RESUMO
Targeted detection programmes are recommended to identify subjects affected by severe alpha1-antitrypsin deficiency (AATD). Guidelines are available to address physicians towards subjects at high risk for AATD. We wanted to investigate the clinical characteristics of subjects enrolled in the programme, who result as not being affected by severe AATD; this information is not available in the present literature. We elaborated data contained in the questionnaires accompanying the samples of 2127 Italian subjects submitted for AATD detection in a period spanning 11 years (1996-2006). A total of 588 subjects were eligible to enter this study: PI*MM subjects and subjects with intermediate AATD, referred for lung disease, were characterised by a relatively young mean age, and a high proportion (31.2% and 28.6%, respectively) were never smokers. Fifty percent or more had symptoms of chronic bronchitis, but without obstruction. Only a minority belonged to most severe GOLD stages. The mean levels of AAT varied as a function of the presence or absence of airflow obstruction in intermediate AATD subjects, but not in PI*MM. Individuals enrolled in AATD detection programmes represent an interesting cohort both for public health and research purposes.
Assuntos
Deficiência de alfa 1-Antitripsina/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pneumopatias Obstrutivas/etiologia , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Fatores de Risco , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (k(obs)) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (k(a)). A proflavine displacement assay was used to determine the deacylation rate constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and a low k(d). Because the number of turnovers per inactivation of Er was congruent with 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a k(d) only, whereas for FE, in addition to the k(d), an alkylation constant (k(2)) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Eritromicina/farmacologia , Elastase de Leucócito/antagonistas & inibidores , Acilação , Bioquímica/métodos , Ativação Enzimática/efeitos dos fármacos , Eritromicina/análogos & derivados , Humanos , Relação Estrutura-AtividadeRESUMO
A capillary isoelectric focusing (CIEF) method using bare fused-silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5-5.0 range has been developed for the identification of alpha1-antitrypsin (alpha1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose alpha1AT phenotype was previously identified and of twelve subjects whose alpha1AT phenotype was unknown. The results obtained allowed us to confirm or identify the alpha1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of alpha1AT variants associated with diseases of clinical relevance.
Assuntos
alfa 1-Antitripsina/genética , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Variação Genética , Humanos , Focalização Isoelétrica/métodos , Elastase de Leucócito/antagonistas & inibidores , Fenótipo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , alfa 1-Antitripsina/análiseRESUMO
Aspergillus fumigatus produces a variety of extracellular proteinases that are believed to be virulence factors towards Aspergillus-related lung disease. Among Aspergillus proteinases, the serine proteinase is thought to play a major virulent role because of its widespread production. Nevertheless, evidence of direct pulmonary injury caused by the A. fumigatus serine proteinase is still lacking. The purpose of our work was: (1) to provide evidence for a pivotal role of A. fumigatus serine proteinase in producing lung injury in an animal model, and (2) to investigate the broadness of the substrate specificity of the proteinase towards extracellular matrix components. To achieve this aim, the proteinase from an A. fumigatus strain isolated from human airways was purified by a four-step procedure, including cation exchange and hydrophobic interaction. High-performance capillary electrophoresis, SDS-PAGE, determination of K(m) towards synthetic substrates, and inhibitory studies were used to further characterize the A. fumigatus serine proteinase. With reference to extracellular matrix components, the A. fumigatus serine proteinase was shown to degrade human lung elastin at a higher rate than an equimolar amount of human neutrophil elastase. Human lung collagen, type I and type III collagens, as well as fibronectin, were quickly digested by the A. fumigatus serine proteinase. Finally, mice intratracheally injected with the proteinase showed a significant degree of lower respiratory tract destruction. We conclude that the A. fumigatus serine proteinase is capable per se of hydrolyzing the major structural barriers of the lung.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Pneumopatias/induzido quimicamente , Pulmão/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Fibronectinas/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Serina Endopeptidases/isolamento & purificação , Especificidade por SubstratoRESUMO
Type I and type III are the most abundant collagens in the lung. The aim of our study was to compare type I and III procollagen peptides in sera of sarcoid patients. Sixty eight patients with sarcoidosis were studied (19 with newly recognized disease, 7 with relapsing disease, 15 with chronic disease, and 27 in stable remission). Thirty healthy volunteers served as controls. The levels of procollagen I and III peptides were determined by radioimmunoassay. Angiotensin-converting enzyme (ACE) level was evaluated by means of a colorimetric assay. In patients with newly recognized sarcoidosis, both serum procollagen I and III peptide levels were increased with respect to controls (p=0.0014 and p<0.00001, respectively). There was a poor correlation between levels of procollagen I and III (r=0.26), whereas there was a closer correlation between procollagen III and ACE (r=0.69). Procollagen I peptide level did not identify patients in roentgenological stage III. In conclusion, in patients with newly recognized sarcoidosis there is a significant increase in the serum level of procollagen I peptide. However, procollagen I peptide is not a marker of sarcoid patients with fibrosis, ie. stage III disease. Its clinical usefulness seems to be weaker than that of procollagen III peptide.
