Immunization with a Mu-class glutathione transferase from Echinococcus granulosus induces efficient antibody responses and confers long-term protection against secondary cystic echinococcosis.

Cystic echinococcosis, a zoonosis caused by cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) genetic complex, affects humans and diverse livestock species. Although a veterinary vaccine exhibiting high levels of antibody-mediated protection has successfully reached the market, the large genetic diversity among parasite isolates and their particular host preferences, makes still necessary the search for novel vaccine candidates. Glutathione transferases (GSTs) constitute attractive targets for immunoprophylaxis due to their outstanding relevance in helminth detoxification processes, against both exogenous and endogenous stressors. Among the six GSTs known to be expressed in E. granulosus s.l., EgGST1 (Mu-class), EgGST2 (Sigma-class), and EgGST3 (a still non-classifiable isoenzyme), show the highest proteomic expression. Therefore, their recombinant forms -rEgGST1, rEgGST2 and rEgGST3- were herein analyzed regarding their potential to induce long-term antiparasite protection in mice. Only immunization with rEgGST1 induced long-lasting protection; and accordingly, rEgGST1-specific antibodies enhanced the parasite killing through both the classical activation of the host complement system and the antibody-dependent cellular cytotoxicity by macrophages. These results support further testing of rEgGST1 as a vaccine candidate in diverse hosts due to the broad expression of EgGST1 in different parasite stages and tissues.

Recombinant CD5 and CD6 Ectodomains Induce Antiparasitic and Immunomodulatory Effects in Secondary Cystic Echinococcosis.

Scavenger receptors participate in a wide range of biological functions after binding to multiple non-self or altered self-ligands. Among them, CD5 and CD6 are lymphocyte scavenger receptors known to interact with different microbial-associated molecular patterns, and the administration of the recombinant soluble ectodomains of human CD5 (rshCD5) and/or CD6 (rshCD6) has shown therapeutic/prophylactic potential in experimental models of fungal, bacterial and echinococcal infections. The latter is a zoonosis caused by the larval stage of the cestode parasite Echinococcus granulosus sensu lato, which in humans can induce secondary cystic echinococcosis (CE) after the spillage of protoscoleces contained within fertile cysts, either spontaneously or during surgical removal of primary hydatid cysts. Herein, we have analysed the mechanisms behind the significant protection observed in the mouse model of secondary CE following prophylactic administration of rshCD5 or rshCD6. Our results show that both molecules exhibit intrinsic antiparasitic activities in vitro, as well as immunomodulatory functions during early secondary CE, mainly through Th1/Th17 cytokine bias and promotion of peritoneal polyreactive antibodies. These data support the relevance of the parasite components bound by rshCD5 and rshCD6, as well as the potential of their prophylactic administration as a useful strategy to reduce secondary CE in patients.

Mouse model of secondary cystic echinococcosis.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the cestode Echinococcus granulosus sensu lato (s. l.), a genetic complex composed of five species: E. granulosus sensu stricto (s. s.), E. equinus, E. ortleppi, E. canadensis, and E. felidis. The parasite requires two mammalian hosts to complete its life cycle: a definitive host (mainly dogs) harboring the adult parasite in its intestines, and an intermediate host (mostly farm and wild ungulates) where hydatid cysts develop mainly in the liver and lungs. Humans are accidental intermediate hosts, being susceptible to either primary or secondary forms of CE; the first one due to the ingestion of oncospheres, and the second one because of the spillage of protoscoleces (PSC) contained within a primary cyst. Secondary CE is a serious medical problem, and can be modeled in immunocompetent mice (a non-natural intermediate host) through the intraperitoneal inoculation of viable PSC from E. granulosus s. l. This model is useful to study not only the immunobiology of CE, but also to test new chemotherapeutics or therapeutical protocols, to explore novel vaccine candidates, and to evaluate alternative diagnostic and/or follow-up tools. The mouse model of secondary CE involves two sequential stages: an early stage of parasite pre-encystment (PSC develop into hydatid cysts in the peritoneal cavity of mice), and a late or chronic stage of parasite post-encystment (already differentiated cysts slowly grow during the whole host lifespan). This model is a time-consuming infection, whose outcome depends on several factors like the parasite infective dose, the mouse strain, and the parasite species/genotype. Thus, such variables should always be adjusted according to the research objectives. Herein, the general materials and procedures needed to establish secondary CE in mice are described, as well as several useful tips and recommendations.

Using the model cestode Taenia crassiceps for the study of cysticercosis.

