RESUMO
After decades of cancer vaccine efforts, there is an imperious necessity for novel ideas that may result in better tumor control in patients. We have proposed the use of a novel Variable Epitope Library (VEL) vaccine strategy, which incorporates an unprecedented number of mutated epitopes to target antigenic variability and break tolerance against tumor-associated antigens. Here, we used an oncofetal antigen/immature laminin receptor protein-derived sequence to generate 9-mer and 43-mer VEL immunogens. 4T1 tumor-bearing mice developed epitope-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell responses after treatment. Tumor and lung analysis demonstrated that VELs could increase the number of tumor-infiltrating lymphocytes with diverse effector functions while reducing the number of immunosuppressive myeloid-derived suppressor and regulatory T cells. Most importantly, VEL immunogens inhibited tumor growth and metastasis after a single dose. The results presented here are consistent with our previous studies and provide evidence for VEL immunogens' feasibility as promising cancer immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Receptores de Laminina/imunologia , Animais , Vacinas Anticâncer/farmacologia , Modelos Animais de Doenças , Mapeamento de Epitopos/métodos , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Extracellular and intraneuronal formation of amyloid-beta (Abeta) deposits have been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of Abeta neurotoxicity is not completely understood. Previous studies suggest that binding of Abeta with a number of targets have deleterious effects on cellular functions. It has been shown that Abeta directly interacted with intracellular protein ERAB (endoplasmic reticulum amyloid beta-peptide-binding protein) also known as ABAD (Abeta-binding alcohol dehydrogenase) resulting in mitochondrial dysfunction and cell death. In the present study we have identified another mitochondrial enzyme, ND3 of the human complex I, that binds to Abeta1-42 by the screening of a human brain cDNA library expressed on M13 phage. Our results indicated a strong interaction between Abeta and a phage-displayed 25 amino acid long peptide TTNLPLMVMSSLLLIIILALSLAYE corresponding to C-terminal peptide domain of NADH dehydrogenase, subunit 3 (MTND3) encoded by mitochondrial DNA (mtDNA). This interaction may explain, in part, the inhibition of complex I activity in astrocytes and neurons in the presence of Abeta, described recently. To our knowledge, the present study is the first demonstration of interaction between Abeta and one of the subunits of the human complex I.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Biblioteca Gênica , Testes Genéticos/métodos , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Southern Blotting/métodos , Fragmentação do DNA/fisiologia , Complexo I de Transporte de Elétrons , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.
Assuntos
Mamastrovirus/metabolismo , Fases de Leitura Aberta/genética , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas não Estruturais Virais/metabolismo , Células CACO-2 , Humanos , Mamastrovirus/classificação , Mamastrovirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sorotipagem , Proteínas não Estruturais Virais/genéticaRESUMO
Human astroviruses are an important cause of gastroenteritis. As part of a molecular epidemiological study carried out in Mexico a human astrovirus isolate, Yuc-8, was adapted to grow in CaCo-2 cells, and its entire genome was sequenced. A 15 amino acid deletion in ORF1a, which has been associated with adaptation of astroviruses to grow in cells other than CaCo-2, was present in Yuc-8. Comparative sequence analysis of the Yuc-8 ORF2 with reported human astrovirus sequences revealed that this isolate belongs to genotype (serotype) 8. Two distinct domains in ORF2 were observed: an amino-terminal domain (residues 1 to 415), with identities higher than 81% among the strains analysed, and a carboxy-terminal domain (residues 416 to 782) with identities between 36 and 60%. Two non-superimposable phylogenetic trees were generated by separate analysis of these two domains, suggesting that a differential selective pressure is exerted along the structural polyprotein.