RESUMO
Angiostrongylus cantonensis is the main cause of human eosinophilic meningitis. Humans are accidental hosts, becoming infected due to ingestion of raw intermediate (snails and slugs) or paratenic hosts. Once ingested, the larvae migrate towards the brain where they die, causing the disease. To develop better mollusk control strategies, it is important to first understand what happens in the snail during infection, therefore our purpose was to characterize proteomic, metabolic and immunologic changes in Biomphalaria glabrata 24 h after infection with A. cantonensis. For this purpose, proteins were extracted from infected and uninfected snails and analyzed through mass spectrometry. Hemolymph was also collected, the number of hemocytes was counted and urea, nitric oxide, calcium, glycogen levels as well as alanine and aspartate aminotransferases activities were assessed. The cephalopodal region and gonad-digestive gland complex were dissected and their glycogen content was measured. After infection with A. cantonensis, we observed an increase of hemocytes and granulocytes as well as an increase in hemoglobin type 2 proteins. Temptin-like protein was also found up-regulated in infected snails. Several proteins with structural function (such as myosin heavy chain - striated muscle - like and protein LOC106059779 with ADAM/reprosolin domain) were also differentially expressed, suggesting loss/damage of internal tissues. Increase in phosphoglycerate mutase indicates an increase in glycolysis, possible to compensate the increase in energetic needs. Consequently, there is a decrease in glycogen reserves, particularly in the gonad - digestive gland complex.
Assuntos
Biomphalaria/parasitologia , Proteômica/métodos , Infecções por Strongylida/metabolismo , Animais , Hemolinfa/química , Interações Hospedeiro-Parasita , Humanos , Ratos , Ratos WistarRESUMO
BACKGROUND: Schistosomiasis chemotherapy is largely based on praziquantel (PZQ). Although PZQ is very safe and tolerable, it does not prevent reinfection and emerging resistance is a primary concern. Recent studies have shown that the targeting of epigenetic machinery in Schistosoma mansoni may result in severe alterations in parasite development, leading to death. This new route for drug discovery in schistosomiasis has focused on classes of histone deacetylases (HDACs) and histone acetyltransferases (HATs) as epigenetic drug targets. Schistosoma histone demethylases also seem to be important in the transition of cercariae into schistosomula, as well as sexual differentiation in adult worms. METHODS: The Target-Pathogen database and molecular docking assays were used to prioritize the druggability of S. mansoni histone demethylases. The transcription profile of Smp_03400 was re-analyzed using available databases. The effect of GSK-J4 inhibitor in schistosomula and adult worms' motility/viability/oviposition was assessed by in vitro assays. Ultrastructural analysis was performed on adult worms exposed to GSK-J4 by scanning electron microscopy, while internal structures and muscle fiber integrity was investigated by confocal microscopy after Langeron's carmine or phalloidin staining. RESULTS: The present evaluation of the potential druggability of 14 annotated S. mansoni demethylase enzymes identified the S. mansoni ortholog of human KDM6A/UTX (Smp_034000) as the most suitable druggable target. In silico analysis and molecular modeling indicated the potential for cofactor displacement by the chemical probe GSK-J4. Our re-analysis of transcriptomic data revealed that Smp_034000 expression peaks at 24 h in newly transformed schistosomula and 5-week-old adult worms. Moreover, this gene was highly expressed in the testes of mature male worms compared to the rest of the parasite body. In in vitro schistosome cultures, treatment with GSK-J4 produced striking effects on schistosomula mortality and adult worm motility and mortality, as well as egg oviposition, in a dose- and time-dependent manner. Unexpectedly, western blot assays did not demonstrate overall modulation of H3K27me3 levels in response to GSK-J4. Confocal and scanning electron microscopy revealed the loss of original features in muscle fibers and alterations in cell-cell contact following GSK-J4 treatment. CONCLUSIONS: GSK-J4 presents promising potential for antischistosomal control; however, the underlying mechanisms warrant further investigation.
Assuntos
Anti-Helmínticos/farmacologia , Benzazepinas/farmacologia , Descoberta de Drogas/métodos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Modelos Moleculares , Pirimidinas/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Biologia Computacional , Epigênese Genética/efeitos dos fármacos , Feminino , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/tratamento farmacológico , TranscriptomaRESUMO
SCOPE: Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. METHODS AND RESULTS: Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. CONCLUSION: Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.
