RESUMO
Chronic obstructive pulmonary disease (COPD) is generally thought to depend on an aberrant immune response to a noxious or infectious agent, which may cause chronic inflammation. However, the initiation of this abnormal response is not fully understood. Here, we propose a new hypothesis for the beginning of COPD, that the immune response to inhaled agents, mainly cigarette smoke, is directed toward the airway epithelium, due to oxidative DNA damage of the lung epithelial barrier cells (LEBCs). The steps of this model are as follows. 1) Cigarette smoke induces oxidative DNA damage of LEBCs. 2) The acquired mutations are expressed at the microsatellite DNA level of LEBCs. 3) The altered LEBCs are recognised by dendritic cells (DCs) as "nonself". DCs travel, with the new information, to the lymph nodes, presenting it to the naïve T-lymphocytes. 4) A predominant CD8+ cytotoxic T-lymphocyte proliferation occurs. The CD8+ cells, by releasing perforin and granzymes, attack the altered LEBCs activating cell death cascades. Obviously, the above scenario needs further experimental exploration. However, it is an attractive model for the initiation of the abnormal inflammation in COPD, comprising oxidative DNA damage of LEBCs and host immune response.
Assuntos
Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/terapia , Linfócitos T CD8-Positivos/metabolismo , Morte Celular , Dano ao DNA , Células Dendríticas/citologia , Epitélio/patologia , Feminino , Humanos , Inflamação , Pulmão/patologia , Masculino , Repetições de Microssatélites/genética , Mutação , Oxigênio/química , Oxigênio/metabolismo , Fumar , Linfócitos T Citotóxicos/citologiaRESUMO
Increased frequency of microsatellite DNA instability (MSI) has been detected in the sputum of chronic obstructive pulmonary disease (COPD) patients. The aim of the present study was to investigate the relationship between MSI in sputum cells and exacerbation frequency, which is an important parameter in the clinical course of the disease. Induced sputum samples and peripheral blood obtained from 36 patients with COPD at stable state were analysed. The control group consisted of 30 nonsmoking healthy subjects. DNA was extracted and analysed for MSI using the following microsatellite markers: RH70958, D5S207, D6S2223, D6S344, D6S263, G29802, D13S71, D14S588, D14S292 and D17S250. Following MSI analysis, exacerbations were recorded for 3 yrs in total. No MSI was detected in healthy nonsmokers. A total of 18 (50%) out of 36 patients exhibited MSI in their sputum cells. Patients who exhibited MSI showed significantly increased frequency of exacerbations compared with patients that did not. In addition, a significantly increased frequency of purulent and of severe type exacerbations was found in patients exhibiting MSI. Patients positive for marker G29802, D13S71 or D14S588 presented increased exacerbation frequency. The significant association between microsatellite DNA instability and chronic obstructive pulmonary disease exacerbations indicates that somatic mutations could be involved in the pathogenesis and natural history of the disease.
Assuntos
Instabilidade de Microssatélites , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Escarro/citologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , Fumar/genéticaRESUMO
Distinguishing malignant from benign pleural effusions using routine cytology is a common diagnostic problem. Recently, genetic alterations, including microsatellite instability (MSI) and loss of heterozygosity (LOH), have been described in malignant pleural effusions and proposed as methods improving diagnostics. The purpose of this study was to evaluate a panel of molecular markers for the detection of genetic alterations of cells in pleural effusions and to determine their diagnostic value as an additional test to cytologic examination. Pleural fluid and peripheral blood from 48 patients (36 male and 12 female, median age 71 years) were analyzed. Twenty-six patients had malignant pleural effusion, including 23 lung cancer and three metastatic non-pulmonary carcinoma. The control group consisted of 22 patients with benign pleural effusions. Only 14 malignancy-associated pleural effusions were cytology-positive for malignant cells (54%), whereas all benign pleural effusions were negative. DNA was extracted from all the samples and analysed for MSI and/or LOH using the following microsatellite markers: D3S1234, D9S171, D12S363, D17S250, D5S346 and TP53Alu, located at five chromosomal regions: 3p, 9p, 12q, 17q, 5q. Microsatellite analysis of the pleural fluid pellet exhibited genetic alterations in two neoplastic pleural fluid cases and in one inflammatory case. Two out of 26 (7.6%) patients with malignant pleural effusion showed genetic alterations. One exhibited MSI in three different microsatellite markers (D17S250, D9S171, D3S134) and the other showed LOH in marker D3S134. One out of 22 (4.5%) patients with benign pleural effusion showed LOH in marker D3S134. In conclusion, genetic alterations at the level of microsatellite DNA, were detected only in very few cases of malignant pleural effusions, and in one case of benign pleural effusion. Thus, our data suggest that microsatellite DNA analysis does not facilitate the diagnosis of malignant pleural effusion.
