RESUMO
Immunoglobulin Y (IgY) could be used in serological diagnosis focused on several infectious agents. This study aims to produce IgY anti-hepatitis B virus surface antigen (anti-HBs) and to assess its use in enzyme immunoassays. Antibodies were produced by immunizing chickens with Hepatitis B vaccine associated (group A), or not, with adjuvant CpG-ODN (group B). Eggs were collected for 20 weeks, yolks were purified based on using polyethylene glycol and affinity chromatography. IgY anti-HBs was featured based on SDS-PAGE and Western Blot techniques. Total protein concentration was measured through spectrophotometry. In-house ELISA used to detect HBsAg was developed based on using IgG/HRP conjugate and IgY-anti-HBs sensitized microplates. Thus, IgY anti-HBs were confirmed through molecular pattern based on SDS-PAGE, whereas specificity of anti-HBs was confirmed through Western Blot. Mean total protein reached 3.27 ± 3.00 mg/mL and 3.11 ± 3.12 mg/mL in groups A and B, respectively. In-house ELISA was developed based on using a panel of HBV positive and negative serum samples; it recorded 100 % sensitivity and 78.9 % specificity to detect HBsAg. In conclusion, it was possible producing anti-HBs IgY by immunizing chickens with HBV vaccine; this molecule could be used as capture antibody to help detecting HBsAg in-house ELISA.
Assuntos
Antígenos de Superfície da Hepatite B , Hepatite B , Animais , Galinhas , Vacinas contra Hepatite B , Hepatite B/diagnóstico , Antígenos E da Hepatite B , Anticorpos Anti-Hepatite BRESUMO
The emergence of clinically relevant mutations in the hepatitis B virus (HBV) genome has been a matter of great debate because of the possibility of escape from the host's immune system, the potential to cause more severe progression of liver diseases and the emergence of treatment-resistant variants. Here we characterized the circulating variants of HBV in Rondônia State, in the north of Brazil. Serum samples of 62 chronic HBV carriers were subjected to PCR assays and clinical data were collected. Mutations and genotypes were characterized through direct sequencing. The findings show the presence of subgenotypes A1 (54.83%, 34/62), D3 (16.13%, 10/62), F2 (16.13%, 10/62), A2 (4.84%, 3/62), D2 (3.23%, 2/62), D1 (1.61%, 1/62), D4 (1.61%, 1/62) and F4 (1.61%, 1/62). Deletions in the pre-S2 region were found in 13.79% (8/58) of the samples, mutations in the S gene in 59.68% (37/62) and RT mutations in 48.39% (30/62). We found a variable genotypic distribution in different locations and important mutations related to immune escape and drug resistance in Western Amazonia, which contributed to genetic surveillance and provided important information to help control the disease.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B/genética , Hepatite B Crônica/epidemiologia , Brasil/epidemiologia , DNA Viral/genética , Genótipo , Mutação , Genômica , Hepatite B/epidemiologia , Filogenia , Antígenos de Superfície da Hepatite B/genéticaRESUMO
Immunoglobulin yolk (IgY) is therapeutic antibodies presented in yolk eggs of birds, reptiles, and amphibians. These proteins produced by the immune system of the animal, are capable of neutralizing antigenic molecules, including viral antigens, fulfilling a role in the body defense. The specificity of these antibodies and the facility for their production, make these molecules capable of being used as tools for diagnosis and immunotherapy. Regarding this last aspect, it is common knowledge that the field of virology, is racing against time in the development of new drugs and vaccines to try to contain pandemics and local epidemics and, in counterproposal, avian antibodies are neutralizing molecules that can help in the control and spread of disease. These molecules have been explored for years and currently chicken eggs are produced in large quantities from the animal's immunization against a specific pathogen. Thus, on this subject, this review made a survey of these researches and presents a summary of all the successful cases and perspectives in the use of IgYs as tools for viral immunization.
