/day to 5 days/wk consisting of treadmill running at 32 m/min for a 10% slope), for a total duration of 8 weeks. Separately, polyphenol supplementation and exercise decreased the quantity of all adipose tissue depots and mesenteric inflammation. Exercise reduced adipocytes' size in all fat stores. Interestingly, combining exercise to polyphenol intake presents no more cumulative benefit on adipose tissue alterations than exercise alone. Insulin sensitivity was improved at systemic, epididymal, and inguinal adipose tissues levels in trained rats thus indicating that despite their effects on adipocyte morphological/metabolic changes, polyphenols at nutritional doses remain less effective than exercise in fighting insulin resistance.

Visceral leishmaniasis is not endemic in West Africa. However, high seroprevalence of Leishmania infantum infection (one of the Leishmania species that cause visceral leishmaniasis) was detected in dogs and humans in the Mont Rolland community (close to Thiès, Senegal), despite the lack of reports concerning human clinical cases. Our aim was to genetically characterize this L. infantum population and identify its origin. We thus conducted seven field surveys in 25 villages of the Mont Rolland community between 2005 and 2009 and blood samples were collected from 205 dogs. Serological testing indicated that 92 dogs (44.9%) were positive for Leishmania infection. L. infantum was identified as the cause of infection. Analysis of 29 L. infantum isolates from these dogs by multilocus microsatellite typing and multilocus sequence typing indicated that this population had very limited genetic diversity, low level of heterozygosity and only seven different genotypes (79.3% of all isolates had the same genotype). Multilocus sequence typing showed that the Mont Rolland isolates clustered with strains from the Mediterranean basin and were separated from East African and Asian strains. Therefore, our data suggest a quite recent and unique introduction into Senegal of a L. infantum strain from the Mediterranean basin.

WOS: 000372567300063, PubMed ID: 26900688, Background The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. Methods/Principal Findings A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L. infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. Conclusions Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories., EU grantEuropean Union (EU) [FP7-261504 EDENext]; Fundacao para a Ciencia e a Tecnologia, Ministerio da Educacao e Ciencia, Portugal [SFRH/BPD/44082/2008], This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext376 (http://www.edenext.eu). The work of CM was done under the fellowship SFRH/BPD/44082/2008 from Fundacao para a Ciencia e a Tecnologia, Ministerio da Educacao e Ciencia, Portugal. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; E. Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension.Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventri-cle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca 2ϩ handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca 2ϩ transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(en-do)plasmic reticular Ca 2ϩ-ATPase activity, increased sarcoplasmic reticular Ca 2ϩ-release fraction, and increased Ca 2ϩ spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca 2ϩ handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs. electrocardiography; monocrotaline; calcium signaling; voltage-sensitive dye imaging; alternans ALTHOUGH RIGHT VENTRICULAR (RV) failure most often develops as a consequence of left ventricular (LV) failure, RV failure occurs in many, increasingly common, diseases associated with dysfunction of the pulmonary circulation (6, 18, 58). This includes pulmonary hypertension, where RV failure is the major cause of death in patients (6). Pulmonary hypertension is associated with RV electrical remodeling (23, 26, 27) and a higher risk of arrhythmias (17). Sudden death occurs in 30-40% of sufferers (10, 63), and lethal arrhythmias are thought to be one cause, in addition to other mechanisms, such as pulmonary embolism and pulmonary artery dissection. Interestingly, the RV is not currently a target for therapeutic intervention in this disease (19). There are several established mechanisms by which arrhyth-mias can arise. Increased electrical heterogeneity and steeper electrical restitution (1, 32, 53) increase the probability of alternans and arrhythmias. This is because heterogeneity increases the dispersion of refractoriness and steeper restitution decreases the stability of action potential (AP) duration (APD) and diastolic interval adaptation to a change in heart rate, also increasing the dispersion of refractoriness. This makes conduction block and reentry more likely (9, 35, 49, 60). Alternans describe the events where parameters such as APD or intracel-lular Ca 2ϩ concentration ([Ca 2ϩ ] i) transient amplitude alternate in size on a beat-to-beat basis. They are established predictors of sudden cardiac death, and discordant alternans, where the parameter in question is out of phase in different regions of the ventricles, are most likely to generate reentrant arrhythmias (9, 35, 49, 60). Defective Ca 2ϩ handling also leads to the generation of arrhythmias. Increased sarcoplasmic reticulum (SR) Ca 2ϩ leak, SR release fraction, SR Ca 2ϩ content, and slowed SR Ca 2ϩ reuptake are thought to contribute to the generation of Ca 2ϩ transient alternans and thus to APD and T-wave alternans (via modulation of Ca 2ϩ-dependent currents) by facilitating beat-to-beat fluctuations in SR Ca 2ϩ release (38, 49). This is because increased SR Ca 2ϩ content favors SR Ca 2ϩ leak and places activated ryanodine receptors in a refractory state for the next beat, leading to fluctuations in SR Ca 2ϩ release (48). Additionally, elevated SR Ca 2ϩ content and release fraction in the presence of a slowed SR Ca 2ϩ reuptake lead to intra-SR fluctuations in Ca 2ϩ content (13). Structural changes, such as an alteration in connexin expression or increased fibrosis, which disrupts the normal myofiber organization, can slow conduction, create electrical heteroge-neity (31), and contribute to dysfunctional electrical activity (16). Given the number of potential arrhythmic mechanisms, the specific occurrence and relative importance of these mechanisms need to be characterized for any given pathology, if that pathology is to be fully understood and treated.

