Association of IL-9, IL-10, and IL-17 Cytokines With Hepatic Fibrosis in Human Schistosoma mansoni Infection.

This is a case series study to evaluate immunological markers associated with schistosomiasis advanced fibrosis, including 69 patients from an endemic area from the State of Sergipe and from the Hepatology Service of the University Hospital in Sergipe, Brazil. Hepatic fibrosis was classified based on Niamey protocol for ultrasonography (US). Immune response to Schistosoma mansoni antigens was evaluated by stimulating peripheral blood mononuclear cells (PBMCs) from these patients with either adult worm (SWAP-10 µg/ml) or egg (SEA-10 µg/ml) antigens or purified protein derivative of turberculin (PPD-10 µg/ml) or phytohemagglutinin (PHA-1 µg/ml) for 72 h. The levels of IFN-γ, TNF-α, IL-5, IL-10, and IL-17 were measured in these supernatants by ELISA and IL-9 by Luminex. Single nucleotide polymorphisms in IL-17, IL10, and CD209 genes were genotyped using TaqMan probe by qPCR. Higher levels of IL-9, IL-10, and IL-17 were found in PBMC supernatants of patients with advanced hepatic fibrosis. Direct correlations were detected between IL-9 and IL-17 levels with US spleen sizes, portal vein diameters, and periportal thickening. The CD209 rs2287886 AG polymorphism patients produce higher IL-17 levels. Together, these data suggest a role of these cytokines in the immunopathogenesis of advanced fibrosis in human schistosomiasis.

FMRF-NH2 -related neuropeptides in Biomphalaria spp., intermediate hosts for schistosomiasis: Precursor organization and immunohistochemical localization.

Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.

Identification and localization of a gonadotropin-releasing hormone-related neuropeptide in Biomphalaria, an intermediate host for schistosomiasis.

Freshwater snails of the genus Biomphalaria serve as obligatory hosts for the digenetic trematode Schistosoma mansoni, the causative agent for the most widespread form of intestinal schistosomiasis. Within Biomphalaria, S. mansoni larvae multiply and transform into the cercariae form that can infect humans. Trematode development and proliferation is thought to be facilitated by modifications of host behavior and physiological processes, including a reduction of reproduction known as "parasitic castration." As neuropeptides participate in the control of reproduction across phylogeny, a neural transcriptomics approach was undertaken to identify peptides that could regulate Biomphalaria reproductive physiology. The present study identified a transcript in Biomphalaria alexandrina that encodes a peptide belonging to the gonadotropin-releasing hormone (GnRH) superfamily. The precursor and the predicted mature peptide, pQIHFTPDWGNN-NH2 (designated Biom-GnRH), share features with peptides identified in other molluscan species, including panpulmonates, opisthobranchs, and cephalopods. An antibody generated against Biom-GnRH labeled neurons in the cerebral, pedal, and visceral ganglia of Biomphalaria glabrata. GnRH-like immunoreactive fiber systems projected to all central ganglia. In the periphery, immunoreactive material was detected in the ovotestis, oviduct, albumen gland, and nidamental gland. As these structures serve crucial roles in the production, transport, nourishment, and encapsulation of eggs, disruption of the GnRH system of Biomphalaria could contribute to reduced reproductive activity in infected snails.

Schistosoma mansoni infection as a trigger to collapsing glomerulopathy in a patient with high-risk APOL1 genotype.