Assuntos
Proteínas Morfogenéticas Ósseas , Metaloendopeptidases/sangue , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Sarcoidose/sangue , Adulto , Análise de Variância , Proteína Morfogenética Óssea 1 , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sarcoidose/diagnóstico , Sensibilidade e EspecificidadeRESUMO
Sarcoidosis is the disease in which increased levels of serum Angiotensin-converting enzyme (sACE) are most often detected. It has recently been shown that the deletion (D) or the insertion (I) of a 250bp-DNA fragment in the ACE gene accounts for three main ACE genotypes (i.e., II, ID, and DD) and for 47% of total phenotypic variance in sACE level. The aim of our work was to investigate whether or not patients with sarcoidosis have an increased incidence of those ACE genotypes coding for highest sACE levels and to investigate whether or not sACE level in sarcoidosis is related to ACE genotypes. We studied 61 unrelated patients with sarcoidosis (test group) and 80 unrelated healthy control subjects (control group). The ACE I and D alleles were detected with polymerase chain reaction on genomic DNA. In the control group we found an ACE genotype distribution that agreed with the Hardy-Weinberg proportion. The ACE genotype distribution was not significantly different in the test group. There was no correlation between ACE genotype and roentgenologic stage of sarcoidosis. Plotting the sACE level in the control group against ACE genotype, we found a trend of increasing mean sACE value according to the order II < ID < DD. The same trend for ACE genotype was found in the test group, in which it also paralleled the trend of sACE values plotted against roentgenologic stage, according to the order Stage I < Stage II < Stage III. We conclude that in sarcoidosis the ACE genotype distribution is not altered. The trends for increasing sACE values in sarcoidosis according to both ACE genotype and roentgenologic stage would suggest that both mechanisms play a role in determining sACE level.
Assuntos
Peptidil Dipeptidase A/genética , Polimorfismo Genético , Sarcoidose Pulmonar/genética , Adulto , Alelos , Feminino , Genótipo , Humanos , Pulmão/diagnóstico por imagem , Masculino , Peptidil Dipeptidase A/sangue , Reação em Cadeia da Polimerase , Radiografia , Sarcoidose Pulmonar/enzimologiaRESUMO
The novel flavonoid compound 3'-hydroxyfarrerol (IdB 1031) was tested in a number of in vitro experiments in order to ascertain its effects on some functions and products of human phagocytes. We found that IdB 1031 did not depress neutrophil phagocytosis and chemotaxis, whereas at a concentration of 10(-4) M it significantly (p < 0.05) reduced the fMLP-triggered neutrophil production of superoxide anion. At the same concentration, the compound decreased the release of neutrophil elastase and myeloperoxidase from neutrophils (p < 0.05). We also found evidence that IdB 1031 is a non competitive, reversible inhibitor of human neutrophil elastase (Ki 200 microns). Finally, IdB 1031 at the concentration of 10(-5) M significantly reduced the release of tumor necrosis factor-alpha and interleukin-8 from monocytes (p < 0.05). We conclude that, in spite of the moderate activity displayed by IdB 1031, these findings add to our current knowledge on the spectrum of the antiinflammatory activities of flavonoids.
Assuntos
Flavonoides/farmacologia , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interleucina-8/metabolismo , Elastase de Leucócito , Lipopolissacarídeos/farmacologia , Concentração Osmolar , Elastase Pancreática/metabolismo , Peroxidase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Toxic oxygen free radicals are believed to play a role in the pathogenesis of a number of respiratory diseases. In particular, pulmonary emphysema may occur because of the oxidative impairment of alpha 1-proteinase inhibitor (alpha 1-PI). We report in vitro data on a new thiol agent, P 1507 [N-5-(thioxo-L-prolyl)-L-cysteine], obtained in a series of experiments designed in view of its therapeutic potential in these clinical conditions. We found that P 1507 at the concentration of 5 x 10(-6) M was able to almost fully abolish the PMA-triggered PMN-induced oxidative impairment of alpha 1-PI. Protection may be due to the radical scavenger ability of P 1507, that markedly reduced superoxide anion production from PMNs. We also found that P 1507 did not significantly impair other defence mechanisms of PMNs (i.e. phagocytosis, chemotaxis and bactericidal activity). The release of cytokines (TNF-alpha, IL-6 and IL-8) from monocytes was not altered in the presence of P 1507. We conclude that the compound P 1507 may be considered for treatment of clinical conditions characterized by overload of oxidants, on the basis of its ability in preventing the oxidative damage of alpha 1-PI and of a lack of unwanted inhibitory effects towards defence mechanisms of phagocytes.
