RESUMO
ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.
RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.
Assuntos
Humanos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fragmentos de Peptídeos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Metaloproteinase 2 da Matriz/metabolismo , Osteopontina/metabolismo , Melatonina/farmacologia , Osteoblastos/metabolismo , Fragmentos de Peptídeos/genética , RNA Mensageiro/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Osteopontina/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Introducción. Toxocara canis es un nematodo patógeno de cánidos que accidentalmente puede ser transmitido a los humanos. A pesar de la importancia de la serología para el diagnóstico de esta zoonosis, los kits diagnósticos usan antígenos crudos de excreción-secreción, en su mayoría glucoproteínas que no son específicas de especie, por lo cual pueden presentarse reacciones cruzadas con anticuerpos generados contra otros parásitos. Objetivos. Producir el antígeno recombinante TES-30 de T. canis y evaluarlo para el inmunodiagnóstico de la toxocariasis. Materiales y métodos. Se clonó el gen que codifica TES-30 en el vector de expresión pET28a (+), usando oligonucleótidos de cadena sencilla unidos mediante reacción en cadena de la polimerasa (PCR). La proteína rTES-30 se purificó por cromotografia de afinidad (Ni 2+ ). La reacción serológica de rTES-30 se evaluó mediante immunoblot . Teniendo en cuenta que no existe una prueba de referencia , se observó el comportamiento del antigeno en comparación con la prueba de rutina para el inmunodiagnóstico de la toxocariasis, es decir, la técnica ELISA convencional con antígenos de excreción-secreción. Resultados. El rTES-30 se produjo a partir de un cultivo de Escherichia coli LB, con un rendimiento de 2,25 mg/l y 95 % de pureza. La concordancia de la reacción entre el immunoblot rTES-30 y la ELISA convencional, fue de 73 % (46/63) y de 100 % con los 21 sueros no reactivos. De los 21 sueros con diagnóstico de otras parasitosis, 19 fueron reactivos con ELISA, mientras que tan solo siete fueron positivos con el immunoblot rTES-30. La concordancia entre la ELISA y el immunoblot fue moderada (índice kappa de 0,575; IC 95% 0,41-0,74). Conclusiones. Los datos presentados respaldan la utilidad del immunoblot r TES-3 0 para la confirmación de los posibles positivos por ELISA, no solo en los estudios epidemiológicos, sino también, como candidato para el desarrollo de pruebas diagnósticas de la toxocariasis ocular en Colombia.
Introduction: Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites. Objectives: To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis. Materials and methods: The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni 2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens. Results: The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the non-reactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74). Conclusions: The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.
Assuntos
Animais , Humanos , Immunoblotting , Toxocaríase/diagnóstico , Toxocara canis/imunologia , Antígenos de Helmintos/sangue , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Solubilidade , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/análise , Ensaio de Imunoadsorção Enzimática , Sequência de Bases , Toxocaríase/sangue , Infecções Oculares Parasitárias/diagnóstico , Cromatografia de Afinidade , Escherichia coli , Genes Sintéticos , Antígenos de Helmintos/isolamento & purificação , Antígenos de Helmintos/genéticaRESUMO
En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.
In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.
Assuntos
Animais , Embrião de Galinha , Cricetinae , Camundongos , Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Chlorocebus aethiops , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Organismos Livres de Patógenos Específicos , Cultura de Vírus , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genéticaRESUMO
The large use of simple and effective diagnostic tools has significantly contributed to the increase in diagnosis of thyroid cancer over the past years. However, there is compelling evidence that most micropapillary carcinomas have an indolent behavior and may never evolve into clinical cancers. Therefore, there is an urgent need for new tools able to predict which thyroid cancers will remain silent, and which thyroid cancers will present an aggressive behavior. There are a number of well-established clinical predictors of malignancy and recent studies have suggested that some of the patientÆs laboratory data and image methods may be useful. Molecular markers have also been increasingly tested and some of them appear to be very promising, such as BRAF, a few GST genes and p53 polymorphisms. In addition, modern tools, such as immunocytochemical markers, and the measure of the fractal nature of chromatin organization may increase the specificity of the pathological diagnosis of malignancy and help ascertain the prognosis. Guidelines designed to select nodules for further evaluation, as well as new methods aimed at distinguishing carcinomas of higher aggressiveness among the usually indolent thyroid tumors are an utmost necessity.
O uso cada vez mais freqüente de métodos diagnósticos simples e efetivos tem contribuído significativamente para um aumento no diagnóstico de câncer da tiróide nos últimos anos. Entretanto, existem importantes evidências de que muitos dos microcarcinomas papilíferos têm um comportamento indolente e podem nunca evoluir para cânceres clínicos. Existe, portanto, uma necessidade urgente de desenvolver novas ferramentas capazes de predizer quais os tumores tiroidianos que permanecerão silenciosos e quais desenvolverão comportamento agressivo. Há uma série de marcadores clínicos de evolução bem estabelecidos e alguns estudos recentes sugerem que dados laboratoriais e métodos de imagem podem ser úteis. Marcadores moleculares também vêm sendo ativamente investigados e alguns, como BRAF, os genes GST e polimorfismos de p53, parecem promissores. Além disso, marcadores imunocitoquímicos e a medida da natureza fractal da cromatina podem aumentar a especificidade do diagnóstico anatomopatológico e ajudar a predizer o prognóstico. Existe uma necessidade imperiosa de elaborarmos diretrizes destinadas a selecionar os nódulos que merecem prosseguimento em sua avaliação, assim como novos métodos capazes de identificar lesões mais agressivas entre os geralmente indolentes tumores tiroidianos.