BACKGROUND: Schistosoma mansoni schistosomiasis (SM) remains a public health problem in Brazil. Renal involvement is classically manifested as a glomerulopathy, most often membranoproliferative glomerulonephritis or focal and segmental glomerulosclerosis. We report a case of collapsing glomerulopathy (CG) associated with SM and high-risk APOL1 genotype (HRG). CASE REPORT: A 35-year-old male was admitted for hypertension and an eight-month history of lower-limb edema, foamy urine, and increased abdominal girth. He had a recent diagnosis of hepatosplenic SM, treated with praziquantel, without clinical improvement. Laboratory tests revealed serum creatinine 1.89mg/dL, blood urea nitrogen (BUN) 24mg/dL, albumin 1.9g/dL, cholesterol 531mg/dL, low-density lipoprotein 426mg/dL, platelets 115000/mm3, normal C3/C4, antinuclear antibody (ANA), rheumatoid factor (RF), and antineutrophil cytoplasmic antibodies (ANCA), negative serologies for hepatitis C virus (HCV) and human immunodeficiency virus (HIV), HBsAg negative and AntiHBc IgG positive, no hematuria or leukocyturia, 24 hour proteinuria 6.56g and negative serum and urinary immunofixation. Kidney biopsy established the diagnosis of CG. A treatment with prednisone was started without therapeutic response, progressing to end-stage kidney disease 19 months later. Molecular genetics investigation revealed an HRG. CONCLUSIONS: This is the first report of CG associated with SM in the setting of an HRG. This case highlights the two-hit model as a mechanism for CG pathogenesis, where the high-risk APOL1 genotype exerts a susceptibility role and SM infection serves as a trigger to CG.

Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.

BACKGROUND: Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE: The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS: By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS: 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS: Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine.

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Interactions of amphipathic α-helical MEG proteins from Schistosomamansoni with membranes.

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.

Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host.

Sm16 is an immunomodulatory protein that seems to play a key role in the suppression of the cutaneous inflammatory response during Schistosoma mansoni penetration of the skin of definitive hosts. Therefore, Sm16 represents a potential target for protective immune responses induced by vaccination. In this work, we generated the recombinant protein rSm16 and produced polyclonal antibodies against this protein to evaluate its expression during different parasite life-cycle stages and its location on the surface of the parasite. In addition, we analyzed the immune responses elicited by immunization with rSm16 using two different vaccine formulations, as well as its ability to induce protection in Balb/c mice. In order to explore the biological function of Sm16 during the course of experimental infection, RNA interference was also employed. Our results demonstrated that Sm16 is expressed in cercaria and schistosomula and is located in the schistosomula surface. Despite humoral and cellular immune responses triggered by vaccination using rSm16 associated with either Freund's or alum adjuvants, immunized mice presented no reduction in either parasite burden or parasite egg laying. Knockdown of Sm16 gene expression in schistosomula resulted in decreased parasite size in vitro but had no effect on parasite survival or egg production in vivo. Thus, our findings demonstrate that although the vaccine formulations used in this study succeeded in activating immune responses, these failed to promote parasite elimination. Finally, we have shown that Sm16 is not vital for parasite survival in the definitive host and hence may not represent a suitable target for vaccine development.

Influence of a TNF-α Polymorphism on the Severity of Schistosomiasis Periportal Fibrosis in the Northeast of Brazil.

AIMS: The proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) is an essential component in the host immune response to infection, and it has been reported to be an important mediator in severe periportal fibrosis (PPF). We hypothesized that the (-G308A) polymorphism of the TNF-α gene is associated with the severity of PPF and that these polymorphisms influence TNF-α expression. METHODS: In this cross-sectional study, we genotyped these polymorphisms within the TNF-α gene in 256 Brazilian subjects infected with Schistosoma mansoni, with different patterns of PPF. RESULTS: The genotype (-308) AA was associated with a significant increase in the risk to advanced PPF (OR = 4.60; p = 0.009). In addition, median levels of TNF-α were higher in patients with moderate to advanced PPF, compared with mild fibrosis (20 and 17.3 pg/mL, respectively; p = 0.040). There was no association between average serum levels of TNF-α and (-G308A) TNF-α polymorphism. CONCLUSIONS: Our results suggest the (-308) AA genotype may be a risk factor for severity in advanced PPF, in this Brazilian population, and could potentially be used to predict the severity of advanced PPF in schistosomiasis.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection.