Taenia solium is the aetiological agent of taeniasis/cysticercosis, one of the most severe neglected tropical diseases (NTD) according to the World Health Organization (WHO). The life cycle of T. solium alternates between pigs (intermediate host) and humans (definitive host). In addition, humans can act as accidental intermediate hosts if they ingest infective eggs. In this case, the most severe condition of the disease occurs when parasites invade the central nervous system, causing neurocysticercosis (NCC). The complexity of the life cycle of T. solium imposes a barrier to study this pathogen thoroughly. Thus, related species, such as T. crassiceps are commonly used. Due to its capacity to multiply asexually, T. crassiceps can be maintained by serial passage in laboratory mice in standard biosecurity level facilities. In addition, an in vitro system to generate cysticerci in the presence of feeder cells has been recently developed. Despite model species display biological differences with their zoonotic counterparts, they have historically helped to understand the biology of the related pathogenic species and hence, generate improvements in NTD detection and control. In this chapter, we describe the procedures to carry out both in vivo and in vitro systems for T. crassiceps in the laboratory.

A Comparative Analysis of the Protein Cargo of Extracellular Vesicles from Helminth Parasites.

Helminth parasites cause debilitating-sometimes fatal-diseases in humans and animals. Despite their impact on global health, mechanisms underlying host-parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization.

Experimental cystic echinococcosis as a proof of concept for the development of peptide-based vaccines following a novel rational workflow.

Vaccines are among the most important advances in medicine throughout the human history. However, conventional vaccines exhibit several drawbacks in terms of design and production costs. Peptide-based vaccines are attractive alternatives, since they can be designed mainly in silico, can be produced cheaply and safely, and are able to induce immune responses exclusively towards protective epitopes. Yet, a proper peptide design is needed, not only to generate peptide-specific immune responses, but also for them to recognize the native protein in the occurrence of a natural infection. Herein, we propose a rational workflow for developing peptide-based vaccines including novel steps that assure the cross-recognition of native proteins. In this regard, we increased the probability of generating efficient antibodies through the selection of linear B-cell epitopes free of post-translational modifications followed by analyzing the 3D-structure similarity between the peptide in-solution vs. within its parental native protein. As a proof of concept, this workflow was applied to a set of seven previously suggested potential protective antigens against the infection by Echinococcus granulosus sensu lato. Finally, two peptides were obtained showing the capacity to induce specific antibodies able to exert anti-parasite activities in different in vitro settings, as well as to provide significant protection in the murine model of secondary echinococcosis.

Mucins Shed from the Laminated Layer in Cystic Echinococcosis Are Captured by Kupffer Cells via the Lectin Receptor Clec4F.

Cystic echinococcosis is caused by the larval stages (hydatids) of cestode parasites belonging to the species cluster Echinococcus granulosus sensu lato, with E. granulosus sensu stricto being the main infecting species. Hydatids are bladderlike structures that attain large sizes within various internal organs of livestock ungulates and humans. Hydatids are protected by the massive acellular laminated layer (LL), composed mainly of mucins. Parasite growth requires LL turnover, and abundant LL-derived particles are found at infection sites in infected humans, raising the question of how LL materials are dealt with by the hosts. In this article, we show that E. granulosus sensu stricto LL mucins injected into mice are taken up by Kupffer cells, the liver macrophages exposed to the vascular space. This uptake is largely dependent on the intact mucin glycans and on Clec4F, a C-type lectin receptor which, in rodents, is selectively expressed in Kupffer cells. This uptake mechanism operates on mucins injected both in soluble form intravenously (i.v.) and in particulate form intraperitoneally (i.p.). In mice harboring intraperitoneal infections by the same species, LL mucins were found essentially only at the infection site and in the liver, where they were taken up by Kupffer cells via Clec4F. Therefore, shed LL materials circulate in the host, and Kupffer cells can act as a sink for these materials, even when the parasite grows in sites other than the liver.

Identification of Omega-class glutathione transferases in helminths of the Taeniidae family.

Glutathione transferase enzymes (GSTs) are believed to be a major detoxification system in helminth parasites and have been associated with immunomodulation of the host response. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least five different GSTs, but no Omega-class enzymes have been reported in this parasite or in any other cestode. Herein we report the identification of a new member of the GST superfamily in E. granulosus s.l., which is phylogenetically related to the Omega-class: EgrGSTO. Through mass spectrometry, we showed that the 237 amino acids protein EgrGSTO is expressed by the parasite. Moreover, we identified homologues of EgrGSTO in other eight members of the Taeniidae family, including E. canadensis, E. multilocularis, E. oligarthrus, Hydatigera taeniaeformis, Taenia asiatica, T. multiceps, T. saginata and T. solium. A manual sequence inspection and rational modification yielded eight Taeniidae's GSTO sequences, each one encoding for a 237 aa polypeptide showing 80.2% overall identity. To the best of our knowledge, this is the first description of genes encoding for Omega-class GSTs in worms belonging to the Taeniidae family -that at least in E. granulosus s.l. is expressed as a protein- suggesting the gene encodes for a functional protein.