Assuntos
Antivirais/farmacologia , Imunoglobulinas/farmacologia , Animais , Humanos , Imunização , Imunoglobulinas/química , Imunoglobulinas/isolamento & purificação , Viroses/imunologia , Viroses/terapia , Vírus/efeitos dos fármacos , Vírus/imunologiaRESUMO
OBJECTIVE AND DESIGN: The involvement of nitric oxide pathway in the antinociceptive activity of Lonchocarpus araripensis lectin (LAL) was investigated in the model of carragenan-induced hypernociception. METHODS: Swiss mice received LAL (0.01-10 mg/kg; i.v.) 30 min before s.c. injection of carragenan in the paws. For the involvement of nociceptive pathways, animals were previously treated with the blockers: NOS (L-NAME, aminoguanidine, 7-nitroindazole); soluble guanylyl cyclase (ODQ); channels of ATP-dependent K+ (glibenclamide); L-type Ca2+ (nifedipine), or Ca2+-dependent Cl- (niflumic acid). Participation of lectin domain was evaluated by injection of LAL associated with N-acetyl-glucosamine (GlcNAc). nNOS gene relative expression was evaluated in the paw tissues and nNOS immunostaining in dorsal root ganglia. RESULTS: LAL at all doses inhibited carrageenan-induced hypernociception (4.12 ± 0.58 g), being maximal at 10 mg/kg (3 h: 59%), and reversed by GlcNAc. At this time, LAL effect was reversed by nifedipine (39%), niflumic acid (59%), L-NAME (59%), 7-nitroindazole (44%), ODQ (45%), and glibenclamide (34%), but was unaltered by aminoguanidine. LAL increased (95%) nNOS gene expression in mice paw tissues, but not its immunoexpression in the dorsal root ganglia. CONCLUSION: The antinociceptive effect of Lonchocarpus araripensis lectin involves activation of the L-arginine/NO/GMPc/K+ATP pathway.
Assuntos
Analgésicos/farmacologia , Arginina/metabolismo , GMP Cíclico/metabolismo , Fabaceae/química , Canais KATP/metabolismo , Lectinas/farmacologia , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Carragenina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismoRESUMO
This study investigated the effects of the alga lectin Hypnea cervicornis agglutinin (HCA) on rat zymosan-induced arthritis (ZyA). Zymosan (50-500 µg/25 µL) or sterile saline (Sham) was injected into the tibio-tarsal joint of female Wistar rats (180-200 g). Arthritic animals received morphine (4 mg/kg, intraperitoneal), indomethacin (5 mg/kg, intraperitoneal), or 2% lidocaine (100 µL, subcutaneous). HCA (0.3-3 mg/kg) was administered by intravenous route 30 min before or 2 h after zymosan. 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 4 µg, intra-articular) was given 30 min prior HCA. Hypernociception was measured every hour until 6 h, time in which animals were sacrificed for evaluation of leukocytes of the intra articular fluid and gene expression of TNF-α, IL-1, IL-10, and iNOS in the joint tissues using PCR techniques. Hypernociception was responsive to morphine and indomethacin, and its threshold was not altered by lidocaine. The post-treatment of HCA reduced both hypernociception and leukocyte influx. This antinociceptive effect was abolished either by ODQ and glibenclamide. HCA also reduced gene expression of iNOS and TNF-α. In conclusion, the antinociceptive effect of HCA in ZyA involves cyclic GMP signalization and selective modulation of cytokine expression.
Assuntos
Artrite/tratamento farmacológico , GMP Cíclico/metabolismo , Citocinas/biossíntese , Lectinas/uso terapêutico , Rodófitas , Zimosan/toxicidade , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Artrite/induzido quimicamente , Artrite/metabolismo , Expressão Gênica , Lectinas/isolamento & purificação , Lectinas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The Tn antigen is an epitope containing N-acetyl-D-galactosamine present in the extracellular matrix of some carcinoma cells in humans, and it is often used as a biomarker. Lectins are proteins capable of binding to carbohydrates and can be used as a molecular tool to recognize antigens and to differentiate cancer cells from normal cells. In this context, the present work aimed to characterize the interaction of Vatairea guianensis seed lectin with N-acetyl-D-galactosamine and the Tn antigen by molecular dynamics and molecular mechanics/Poisson-Boltzmann solvent-accessible surface area analysis. This study revealed new interacting residues not previously identified in static analysis of the three-dimensional structures of Vatairea lectins, as well as the configuration taken by the carbohydrate recognition domain, as it interacts with each ligand. During the molecular dynamics simulations, Vatairea guianensis lectin was able to bind stably to Tn antigen, which, as seen previously for other lectins, enables its use in cancer research, diagnosis, and therapy. This work further demonstrates the efficiency of bioinformatics in lectinology.