Schistosomiasis remains a disease of major global public health concern since it is a chronic and debilitating illness. The widely distributed Schistosoma mansoni that causes intestinal schistosomiasis represents a great threat. Its world-wide distribution is permitted by the broad geographic range of the susceptible species of its intermediate host, Biomphalaria, which serves as an obligatory host for the larval stage, at which humans get infected. The objectives were to identify the proteins responsible for the snails' compatibility outcome through differentiation between the total proteins among Biomphalaria alexandrina snails at different ages. The work was conducted on snails that differ in age and genetic backgrounds. Four subgroups (F1) from the progeny of self-reproduced susceptible and resistant snails (F0) were studied. Infection rates of these subgroups (young susceptible, adult susceptible, young resistant and adult resistant) were 90 %, 75 %, 40 % and 0 %, respectively. Using Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis (SDS-PAGE), differences in protein expression were evaluated between adult and young snails of different subgroups. Dice similarity coefficient was calculated to determine the percentage of band sharing among the experimental subgroups. The results showed that the combination of similarities between age and compatibility status of the snails, lead to the highest similarity coefficient, followed by the combination of similarities of both genetic origin and age, even though they differ in the compatibility status. On the other hand, the differences in the genetic background, age and compatibility status, lead to the least similarity index. It was also found that the genetic background in young snails plays a major role in the determination of their compatibility, while the internal defense system has the upper hand in determining the level of adult compatibility. In conclusion, the findings of the present work highlight the great impact of the snail age in concomitance with the genetics and the internal defense in the determination of B. alexandrina/S.mansoni compatibility. Future works are recommended, as further characterization of the shared protein bands among the studied subgroups is needed to clarify their role in host-parasite relationship.

Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni.

Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.

Aspectos adaptativos de Schistosoma mansoni na fase esquistossômulo: abordagem in vivo e in vitro

Dentre as formas evolutivas do Schistosoma mansoni, o esquistossômulo é uma das mais estudadas para desenvolvimento de novos fármacos e sistemas diagnósticos. Embora a transformação in vitro seja vantajosa, é importante discutir as limitações do uso de resultados obtidos a partir de parasitos cultivados in vitro em relação aos obtidos no processo natural de infecção. No presente trabalho, esquistossômulos obtidos in vivo e in vitro foram comparados em relação a capacidade de captar sondas fluorescentes, específicas para estruturas internas celulares ou membranas superficiais do parasito. As sondas empregadas para membranas e estruturas internas mostraram marcação similar entre parasitos obtidos in vitro, por transformação mecânica (Mec) e por penetração em pele (Pel). No entanto, diferenças foram observadas quando estes parasitos foram comparados a outros obtidos in vivo pelo método de Clegg (Clegg et al,1965), sendo detectado um aumento da permeabilidade de membranas.

Os dados sugerem que nos esquistossômulos cultivados in vivo o metabolismo é mais ativo nas células superficiais e que durante sua permanência por até 72 horas na pele há um extenso turnover da superfície do parasito, envolvendo moléculas internas e uma umento da liberação de imunógenos. A aumentada permeabilidade pode permitir ainda a captação de moléculas, capazes de estimular o crescimento do parasito. Além disso, bibliotecas de esquistossômulos Mec cultivados em soro portal (SPO3h eSPO12h) e soro periférico de hamster (SPE3h, SPE12h) foram comparadas utilizando sequenciamento de nova geração (NGS) e análise da expressão de genes específicos. Após o sequenciamento e análise dos genes expressos uma alta similaridade entre as réplicas foi encontrada. Comparando as amostras SPO3h versus SPO12h 58 transcritos se mostraram diferencialmente expressos. De acordo com as anotações de termos de ontologia gênica (GO) os genes diferencialmente expressos após 12 horas de contato com o soro portal de hamster, estão ligados a processos importantes ao desenvolvimento até vermes adultos. Assim, este estudo viabilizou um maior entendimento de mecanismos de controle sobre a internalização de moléculas pelo parasito no estádio larvário intravascular, bem como da regulação de sua expressão de genes quando o mesmo se encontra no sistema porta hepático do hospedeiro, onde as formas adultas são essenciais para desenvolvimento da doença

Aspectos adaptativos de Schistosoma mansoni na fase esquistossômulo: abordagem in vivo e in vitro / Adaptive aspects of Schistosoma mansoni in schistosomulum phase: in vivo and in vitro approach