International audience; Background: Phlebotomus ariasi Tonnoir, 1921, is the predominant sand fly species in the Cevennes region and a proven vector of Leishmania infantum, which is the main pathogen of visceral and canine leishmaniasis in the south of France. Even if this species is widely present in Western Mediterranean countries, its biology and ecology remain poorly known. The main goals of this work are to investigate the phenotypic variation of P. ariasi at a local scale in a region characterized by climatic and environmental fluctuations, and to determine if slope and altitude could affect the sand fly phenotypes.Results: Sand flies were captured along a 14 km-long transect in 2011 from May to October. At the same time, environmental data such as altitude and slope were also collected. Morphological analysis of P. ariasi wings was performed by a geometric morphometrics approach. We found morphological variation among local populations of P. ariasi. Strong shape and size variations were observed in the course of the season (particularly in June and July) for both genders. During June, we highlighted differences in wing phenotypes according to altitude for both sexes and to slope and station for females.Conclusions: The phenotypic variations observed in P. ariasi along the studied transect indicated these populations are subjected to environmental pressures. Nevertheless, it seems that sand flies are more sensitive to extrinsic factors in June and July, suggesting a phenotypic plasticity.

Immunotherapy by using multimerized self-peptides has demonstrated a clear protective effect on experimental models of autoimmune diseases. However, the mechanisms involved remain ill-defined. Here we have evaluated the therapeutic efficacy of multimerized self-peptides at the effector phase of autoimmune diabetes and examined their mechanisms of action. Diabetes was induced in rat insulin promoter-hemagglutinin (HA) mice expressing HA in pancreatic β-cells by adoptive transfer of HA 110–119 -specific T helper 1 cells. Complete protection was provided by low doses of the HA 4-mer consisting of four covalently linked linear HA 107–119 peptides. In vivo , the 4-mer appeared to act directly on the pathogenic HA-specific T helper 1 cells and indirectly by activation/recruitment of lymphocytes with regulatory properties so that mice became resistant to a second transfer of diabetogenic T cells. This effect was associated with a recruitment of Foxp3 + CD4 T cells around islets. Moreover, we show that dominant protection from autoimmunity was transferable by spleen cells, and that development of this regulatory population was crucially dependent on the lymphocytes from treated rat insulin promoter-HA mice. Thus, immunotherapy using multimerized epitopes emerges as a promising strategy in view of the current identification of self-epitopes that are major targets of the pathogenic CD4 T cell response in autoimmune diseases.

International audience; Multiple sclerosis (MS) is a chronic inflammatory disease resulting in demyelination and axonal loss within the CNS. An autoimmune reaction directed against myelin antigens contributes to the disease process. As the CNS has long been considered an immune privileged site, how such an immune response can develop locally has remained enigmatic. Recent data, mostly based on the study of animal models for MS, have shown that the CNS is in fact more permissive to the development of immune responses than previously thought. This observation is counterbalanced by the fact that immune tolerance to myelin antigens can be induced outside the CNS. This review focuses on the mechanisms preventing CNS autoimmunity, which act in three separate tissues. In the thymus, expression of CNS autoantigens promotes partial protection, notably through elimination of autoreactive T cells. In the secondary lymphoid organs, the remaining autoreactive T cells are kept under control by the naturally occurring regulatory T cells of the CD4 + Foxp3 + phenotype. In the CNS, multiple mechanisms including the local activation of regulatory T cells further limit autoimmunity. A better understanding of the induction of regulatory T cells, of their mechanisms of action, and of approaches to manipulate them in vivo may offer new therapeutic opportunities for MS patients.