Assuntos
Ácidos Graxos Ômega-3/farmacologia , Resistência à Insulina , Lipodistrofia/complicações , Hepatopatia Gordurosa não Alcoólica/dietoterapia , Adipócitos/metabolismo , Animais , Dieta Hiperlipídica , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Masculino , Camundongos Knockout , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR gama/genéticaRESUMO
BACKGROUND: Schistosomiasis control in endemic areas depends on several factors, including chemotherapy, snail control and adequate sanitation. In this context, the employment of compounds isolated from plants is an important issue regarding infection and snail control. The aim of this study was therefore to evaluate the effects of curcumin (CUR), a compound isolated from Curcuma longa, against snails and embryos of Biomphalaria glabrata, which is the most important intermediate host of schistosomiasis in the Americas, as well as in cercariae, the infecting larval stage of Schistosoma mansoni. RESULTS: CUR presented high activity against B. glabrata embryos and moderate activity against newborn and adult snails. The lethal concentration (LC50 ) values after being exposed for 24 h and evaluated for 7 days were 6.54 (95% confidence interval (CI) 5.86-7.30) µg mL-1 for the embryos and 42.29 (95% CI 33.82-52.87) µg mL-1 and 87.69 (95% CI 68.82-111.7) µg mL-1 for the newborn and adult snails, respectively. Moreover, CUR inhibited the development of embryos and egg hatching, and decreased the fecundity rates of adult snails. CUR also demonstrated cercaricidal activity with LC50 values lower than 10 µg mL-1 at 1, 3, 6, 9 and 12 h, respectively. CONCLUSION: Our data show that CUR has potential molluscicidal and cercaricidal activities. Moreover, as a nutraceutical compound that is toxic to both invertebrate host and parasite, CUR has the potential to be explored as a safe new agent to combat schistosomiasis. © 2019 Society of Chemical Industry.
Assuntos
Biomphalaria , Moluscocidas , Schistosoma mansoni , Animais , Curcumina , Dose Letal MedianaRESUMO
Background: Schistosomiasis chemotherapy is largely based on praziquantel (PZQ). Although PZQ is very safe and tolerable, it does not prevent reinfection and emerging resistance is a primary concern. Recent studies have shown that the targeting of epigenetic machinery in Schistosoma mansoni may result in severe alterations in parasite development, leading to death. This new route for drug discovery in schistosomiasis has focused on classes of histone deacetylases (HDACs) and histone acetyltransferases (HATs) as epigenetic drug targets. Schistosoma histone demethy-lases also seem to be important in the transition of cercariae into schistosomula, as well as sexual diferentiation in adult worms. Methods: The Target-Pathogen database and molecular docking assays were used to prioritize the druggability of S. mansoni histone demethylases. The transcription profle of Smp_03400 was re-analyzed using available databases. The efect of GSK-J4 inhibitor in schistosomula and adult worms’ motility/viability/oviposition was assessed by in vitro assays. Ultrastructural analysis was performed on adult worms exposed to GSK-J4 by scanning electron microscopy, while internal structures and muscle fber integrity was investigated by confocal microscopy after Langeron's carmine or phalloidin staining. Results: The present evaluation of the potential druggability of 14 annotated S. mansoni demethylase enzymes identifed the S. mansoni ortholog of human KDM6A/UTX (Smp_034000) as the most suitable druggable target. In silico analysis and molecular modeling indicated the potential for cofactor displacement by the chemical probe GSK-J4. Our re-analysis of transcriptomic data revealed that Smp_034000 expression peaks at 24 h in newly transformed schisto somula and 5-week-old adult worms. Moreover, this gene was highly expressed in the testes of mature male worms compared to the rest of the parasite body. In in vitro schistosome cultures, treatment with GSK-J4 produced strikingefects on schistosomula mortality and adult worm motility and mortality, as well as egg oviposition, in a dose- and time-dependent manner. Unexpectedly, western blot assays did not demonstrate overall modulation of H3K27me3 levels in response to GSK-J4. Confocal and scanning electron microscopy revealed the loss of original features in muscle fibers and alterations in cell-cell contact following GSK-J4 treatment. Conclusions GSK-J4 presents promising potential for antischistosomal control; however, the underlying mechanisms warrant further investigation.
RESUMO
Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-gama deletion exclusively in adipocytes (A-PPARgama KO) and littermate controls (A-PPARgama WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.
