Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba.

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.

Zika virus RNA excretion in sweat with concomitant detection in other body fluid specimens.

We evaluated sweat, blood and urine specimens obtained from an ongoing cohort study in Brazil. Samples were collected at pre-established intervals after the initial rash presentation and tested for Zika virus (ZIKV) RNA presence by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). From 254 participants with confirmed infection, ZIKV RNA was detected in the sweat of 46 individuals (18.1%). Sweat presented a median cycle threshold (Ct) of 34.74 [interquartile range (IQR) 33.44-36.04], comparable to plasma (Ct 35.96 - IQR 33.29-36.69) and higher than urine (Ct 30.78 - IQR 28.72-33.22). Concomitant detection with other specimens was observed in 33 (72%) of 46 participants who had a positive result in sweat. These findings represent an unusual and not yet investigated virus shedding through eccrine glands.

An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction.

One of the Leishmania species known to be non-infective to humans is Leishmania (Mundinia) enriettii whose vertebrate host is the guinea pig Cavia porcellus. It is a good model for cutaneous leishmaniasis, chemotherapeutic and molecular studies. In the last years, an increased interest has emerged concerning the L. (Mundinia) subgenus after the finding of Leishmania (M.) macropodum in Australia and with the description of other new/putative species such as L. (M.) martiniquensis and 'L. (M.) siamensis'. This review focused on histopathology, glycoconjugates and innate immunity. The presence of Leishmania RNA virus and shedding of extracellular vesicles by the parasite were also evaluated.

Reactive Oxygen Species (ROS) Are Not a Key Determinant for Zika Virus-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells.

INTRODUCTION: ZIKV is a highly neurotropic virus that can cause the death of infected neuroprogenitor cells through mitochondrial damage and intrinsic apoptotic signaling. In this context, the role of reactive oxygen species (ROS) in neuronal cell death caused by ZIKV still remains elusive. OBJECTIVE: We aimed at evaluating the role of these cellular components in the death of human undifferentiated neuroblastoma cell line infected with ZIKV. RESULTS: ZIKV infection resulted in the extensive death of SH-SY5Y cells with the upregulation of several genes involved in survival and apoptotic responses as well as the colocalization of mitochondrial staining with ZIKV Envelope (E) protein. Notably, levels of intracellular reactive oxygen species (ROS) were not altered during ZIKV infection in undifferentiated SH-SY5Y cells, and consistent with these results, the treatment of infected cells with the widely studied ROS scavenger N-acetylcysteine (NAC) did not prevent cell death in these cells. CONCLUSION: Altogether, our results suggest that excessive ROS production is not the main trigger of SH-SY5Y cells death in ZIKV infection.

Immunomodulatory Properties of Leishmania Extracellular Vesicles During Host-Parasite Interaction: Differential Activation of TLRs and NF-κB Translocation by Dermotropic and Viscerotropic Species.

Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral vs. cutaneous). The purpose of this study was to compare macrophage activation using EVs from three Leishmania species from New World including L. infantum, L. braziliensis, and L. amazonensis. EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. NTA analysis did not show any differences in the EV sizes among the strains. EVs from L. braziliensis and L. infantum failed to induce a pro-inflammatory response. EVs from both L. infantum WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with macrophages, suggesting that LPG solely was not determinant for activation. On the other hand, EVs from L. amazonensis were immunomodulatory inducing NO, TNF-α, IL-6, and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-κB p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from L. amazonensis exhibited a highly percentage of cells positive for NF-κB. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species. For L. infantum, LPG was not determinant for the activation.

Proteomics study of human cord blood reticulocyte-derived exosomes.

Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis.

Highlights of the São Paulo ISEV workshop on extracellular vesicles in cross-kingdom communication.

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host-pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite-parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites.

Corrigendum: Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T Cell Subsets' Distribution.

[This corrects the article on p. 131 in vol. 4, PMID: 27900319.].

Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil.

The double stranded RNA (dsRNA) virus Leishmaniavirus (Totiviridae) was first described in Leishmania guyanensis and L. braziliensis (LRV1), and more recently from L. major and L. aethiopica (LRV2). Parasites bearing LRV1 elicit a higher pro-inflammatory profile, arising through activation of Toll like receptor 3(TLR3) interacting with the viral dsRNA. LRV1 is most common in Leishmania from the Amazon region; however data for other regions of Brazil are more limited. Here we applied PCR tests with validated 'universal' LRV1 primers to search for LRV1 in 40 strains of cultured L. braziliensis from several locales within Minas Gerais State, including patients presenting with atypical lesion pathology. All strains were negative however. These data are in agreement with results from other areas of Southeastern Brazil that LRV1 is relatively uncommon.

Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T cell Subsets' Distribution.

Reticulocyte-derived exosomes (rex) are 30-100 nm membrane vesicles of endocytic origin released during the maturation of reticulocytes to erythrocytes upon fusion of multivesicular bodies with the plasma membrane. Combination of CpG-ODN with rex obtained from BALB/c mice infected with the reticulocyte-prone non-lethal P. yoelii 17X malaria strain (rexPy), had been shown to induce survival and long lasting protection. Here, we show that splenectomized mice are not protected upon rexPy+CpG inmunizations and that protection is restored upon passive transfer of splenocytes obtained from animals immunized with rexPy+CpG. Notably, rexPy immunization of mice induced changes in PD1- memory T cells with effector phenotype. Proteomics analysis of rexPy confirmed their reticulocyte origin and demonstrated the presence of parasite antigens. Our studies thus prove, for what we believe is the first time, that rex from reticulocyte-prone malarial infections are associated with splenic long-lasting memory responses. To try extrapolating these data to human infections, in vitro experiments with spleen cells of human transplantation donors were performed. Plasma-derived exosomes from vivax malaria patients (exPv) were actively uptaken by human splenocytes and stimulated spleen cells leading to changes in T cell subsets.

Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples.

Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.

Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.

Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

Zika virus RNA excretion in sweat with concomitant detection in other body fluid specimens

We evaluated sweat, blood and urine specimens obtained from an ongoing cohort study in Brazil. Samples were collected at pre-established intervals after the initial rash presentation and tested for Zika virus (ZIKV) RNA presence by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). From 254 participants with confirmed infection, ZIKV RNA was detected in the sweat of 46 individuals (18.1%). Sweat presented a median cycle threshold (Ct) of 34.74 [interquartile range (IQR) 33.44-36.04], comparable to plasma (Ct 35.96 - IQR 33.29-36.69) and higher than urine (Ct 30.78 - IQR 28.72-33.22). Concomitant detection with other specimens was observed in 33 (72%) of 46 participants who had a positive result in sweat. These findings represent an unusual and not yet investigated virus shedding through eccrine glands.

Extracellular vesicles in parasitic diseases.

Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite-parasite inter-communication as well as in parasite-host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens.

"Omics" in the study of the major parasitic diseases malaria and schistosomiasis.

Genomic, transcriptomic, proteomic, and metabolomic projects exemplify the "omics" era, and have significantly expanded available data for biomedical research. Recently, next generation sequencing technologies have even more greatly expanded DNA and RNA information. The present challenge is mining this information to obtain meaningful data such as that identifying novel drug targets and vaccine candidates. Several bioinformatics tools and new technologies have been used to high-throughput identification of potential candidates. We illustrate the utilization of new strategies in the study of two major parasitic diseases: schistosomiasis and malaria.

Plasma circulating nucleic acids levels increase according to the morbidity of Plasmodium vivax malaria.