First genetic characterization of human cystic echinococcosis in Uruguay.

Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to determine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog-sheep cycle. However, other intermediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active parasite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue determinar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

First genetic characterization of human cystic echinococcosis in Uruguay

[ABSTRACT]. Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to deter- mine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog–sheep cycle. However, other inter- mediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active par- asite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

[RESUMEN]. La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue deter- minar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

[RESUMO]. A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

CD6 deficiency impairs early immune response to bacterial sepsis.

CD6 is a lymphocyte-specific scavenger receptor expressed on adaptive (T) and innate (B1a, NK) immune cells, which is involved in both fine-tuning of lymphocyte activation/differentiation and recognition of bacterial-associated molecular patterns (i.e., lipopolysaccharide). However, evidence on CD6's role in the physiological response to bacterial infection was missing. Our results show that induction of monobacterial and polymicrobial sepsis in Cd6 -/- mice results in lower survival rates and increased bacterial loads and pro-inflammatory cytokine levels. Steady state analyses of Cd6 -/- mice show decreased levels of natural polyreactive antibodies, concomitant with decreased cell counts of spleen B1a and marginal zone B cells. Adoptive transfer of wild-type B cells and mouse serum, as well as a polyreactive monoclonal antibody improve Cd6 -/- mouse survival rates post-sepsis. These findings support a nonredundant role for CD6 in the early response against bacterial infection, through homeostatic expansion and functionality of innate-related immune cells.

Expanding the family of Mu-class glutathione transferases in the cestode parasite Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) perform catalytic and non-catalytic activities, mostly involved in stress-response and cell detoxification. Helminth parasites express several GSTs of multiple classes that are involved in the neutralization of potentially harmful oxidants, and in the inactivation or removal of xenobiotics. Additionally, GSTs participate in immunomodulatory processes that facilitate the parasite establishment and survival within its host. In Echinococcus granulosus sensu lato (s.l.) -the cestode parasite responsible for cystic echinococcosis- only one Mu-class GST has been reported. In the present work, by using bioinformatic and proteomic approaches we searched for novel Mu-class GSTs potentially involved in the parasite oxidative-stress metabolism. In the genome of E. granulosus s.l., 6 GST-related sequences were found to constitute a strongly conserved phylogenetical clade with Mu-class members. Among them, 5 displayed conserved gene structure (exon/intron), as well as specific residues and motifs characteristic of Mu-class enzymes. By proteomic analysis, 3 Mu-GSTs were identified to be expressed in the protoscolex parasite stage, 2 of them being firstly described as Mu-class GSTs here. The existence of more than one productive Mu-GST gene expands the parasite xenobiotic phase II metabolism, which might have beneficial roles on E. granulosus s.l. ability to successfully infect its host.

Unraveling post-translational modifications in Echinococcus granulosus sensu lato.

Echinococcus granulosus sensu lato (s.l.) is the helminth parasite responsible for cystic echinococcosis, a neglected tropical disease currently affecting millions of people worldwide. Incomplete knowledge on the parasite biochemistry contributes, at least partially, to the limited development of useful biotechnological advances for the infection control. In this sense, little information is available regarding post-translational modifications (PTMs) occurring in E. granulosus s.l. proteins, which ultimately may affect the performance of biotechnological products to be developed. Therefore, we report here a proteomic analysis of the parasite PTMs identified through FindMod software applied to a set of tegumental proteins previously characterized by mass spectrometry (MALDI-TOF/TOF) analysis of protein spots from a 2D electrophoresis gel. Manual searches for already annotated proteins exhibiting such PTMs were also performed within proteome databases of E. granulosus s.l. and other platyhelminthes. In addition, key enzymes involved in PTMs modifications were searched for within E. granulosus s.l. proteome. Finally, the presence of selected PTMs was further confirmed by a high-resolution proteomic approach (nanoLC-MS/MS). A set of 22 different PTMs most likely to be present in the parasite was suggested, 9 of them with high confidence as they were identified in the same m/z fragment by both proteomic techniques (acetylation, deamidation, deamidation followed by methylation, mono- and di-hydroxylation, mono- and di-methylation, S-nitrosylation and phosphorylation). Interestingly, 5 PTMs were herein identified for the first time in E. granulosus s.l. proteins. Our results expand the scarcely studied topic of PTMs in platyhelminthes.