Alterations in the intracellular environment lead to decreased frequency of Ca2+ sparks in a model of heart failure despite enhanced ryanodine receptor activity., Abnormalities in cardiomyocyte Ca2+ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca2+ sparks (brief, localized increased in [Ca2+]i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca2+]i transient amplitude and sarcoplasmic reticulum (SR) Ca2+ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca2+ sparks were less frequent, whereas the fractional release and the frequency of Ca2+ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca2+ sparks were more frequent in PMI cells (under conditions of similar SR Ca2+ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca2+ sparks frequency normalized by the SR Ca2+ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca2+ spark frequency. Indeed, using an internal “failing solution” with less ATP (as found in HF), we observed a dramatic decrease in Ca2+ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca2+ load may mask these effects at the Ca2+ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca2+ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.

Leishmania (L.) infantum is the causative agent in an endemic focus of canine leishmaniasis in the Mont-Rolland district (Thiès, Senegal). In this area, the transmission cycle is well established and more than 30% of dogs and 20% of humans are seropositive for L. infantum. However, the sand fly species involved in L. infantum transmission cycle are still unknown. Between 2007 and 2010, 3654 sand flies were collected from different environments (indoor, peridomestic, farming and sylvatic areas) to identify the main L. infantum vector(s). Nine sand fly species were identified. The Phlebotomus genus (n = 54 specimens; Phlebotomus (Ph) duboscqi and Phlebotomus (Ph). rodhaini) was markedly under-represented in comparison to the Sergentomyia genus (n = 3600 specimens; Sergentomyia (Se) adleri, Se. clydei, Se. antennata, Se. buxtoni, Se. dubia, Se. schwetzi and Se. magna). Se. dubia and Se. schwetzi were the dominant species indoor and in peridomestic environments, near humans and dogs. Blood-meal analysis indicated their anthropophilic behavior. Some Se. schwetzi specimens fed also on dogs. The dissection of females in the field allowed isolating L. infantum from sand flies of the Sergentomyia genus (0.4% of Se. dubia and 0.79% of Se. schwetzi females). It is worth noting that one Se. dubia female not engorged and not gravid revealed highly motile metacyclic of L. infantum in the anterior part of the midgut. PCR-based diagnosis and sequencing targeting Leishmania kinetoplast DNA (kDNA) highlighted a high rate of L. infantum-positive females (5.38% of Se. dubia, 4.19% of Se. schwetzi and 3.64% of Se. magna). More than 2% of these positive females were unfed, suggesting the parasite survival after blood-meal digestion or egg laying. L. infantum prevalence in Se. schwetzi was associated with its seroprevalence in dogs and humans and L. infantum prevalence in Se. dubia was associated with its seroprevalence in humans. These evidences altogether strongly suggest that species of the Sergentomyia genus are probably the vectors of canine leishmaniasis in the Mont-Rolland area and challenge one more time the dogma that in the Old World, leishmaniasis is exclusively transmitted by species of the Phlebotomus genus., Author Summary Leishmaniases, neglected tropical vector-borne diseases, remain today a problem of public health. Classically, the sand flies involved in Leishmania transmission belong either to the Phlebotomus genus (Old World) or to the Lutzomyia genus (New World). In the rural community of Mont-Rolland (Senegal, West Africa), Leishmania infantum is the causative agent in an endemic focus of canine leishmaniasis. Recent surveys revealed more than 30% of dogs and 20% of humans with a positive serological test for Leishmania in this community. However, the sand fly species involved in L. infantum transmission were still unknown. Between 2007 and 2010, we carried out a study in this community to identify the sand fly species responsible for L. infantum transmission. We collected nine species belonging mainly to Sergentomyia genus and in low proportion to Phlebotomus genus. The abundance around dogs and humans, the detection of live and mature parasites in anterior midgut, the high rate of L. infantum-positive females using molecular analyses and the identification of dog and human blood in the fed females incriminates Se. schwetzi and Se. dubia as possible vectors of L. infantum. This hypothesis is strongly supported by statistical analyses performed to compare the prevalence of infected sand flies with the seroprevalence data in humans and dogs.

