RESUMO
The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.
Assuntos
Echinococcus granulosus/química , Proteínas de Helminto/farmacologia , Interleucina-12/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Helminto/isolamento & purificação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Proteínas de Helminto/farmacologia , Interferon gama/imunologia , Interleucina-17/imunologia , Vacinas/farmacologia , Animais , Fasciolíase/imunologia , Feminino , Proteínas de Helminto/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas/imunologiaRESUMO
Inflammation is currently considered a hallmark of cancer and plays a decisive role in different stages of tumorigenesis, including initiation, promotion, progression, metastasis and resistance to antitumor therapies. Colorectal cancer is a disease widely associated with local chronic inflammation. Additionally, extrinsic factors such as infection may beneficially or detrimentally alter cancer progression. Several reports have noted the ability of various parasitic infections to modulate cancer development, favoring tumor progression in many cases and inhibiting tumorigenesis in others. The aim of our study was to determine the effects of excreted/secreted products of the helminth Taenia crassiceps (TcES) as a treatment in a murine model of colitis-associated colon cancer (CAC). Here, we found that after inducing CAC, treatment with TcES was able to reduce inflammatory cytokines such as IL-1ß, TNF-α, IL-33 and IL-17 and significantly attenuate colon tumorigenesis. This effect was associated with the inhibition of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation. Furthermore, we determined that TcES interfered with LPS-induced NF-κB p65 activation in human colonic epithelial cell lines in a Raf-1 proto-oncogene-dependent manner. Moreover, in three-dimensional cultures, TcES promoted reorganization of the actin cytoskeleton, altering cell morphology and forming colonospheres, features associated with a low grade of aggressiveness. Our study demonstrates a remarkable effect of helminth-derived molecules on suppressing ongoing colorectal cancer by downregulating proinflammatory and protumorigenic signaling pathways.
Assuntos
Anti-Inflamatórios/administração & dosagem , Azoximetano/efeitos adversos , Colite/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Proteínas de Helminto/administração & dosagem , Taenia/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colite/induzido quimicamente , Colite/complicações , Neoplasias do Colo/etiologia , Modelos Animais de Doenças , Feminino , Proteínas de Helminto/farmacologia , Humanos , Interleucina-1beta/metabolismo , Interleucina-33/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proto-Oncogene Mas , Fator de Transcrição STAT3/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.
Assuntos
Inibidores da Angiogênese/farmacologia , Fator Xa/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Fator X/farmacologia , Fator X/uso terapêutico , Fator Xa/uso terapêutico , Proteínas de Helminto/farmacologia , Proteínas de Helminto/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Receptor PAR-1/metabolismo , Peixe-ZebraRESUMO
Helminths secrete several molecules that can modulate the immune responses, favoring their evasion and perpetuate their survival in the host. These molecules interfere with antigen presentation, cell proliferation and activation, antibody production, cause cell death, and stimulate regulatory responses. Here, we focus on some helminth products and address their immunomodulatory effects in the host immune system and, also, we describe some anti-inflammatory properties of an Ascaris suum-derived immunomodulatory molecule, named PAS-1. This protein is a 200-kDa molecule isolated by affinity chromatography using MAIP-1 (monoclonal antibody which recognizes PAS-1), coupled to Sepharose 4B. It suppresses the inflammatory responses in murine models of delayed-type hypersensitivity, lung allergic inflammation and LPS-induced inflammation into air pouches. PAS-1 also stimulates the secretion of regulatory cytokines such as IL-10 and TGF-beta and primes IFN-gamma-secreting CD8+ and IL-10/ TGF-beta-secreting CD4+CD25+ cell clones that avoid the lung inflammation. Thus, this protein is a potent immunomodulatory component that may be used for therapeutic interventions in inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Helminto/farmacologia , Helmintos/metabolismo , Imunidade/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Inflamação/tratamento farmacológico , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Ascaris suum/metabolismo , Citocinas/biossíntese , Citocinas/farmacologia , Citocinas/uso terapêutico , Proteínas de Helminto/biossíntese , Proteínas de Helminto/uso terapêutico , Fatores Imunológicos/biossíntese , Fatores Imunológicos/uso terapêutico , Inflamação/imunologia , Linfócitos T/imunologiaRESUMO
A mycobacterial codon-optimized gene encoding the Sm14 antigen of Schistosoma mansoni was generated using oligonucleotide assembly. This synthetic gene enhanced approximately fourfold the protein expression level in recombinant Mycobacterium bovis Bacille Calmette-Guérin (rBCG) when compared to that obtained using the native gene in the same expression vector. Immunization of mice with rBCG expressing Sm14 via the synthetic gene induced specific cellular Th1-predominant immune responses, as determined by interferon-gamma production of Sm14-stimulated splenocytes, which were comparable to those recorded in animals immunized with an rBCG strain expressing the native gene. Administration of a single dose of the rBCG-Sm14 construct carrying the synthetic gene conferred protection against cercarial challenge in outbred Swiss mice, at a level equivalent to those provided by either a single dose of rBCG expressing the native gene or three doses of Escherichia coli-derived recombinant Sm14. Our data demonstrated that despite improving the level of antigen expression, the codon optimization strategy did not result in enhanced immunity or protection against cercarial S. mansoni challenge.