Assuntos
Feminino , Humanos , Masculino , Carcinoma Papilar/patologia , Neoplasias da Glândula Tireoide/patologia , Biomarcadores Tumorais/metabolismo , Fatores Etários , Carcinoma Papilar/etiologia , Carcinoma Papilar/metabolismo , Exposição Ambiental/efeitos adversos , Metástase Linfática , Mucinas/genética , Mucinas/metabolismo , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Risco , Lesões por Radiação/mortalidade , Fatores de Tempo , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/metabolismo , Tireotropina/sangueRESUMO
The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70 percent, compared to 64 percent for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85 percent, compared to 88 percent for GWGR. The assessment of RFLP revealed 68 percent sensitivity and 94 percent specificity for the B-GPGR strain compared to 84 and 90 percent for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.
Assuntos
Humanos , Masculino , Feminino , Adulto , /genética , Infecções por HIV/virologia , HIV-1 , Leucócitos Mononucleares/virologia , Fragmentos de Peptídeos/genética , Sequência de Aminoácidos , DNA Viral/análise , HIV-1 , Técnicas Imunoenzimáticas , Polimorfismo de Fragmento de Restrição , Provírus/genética , Sensibilidade e Especificidade , SorotipagemRESUMO
Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.
Assuntos
Feminino , Humanos , Masculino , Especificidade de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , /imunologia , HIV-1 , Fragmentos de Peptídeos/imunologia , Vacinas contra a AIDS , Especificidade de Anticorpos/genética , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Genótipo , /genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Testes de Neutralização/métodos , Fragmentos de Peptídeos/genéticaRESUMO
For effective FMD control programme, India needs large quantities of cheaper diagnostics in addition to vaccine. Diagnostic reagents produced through conventional methods may not be able to meet such requirements. Alternatively, rDNA technology using suitable heterologous systems that permit production of recombinant antigens to the most native form may be exploited. Studies conducted in our laboratory have led us to select carboxy terminal part of VP1 for expression and evaluation. The protein, which was purified from E.coli under denaturing conditions, was renatured and its reactivity was compared with the protein expressed in insect cells through recombinant baculovirus. The expressed protein in the insect cell whole lysate reacted more efficiently with antibodies raised against whole virus than the purified and renatured protein produced in E.coli. But for its lower reactivity, protein produced from E.coli was found to be suitable in type detection. In addition, the size of the protein is small (16 kD) and production and purification of it from E.coli may be cost effective. Hence, it may be exploited for FMDV typing.
Assuntos
Animais , Antígenos Virais/genética , Aphthovirus/genética , Sequência de Bases , Capsídeo/genética , Proteínas do Capsídeo , Linhagem Celular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/genética , SpodopteraRESUMO
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
Assuntos
Bovinos , Embrião de Galinha , Camundongos , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/genética , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Córion/patologia , Córion/efeitos dos fármacos , Dicroísmo Circular , Colágeno/farmacologia , Colágeno/isolamento & purificação , Colágeno/genética , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/citologia , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/genética , Dobramento de Proteína , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Solubilidade , Leveduras/genéticaRESUMO
We have synthesized a tris-sulfotyrosyl dodecapeptide (3S-peptide-I) that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit and showed that it potently inhibited insulin receptor dephosphorylation by protein tyrosine phosphatases (PTPases) in vitro. 3S-peptide-I also inhibited tyrosine dephosphorylation of a synthetic peptide by the recombinant PTPase PTP-1B, indicating that 3S-peptide-I interacts directly with PTPase, causing its inactivation. The peptide had no effect on the activity of serine/threonine phosphatases, PP-1 and PP-2A, or alkaline phosphatase. Furthermore, we found that the introduction of a N-stearyl derivative of 3S-peptide-I in CHO/HIRc cells caused a significant increase in insulin-stimulated phosphorylation of the insulin receptor. In contrast, ligand-stimulated phosphorylation of epidermal growth factor (EGF) receptor in CHO cells overexpressing EGF receptors was not affected by the presence of N-stearyl-3S-peptide-I. These data suggest that by inhibiting dephosphorylation of the insulin receptor in intact cells, 3S-peptide-I may specifically enhance insulin signalling.
Assuntos
Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Inibidores Enzimáticos/farmacologia , Humanos , Fragmentos de Peptídeos/genética , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptores ErbB/genética , Receptor de Insulina/química , Proteínas Recombinantes/genética , Transdução de SinaisRESUMO
The specificity of murine antibodies raised against structurally related peptides derived from a malaria parasite membrane protein was studied. The peptides were conjugated to bovine serum albumin (BSA) with 6-maleimido caproic acyl N-hydroxysuccinimide ester before immunization. Conjugation to BSA through a C-terminal or an internal cysteine residue elicited antibodies with noticeably different specificities. An N-terminal tripeptide sequence arginine-asparagine-asparagine had a dominant influence on the immunogenicity of the peptides. Such factors need to be taken into consideration while designing peptide-based immunogens.