Alteration in intracellular calcium handling contributes to contractile dysfunction in heart failure (HF). In vitro lipid bilayer studies with reconstituted ryanodine receptor (RyR) reveal that these channels are more spontaneously active in HF compared to control.Here, spontaneous Ca2+ sparks have been characterized to evaluate RyR cluster activity in situ in a mice post-myocardial infarction (PMI) HF model. PMI mice showed reduced ejection fraction and their cardiomycytes were hypertrophied. Ca2+ transient amplitude and SR Ca2+ load were depressed, as well as the spontaneous Ca2+ spark frequency in intact cardiomycytes of PMI mice. However, both the fractional release and Ca2+ wave frequency were elevated, suggesting an increased RyR activity. In agreement, in permeabilized cardiomyocytes, which enabled internal solution controlling, Ca2+ sparks displayed a higher frequency in HF cells, whereas SR Ca2+ content were comparable under these conditions. To resolve the discrepancy, Ca2+ spark frequency in intact cells was normalized to SR Ca2+ load in each cell. While the reduced SR Ca2+ content in intact cells might account for the reduced Ca2+ spark frequency, it could also be attributable to the cytosolic environment in intact cells. In fact, HF is associated to metabolic changes that may affect RyR function. This was further strengthened by the observation that Ca2+ spark frequency was remarkably decreased by using an internal failing solution containing less ATP as found in HF models.Together, although the RyR channels isolated from failing heart may display more activity, our results suggest that in intact cells, concomitant alterations in intracellular media composition together with SR Ca2+ load are likely to mask those effects at the Ca2+ spark level. However, once one spark is generated, the probability of triggering an arrhythmogenic Ca2+ wave is elevated in PMI cardiomyocytes.

International audience; RATIONALE: TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.OBJECTIVES: We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4-/-) model.METHODS AND RESULTS: Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.CONCLUSIONS: TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Aims Clinical studies failed to prove convincingly efficiency of intravenous infusion of neseritide during heart failure and evidence suggested a pro-adrenergic action of B-type natriuretic peptide (BNP). However, subcutaneous BNP therapy was recently proposed in heart failure, thus raising new perspectives over what was considered as a promising treatment. We tested the efficiency of a combination of oral β1-adrenergic receptor blocker metoprolol and subcutaneous BNP infusion in decompensated heart failure. Methods and results The effects of metoprolol or/and BNP were studied on cardiac remodelling, excitation–contraction coupling and arrhythmias in an experimental mouse model of ischaemic heart failure following postmyocardial infarction. We determined the cellular and molecular mechanisms involved in anti-remodelling and antiarrhythmic actions. As major findings, the combination was more effective than metoprolol alone in reversing cardiac remodelling and preventing ventricular arrhythmia. The association of the two molecules improved cardiac function, reduced hypertrophy and fibrosis, and corrected the heart rate, sympatho-vagal balance (low frequencies/high frequencies) and ECG parameters (P to R wave interval (PR), QRS duration, QTc intervals). It also improved altered Ca 2+ cycling by normalising Ca 2+ -handling protein levels (S100A1, SERCA2a, RyR2), and prevented pro-arrhythmogenic Ca 2+ waves derived from abnormal Ca 2+ sparks in ventricular cardiomyocytes. Altogether these effects accounted for decreased occurrence of ventricular arrhythmias. Conclusions Association of subcutaneous BNP and oral metoprolol appeared to be more effective than metoprolol alone. Breaking the deleterious loop linking BNP and sympathetic overdrive in heart failure could unmask the efficiency of BNP against deleterious damages in heart failure and bring a new potential approach against lethal arrhythmia during heart failure.

International audience; Cardiac actions of Epac (exchange protein directly activated by cAMP) are not completely elucidated. Epac induces cardiomyocytes hypertrophy, Ca(2+)/calmodulin protein kinase II (CaMKII) and excitation-transcription coupling in rat cardiac myocytes. Here we aimed to elucidate the pathway cascade involved in Epac sustained actions, as during the initiation of hypertrophy development, where β-adrenergic signaling is chronically stimulated. Rats were treated with the Epac selective activator 8-pCPT during 4weeks and Ca(2+) signaling was analyzed in isolated cardiac myocytes by confocal microscopy. We observed a positive inotropic effect manifested by increased [Ca(2+)](i) transient amplitudes. In order to further analyze these actions, we incubated adult cardiomyocytes in the presence of 8-pCPT. The effects were similar to those obtained in-vivo and are blunted by Epac1 knock down. Interestingly, the increase in [Ca(2+)] transients was abolished by protein synthesis blockade or when the downstream effectors of calmodulin (CaMKII or calcineurin) were inhibited, pointing to calmodulin (CaM) as an important downstream protein in Epac sustained actions. In fact, CaM expression was enhanced by 8-pCPT treatment in isolated cells, as found by Western blots. Moreover, the 8-pCPT-induced, PKA-independent, positive inotropic effect was favored by enhanced extracellular Ca(2+) influx via L-type Ca(2+) channels. However, 8-pCPT also induced aberrant Ca(2+) release as Ca(2+) waves and extra [Ca(2+)](i) transients, suggesting proarrhythmic effect. These results provide new insights regarding Epac cardiac actions, suggesting an important role in the initial compensation of the heart to pathological stimuli during the initiation of cardiac hypertrophy, favoring contraction but also arrhythmia risk.