Assuntos
DNA de Neoplasias/genética , DNA/genética , Repetições de Microssatélites , Derrame Pleural/fisiopatologia , Neoplasias Pleurais/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Mapeamento Cromossômico , Cromossomos Humanos , Feminino , Instabilidade Genômica , Humanos , L-Lactato Desidrogenase/análise , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Proteínas/análiseRESUMO
STUDY OBJECTIVES: Cigarette smoking is the prime cause of COPD; however, only a few smokers develop the disease. In a previous study, we demonstrated that microsatellite DNA instability (MSI) is a detectable phenomenon in sputum cells of COPD patients. Therefore, we hypothesize that this genetic alteration may indicate susceptibility to COPD. DESIGN: In order to investigate this hypothesis, we compared smokers who developed COPD with smokers who did not develop COPD (referred to as non-COPD smokers). SETTING: Seven highly polymorphic microsatellite markers were targeted on the DNA of sputum cells and of WBCs. PATIENTS AND PARTICIPANTS: We studied 60 non-COPD smokers and 59 severe COPD patients with a similar smoking history (mean +/- SD) of 48+/-25 and 54+/-33 pack-years, respectively (p = 0.77). Non-COPD smokers were tested once; COPD smokers were tested twice, with an interval of 24 months between tests. RESULTS: MSI was detected in 14 COPD patients (24%) but in none of the non-COPD smokers. In 10 COPD patients, MSI was exhibited by one microsatellite marker; in the remaining 4 COPD patients, MSI was exhibited by two different alleles. The most commonly affected marker was THRA1 on chromosome 17 (43%). No significant differences were found between MSI-positive and MSI-negative COPD patients for clinical or laboratory parameters, survival, and development of lung cancer. No change in the microsatellite alleles was found between the tests performed with a 24-month interval. CONCLUSIONS: This study demonstrated that MSI was found exclusively in the sputum cells of smokers with COPD. The results support the hypothesis that MSI could be part of the complex genetic basis of COPD, and it could be a marker of the genetic alteration caused by smoking that allows COPD to develop.
Assuntos
Pneumopatias Obstrutivas/genética , Repetições de Microssatélites , Fumar/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escarro/citologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is a relatively common disease, affecting mainly males in the western world. Although substantial data are available as regards the clinicopathological characterization of COPD, little is known of the molecular basis of the disease. In the present study we analysed the incidence of microsatellite instability (MI) in cytological specimens from patients with COPD. MI reflects increased mutational rate and is associated with decreased accuracy in the DNA repair, resulting in the accumulation of somatic mutations in cells manifesting this genetic alteration. Among 31 specimens tested, 7 (23%) exhibited MI in at least one among 6 microsatellite markers tested. 5 cases were affected in only one marker while the remaining two cases exhibited evidence of MI in two microsatellite markers. These data suggest that an elevated mutational rate as reflected by the increased incidence of MI is associated with the development of the disease.
RESUMO
COPD is a common disease and its major risk factor, cigarette smoking, has been identified. However, only a minority of smokers develop clinically relevant disease. Although, the current understanding of the pathogenesis includes an "abnormal inflammation" as a response to various noxious agents, its various pathways are not clear. Oxidative stress, inflammation, tissue damage and tissue repair (remodeling) are parts of the complex procedure leading to COPD. This is a review of the available literature concerning the "susceptible" smoker. An epidemiological model is discussed, putting emphasis on the timing of the exposure to cigarette smoke. There are evidences that respiratory adenoviral infection in early life could be also an important factor. Differences in nutrition could also play a role in protecting against the oxidative stress. Airway hyperresponsiveness failed to clarify the whole picture and is still open for debate. Genetic differences are the most likely explanations to describe the "susceptible" smoker. However, the only well-established genetic risk factor is the alpha-l-antitrypsin. Other candidate genes were reviewed, alpha-l-antichymotrypsin, blood group antigens, vitamin-D binding protein, a2-macroglobulin, immunoglobulin deficiency, extracellular superoxide dismutase, secretory leukocyte proteinase inhibitor, cathepsin G, tumor necrosis factor-a gene and others. Microsatellite DNA instability in COPD could be a useful tool to identify the locus of genetic alterations leading to COPD. Thus, in addition to exposure to exogenous factors, host factors, most likely several genes, are involved and affect various pathways of the pathogenesis of COPD.