Assuntos
Antioxidantes/farmacologia , Dipeptídeos/farmacologia , Fagócitos/efeitos dos fármacos , alfa 1-Antitripsina/efeitos dos fármacos , Candida albicans/fisiologia , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Pneumopatias Obstrutivas/tratamento farmacológico , Fagocitose/efeitos dos fármacos , alfa 1-Antitripsina/metabolismoRESUMO
MR889 is a synthetic inhibitor of human neutrophil elastase with potential in clinical conditions characterized by a high load of this proteolytic enzyme, mainly chronic obstructive pulmonary disease. On the basis of its mechanism of inhibition, it has been suggested that MR889, upon reaction with elastase, would generate new free thiol groups. The aim of this study was to investigate whether MR889, upon reaction with elastase, may modify rheologic properties, i.e. apparent viscosity and elasticity, of both human sputum and porcine stomach mucus, in ex-vivo and in vitro experiments. MR889 10(-4) M alone had no effect on rheologic properties of samples, whereas an agent with free thiol group, n-acetylcysteine 10(-4) M reduced both viscosity and elasticity. MR889 10(-4) M upon reaction with elastase 0.03 nMol (giving 83% of elastase inhibition) had no effect on rheologic properties of samples, whereas upon reaction with elastase 0.15 nMol (49% inhibition) yielded a marked decrease in viscosity. This result would suggest that MR889, in the presence of a high load of elastase, may acquire direct mucus modifying capacity, likely because of its greater utilization upon reaction with the enzyme.
Assuntos
Bronquite/enzimologia , Muco/efeitos dos fármacos , Elastase Pancreática/antagonistas & inibidores , Escarro/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Bronquite/tratamento farmacológico , Doença Crônica , Elasticidade/efeitos dos fármacos , Mucosa Gástrica/enzimologia , Humanos , Técnicas In Vitro , Elastase de Leucócito , Muco/enzimologia , Escarro/enzimologia , Suínos , Viscosidade/efeitos dos fármacos , alfa 1-Antitripsina/farmacologiaRESUMO
An association between arterial blood pressure and blood viscosity has been suggested in healthy and in diabetic subjects, and that the hemorheological pattern may be influenced by blood lipid alterations. In diabetic patients a relationship between arterial hypertension and blood lipid changes may therefore be suggested. This study concerns 19 type II diabetics with hyperlipidemia (triglycerides = 3.2 +/- 1 mmol/l; total cholesterol = 6.1 +/- 1.2 mmol/l; HDL-cholesterol = 0.92 +/- 0.27 mmol/l; VLDL = 29 +/- 5%) (group A), and 19 normolipidemic type II diabetics (triglycerides = 1.15 +/- 0.5 mmol/l; total cholesterol = 5.1 +/- 1 mmol/l; HDL-cholesterol = 1.25 +/- 0.38 mmol/l; VLDL = 20 +/- 5%) (group B). No differences concerning age, body weight, duration of diabetes and glycemic control were found in hyperlipidemic compared to normolipidemic diabetics. On the contrary, higher systolic and diastolic blood pressure levels were demonstrated in group A (167 +/- 14 mmHg and 101 +/- 5.2 mmHg, respectively) than in group B (144 +/- 15 mmHg, p less than 0.001 and 87 +/- 6.9 mmHg, p less than 0.001, respectively). An increase of plasma apolipoprotein B level (163 +/- 27 mg/dl vs 102 +/- 21 mg/dl, p less than 0.001), of plasma viscosity (1.81 +/- 0.08 mPas vs 1.51 +/- 0.07 mPas, p less than 0.001) and of blood viscosity (5.37 +/- 0.33 mPas vs 5.07 +/- 0.04 mPas, p less than 0.01, at shear-rate of 90 s-1; 18.4 +/- 1 mPas vs 14.1 +/- 0.9 mPas, p less than 0.001 at shear-rate of 2.25 s-1) was found in group A, compared to group B.(ABSTRACT TRUNCATED AT 250 WORDS)