Aims During heart failure (HF), the left ventricle (LV) releases B-type natriuretic peptide (BNP), possibly contributing to adverse cardiovascular events including ventricular arrhythmias (VAs) and LV remodelling. We investigated the cardiac effects of chronic BNP elevation in healthy mice and compared the results with a model of HF after myocardial infarction (PMI mice). Methods and results Healthy mice were exposed to circulating BNP levels (BNP-Sham) similar to those measured in PMI mice. Telemetric surface electrocardiograms showed that in contrast with fibrotic PMI mice, electrical conduction was not affected in BNP-Sham mice. VAs were observed in both BNP-Sham and PMI but not in Sham mice. Analysis of heart rate variability indicated that chronic BNP infusion increased cardiac sympathetic tone. At the cellular level, BNP reduced Ca2+ transients and impaired Ca2+ reuptake in the sarcoplasmic reticulum, in line with blunted SR Ca2+ ATPase 2a and S100A1 expression. BNP increased Ca2+ spark frequency, reflecting Ca2+ leak through ryanodine receptors, elevated diastolic Ca2+, and promoted spontaneous Ca2+ waves. Similar effects were observed in PMI mice. Most of these effects were reduced in BNP-Sham and PMI mice by the selective β1-adrenergic blocker metoprolol. Conclusion Elevated BNP levels, by inducing sympathetic overdrive and altering Ca2+ handling, promote adverse cardiac remodelling and VAs, which could account in part for the progression of HF after MI. The early use of β-blockers to prevent the deleterious effects of chronic BNP exposure may be beneficial in HF.

Background Controlled mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction, which impedes weaning from mechanical ventilation. To design future clinical trials in humans, a better understanding of the molecular mechanisms using knockout models, which exist only in the mouse, is needed. The aims of this study were to ascertain the feasibility of developing a murine model of ventilator-induced diaphragmatic dysfunction and to determine whether atrophy, sarcolemmal injury, and the main proteolysis systems are activated under these conditions. Methods Healthy adult male C57/BL6 mice were assigned to three groups: (1) mechanical ventilation with end-expiratory positive pressure of 2-4 cm H2O for 6 h (n=6), (2) spontaneous breathing with continuous positive airway pressure of 2-4 cm H2O for 6 h (n=6), and (3) controls with no specific intervention (n=6). Airway pressure and hemodynamic parameters were monitored. Upon euthanasia, arterial blood gases and isometric contractile properties of the diaphragm and extensor digitorum longus were evaluated. Histology and immunoblotting for the main proteolysis pathways were performed. Results Hemodynamic parameters and arterial blood gases were comparable between groups and within normal physiologic ranges. Diaphragmatic but not extensor digitorum longus force production declined in the mechanical ventilation group (maximal force decreased by approximately 40%) compared with the control and continuous positive airway pressure groups. No histologic difference was found between groups. In opposition with the calpains, caspase 3 was activated in the mechanical ventilation group. Conclusion Controlled mechanical ventilation for 6 h in the mouse is associated with significant diaphragmatic but not limb muscle weakness without atrophy or sarcolemmal injury and activates proteolysis.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca 2+ and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca 2+ homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca 2+ leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca 2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca 2+ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α–induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca 2+ leak trough normalization of RyR2 function is cardioprotective.

International audience; Isolated cardiomyocyte contractility was explored in a model of metabolic syndrome (the fructose-fed rat) and the effect of a treatment by a total red wine polyphenolic extract (RWPE) determined. Fructose-enriched diet impaired cardiomyocyte maximal shortening and velocities of contraction and relaxation, while RWPE was able to prevent those changes. Those studies suggest that red wine polyphenols are able to prevent the development of a cardiac phenotype commonly observed at an early stage of heart failure.

International audience; Abstract Cardiovascular disease is a frequent complication inducing mortality in chronic kidney disease (CKD) patients, which can be determined by both traditional risk factors and non-traditional risk factors such as malnutrition and oxidative stress. This study aimed to investigate the role of oxidative stress in uremia-induced cardiopathy in an experimental CKD model. CKD was induced in Sprague-Dawley rats by a 4-week diet supplemented in adenine, calcium and phosphorous and depleted in proteins. CKD was associated with a 3-fold increase in superoxide anion production from the NADPH oxidase in the left ventricle, but the maximal activity of mitochondrial respiratory chain complexes was not different. Although manganese mitochondrial SOD activity decreased, total SOD activity was not affected and catalase or GPx activities were increased, strengthening the major role of NADPH oxidase in superoxide anion output. Superoxide anion output was associated with enhanced expression of osteopontin (×7.7) and accumulation of pro-collagen type I (×3.7). To conclude, the increased activity of NADPH oxidase during CKD is associated with protein modifications which could activate a pathway leading to cardiac remodelling.