First genetic characterization of human cystic echinococcosis in Uruguay / Primera caracterización genética de la equinococosis quística humana en Uruguay / Primeira caracterização genética da equinococose cística humana no Uruguai

ABSTRACT Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to determine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog-sheep cycle. However, other intermediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active parasite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

RESUMEN La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue determinar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

RESUMO A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

Ultrastructural characterization of the tegument in protoscoleces of Echinococcus ortleppi.

Cystic echinococcosis is a globally distributed zoonosis caused by cestodes of the Echinococcus granulosus sensu lato (s.l.) complex, with Echinococcus ortleppi mainly involved in cattle infection. Protoscoleces show high developmental plasticity, being able to differentiate into either adult worms or metacestodes within definitive or intermediate hosts, respectively. Their outermost cellular layer is called the tegument, which is important in determining the infection outcome through its immunomodulating activities. Herein, we report an in-depth characterization of the tegument of E. ortleppi protoscoleces performed through a combination of scanning and transmission electron microscopy techniques. Using electron tomography, a three-dimensional reconstruction of the tegumental cellular territories was obtained, revealing a novel structure termed the 'tegumental vesicular body' (TVB). Vesicle-like structures, possibly involved in endocytic/exocytic routes, were found within the TVB as well as in the parasite glycocalyx, distal cytoplasm and close inner structures. Furthermore, parasite antigens (GST-1 and AgB) were unevenly localised within tegumental structures, with both being detected in vesicles found within the TBV. Finally, the presence of host (bovine) IgG was also assessed, suggesting a possible endocytic route in protoscoleces. Our data forms the basis for a better understanding of E. ortleppi and E. granulosus s.l. structural biology.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

Polyreactive antibodies as potential humoral biomarkers of host resistance to cystic echinococcosis.

Polyreactive antibodies (pAb) bind to a broad range of unrelated structures, providing hosts with functional components able to rapidly recognize and protect against different pathogens. However, their roles against helminth parasites are still unexplored. Here, pAb profiles were analysed in cystic echinococcosis (CE), a zoonosis caused by the cestode Echinococcus granulosus sensu lato. Levels of anti-DNP (2,4-dinitrophenyl-hapten) antibodies were measured as a surrogate parameter of pAb in different biological settings. Firstly, levels of serum and peritoneal pAb were measured during early experimental secondary CE, using both high (Balb/c) and low (C57Bl/6) susceptible mouse strains. Serum pAb mostly differed in normal mice, being pAb levels of IgG subclasses with poor anti-parasite activities predominant in Balb/c animals. Conversely, peritoneal pAb isotypes/subclasses with efficient anti-parasite activities predominated in normal and infected C57Bl/6 mice. Secondly, sera from potentially resistant patients, susceptible individuals and healthy donors were analysed, showing higher pAb levels of the IgA and IgG-particularly IgG1-isotypes in potentially resistant individuals compared to control groups. Finally, since remarkable differences were observed in pAb profiles according to the intrinsic host susceptibility to the infection, we proposed here that pAb might be considered as potential humoral biomarkers for host resistance to CE.

The Lymphocytic Scavenger Receptor CD5 Shows Therapeutic Potential in Mouse Models of Fungal Infection.

Invasive fungal diseases represent an unmet clinical need that could benefit from novel immunotherapeutic approaches. Host pattern recognition receptors (e.g., Toll-like receptors, C-type lectins, or scavenger receptors) that sense conserved fungal cell wall constituents may provide suitable immunotherapeutic antifungal agents. Thus, we explored the therapeutic potential of the lymphocyte class I scavenger receptor CD5, a nonredundant component of the antifungal host immune response that binds to fungal ß-glucans. Antifungal properties of the soluble ectodomain of human CD5 (shCD5) were assessed in vivo in experimental models of systemic fungal infection induced by pathogenic species (Candida albicans and Cryptococcus neoformans). In vitro mechanistic studies were performed by means of fungus-spleen cell cocultures. shCD5-induced survival of lethally infected mice was dose and time dependent and concomitant with reduced fungal load and increased leukocyte infiltration in the primary target organ. Additive effects were observed in vivo after shCD5 was combined with suboptimal doses of fluconazole. Ex vivo addition of shCD5 to fungus-spleen cell cocultures increased the release of proinflammatory cytokines involved in antifungal defense (tumor necrosis factor alpha and gamma interferon) and reduced the number of viable C. albicans organisms. The results prompt further exploration of the adjunctive therapeutic potential of shCD5 in severe invasive fungal diseases.