Dentre as formas evolutivas do Schistosoma mansoni, o esquistossômulo é uma das mais estudadas para desenvolvimento de novos fármacos e sistemas diagnósticos. Embora a transformação in vitro seja vantajosa, é importante discutir as limitações do uso de resultados obtidos a partir de parasitos cultivados in vitro em relação aos obtidos no processo natural de infecção. No presente trabalho, esquistossômulos obtidos in vivo e in vitro foram comparados em relação a capacidade de captar sondas fluorescentes, específicas para estruturas internas celulares ou membranas superficiais do parasito. As sondas empregadas para membranas e estruturas internas mostraram marcação similar entre parasitos obtidos in vitro, por transformação mecânica (Mec) e por penetração em pele (Pel). No entanto, diferenças foram observadas quando estes parasitos foram comparados a outros obtidos in vivo pelo método de Clegg (Clegg et al,1965), sendo detectado um aumento da permeabilidade de membranas...

Os dados sugerem que nos esquistossômulos cultivados in vivo o metabolismo é mais ativo nas células superficiais e que durante sua permanência por até 72 horas na pele há um extenso turnover da superfície do parasito, envolvendo moléculas internas e uma umento da liberação de imunógenos. A aumentada permeabilidade pode permitir ainda a captação de moléculas, capazes de estimular o crescimento do parasito. Além disso, bibliotecas de esquistossômulos Mec cultivados em soro portal (SPO3h eSPO12h) e soro periférico de hamster (SPE3h, SPE12h) foram comparadas utilizando sequenciamento de nova geração (NGS) e análise da expressão de genes específicos. Após o sequenciamento e análise dos genes expressos uma alta similaridade entre as réplicas foi encontrada. Comparando as amostras SPO3h versus SPO12h 58 transcritos se mostraram diferencialmente expressos. De acordo com as anotações de termos de ontologia gênica (GO) os genes diferencialmente expressos após 12 horas de contato com o soro portal de hamster, estão ligados a processos importantes ao desenvolvimento até vermes adultos. Assim, este estudo viabilizou um maior entendimento de mecanismos de controle sobre a internalização de moléculas pelo parasito no estádio larvário intravascular, bem como da regulação de sua expressão de genes quando o mesmo se encontra no sistema porta hepático do hospedeiro, onde as formas adultas são essenciais para desenvolvimento da doença...

Human plasma lipid modulation in schistosomiasis mansoni depends on apolipoprotein E polymorphism.

BACKGROUND: Schistosomiasis mansoni is a parasitic liver disease, which causes several metabolic disturbances. Here, we evaluate the influence of Apolipoprotein E (APOE) gene polymorphism, a known modulator of lipid metabolism, on plasma lipid levels in patients with hepatosplenic schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were used for APOE genotyping and to measure total cholesterol (TC), LDL-C, HDL-C and triglycerides. Schistosomiasis patients had reduced TC, LDL-C and triglycerides (25%, 38% and 32% lower, respectively; P<0.0001) compared to control individuals, whereas HDL-C was increased (10% higher; P = 0.0136). Frequency of the common alleles, ε2, ε3 and ε4, was similar (P = 0.3568) between controls (n = 108) and patients (n = 84), implying that APOE genotype did not affect susceptibility to the advanced stage of schistosomiasis. Nevertheless, while patient TC and LDL-C levels were significantly reduced for each allele (except TC in ε2 patients), changes in HDL-C and triglycerides were noted only for the less common ε2 and ε4 alleles. The most striking finding, however, was that accepted regulation of plasma lipid levels by APOE genotype was disrupted by schistosomiasis. Thus, while ε2 controls had higher TC and LDL-C than ε3 carriers, these parameters were lower in ε2 versus ε3 patients. Similarly, the inverse relationship of TG levels in controls (ε2>ε3>ε4) was absent in patients (ε2 or ε4>ε3), and the increase in HDL-C of ε2 or ε4 patients compared to ε3 patients was not seen in the control groups. CONCLUSION/SIGNIFICANCE: We confirm that human schistosomiasis causes dyslipidemia and report for the first time that certain changes in plasma lipid and lipoprotein levels depend on APOE gene polymorphism. Importantly, we also concluded that S. mansoni disrupts the expected regulation of plasma lipids by the different ApoE isoforms. This finding suggests ways to identify new metabolic pathways affected by schistosomiasis and also potential molecular targets to treat associated morbidities.