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca 2+ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca 2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca 2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca 2+ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca 2+ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca 2+ leak prevents fatal sudden cardiac arrhythmias in DMD.

Les arythmies ventriculaires gauches representent une des principales causes de deces chez les patients atteint de la Dystrophie Musculaire de Duchenne (DMD). Dans le muscle squelettique, nous avons mis evidence une augmentation de la s-nytrosylation du canal calcique de type 1 du reticulum sarcoplasmique (RS), le recepteur a la ryanodine (RyR1), a l’origine d’une augmentation de la fuite spontanee du Ca2+ du RS (Bellinger et al (2009), Nature Med,). L’objectif de ce travail, a ete de determiner si une s-nytrolsylation accrue de l’isoforme cardiaque du RyR (RyR2) pouvait rendre compte des arythmies ventriculaires chez des souris deficientes en dystrophine, les souris mdx. En western blot, l’augmentation de la s-nytrosylation du RyR2 dans les cœurs de souris mdx apparait des l’ages de 40 jours. Cette s-nytrosylation est associee a la dissociation de la sous-unite regulatrice du RyR2, la calstabin 2 (ou FKBP12.6), et est a l’origine d’une augmentation de la fuite spontanee du Ca2+ du RS, mesuree par microscopie confocale (Fluo-4) sur des cardiomyocytes ventriculaires isoles. 10 jours de traitement par voie orale avec le S107 (Rycal) qui stabilise la liaison de la calstabin 2 avec le RyR2, empeche la fuite du Ca2+ du RS, normalise la signalisation Ca2+, et previent le declenchement d’arythmies induit par une stimulation adrenergique que ce soit au niveau cellulaire (Isoprenaline : 100 nM) ou in vivo (Isoprenaline : 2 mg/kg). En conclusion, des les premiers stades de la pathologie, la s-nytrosylation du RyR2 induit la dissociation de la calstabin 2 du RyR2 et augmente la fuite diastolique de Ca2+ ce qui dans des conditions de stress augmente la prevalence des arythmies. Une normalisation precoce de la fonction du RyR2 apparait comme une cible therapeutique nouvelle dans la prevention des phenomenes arythmogenes au cours de la DMD.

Thymus-derived regulatory T cells (Tregs) expressing CD4, CD25, and the transcription factor Foxp3 play major roles in preventing autoimmunity. The Treg population is enriched in T cells expressing high-avidity self-reactive T cell receptors, and thymic epithelial cells expressing self-antigens (Ag) have been implicated in their induction and/or selection. However, the thymic selection events leading to Treg lineage commitment remain unclear. We followed the thymic development of self-Ag-specific Tregs in double-transgenic mice coexpressing a neo-self-Ag, hemagglutinin (HA) under the control of a neural tissue-specific promoter, and a transgenic class II-restricted T cell antigen receptor specific for HA111-119. Our data show that the promiscuous expression of the HA transgene in thymic epithelial cells is involved in the selective induction and/or expansion of HA-specific Foxp3 + Treg thymic precursors as early as the double-positive stage.

Introduction Nous avons montre que le syndrome metabolique (SM), induit experimentalement par une alimentation enrichie en fructose 60% (F), etait associe a une augmentation de la pression arterielle, une fibrose cardiaque et une hyperproduction d'especes oxygenees reactives, et que ces manifestations etaient prevenues par l'administration d'un extrait polyphenolique total de vin (EPV, 21mg/kg/jour) ou de polyphenols purs (2mg/kg/jour). Cependant, aucune alteration fonctionnelle ou morphologique n'a ete retrouvee in vivo par echocardiographie. Toutefois, nos donnees recentes indiquent que la contractilite des cardiomyocytes isoles peut etre alteree, meme en absence de manifestations echocardiographiques (Andre et al., 2010). Nous avons donc determine les parametres-cles du couplage excitation/contraction de cardiomyocytes isoles apres 2, 8 et 13 semaines de regime F, ainsi que les effets preventifs eventuels de l'EPV. Materiels et methodes La fraction de raccourcissement cellulaire (FRC) et les modifications du Ca2 + cytosolique (Indo-1) des cardiomyocytes ont ete determinees simultanement sous stimulations en champ (0,5 Hz, 22°C, 1,8mM Ca 2+ ). Resultats Chez les animaux temoins (C), la FRC ete similaire a 2 et 8 semaines, avant de decroitre spontanement a 16 semaines. Chez les animaux F, la FRC a ete significativement diminuee par rapport au groupe C a 2, 8 et 13 semaines, la difference entre C et F etant maximale a 8 semaines. De plus, chez les rats F, les vitesses de contraction et de relaxation ont ete diminuees et le re-captage du Ca2 + cytosolique ralenti (augmentation de Tau), suggerant une alteration des pompes Serca2a. Chez les rats C, l'EPV diminue la vitesse de relaxation et le recaptage calcique a 8 semaines. Chez les rats F, l'EPV previent l'alteration du FRC et le ralentissement de contraction et de relaxation, tout en accentuant le ralentissement du recaptage calcique. Conclusion La contractilite et la cinetique calcique des cardiomyocytes sont alterees precocement par un regime inducteur du SM. Un EPV anti-oxydant previent l'alteration de contractilite.