Assuntos
Vacina BCG/imunologia , Proteínas de Transporte de Ácido Graxo/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Schistosoma mansoni/química , Esquistossomose mansoni/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Códon/genética , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/imunologia , Proteínas de Transporte de Ácido Graxo/uso terapêutico , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Schistosoma mansoni/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas SintéticasRESUMO
BACKGROUND: Recently, we identified a 200 kDa protein (PAS-1) from Ascaris suum worms, that suppresses the humoral immune response. Here, the effect of PAS-1 on inflammatory leukocyte migration induced by bacterial lipopolysaccharide (LPS) was investigated. METHODS: Cellular migration and cytokine release, stimulated by LPS or LPS+PAS-1, were analyzed in air pouches induced in the shaved back of BALB/c mice. Cytokines were determined by ELISA and RT-PCR on air pouch exudates and in vitro stimulated peritoneal macrophages. RESULTS: The significant cellular influx induced by LPS, consisting predominantly of neutrophils, was highly suppressed in the presence of PAS-1, but not a non-related protein. PAS-1 led also to a marked reduction of TNF-alpha, IL-1 beta and IL-6 levels in both LPS-stimulated air pouches and peritoneal macrophage cultures. In contrast, PAS-1 induced a significant increase of IL-10 and TGF-beta production. CONCLUSIONS: These results demonstrate that PAS-1 has a potent anti-inflammatory activity, probably due to the stimulation of regulatory cytokines in macrophages, thus leading to the inhibition of pro-inflammatory cytokine production.
Assuntos
Anti-Inflamatórios/farmacologia , Ascaris suum/química , Proteínas de Helminto/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Feminino , Proteínas de Helminto/isolamento & purificação , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.
Assuntos
Ascaris suum/química , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Animais , Interferon gama/deficiência , Interferon gama/imunologia , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-12/imunologia , Camundongos , Camundongos Knockout , Modelos Moleculares , Peso Molecular , Ovalbumina/imunologiaRESUMO
We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.
Assuntos
Proteínas de Helminto/isolamento & purificação , Lectina de Ligação a Manose/isolamento & purificação , Schistosoma mansoni/química , Animais , Degranulação Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Cromatografia de Afinidade , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Interações Hospedeiro-Parasita , Masculino , Lectina de Ligação a Manose/metabolismo , Lectina de Ligação a Manose/farmacologia , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos WistarRESUMO
The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa , Proteínas de Helminto/isolamento & purificação , Sanguessugas/química , Saliva/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Inibidores de Serina Proteinase/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Compostos Cromogênicos/metabolismo , Proteínas de Helminto/farmacologia , Hemeproteínas/química , Hemeproteínas/farmacologia , Hemeritrina/análogos & derivados , Hemeritrina/química , Humanos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/farmacologia , Especificidade da Espécie , Especificidade por Substrato , Tromboplastina/antagonistas & inibidoresRESUMO
Whole worm homogenates and excretory/secretory (E/S) products of adult Nippostrongylus brasiliensis significantly decreased the amplitude of contractions of segments of uninfected rat intestine maintained in an organ bath, with significantly larger volumes of E/S products being required to bring about a similar decrease to that caused by the whole worm homogenate. Boiled samples of homogenate and E/S products significantly decreased the amplitude of contractions of uninfected rat intestine, but larger volumes were needed than with unboiled samples. Frequency of contraction was unaltered by homogenates or E/S products. When electric eel AChE was injected into the lumen of segments of uninfected rat intestine maintained in an organ bath there was no significant decrease in the amplitude of contractions. These results suggest that substances present in the E/S products of N. brasiliensis significantly reduce the amplitude of contractions of uninfected rat intestine in vitro and that the biochemical holdfast responsible for this phenomenon may not be AChE.
Assuntos
Acetilcolinesterase/farmacologia , Proteínas de Helminto/farmacologia , Intestinos/efeitos dos fármacos , Nippostrongylus/metabolismo , Animais , Electrophorus , Feminino , Intestinos/fisiologia , Contração Muscular/efeitos dos fármacos , Concentração Osmolar , Ratos , Ratos WistarRESUMO
Samples of Schistosoma mansoni soluble adult worm proteins (SWAP) were iodinated with 4-15 µmolI/mg protein using iodine monochloride. The capacity to elicit immediate hypersensitivity reactions of the iodinated derivatives was compared to that of the native SWAP preparations. The degranulation of mast clls from infected mice decreased with increasing iodine incorporation and was absent in fully iodinated samples containing 15 µmol I/mg protein. The response of guinea pigs and humans to the intradermal test with iodinated SWAP also decrease in proportion to iodine incorporation, and no responses were obtained with fully iodinated samples. No false-positive tests were observed. Antibodies to the folly iodinated extracts generated in C57BL/10 mice reacted by ELISA with the unmodified proteins and by immunoprecipitation on agar gel. The immunoprecicpitation pattern suggested that some epitopes were altered by iodination