Targeting endothelial thioredoxin-interacting protein (TXNIP) protects from metabolic disorder-related impairment of vascular function and post-ischemic revascularisation.

INTRODUCTION: Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biological functions, the contribution of endothelial TXNIP has not been well-defined in regards to endothelial and vascular function or in post-ischemic revascularisation. We postulated that inhibition of endothelial TXNIP with siRNA or in a Cre-LoxP system could be involved in protection from high fat, high protein, low carbohydrate (HFHPLC) diet-induced oxidative stress and endothelial dysfunction, leading to vascular damage and impaired revascularisation in vivo. METHODS AND RESULTS: To investigate the role of endothelial TXNIP, the TXNIP gene was deleted in endothelial cells using anti-TXNIP siRNA treatment or the Cre-LoxP system. Murine models were fed a HFHPLC diet, known to induce metabolic disorders. Endothelial TXNIP targeting resulted in protection against metabolic disorder-related endothelial oxidative stress and endothelial dysfunction. This protective effect mitigates media cell loss induced by metabolic disorders and hampered metabolic disorder-related vascular dysfunction assessed by aortic reactivity and distensibility. In aortic ring cultures, metabolic disorders impaired vessel sprouting and this alteration was alleviated by deletion of endothelial TXNIP. When subjected to ischemia, mice fed a HFHPLC diet exhibited defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. However, reducing endothelial TXNIP rescued metabolic disorder-related impairment of ischemia-induced revascularisation. CONCLUSION: Collectively, these results show that targeting endothelial TXNIP in metabolic disorders is essential to maintaining endothelial function, vascular function and improving ischemia-induced revascularisation, making TXNIP a potential therapeutic target for therapy of vascular complications related to metabolic disorders.

Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms.

Currently, most nonviral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene-silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene-silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using small-angle X-ray scattering (SAXS) synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allows for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene-silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after resuspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA-delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIPfl/fl cdh5cre). Control (TXNIPfl/fl) and TXNIPfl/fl cdh5cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIPfl/fl and TXNIPfl/fl cdh5cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIPfl/fl cdh5cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1ß. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Analysis of the evolution of the detection limits of electrochemical nucleic acid biosensors II.

This critical review of electrochemical biosensors allowing direct detection of nucleic acid targets reports on different transduction pathways and their latest breakthroughs. A classification of the various strategies based on the nature of the electrochemical transduction is established to emphasize the efficiency of each of them. It provides an overall picture of the detection limit of the various approaches developed during the last two decades. Graphical Abstract Detection limits evolutions of electrochemical DNA biosensors.

Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family.

Characterization of an A-kinase anchoring protein-like suggests an alternative way of PKA anchoring in Plasmodium falciparum.

BACKGROUND: The asexual intra-erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regulation and signaling pathways. The activity of cAMP-dependent protein kinase (PKA) depends on A-kinase anchoring proteins (AKAPs) through protein interactions. While several components of the cAMP dependent pathway-including the PKA catalytic and regulatory subunits-have been characterized in P. falciparum, whether AKAPs are involved in this pathway remains unclear. Here, PfAKAL, an open reading frame of a potential AKAP-like protein in the P. falciparum genome was identified, and its protein partners and putative cellular functions characterized. METHODS: The expression of PfAKAL throughout the erythrocytic cycle of the 3D7 strain was assessed by RT-qPCR and the presence of the corresponding protein by immunofluorescence assays. In order to study physical interactions between PfAKAL and other proteins, pull down experiments were performed using a recombinant PfAKAL protein and parasite protein extracts, or with recombinant proteins. These interactions were also tested by combining biochemical and proteomic approaches. As phosphorylation could be involved in the regulation of protein complexes, both PfAKAL and Pf14-3-3I phosphorylation was studied using a radiolabel kinase activity assay. Finally, to identify a potential function of the protein, PfAKAL sequence was aligned and structurally modeled, revealing a conserved nucleotide-binding pocket; confirmed by qualitative nucleotide binding experiments. RESULTS: PfAKAL is the first AKAP-like protein in P. falciparum to be identified, and shares 23 % sequence identity with the central domain of human AKAP18δ. PfAKAL is expressed in mature asexual stages, merozoites and gametocytes. In spite of homology to AKAP18, biochemical and immunochemical analyses demonstrated that PfAKAL does not interact directly with the P. falciparum PKA regulatory subunit (PfPKA-R), but instead binds and colocalizes with Pf14-3-3I, which in turn interacts with PfPKA-R. In vivo, these different interactions could be regulated by phosphorylation, as PfPKA-R and Pf14-3-3I, but not PfAKAL, are phosphorylated in vitro by PKA. Interestingly, PfAKAL binds nucleotides such as AMP and cAMP, suggesting that this protein may be involved in the AMP-activated protein kinase (AMPK) pathway, or associated with phosphodiesterase activities. CONCLUSION: PfAKAL is an atypical AKAP that shares common features with human AKAP18, such as nucleotides binding. The interaction of PfAKAL with PfPKA-R could be indirectly mediated through a join interaction with Pf14-3-3I. Therefore, PfPKA localization could not depend on PfAKAL, but rather involves other partners.

SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor ß.

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type ß (TGF-ß) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-ß/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-ß. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-ß in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-ß-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-ß-mediated CTGF expression.

Suprachoroidal electrotransfer: a nonviral gene delivery method to transfect the choroid and the retina without detaching the retina.

Photoreceptors and retinal pigment epithelial cells (RPE) targeting remains challenging in ocular gene therapy. Viral gene transfer, the only method having reached clinical evaluation, still raises safety concerns when administered via subretinal injections. We have developed a novel transfection method in the adult rat, called suprachoroidal electrotransfer (ET), combining the administration of nonviral plasmid DNA into the suprachoroidal space with the application of an electrical field. Optimization of injection, electrical parameters and external electrodes geometry using a reporter plasmid, resulted in a large area of transfected tissues. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month when using a cytomegalovirus (CMV) promoter. No ocular complications were recorded by angiographic, electroretinographic, and histological analyses, demonstrating that under selected conditions the procedure is devoid of side effects on the retina or the vasculature integrity. Moreover, a significant inhibition of laser induced-choroidal neovascularization (CNV) was achieved 15 days after transfection of a soluble vascular endothelial growth factor receptor-1 (sFlt-1)-encoding plasmid. This is the first nonviral gene transfer technique that is efficient for RPE targeting without inducing retinal detachment. This novel minimally invasive nonviral gene therapy method may open new prospects for human retinal therapies.

First-trimester Plasmodium falciparum infections display a typical "placental" phenotype.

BACKGROUND: Plasmodium falciparum-infected erythrocytes (IEs) adhere to host cell receptors, allowing parasites to sequester into deep vascular beds of various organs. This defining phenomenon of malaria pathogenesis is key to the severe clinical complications associated with cerebral and placental malaria. The principal ligand associated with the binding to chondroitin sulfate A (CSA) that allows placental sequestration of IEs is a P. falciparum erythrocyte membrane protein 1 (PfEMP1) family member encoded by the var2csa gene. METHODS: Here, we investigated the transcription pattern of var genes by real-time polymerase chain reaction, the expression of VAR2CSA, protein by flow cytometry, and the CSA-binding ability of IEs collected at different stages of pregnancy using a static-based Petri dish assay. RESULTS: Through comparison with the profiles of isolates from nonpregnant hosts, we report several lines of evidence showing that parasites infecting women during pregnancy preferentially express VAR2CSA protein, and that selection for the capacity to adhere to CSA via VAR2CSA expression occurs early in pregnancy. CONCLUSIONS: Our data suggest that the placental tropism of P. falciparum is already established in the first trimester of pregnancy, with consequent implications for the development of the pathology associated with placental malaria.

Development and Application of an Assay to Evaluate the Anti-Parasitic Effect of Humoral Responses against Trypanosoma cruzi.

Mounting a balanced and robust humoral immune response is of utmost importance for reducing the infectivity of Trypanosoma cruzi. While the role of such a response in controlling the infection is well known, there is a lack of tools that can be used to quickly evaluate it. We developed a serum parasite inhibition assay (to evaluate changes in the parasite infection after exposing infective T. cruzi trypomastigotes to serum samples from infected patients). It is based on Vero cells as the hosts and the Tulahuen ß-galactosidase parasite strain, genetically engineered to be quantifiable by spectrophotometry. In parallel, we developed an in-house ELISA to correlate the anti-T. cruzi antibody titres of the clinical samples with their observed anti-parasitic effect in the serum parasite inhibition assay. Serum samples from chronically T. cruzi-infected patients significantly inhibited parasite invasion in a titre-dependant manner, regardless of the patient's clinical status, compared to samples from the non-infected controls. In addition, there was a clear correlation between the reactivity of the samples to the whole-parasite lysates by ELISA and the inhibitory effect. The results of this work confirm the previously described anti-parasitic effect of the serum of individuals exposed to T. cruzi and present a framework for its large-scale evaluation in further studies. The serum parasite inhibition assay represents a reproducible way to evaluate the intensity and anti-parasitic effect of humoral responses against T. cruzi, which could be applied to the evaluation of candidate antigens/epitopes in the design of Chagas disease vaccine candidates.

The NTS-DBL2X region of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A.

BACKGROUND: Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS: Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS: Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS: This study highlights an important step achieved toward development of a protective vaccine against placental malaria.

Computer-Aided Analysis of West Sub-Saharan Africa Snakes Venom towards the Design of Epitope-Based Poly-Specific Antivenoms.