International audience; Scope: We compared the effects of two dealcoholized wines, persimmon (P) and Merlot (M), in hypercholesterolemic hamsters.Methods and results: Four groups of hamsters received a standard (ST) or an atherogenic diet (AT) for 12 weeks. AT animals received either dealcoholized persimmon wine (AT + P) or Merlot wine (AT + M) by gavage, while controls received water (AT and ST). Plasma cholesterol, triglycerides and glucose and paraoxonase activity were measured. Oxidative stress was assessed by aortic O(2)°(-) production, and vascular function was evaluated in aortic rings. The atherogenic diet led to higher plasma triglycerides (246%), total cholesterol (142%), LDL-cholesterol (91%) and HDL-cholesterol (49%). Aortic production of O(2)°(-) also increased (207%) and vascular reactivity was modified with altered endothelial function as assessed by acetylcholine-dependent vasorelaxation. The two wines partially prevented these alterations, reducing O(2)°(-) production and improving vascular reactivity without altering endothelial function. There was no difference between the P and M groups, although the procyanidin composition of the two dealcoholized fractions differed significantly, and only dimer concentrations were similar.Conclusion: These findings indicate that polyphenols are responsible, at least in part, for the antiatherogenic/antioxidant effects of wines.

Les effets bénéfiques des inhibiteurs de l’enzyme de conversion de l’angiotensine (IEC) sur la mortalité, la morbidité et la qualité de vie des patients en insuffisance cardiaque ont largement été démontrés. Toutefois, le rôle des IEC sur l’incidence des arythmies ventriculaires n’a pas été clairement établi. Le but de ce travail a été d’évaluer les effets du delapril (un IEC) sur les arythmies ventriculaires et les troubles de conduction dans un modèle murin d’infarctus du myocarde (IM).Méthodes et RésultatsDes souris en insuffisance cardiaque, après ligature de la coronaire gauche (souris IM), ont été comparées à des souris contrôles. Les souris IM sont caractérisées par une hypertrophie cardiaque (échocardiographie) et une augmentation significative de la mortalité. Les analyses des coupes histologiques révèlent une augmentation de la fibrose interstitielle associée à une augmentation des ARNm codants pour le collagène I et III. Les mesures électrophysiologiques in vivo (ECG de surface, explorations endocavitaires) montrent un allongement des intervalles QTc et QRS, une augmentation du nombre d’arythmies ventriculaires et des blocs atrio-ventriculaires (BAV), ainsi qu’une altération des intervalles Atrio- Hissien (AH) et Hissien-Ventriculaire (HV). Les mesures réalisées en patch-clamp sur des cardiomyocytes ventriculaires isolés révèlent une hypertrophie cellulaire, une dépolarisation du potentiel de membrane et un allongement du potentiel d’action, en accord avec une diminutiondu courant rectifiant entrant potassique de fond (Ik1).Le traitement des souris IM avec une dose non-hypotensive (6mg/kg/jour) de delapril, 24h après la chirurgie et ce, pendant 6 semaines : (i) améliore la survie (87% chez les souris IM-delapril vs. 62%chez les souris IM; P

International audience; Here we identified Ito cells (hepatic stellate cells, HSC), known for storage of vitamin A and participation in hepatic fibrosis, as professional liver-resident antigen-presenting cells (APC). Ito cells efficiently presented antigens to CD1-, major histocompatibility complex (MHC)-I-, and MHC-II-restricted T cells. Ito cells presented lipid antigens to CD1-restricted T lymphocytes such as natural killer T (NKT) cells and promoted homeostatic proliferation of liver NKT cells through interleukin-15. Moreover, Ito cells presented antigenic peptides to CD8 + and CD4 + T cells and mediated crosspriming of CD8 + T cells. Peptide-specific T cells were activated by transgenic Ito cells presenting endogenous neoantigen. Upon bacterial infection, Ito cells elicited antigen-specific T cells and mediated protection. In contrast to other liver cell types that have been implicated in induction of immunological tolerance, our data identify Ito cells as professional intrahepatic APCs activating T cells and eliciting a multitude of T cell responses specific for protein and lipid antigens.