Identification of a vaccine against schistosomiasis using bioinformatics and molecular modeling tools.

Schistosomiasis is a serious public health problem in Brazil and worldwide. Although the drugs used to treatment schistosomiasis are effective, the disease continues to expand in all endemic countries due to constant reinfection, poor sanitation, and the lack of effective programs for disease control. However, advances generated through genome projects have provided important information that has improved the understanding of the biology of this parasite. These advances, associated with the advent of bioinformatic analysis, are becoming an important tool in reverse vaccinology. Through database access to the DNA and protein sequences of Schistosoma mansoni and the use of bioinformatics programs, fourteen epitopes were identified. Five epitopes were obtained from proteins whose immunogenic potential had already been assessed in other studies (KP), and nine whose immunogenic potential is unknown (UP). To improve stimulation of the host immune system, the selected epitopes were modeled with a sugar moiety. After this addition, all of the epitopes showed structures similar to those observed in the native proteins, but only eleven of the peptides presented thermodynamically stable structures. Prediction analysis and molecular modeling showed that the glycopeptides presented here are important targets in the search for a vaccine against schistosomiasis. Additionally, they suggest that these molecules may be used in immunological assays to evaluate the level of protection, the effect on pathology reduction and the profile of cytokines and antibodies induced by them.

Estudo funcional da proteína quinase JNK de Schistosoma mansoniatravés de expressão heteróloga no organismo modelo Caenorhabditis elegans

A identificação e caracterização dos mecanismos e moléculas envolvidos em sinalização celular são essenciais para o entendimento da biologia parasitária do S. mansoni. Proteína quinases desempenham papel chave em vias de sinalização e tem sido propostas como potenciais alvos para o desenvolvimento de novas drogas anti-Schistosoma. Visto que a caracterização funcional em S. mansoni é dificultada por limitações nos métodos de transformação genética deste parasito, o presente estudo propõe o uso de C. elegans como um modelo para a expressão heteróloga de genes de S. mansoni que codificam proteínas quinases. Genes que codificam proteína quinase sem S. mansoni,homólogos aos identificados em C. elegans, foram selecionados pelo nosso grupo a partir do proteoma do parasito através de uma abordagem filogenômica. Inicialmente, foi selecionada a proteína quinase JNK, que participa da via de sinalização das MAP quinases para a realização das análises experimentais. Em C. elegans, a proteína JNK está relacionada ao aumento de longevidade e da resistência aos estresses térmico e oxidativo.

Oligonucleotídeos específicos foram desenhados paraamplificar a região promotora do gene emC. elegansbem como as regiões codificantes(CDS) em ambos os organismos. A região promotora foi amplificada a partir do DNAgenômico extraído deC. elegansadultos. O RNA total foi extraído de esquistossômuloseC. elegansadultos. As CDS foram amplificadas a partir do cDNA sintetizado e osfragmentos de DNA resultantes foram clonados emE. coliDH5α. As construçõesobtidas foram digeridas com enzimas de restrição selecionadas de forma a linearizar ovetor contendo a região promotora e recuperar as CDS. Posteriormente, foramrealizadas subclonagens através da ligação das CDS deC. eleganseS. mansonicom aconstrução contendo a região promotora.C. elegansN2 receberam as construçõesfinais através de microinjeção. Foram obtidas três linhagens que expressam Ce_JNK-1,denominadas N2Ex01[Ce_jnk-1], N2Ex02[Ce_jnk-1]e N2Ex03[Ce_jnk-1]e duaslinhagens expressando Sm_JNK, denominadas N2Ex04[Sm_jnk]e N2Ex05[Sm_jnk].Os níveis de expressão de Sm_JNK e Ce_JNK-1 nas linhagens transgênicas foramavaliados por RT-PCR quantitativo. Apesar do aumento da expressão de JNKobservado nas linhagens transgênicas, não houve aumento na longevidade dasmesmas. Embora esta seja a primeira utilização de expressão heteróloga emC. eleganspara investigar a função de genes deS. mansoni, esta técnica tem sido utilizada comsucesso para nematóides parasitos e pode tornar-se uma abordagem alternativa para osestudos funcionais em outros parasitos