RESUMO
BACKGROUND: Cachexia is a paraneoplastic syndrome related with poor prognosis. The tumour micro-environment contributes to systemic inflammation and increased oxidative stress as well as to fibrosis. The aim of the present study was to characterise the inflammatory circulating factors and tumour micro-environment profile, as potentially contributing to tumour fibrosis in cachectic cancer patients. METHODS: 74 patients (weight stable cancer n = 31; cachectic cancer n = 43) diagnosed with colorectal cancer were recruited, and tumour biopsies were collected during surgery. Multiplex assay was performed to study inflammatory cytokines and growth factors. Immunohistochemistry analysis was carried out to study extracellular matrix components. RESULTS: Higher protein expression of inflammatory cytokines and growth factors such as epidermal growth factor, granulocyte-macrophage colony-stimulating factor, interferon-α, and interleukin (IL)-8 was observed in the tumour and serum of cachectic cancer patients in comparison with weight-stable counterparts. Also, IL-8 was positively correlated with weight loss in cachectic patients (P = 0.04; r = 0.627). Immunohistochemistry staining showed intense collagen deposition (P = 0.0006) and increased presence of α-smooth muscle actin (P < 0.0001) in tumours of cachectic cancer patients, characterizing fibrosis. In addition, higher transforming growth factor (TGF)-ß1, TGF-ß2, and TGF-ß3 expression (P = 0.003, P = 0.05, and P = 0.047, respectively) was found in the tumour of cachectic patients, parallel to p38 mitogen-activated protein kinase alteration. Hypoxia-inducible factor-1α mRNA content was significantly increased in the tumour of cachectic patients, when compared with weight-stable group (P = 0.005). CONCLUSIONS: Our results demonstrate TGF-ß pathway activation in the tumour in cachexia, through the (non-canonical) mitogen-activated protein kinase pathway. The results show that during cachexia, intratumoural inflammatory response contributes to the onset of fibrosis. Tumour remodelling, probably by TGF-ß-induced transdifferentiation of fibroblasts to myofibroblasts, induces unbalanced inflammatory cytokine profile, angiogenesis, and elevation of extracellular matrix components (EMC). We speculate that these changes may affect tumour aggressiveness and present consequences in peripheral organs.
Assuntos
Caquexia/etiologia , Caquexia/metabolismo , Neoplasias/complicações , Neoplasias/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Idoso , Biomarcadores , Biópsia , Composição Corporal , Índice de Massa Corporal , Caquexia/patologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos , Fibrose , Expressão Gênica , Humanos , Hipóxia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Estresse Oxidativo , Microambiente TumoralRESUMO
BACKGROUND: The surface of infected red blood cells (iRBCs) has been widely investigated because of the molecular complexity and pathogenesis mechanisms involved. Asymptomatic individuals are important in the field because they can perpetuate transmission as natural reservoirs and present a challenge for diagnosing malaria because of their low levels of circulating parasites. Recent studies of iRBC antibody recognition have shown that responses are quantitatively similar in symptomatic and asymptomatic infections, but no studies have characterised the plasmodial proteins targeted by this response. OBJECTIVES: Our main objective was to identify Plasmodium falciparum proteins associated with iRBC ghosts recognised by antibodies in the sera of symptomatic and asymptomatic individuals in the Brazilian Amazon. METHODS: We collected symptomatic and asymptomatic sera from patients residing in the Brazilian Amazon and P. falciparum iRBC ghosts to identify the proteins involved in natural antibody recognition by 2D-electrophoresis, western blotting, and high- resolution mass spectrometry. FINDINGS: 2D gel-based immunoproteome analysis using symptomatic and asymptomatic sera identified 11 proteins with at least one unique peptide, such as chaperones HSP70-1 and HSP70-x, which likely are components of the secretion machinery/PTEX translocon. PfEMP1 is involved in antigenic variation in symptomatic infections and we found putative membrane proteins whose functions are unknown. MAIN FINDINGS: Our results suggest a potential role of old and new proteins, such as antigenic variation proteins, iRBC remodelling, and membrane proteins, with no assigned functions related to the immune response against P. falciparum, providing insights into the pathogenesis, erythrocyte remodelling, and secretion machinery important for alternative diagnosis and/or malaria therapy.
Assuntos
Anticorpos Antiprotozoários/genética , Antígenos de Protozoários/genética , Membrana Eritrocítica/parasitologia , Plasmodium falciparum/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Infecções Assintomáticas , Western Blotting , Eletroforese em Gel Bidimensional , Membrana Eritrocítica/imunologia , Humanos , Espectrometria de Massas , Plasmodium falciparum/genética , ProteômicaRESUMO
The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings.
Assuntos
Anticorpos Antiprotozoários/sangue , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Antiprotozoários/metabolismo , Variação Antigênica , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Brasil/epidemiologia , Células CHO , Criança , Pré-Escolar , Cricetinae , Cricetulus , Estudos Transversais , Doenças Endêmicas/estatística & dados numéricos , Variação Genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/imunologia , Adulto JovemRESUMO
Several signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor ß, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated with DAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.