Assuntos
Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Bronquite Crônica/etiologia , Suscetibilidade a Doenças , Humanos , Neutrófilos/fisiologia , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/imunologia , Enfisema Pulmonar/etiologia , Fatores de Risco , Fumar/imunologia , Linfócitos T/fisiologiaRESUMO
We have shown previously that experienced readers can use the palpation or pen method for reading the Mantoux tuberculin skin test with accuracy. In order to investigate if the inexperienced reader can use both methods with the same degree of accuracy, we performed Mantoux tests in 539 healthy subjects, aged 20-57 years (mean 33 years), using the PPD-RT 23 in dose 2 I.U. (1/5000). In random order and a double-blind fashion, an experienced and an inexperienced reader measured the induration after 48 h by the palpation method initially and then by the pen method, without knowing the identity of the subject. Results of the study did not show any significant difference in any range of measurements by the two readers, using either the pen or palpation method (P greater than 0.1). A highly statistically significant linear relationship was found between the measurements by the pen method used by experienced and inexperienced readers (r = 0.823, P less than 0.0001) and between the palpation measurements by the two readers (r = 0.939, P less than 0.0001). We conclude that the effects of an inexperienced examiner in the measurement of of tuberculin skin induration are minimal. Inexperienced readers can use the pen method as accurately as the traditional palpation method.
Assuntos
Teste Tuberculínico/métodos , Adulto , Método Duplo-Cego , Humanos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Palpação , Estudos Prospectivos , Distribuição Aleatória , Reprodutibilidade dos TestesRESUMO
Previous studies have shown that microsatellite (MS) DNA instability (MSI) is detectable in sputum cells in chronic obstructive pulmonary disease (COPD) and asthma. The aim of the present study was to investigate whether asthma and COPD could be distinguished at the MS DNA level. DNA was extracted from sputum cells and white blood cells from 63 COPD patients, 60 non-COPD smokers, 36 asthmatics and 30 healthy nonsmokers. Ten MS markers located on chromosomes 2p, 5q, 6p, 10q, 13q, 14q and 17q were analysed. No MSI was detected in non-COPD smokers or healthy nonsmokers. A significantly higher proportion of COPD patients exhibited MSI (49.2%) compared to asthmatics (22.2%). MSI was detected even in the mild stages of COPD (33.3%) and asthma (22.2%). No relationship was found between MSI and COPD severity. The most frequently affected marker was D14S588 (17.5% in COPD and 2.7% in asthma). The markers D6S344, G29802 and D13S71 showed alterations only in COPD, and G29802 was associated with a significantly decreased forced expiratory volume in one second FEV1 (% predicted), whereas MSI in D6S344 was associated with a significantly higher FEV1 (% pred). The frequency of microsatellite instability was higher in chronic obstructive pulmonary disease than in asthma, and microsatellite instability in three workers showed chronic obstructive pulmonary disease specificity. However, further studies are needed to verify the differences between chronic obstructive pulmonary disease and asthma at the microsatellite level.
Assuntos
Asma/diagnóstico , Instabilidade de Microssatélites , Repetições de Microssatélites , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Adulto , Idoso , Asma/genética , Biomarcadores/análise , DNA/análise , Diagnóstico Diferencial , Feminino , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/genética , Escarro/químicaRESUMO
Genetic alterations, such as loss of heterozygosity (LOH) or microsatellite instability (MI), have been reported in both malignant and benign disorders. In order to identify loci of deoxyribonucleic acid (DNA) mutation in asthma, MI and LOH were studied in sputum cells. DNA was extracted from cells in the sputum and blood cells of 22 patients with moderate asthma. Cells were analysed for MI and LOH using 18 polymorphic markers on chromosome 5q, 6p, 11q, 14q. Microsatellite analysis was also performed in six healthy subjects. None of the healthy individuals exhibited any genetic alteration. Genetic alterations were found in 16 of 22 asthmatic patients (73%). In total, 12 (54.5%) patients exhibited LOH only, one (4.5%) MI only, while three showed both MI and LOH. The highest incidence of LOH and MI was found on chromosome 14q. Mean immunoglobulin E and blood eosinophil levels were significantly higher in asthmatics with three or more genetic alterations. A high incidence of genetic alterations in the deoxyribonucleic acid of the sputum cells was found in asthmatic patients. Further studies are needed to identify the role of loss of heterozygosity and microsatellite instability in the investigation of genetic susceptibility of asthma and thus, in its pathogenesis.