Pertussis toxin (PTx) is a bacterial toxin used to enhance the severity of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis. It is known to promote permeabilization of the blood-brain barrier, maturation of APC, activation of autoreactive lymphocytes and alteration of lymphocyte migration. In this study, we show that i.v. injection of PTx in mice induces a decrease in the number of splenic CD4+CD25+ regulatory T cells (Treg cells). Furthermore, PTx not only induces a depletion of the dominant CD4+CD25+Foxp3+ subpopulation of splenic Treg cells, but also reduces to a similar extent the CD4+CD25−Foxp3+ subpopulation. On a per cell basis, the suppressive properties of the remaining Treg cells are not modified by PTx treatment. The reduction in splenic Treg cells is associated with preferential migration of these cells to the liver. Additionally, Treg cells exhibit a high sensitivity to PTx-mediated apoptosis in vitro. Finally, in vivo depletion of Treg cells by injection of an anti-CD25 Ab, and PTx treatment, present synergistic experimental autoimmune encephalomyelitis exacerbating effects. Therefore, we identify a new effect of PTx and provide an additional illustration of the influence of microbial components on the immune system affecting the balance between tolerance, inflammation and autoimmunity.

International audience; Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.

Coronary vasculature is required to maintain cardiomyocyte survival, via delivery of oxygen and nutrients, and consequently myocardial architecture and cardiac function. Ischemic heart disease following myocardial infarction causes irreversible cell loss and scaring and is a major cause of morbidity and mortality. Revascularization of injured, ischemic and regenerating organs is essential to restore organ function and requires the formation of new vessels by the mechanisms of vasculogenesis, angiogenesis or arteriogenesis. With the objective of studying vascular remodeling during myocardial infarction (MI), we have performed permanent left coronary ligation on Connexin40-GFP (Cx40GFP/+) mice. Cx40 encodes a gap junction protein and is expressed in endothelial cells of large vessels. In the heart, Cx40-GFP expression is detected in coronary arteries but not in veins, capillaries or endocardium. After two weeks of ligation, MI was detected in left ventricle by echocardiography and anatomical examination of these hearts revealed the presence of an extensive network of GFP-positive vasculature within the infarct area. These vessels follow a tortuous route in the remaining ventricular wall and some communicate with the left ventricular lumen forming a crater covered with GFP and VEGF-R2 positive endothelial cells at the endocardial surface. To determine whether these vessels result from neo-vascularization or coronary artery remodeling, we carried out genetic lineage tracing of coronary endothelial cells using an inducible Cx40-cre allele. Our results show that GFP positive endothelial cells forming the endocardial carters are not always derived from preexisting coronary arteries, suggesting that endocardium may also contribute to the generation of new vessels during vascular remodeling in the adult heart by angiogenesis.

Duchenne muscular dystrophy (DMD) affects one in 3500 newborn males and usually leads to death from respiratory or cardiac failure by age 30. Interestingly, the severity of cardiomyopathy is not proportional to the severity of skeletal muscle disorder. Among DMD patients, the cardiac phenotype varies with age from no discernable cardiac left ventricular enlargement or dysfunction to early onset of dilated cardiomyopathy (DCM) with heart failure. The incidence of DCM in DMD patients has been estimated to be 25% by 6 years of age, 59% at 10 years of age and ∼100% in adults. DMD patients often exhibit electrocardiographic abnormalities and frequent premature ventricular contractions. As the cardiomyopathy progresses, ventricular arrhythmias (VA) increase, often leading to sudden death. Most of the electrical and functional abnormalities have been attributed to cardiac fibrosis. However, electrical abnormalities may occur in the absence of overt cardiac histopathology and ECG changes are similar in patients with DMD regardless of presence of DCM.Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6) resulting in “leaky” RyR2 channels and diastolic SR Ca2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the calcium channel stabilizer, S107 (“rycal”), inhibited the SR Ca2+ leak, restored normal Ca2+ transients, inhibited isoproterenol induced aberrant depolarizations in isolated cardiomyocytes and prevented arrhythmias in vivo.Thus, diastolic SR Ca2+ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Prevention of the RyR2-mediated diastolic SR Ca2+ leak may provide a novel cardiac therapeutic approach in DMD.