BACKGROUND: Given the increasing evidence of Plasmodium vivax infections associated with severe and fatal disease, the identification of sensitive and reliable markers for vivax severity is crucial to improve patient care. Circulating nucleic acids (CNAs) have been increasingly recognized as powerful diagnostic and prognostic tools for various inflammatory diseases and tumors as their plasma concentrations increase according to malignancy. Given the marked inflammatory status of P. vivax infection, we investigated here the usefulness of CNAs as biomarkers for malaria morbidity. METHODS AND FINDINGS: CNAs levels in plasma from twenty-one acute P. vivax malaria patients from the Brazilian Amazon and 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the human telomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometric dsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as compared to healthy donors (p<0.0001). Importantly, plasma CNAs levels were strongly associated with vivax morbidity (p<0.0001), including a drop in platelet counts (p = 0.0021). These findings were further sustained when we assessed CNAS levels in plasma samples from 14 additional P. vivax patients of a different endemic area in Brazil, in which CNAS levels strongly correlated with thrombocytopenia (p = 0.0072). We further show that plasma CNAs levels decrease and reach physiological levels after antimalarial treatment. Although we found both host and parasite specific genomic sequences circulating in plasma, only host CNAs clearly reflected the clinical spectrum of P. vivax malaria. CONCLUSIONS: Here, we provide the first evidence of increased plasma CNAs levels in malaria patients and reveal their potential as sensitive biomarkers for vivax malaria morbidity.

Aprimoramento da anotação N-terminal de proteínas através da predição de peptídeo sinal em proteínas ortólogase desenvolvimento de uma ferramenta automática para a identificação de grupos ortólogos contendo erros deanotação

O peptídeo sinal é um motivo encontrado, geralmente, na extremidade N-terminal deproteínas e a sua presença determina a entrada na via clássica de transporte intracelular,após a translocação da proteína para o lúmen do retículo endoplasmático. Portanto, apresença ou ausência do peptídeo sinal influencia a função biológica de uma proteína ao serum fator determinante da sua localização subcelular. Como a conservação de função entreproteínas ortólogas é esperada, foi hipotetizado que a localização subcelular e,consequentemente, a presença do peptídeo sinal deveriam, também, se apresentarconservadas. Partindo desta premissa, as predições de peptídeo sinal em proteínasortólogas de cinco espécies de Plasmodium foram analisadas.Predições de peptídeo sinal (SignalP) e informações de ortologia (OrthoMCL-DB)para proteínas de cinco espécies do gênero Plasmodium (Plasmodium falciparum,Plasmodium vivax, Plasmodium knowlesi, Plasmodium berguei e Plasmodium yoelii) foramcombinadas em uma estratégia inovadora, visando a identificação de grupos de proteínasortólogas que apresentam predições de peptídeo sinal divergentes (grupos Mistos).

Asproteínas pertencentes a estes grupos foram submetidas a uma análise comparativabaseada na inspeção visual de alinhamentos múltiplos e de modelos gênicos e regiõesgenômicas flanqueadoras da extremidade N-terminal. Novos modelos gênicos foramsugeridos para aquelas proteínas que apresentavam prováveis erros de anotação desequência, especialmente na região N-terminal. Alguns dos novos modelos gênicos foramvalidados por RT-PCR. Os resultados da inspeção visual foram usados para treinar umaMáquina de Suporte de Vetores (Support Vector Machine) com o objetivo de classificargrupos Mistos em: (1) Com erros de anotação ou (2) Sem erros de anotação. O SVM foiaplicado para classificar os grupos Mistos de cinco bancos de dados, montados a partir devinte e duas espécies.Os grupos contendo proteínas com predições de peptídeo sinal divergentesapresentaram uma alta taxa de erros de anotação. Um total de 478 proteínas dePlasmodium foram reanotadas sendo que a maioria apresentou inversões das suaspredições de peptídeo sinal originais, representando um impacto significativo no conjuntofinal de proteínas destinadas à via clássica de transporte intracelular, principalmente paraPlasmodium vivax e Plasmodium yoelii. O classificador baseado nos dados da inspeçãovisual se mostrou bastante flexível e robusto, apresentando uma performance boa econsistente mesmo frente a cenários variados de agrupamento de espécies.A metodologia proposta introduz uma abordagem simples, porém promissora, para arealização de tarefas de curadoria e controle de qualidade dos dados de anotação desequências proteicas em uma escala genômica. Os resultados do classificador definem a base para seu desenvolvimento em uma ferramenta computacional e os resultados dasreanotações em Plasmodium impactarão a busca por novos alvos vacinais equimioterápicos.