Estudo funcional da proteína quinase JNK de Schistosoma mansoniatravés de expressão heteróloga no organismo modelo Caenorhabditis elegans / Functional studies of the protein kinase JNK Schistosoma mansoni by heterologous expression in the model organism C. elegans

A identificação e caracterização dos mecanismos e moléculas envolvidos em sinalização celular são essenciais para o entendimento da biologia parasitária do S. mansoni. Proteína quinases desempenham papel chave em vias de sinalização e tem sido propostas como potenciais alvos para o desenvolvimento de novas drogas anti-Schistosoma. Visto que a caracterização funcional em S. mansoni é dificultada por limitações nos métodos de transformação genética deste parasito, o presente estudo propõe o uso de C. elegans como um modelo para a expressão heteróloga de genes de S. mansoni que codificam proteínas quinases. Genes que codificam proteína quinase sem S. mansoni,homólogos aos identificados em C. elegans, foram selecionados pelo nosso grupo a partir do proteoma do parasito através de uma abordagem filogenômica. Inicialmente, foi selecionada a proteína quinase JNK, que participa da via de sinalização das MAP quinases para a realização das análises experimentais. Em C. elegans, a proteína JNK está relacionada ao aumento de longevidade e da resistência aos estresses térmico e oxidativo. Oligonucleotídeos específicos foram desenhados paraamplificar a região promotora do gene emC. elegansbem como as regiões codificantes(CDS) em ambos os organismos. A região promotora foi amplificada a partir do DNAgenômico extraído deC. elegansadultos. O RNA total foi extraído de esquistossômuloseC. elegansadultos. As CDS foram amplificadas a partir do cDNA sintetizado e osfragmentos de DNA resultantes foram clonados emE. coliDH5α. As construçõesobtidas foram digeridas com enzimas de restrição selecionadas de forma a linearizar ovetor contendo a região promotora e recuperar as CDS. Posteriormente, foramrealizadas subclonagens através da ligação das CDS deC. eleganseS. mansonicom aconstrução contendo a região promotora.C. elegansN2 receberam as construçõesfinais através de microinjeção. Foram obtidas três linhagens que expressam Ce_JNK-1,denominadas N2Ex01[Ce_jnk-1], N2Ex02[Ce_jnk-1]e N2Ex03[Ce_jnk-1]e duaslinhagens expressando Sm_JNK, denominadas N2Ex04[Sm_jnk]e N2Ex05[Sm_jnk].Os níveis de expressão de Sm_JNK e Ce_JNK-1 nas linhagens transgênicas foramavaliados por RT-PCR quantitativo. Apesar do aumento da expressão de JNKobservado nas linhagens transgênicas, não houve aumento na longevidade dasmesmas. Embora esta seja a primeira utilização de expressão heteróloga emC. eleganspara investigar a função de genes deS. mansoni, esta técnica tem sido utilizada comsucesso para nematóides parasitos e pode tornar-se uma abordagem alternativa para osestudos funcionais em outros parasitos.

Functional polymorphisms in IL13 are protective against high Schistosoma mansoni infection intensity in a Brazilian population.

