RESUMO
Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.
Assuntos
Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Lectinas Tipo C/química , Lectinas Tipo C/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Biblioteca de Peptídeos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/genéticaRESUMO
Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.
Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Hemaglutininas/genética , Vírus da Influenza A/isolamento & purificação , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Anticorpos Antivirais/sangue , Embrião de Galinha , Galinhas , Patos , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Inibição da Hemaglutinação/veterinária , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Nucleoproteínas/química , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , VirulênciaRESUMO
A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.