Assuntos
Fabaceae/química , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Humanos , Neoplasias , Domínios ProteicosRESUMO
Lonchocarpus campestris (tribe Dalbergieae) possess a mannose biding lectin (LCaL) purified by ion exchange chromatography on DEAE-Sephacel, HiTrap DEAE FF and TSKgel engaged in AKTA-HPLC system. LCaL agglutinates trypsinized rabbit erythrocytes and its activity was maintained after incubation in a wide range of temperature (4-100⯰C) and pH (4-9). The lectin had its apparent molecular weight evaluated by size-exclusion chromatography and SDS-PAGE and presented a profile of 10â¯kDa and 25â¯kDa in denaturing and native conditions, respectively. LCaL injected by intravenous route in mice showed antinociceptive activity in the behavioral tests of Formalin and Writhing. In the formalin test LCaL inhibited the licking time by 37% in the neurogenic phase and by 73% in the inflammatory phase. In the acetic acid-induced writhing test LCaL showed inhibitory effect at 0.1â¯mg/kg (72%), 1â¯mg/kg (74%) and 10â¯mg/kg (70%). The lectin also inhibited the increase in vascular permeability at 10â¯mg/kg and leukocyte migration at 0.1, 1 and 10â¯mg/kg concentrations. Additionally, LCaL inhibited paw edema (mainly from 1 to 3â¯h by 46%) and hyperalgesia (1â¯h: 82%; 3â¯h: 63%) induced by carrageenan. In conclusion, LCaL presents an antinociceptive action mainly via inhibition of inflammation.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Fabaceae/química , Lectinas/isolamento & purificação , Nociceptividade/efeitos dos fármacos , Sementes/química , Animais , Hemaglutinação , Lectinas/química , Masculino , Camundongos , Peso MolecularRESUMO
Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe.
Assuntos
Fabaceae/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Lectinas de Plantas/química , Sementes/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Modelos Animais de Doenças , Edema/tratamento farmacológico , Edema/etiologia , Edema/patologia , Metais/química , Camundongos , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dioclea/química , Glioma/patologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Animais , Antineoplásicos/metabolismo , Metabolismo dos Carboidratos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Simulação de Acoplamento Molecular , Lectinas de Plantas/metabolismo , Conformação Proteica , Ratos , Sementes/químicaRESUMO
The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.
Assuntos
Fabaceae/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Polissacarídeos/química , Cristalografia por Raios XRESUMO
Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Analgésicos/administração & dosagem , Analgésicos/isolamento & purificação , Fabaceae/química , Dor/tratamento farmacológico , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/isolamento & purificação , Analgésicos/química , Analgésicos/farmacologia , Animais , Cromatografia de Afinidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Testes de Hemaglutinação , Concentração de Íons de Hidrogênio , Manose/farmacologia , Camundongos , Peso Molecular , Dor/etiologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Estabilidade Proteica , Coelhos , Sacarose/farmacologia , TemperaturaRESUMO
The anti-inflammatory activity of Canavalia seed lectins (Canavalia gladiata [CGL], Canavalia maritima [ConM] and Canavalia brasiliensis [ConBr]) was evaluated by intravenous administration in rats. In non-sensitized rats, cellular edema elicited by carrageenan was reduced (45-51 %) by ConM and (44-59 %) by CGL. Osmotic edema elicited by dextran was reduced by ConM and CGL in 27 % and 29 %. ConM and CGL reduced the edema elicited by L-arginine in 53 % and that of prostaglandin E2 in 48 % and 36 %. Leukocyte migration elicited by carrageenan was reduced in 49 % by ConM and in 55 % by CGL (attenuated in 4× by glucose) and peritoneal TNF-α content in 82 %. In rats sensitized, ConM inhibited the paw edema and leukocyte migration elicited by ovalbumin in 34 % and 70 %. ConM and CGL are anti-inflammatory, mainly in cellular events mediated by prostaglandin E2, nitric oxide and TNF-α in non-sensitized rats. However, only ConM is anti-inflammatory in sensitized rats. CGL effect involves the lectin domain.