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1.
Inflamm Bowel Dis ; 19(11): 2273-81, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23949620

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.


Assuntos
Anticorpos/administração & dosagem , Colite/tratamento farmacológico , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Inflamação/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos/farmacologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Bovinos , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana/toxicidade , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/farmacologia , Inflamação/etiologia , Inflamação/patologia , Infliximab , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Trinitrobenzenossulfônico/toxicidade , Fator de Necrose Tumoral alfa/imunologia
2.
Vaccine ; 29(31): 5031-9, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21616113

RESUMO

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products.


Assuntos
Química Farmacêutica/métodos , Vacina contra Sarampo/química , Vírus do Sarampo/efeitos dos fármacos , Vírus do Sarampo/efeitos da radiação , Estabilidade de Medicamentos , Excipientes/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Vírus do Sarampo/patogenicidade , Temperatura
3.
Proc Natl Acad Sci U S A ; 103(15): 5953-8, 2006 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-16585504

RESUMO

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.


Assuntos
Globinas/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , Sequência de Bases , Inativação Gênica , Genes Reporter , Células HeLa , Humanos , Luciferases de Renilla/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
J Virol ; 78(10): 5079-87, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15113889

RESUMO

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertion-tolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.


Assuntos
Epitopos , Glicoproteínas de Membrana/química , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Linhagem Celular , Cricetinae , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/imunologia , Replicação Viral
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