RESUMO
Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.
Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Técnicas de Visualização da Superfície Celular , Citocinas/metabolismo , Humanos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Modelos Moleculares , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Nanomedicina Teranóstica , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologiaRESUMO
AIM: The dengue virus is responsible for a high worldwide incidence of infections, aggravated by late diagnosis, and often confused with other tropical diseases. Results/methodology: Oligonucleotide aptamers binding to the 5'-UTR from dengue virus selected after eight rounds by systematic evolution of ligands by exponential enrichment technology were analyzed by dot-blot assay and in silico prediction of secondary structures, demonstrating the presence of stem-loops that may have the potential for interaction with the viral genome, which can lead to loss of their original conformation. CONCLUSION: This is the first description of RNA aptamers against functional RNA elements of the dengue virus genome with implications for disease control, which may have potential as tools in the future of antiviral therapies and for diagnostics.
Assuntos
Regiões 5' não Traduzidas/efeitos dos fármacos , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Vírus da Dengue/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Regiões 5' não Traduzidas/genética , Antivirais/química , Aptâmeros de Nucleotídeos/química , Sítios de Ligação/efeitos dos fármacos , Vírus da Dengue/genética , Ligantes , Testes de Sensibilidade Microbiana , Oligonucleotídeos/química , Relação Estrutura-AtividadeRESUMO
Strongyloidiasis is one of the major intestinal infections in humans, and a neglected tropical disease whose diagnosis still poses a challenge. We hypothesized that diagnostic tests based on short peptides containing major epitopes may represent a promising strategy to improve strongyloidiasis detection due to reduced cross-reactivity and higher sensitivity. Our aim was to evaluate two synthetic peptides selected by phage display (C10 and D3) as potential tools for serodiagnosis of strongyloidiasis, and to predict their putative antigen target. To investigate their diagnostic potential, we have tested different panels of serum samples (n=120) by enzyme linked immunosorbent assay (ELISA) to detect specific IgG, and their diagnostic parameters were calculated. Similarities with proteins from Strongyloides stercoralis were searched and conformational epitopes were predicted and aligned to known protein structures. Both C10 and D3 achieved sensitivity of 95%, and specificities were 89.2% and 92.5%, respectively. D3 presented the highest diagnostic efficiency (93.3%). Epitope prediction for both C10 and D3 led to the alignment with the cytochrome c oxidase subunit 1 structure. In brief, we propose two synthetic peptides as new biomarkers for serodiagnosis of strongyloidiasis, which can be promptly used for ELISA and in future field sensor platforms.
Assuntos
Epitopos de Linfócito B/imunologia , Doenças Negligenciadas/imunologia , Fragmentos de Peptídeos/imunologia , Strongyloides stercoralis/imunologia , Estrongiloidíase/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Biomarcadores/metabolismo , Brasil , Simulação por Computador , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Fragmentos de Peptídeos/síntese química , Valor Preditivo dos Testes , Prognóstico , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
The transforming growth factor beta 1 (TGF-ß1) is a pleiotropic cytokine with multiple roles in development, wound healing, and immune regulation. TGF-ß1-mediated immune dysfunction may lead to pathological conditions, such as inflammation. Chronic inflammatory process is characterized by a continuous release of pro-inflammatory cytokines, and the inhibition or the blockage of these cytokines signaling pathways are considered a target treatment. In this context, despite the high numbers of TGF-ß-targeted pathways, the inducible regulatory T cells (iTreg) to control inflammation seems to be a promising approach. Our aim was to develop novel peptides through phage display (PhD) technology that could mimic TGF-ß1 function with higher potency. Specific mimetic peptides were obtained through a PhD subtraction strategy from whole cell binding using TGF-ß1 recombinant as a competitor during elution step. We have selected a peptide that seems to play an important role on cellular differentiation and modulation of TNF-α and IL-10 cytokines. The synthetic pm26TGF-ß1 peptide tested in PBMC significantly down-modulated TNF-α and up-regulated IL-10 responses, leading to regulatory T cells (Treg) phenotype differentiation. Furthermore, the synthetic peptide was able to decrease leukocytes rolling in BALB/C mice and neutrophils migration during inflammatory process in C57BL/6 mice. These data suggest that this peptide may be useful for the treatment of inflammatory diseases, especially because it displays potent anti-inflammatory properties and do not exhibit neutrophils' chemoattraction.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Materiais Biomiméticos/farmacologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Neutrófilos/imunologia , Peptídeos/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Materiais Biomiméticos/química , Feminino , Humanos , Interleucina-10/imunologia , Migração e Rolagem de Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/patologia , Peptídeos/química , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta1/química , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.
Assuntos
Antígenos de Neoplasias/genética , Aptâmeros de Nucleotídeos/metabolismo , Neoplasias da Próstata/diagnóstico , Anticorpos Antinucleares/imunologia , Antígenos de Neoplasias/sangue , Sequência de Bases , DNA de Neoplasias/sangue , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/imunologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Conformação de Ácido Nucleico , Próstata/metabolismo , Próstata/patologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA/química , RNA/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise Serial de TecidosRESUMO
Nanotechnological tools and biomarkers for diagnosis and prognosis, as well as strategies for disease control and monitoring populations at higher risk, are continuous worldwide challenges for infectious diseases. Phage display and monoclonal antibody combinatorial libraries are important sources for biomarker discovery and for improved diagnostic strategies. Mimetic peptides were selected against polyclonal antibodies from patients with dengue fever, leprosy, and leishmaniasis as model diseases, and from immunized chickens with total antigens from all three pathogens. Selected single or combined multi-epitope peptide biomarkers were further associated with four different sensor platforms, classified as affinity biosensors, that may be suitable as general protocols for field diagnosis. We have also developed two methods for nanoparticle agglutination assays (a particle gel agglutination test and a magnetic microparticle [MMP]-enzyme-linked immunosorbent assay [ELISA]) and two electrochemical biosensors (impedimetric and amperometric) for DNA and antibody detection. For the agglutination tests, micro- and nanoparticles were coupled with filamentous bacteriophages displaying the selected mimotopes on their surfaces, which has favored the formation of the antigen-antibody or peptide-protein complexes, amplifying the optical detection in ELISA assays or after the chromatographic separation of the microagglutinates. We have also demonstrated a proof-of-concept for the electrochemical biosensors by using electrodes modified with novel functionalized polymers. These electrochemical biosensors have proven to be fast, very sensitive, and specific for the detection of pathogen DNA and circulating antibodies of patients, which may become important in a wide range of diagnostic devices for many infectious agents.