Aprimoramento da anotação N-terminal de proteínas através da predição de peptídeo sinal em proteínas ortólogase desenvolvimento de uma ferramenta automática para a identificação de grupos ortólogos contendo erros deanotação / Improved annotation of N-terminal protein through the prediction of signal peptide in orthologous proteins and developing a tool for automatic Identification of orthologs groups containing errors annotation

O peptídeo sinal é um motivo encontrado, geralmente, na extremidade N-terminal deproteínas e a sua presença determina a entrada na via clássica de transporte intracelular,após a translocação da proteína para o lúmen do retículo endoplasmático. Portanto, apresença ou ausência do peptídeo sinal influencia a função biológica de uma proteína ao serum fator determinante da sua localização subcelular. Como a conservação de função entreproteínas ortólogas é esperada, foi hipotetizado que a localização subcelular e,consequentemente, a presença do peptídeo sinal deveriam, também, se apresentarconservadas. Partindo desta premissa, as predições de peptídeo sinal em proteínasortólogas de cinco espécies de Plasmodium foram analisadas.Predições de peptídeo sinal (SignalP) e informações de ortologia (OrthoMCL-DB)para proteínas de cinco espécies do gênero Plasmodium (Plasmodium falciparum,Plasmodium vivax, Plasmodium knowlesi, Plasmodium berguei e Plasmodium yoelii) foramcombinadas em uma estratégia inovadora, visando a identificação de grupos de proteínasortólogas que apresentam predições de peptídeo sinal divergentes (grupos Mistos).

Asproteínas pertencentes a estes grupos foram submetidas a uma análise comparativabaseada na inspeção visual de alinhamentos múltiplos e de modelos gênicos e regiõesgenômicas flanqueadoras da extremidade N-terminal. Novos modelos gênicos foramsugeridos para aquelas proteínas que apresentavam prováveis erros de anotação desequência, especialmente na região N-terminal. Alguns dos novos modelos gênicos foramvalidados por RT-PCR. Os resultados da inspeção visual foram usados para treinar umaMáquina de Suporte de Vetores (Support Vector Machine) com o objetivo de classificargrupos Mistos em: (1) Com erros de anotação ou (2) Sem erros de anotação. O SVM foiaplicado para classificar os grupos Mistos de cinco bancos de dados, montados a partir devinte e duas espécies.Os grupos contendo proteínas com predições de peptídeo sinal divergentesapresentaram uma alta taxa de erros de anotação. Um total de 478 proteínas dePlasmodium foram reanotadas sendo que a maioria apresentou inversões das suaspredições de peptídeo sinal originais, representando um impacto significativo no conjuntofinal de proteínas destinadas à via clássica de transporte intracelular, principalmente paraPlasmodium vivax e Plasmodium yoelii. O classificador baseado nos dados da inspeçãovisual se mostrou bastante flexível e robusto, apresentando uma performance boa econsistente mesmo frente a cenários variados de agrupamento de espécies.A metodologia proposta introduz uma abordagem simples, porém promissora, para arealização de tarefas de curadoria e controle de qualidade dos dados de anotação desequências proteicas em uma escala genômica. Os resultados do classificador definem a base para seu desenvolvimento em uma ferramenta computacional e os resultados dasreanotações em Plasmodium impactarão a busca por novos alvos vacinais equimioterápicos.