Modulatory Effect of Trypanosoma cruzi Infective Stages in Different Dendritic Cell Populations in vitro

Trypanosoma cruzi is a protozoan parasite that infects at least 7 million persons in the world (OMS, 2019). In endemic areas, infection normally occurs by vectorial transmission; however, outside, it normally happens by blood and includes congenital transmission. The persistence of T. cruzi during infection suggests the presence of immune evasion mechanisms and the modulation of the anti-parasite response to a profile incapable of eradicating the parasite. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) that patrol tissues with a key role in mediating the interface between the innate and adaptive immune response. Previous results from our lab and other groups have demonstrated that T. cruzi modulates the functional properties of DCs, in vitro and in vivo. During vectorial transmission, metacyclic (m) trypomastigotes (Tps) eliminated along with the insect feces reach the mucous membranes or injured skin. When transmission occurs by the hematic route, the parasite stage involved in the infection is the circulating or blood (b) Tp. Here, we studied in vitro the effect of both infective mTp and bTp in two different populations of DCs, bone marrow–derived DCs (BMDCs) and XS106, a cell line derived from epidermal DCs. Results demonstrated that the interaction of both Tps imparts a different effect in the functionality of these two populations of DCs, suggesting that the stage of T. cruzi and DC maturation status could define the immune response from the beginning of the ingress of the parasite, conditioning the course of the infection.Fil: Gutierrez, Brenda Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Lammel, Estela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ramirez, Marcel Ivan. Fundación Oswaldo Cruz; Brasil. Universidade Federal do Paraná; BrasilFil: González, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Poncini, Carolina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From

is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from . The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells . Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis. [Abstract copyright: Copyright © 2020 Gavinho, Sabatke, Feijoli, Rossi, da Silva, Evans-Osses, Palmisano, Lange and Ramirez.

Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy

Extracellular vesicles (EVs) are a unique and heterogeneous class of lipid bilayer nanoparticles secreted by most cells. EVs are regarded as important mediators of intercellular communication in both prokaryotic and eukaryotic cells due to their ability to transfer proteins, lipids and nucleic acids to recipient cells. In addition to their physiological role, EVs are recognized as modulators in pathological processes such as cancer, infectious diseases, and neurodegenerative disorders, providing new potential targets for diagnosis and therapeutic intervention. For a complete understanding of EVs as a universal cellular biological system and its translational applications, optimal techniques for their isolation and characterization are required. Here, we review recent progress in those techniques, from isolation methods to characterization techniques. With interest in therapeutic applications of EVs growing, we address fundamental points of EV‐related cell biology, such as cellular uptake mechanisms and their biodistribution in tissues as well as challenges to their application as drug carriers or biomarkers for less invasive diagnosis or as immunogens.This article is categorized under: Diagnostic Tools > BiosensingTherapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic DiseaseTherapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Diseas

Cell adhesion and Ca2+ signaling activity in stably transfected trypanosoma cruzi epimastigotes expressing the metacyclic stage-specific surface molecule gp82

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca2+ mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca2+ response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca2+ response in HeLa cells, in the same manner as the metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in metacyclic forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilWeb of Scienc

Involvement of the stage-specific 82-kilodalton adhesion molecule of Trypanosoma cruzi metacyclic trypomastigotes in host cell invasion

This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibod

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

Chagas disease is a neglected tropical disease caused by , which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, expresses different molecules on its surface and releases extracellular vesicles (EVs). Here, we have selected a population of epimastigotes (a replicative form) from through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population. The 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells. Altogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

IntroductionChagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs).MethodsHere, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population.ResultsThe 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells.ConclusionsAltogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years