RESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis. METHODS: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope. RESULTS: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases. CONCLUSION: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.
Asunto(s)
Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Anhidrasa Carbónica III/sangre , Técnicas de Visualización de Superficie Celular/métodos , Modelos Animales de Enfermedad , Imitación Molecular/fisiología , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/genética , Anhidrasa Carbónica III/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5 days of a protein and amino acid-free diet followed by 5 days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (-48% and -64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (-66%) and GSTs (-26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions.
Asunto(s)
Proteínas en la Dieta/administración & dosificación , Hígado/metabolismo , Hígado/patología , Desnutrición/metabolismo , Estrés Oxidativo/efectos de los fármacos , Alanina Transaminasa/sangre , Alanina Transaminasa/efectos de los fármacos , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/efectos de los fármacos , Aspartato Aminotransferasas/metabolismo , Anhidrasa Carbónica III/efectos de los fármacos , Anhidrasa Carbónica III/metabolismo , Catalasa/efectos de los fármacos , Catalasa/metabolismo , Ácido Graso Sintasas/efectos de los fármacos , Ácido Graso Sintasas/metabolismo , Femenino , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/efectos de los fármacos , Glutatión Reductasa/metabolismo , Glutatión Transferasa/efectos de los fármacos , Glutatión Transferasa/metabolismo , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/efectos de los fármacos , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Peroxidación de Lípido , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Carbonilación Proteica/efectos de los fármacosRESUMEN
The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.