RESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is caused by alterations in expression of proteins involved in multiple pathways, including matrix deposition, inflammation, injury, and repair. OBJECTIVES: To understand the pathogenic changes in lung protein expression in IPF and to evaluate apolipoprotein (Apo) A-I as a candidate therapeutic molecule. METHODS: Two-dimensional electrophoresis was adopted for differential display proteomics. Reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemical staining, and ELISA were performed for identification and quantitative measurement of Apo A-I in bronchoalveolar lavage fluids from subjects with IPF and experimental bleomycin-induced mice. MEASUREMENTS AND MAIN RESULTS: Sixteen protein spots showed differences in relative intensity between IPF (n = 14) and healthy control subjects (n = 8). Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increase of haptoglobulin and decrease of alpha(1)-antitrypsin, alpha(1)-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, Apo A-I, clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A in IPF compared with normal control subjects (P = 0.006-0.044). Apo A-I concentrations were lower in bronchoalveolar lavage fluids from subjects with IPF (n = 28) than in normal control subjects (n = 18; P < 0.01). In bleomycin-treated mice, Apo A-I protein in BALF was lower than that in sham-treated control animals. Immunohistochemical analysis showed positive staining on intraalveolar macrophages and epithelial cells of the lungs. Intranasal treatment with Apo A-I protein reduced the bleomycin-induced increases in number of inflammatory cells and collagen deposition in sham-treated mice in a dose-dependent manner. CONCLUSIONS: Alterations of several inflammatory and antiinflammatory proteins in the lungs may be related to the pathogenesis of IPF, and local treatment with Apo A-I is very effective against the development of experimental lung injury and fibrosis.
Assuntos
Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/uso terapêutico , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Idoso , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Bleomicina , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteômica/métodos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: Brucellosis is one of the most common zoonoses in the world, and occurs mainly in farmers, slaughterhouse workers, and veterinarians via direct or indirect contact with infected animals or their products. The clinical symptoms of human brucellosis are nonspecific, such as fever, headache, chills, and sweating. Diagnosis and treatment of brucellosis requires laboratory tests. Although the serum tube agglutination test (SAT) is the standardized gold method, it is laborious, time consuming, and requires a number of reagents. A microagglutination test (MAT) variant of the SAT or enzyme-linked immunosorbent assay (ELISA) is recommended for serological diagnoses. For the simple and rapid diagnosis of brucellosis, the MAT was standardized using samples for the SAT to define positive and negative categories, and we then compared the sensitivity and specificity of the MAT and ELISA. METHODS: Thirty SAT-positive sera and 60 SAT-negative sera were used in this study. Antibody titers of ≥1:160 were considered positive readings in both the SAT and MAT. Brucella abortus antigens and Brucella-positive control antiserum were used in the SAT and MAT. ELISAs of IgM and IgG were performed according to the manufacturers' instructions. RESULTS: The titers of the MAT differed according to antigen concentration. The optimal concentration of B abortus antigen was determined to compare the sensitivity and specificity between the MAT and SAT. The sensitivity and specificity of the MAT were 93.3% and 96.7%, respectively, for IgG with reference to ELISA, and 96.7% and 98.3%, respectively, for IgM. CONCLUSIONS: The optimal concentration of antigen for the MAT was 1:10. The MAT is less time consuming and requires less antigen and serum than the SAT. The results of the MAT showed good agreement with those of ELISA. The results of this study suggest that the MAT could be useful for diagnosis of brucellosis.
RESUMO
BACKGROUND: The peroxisome proliferator-activated receptors (PPAR) are the nuclear hormone receptor superfamily of ligand-activated transcriptional factors. PPAR-gamma (PPARG) activation downregulates production of Th2 type cytokines and eosinophil function. Additionally, treatment with a synthetic PPARG ligand can reduce lung inflammation and IFN-gamma, IL-4, and IL-2 production in experimental allergic asthma. In patients with asthma, PPARG gene expression is known to be associated with the airway inflammatory and remodeling responses. Thus, genetic variants of PPARG may be associated with the development of asthma. METHODS: We genotyped two single nucleotide polymorphisms on the PPARG gene, +34C>G (Pro12Ala) and +82466C>T (His449His), in Korean subjects (839 subjects with asthma and 449 normal controls). RESULTS: Association analysis using logistic regression analysis showed that +82466C>T and haplotypes 1(CC) and 2(CT) were associated with the development of asthma (p=0.01-0.04). The frequency of PPARG-ht2 was significantly lower in the patients with asthma compared to the normal controls in codominant and dominant models (p=0.01, p(corr)=0.03 and p=0.02, p(corr)=0.03, respectively). Conversely, the frequency of PPARG-ht1 was significantly higher in the patients with asthma compared to the normal controls in the codominant model [p=0.04, OR: 1.27 (1.01-1.6)]. In addition, the rare allele frequency of +82466C>T was significantly lower in patients with asthma in comparison to normal controls in the codominant model (OR: 0.78, p=0.04). Thus, polymorphism of the PPARG gene may be linked to an increased risk of asthma development.