Snakebite envenomation is a neglected tropical disease that causes over 100,000 deaths each year. The only effective treatment consists of antivenoms derived from animal sera, but these have been deemed with highly variable potency and are usually inaccessible and too costly for victims. The production of antivenoms by venom-independent techniques, such as the immunization with multi-epitope constructs, could circumvent those drawbacks. Herein, we present a knowledge-based pipeline to prioritize potential epitopes of therapeutic relevance from toxins of medically important snakes in West Sub-Saharan Africa. It is mainly based on sequence conservation and protein structural features. The ultimately selected 41 epitopes originate from 11 out of 16 snake species considered of highest medical importance in the region and 3 out of 10 of those considered as secondary medical importance. Echis ocellatus, responsible for the highest casualties in the area, would be covered by 12 different epitopes. Remarkably, this pipeline is versatile and customizable for the analysis of snake venom sequences from any other region of the world.

Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.

Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.

IgG acquisition against PfEMP1 PF11_0521 domain cassette DC13, DBLß3_D4 domain, and peptides located within these constructs in children with cerebral malaria.

The Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7_1150400/PF11_0521) contains both domain cassette DC13 and DBLß3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7_1150400/PF11_0521 DC13 and DBLß3_D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7_D2 and DBLß3_D4 domains. We found significant IgG responses against the entire DC13, PF11_0521_DBLß3_D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLß3 B3-34 peptide that contains essential residues involved in the interaction between PF11_0521 DBLß3_D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11_0521 DBLß3_D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7_1150400/PF11_0521 structures to prevent CM.

How to make siRNA lipoplexes efficient? Add a DNA cargo.

BACKGROUND: We recently reported an efficient formulation of siRNA targeting TNF-alpha, that was able to restore immunological balance in a mouse arthritis model following intravenous injection. METHOD: Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect. RESULTS: We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate. CONCLUSIONS: These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.

The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins.

BACKGROUND: We have developed a nonviral gene therapy method based on the electrotransfer of plasmid in the ciliary muscle. These easily accessible smooth muscle cells could be turned into a biofactory for any therapeutic proteins to be secreted in a sustained manner in the ocular media. METHODS: Electrical conditions, design of electrodes, plasmid formulation, method and number of injections were optimized in vivo in the rat by localizing ß-galactosidase expression and quantifying reporter (luciferase) and therapeutic (anti-tumor necrosis factor) proteins secretion in the ocular media. Anatomical measurements were performed via human magnetic resonance imaging to design a human eye-sized prototype that was tested in the rabbit. RESULTS: In the rat, transscleral injection of 30 µg of plasmid diluted in half saline (77 mM NaCl) followed by application of eight square-wave electrical pulses (15 V, 10 ms, 5.3 Hz) using two platinum/iridium electrodes, an internal wire and an external sheet, delivered plasmid efficiently to the ciliary muscle fibers. Gene transfer resulted in a long-lasting (at least 5 months) and plasmid dose-/injection number- dependent secretion of different molecular weight proteins mainly in the vitreous, without any systemic exposure. Because ciliary muscle anatomical measurements remained constant among ages in adult humans, an integrated device comprising needle-electrodes was designed and manufactured. Its usefulness was validated in the rabbit. CONCLUSIONS: Plasmid electrotransfer to the ciliary muscle with a suitable medical device represents a promising local and sustained protein delivery system for treating posterior segment diseases, avoiding repeated intraocular injections.

Design of an Epitope-Based Vaccine Ensemble for Animal Trypanosomiasis by Computational Methods.

African animal trypanosomiasis is caused by vector-transmitted parasites of the genus Trypanosoma. T. congolense and T. brucei brucei are predominant in Africa; T. evansi and T. vivax in America and Asia. They have in common an extracellular lifestyle and livestock tropism, which provokes huge economic losses in regions where vectors are endemic. There are licensed drugs to treat the infections, but adherence to treatment is poor and appearance of resistances common. Therefore, the availability of a prophylactic vaccine would represent a major breakthrough towards the management and control of the disease. Selection of the most appropriate antigens for its development is a bottleneck step, especially considering the limited resources allocated. Herein we propose a vaccine strategy based on multiple epitopes from multiple antigens to counteract the parasites´ biological complexity. Epitopes were identified by computer-assisted genome-wide screenings, considering sequence conservation criteria, antigens annotation and sub-cellular localization, high binding affinity to antigen presenting molecules, and lack of cross-reactivity to proteins in cattle and other breeding species. We ultimately provide 31 B-cell, 8 CD4 T-cell, and 15 CD8 T-cell epitope sequences from 30 distinct antigens for the prospective design of a genetic ensemble vaccine against the four trypanosome species responsible for African animal trypanosomiasis.

Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Corrigendum: In silico Design of an Epitope-Based Vaccine Ensemble for Chagas Disease.

[This corrects the article DOI: 10.3389/fimmu.2019.02698.].

In silico Design of an Epitope-Based Vaccine Ensemble for Chagas Disease.

Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ~30% of those chronically infected. By then, the drugs' efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8+ T cell, CD4+ T cell, and B cell epitopes that have <70% identity to human or human microbiome protein sequences and represent the basis toward the development of an epitope-based vaccine against T. cruzi.