BACKGROUND: IL-13 is a signature cytokine of the helper T cell type 2 (TH2) pathway which underlies host defense to helminthic infection and activates production of IgE in both parasitized populations and in urban settings after allergen exposure. METHODOLOGY/PRINCIPAL FINDINGS: Two functional polymorphisms in IL13, rs1800925 (or c.1-1111C>T) and rs20541 (or R130Q) were previously found to be associated with Schistosoma hematobium infection intensity. They have not been thoroughly explored in S. mansoni-endemic populations, however, and were selected along with 5 tagging SNPs for genotyping in 812 individuals in 318 nuclear families from a schistosomiasis-endemic area of Conde, Bahia, in Brazil. Regression models using GEE to account for family membership and family-based quantitative transmission disequilibrium tests (QTDT) were used to evaluate associations with total serum IgE (tIgE) levels and S. mansoni fecal egg counts adjusted for non-genetic covariates. We identified a protective effect for the T allele at rs20541 (P = 0.005) against high S. mansoni egg counts, corroborated by QTDT (P = 0.014). Our findings also suggested evidence for protective effects for the T allele at rs1800925 and A allele at rs2066960 after GEE analysis only (P = 0.050, 0.0002). CONCLUSIONS/SIGNIFICANCE: The two functional variants in IL13 are protective against high S. mansoni egg counts. These markers showed no evidence of association with tIgE levels, unlike tIgE levels previously studied in non-parasitized or atopic study populations.

Seleção de um painel de antígenos biomarcadores de Schistosoma mansoni através de análises do proteoma sorológico

Apesar do grande esforço na tentativa de controlar a esquistossomose, esta doença continua sendo uma das mais prevalentes no mundo. Novas intervenções são uma prioridade importante para a eliminação da esquistossomose, uma vez que o controle da doença tem sido baseado essencialmente na quimioterapia, a qual não previne a reinfecção. O desenvolvimentode uma vacina para a esquistossomose para proteção a longo prazo, bem como de um novo teste de diagnóstico, constituirá um grande avanço para o controle da doença. A compreensão da resposta imunológica associada com os estados de infecção/proteção pode constituir a base da descoberta de novos antígenos biomarcadores para vacina e diagnóstico para a esquistossomose. Recentes avanços na área pós-genômica têm permitido uma busca mais racional por biomarcadores. Inicialmente, eletroforese bidimensional (2-DE) de extratoproteico total de diferentes fases de desenvolvimento do Schistosoma mansoni foi realizada, obtendo-se um perfil de separação de spots proteicos com boa resolução com os extratos de todas as fases, mas com um perfil distinto entre eles.

Posteriormente, proteínas do extratoproteico de verme adulto, total e de tegumento, foram separaradas por 2-DE e, então, incubadas com amostras de soro de indivíduos infectados (INF) e não infectados de área endêmica (NE) e de indivíduos não infectados de área não endêmica (NI) para esquistossomose em experimento de Western-blotting bidimensional (2D-WB). No total, 47 proteínas imunogênicas foram identificadas por espectrometria de massas. Embora a maioria dos spots proteicos sejam imunogênicos aos diferentes pools de soro, nove spots proteicos reagiram exclusivamente com o pool de soro INF e um com o pool de soro NE. Algumas glicoproteínas foram identificadas no extrato proteico total de verme adulto de S. mansoni usando o método Periodic Acid-Schiff e lectina-blotting. No entanto, o tratamento com periodato/borohidreto indicou que a porção glicídica não tem influência sobre a reatividade das proteínas aos diferentes pools de soro utilizados nos experimentos de 2D-WB. Westernblotting de duas proteínas recombinantes, selecionadas dos experimentos de 2D-WB, mostrou um perfil de reconhecimento pelos diferentes pools de soro semelhante ao das proteínas nativas. Dentre as proteínas imunogênicas identificadas nos experimentos de 2D-WB, 27 foram expressas in vitro com sucesso, as quais serão utilizadas em experimentos futuros em um microarranjo de proteínas. A associação de eletroforese bidimensional e Western-blotting permitiu a seleção de um painel de antígenos proteicos capazes de distinguir os estados de suscetibilidade e resistência à esquistossomose mansônica. Estes antígenos poderão ser utilizados como biomarcadores no desenvolvimento de uma vacina e/ou de um novo teste diagnóstico para a doença.