Mutational spectra of a 100-base pair mitochondrial DNA target sequence in bronchial epithelial cells: a comparison of smoking and nonsmoking twins.

Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmokers. Among the individuals sampled were three pairs of monozygotic twins in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years. Individual point mutations present at frequencies as low as 10(-6) were detected. Partially denaturing electrophoresis was used to separate mutant from nonmutant sequences on the basis of their melting temperatures, and the target sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch intermediates or DNA adducts present in the cellular DNA were converted to mutants during PCR. Hot spot mutations were clearly observed in bronchial epithelial cells, and the same hot spots were observed consistently in different samples. Significant numerical variability in the mutant fractions for individual mutants was observed among samples and are ascribed to unequal mitochondrial segregation in stem and transition cells. The mutational spectra in smokers' samples did not differ significantly from the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA. No smoking-specific hot spots were detected. The overall mutant fractions in smokers' samples were not elevated compared to those of nonsmokers. As much variability was observed between two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker. These findings demonstrate that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial epithelial cells. Endogenous factors (e.g., DNA replication errors or DNA damage by endogenous reactive chemicals) are suggested to be more likely to represent the most important contributors to mitochondrial mutagenesis.

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Probability of a positive transbronchial lung biopsy result in sarcoidosis.

Forty-one consecutive patients with suspected sarcoidosis underwent pulmonary function testing and transbronchial lung biopsy at three community teaching hospitals. Transbronchial biopsy disclosed noncaseating granulomas in 22 of 23 patients (96%) in whom parenchymal disease was roentgenographically apparent and in eight of 18 patients (44%) in whom it was not. The stage of the disease as determined by the chest roentgenogram was the most reliable determinant for a positive biopsy result. Not the presence of cough, dyspnea, or constitutional symptoms or pulmonary function as measured by forced vital capacity and carbon monoxide diffusing capacity served to predict a positive transbronchial biopsy finding any more accurately than did the roentgenographic staging of the disease itself. This study suggests that while transbronchial lung biopsy may be an acceptable initial diagnostic procedure in suspected sarcoid patients without parenchymal lung disease, clinical symptoms and pulmonary function abnormalities are not helpful in predicting the liklihood of a positive biopsy result.

Predictors of mortality in the immunocompromised patient with pulmonary infiltrates.

To determine predictors of mortality in immunocompromised patients with pulmonary infiltrates, we reviewed the records of all such patients admitted to two community teaching hospitals who underwent a lung biopsy over a ten-year period. We examined the consequences of advancing age, primary disease, fever, neutropenia, immunosuppressive corticosteroid therapy, previous lung radiation, roentgenographic pattern, result of lung biopsy, room air arterial oxygen pressure (Pao2), early mechanical ventilation, and the presence of a comorbid disease on eventual outcome. We identified 104 episodes in 99 patients. Sixty-seven (64%) survived and 37 died. By both discriminant analysis and logistic regression statistical methods, mechanical ventilation, the initial room air Pao2, and corticosteroid therapy were the dominant independent variables, in that order, to significantly predict mortality. No patient survived who simultaneously had a room air Pao2 less than or equal to 50 mm Hg, was on corticosteroids, and was mechanically ventilated. Eighty-three percent of survivors had either none or, at most, one of these three variables present. We conclude that hypoxia, immunosuppression by corticosteroids, and the necessity for mechanical ventilation within 72 hours of hospitalization indicate a poor prognosis in the immunocompromised patient with pulmonary infiltrates who has undergone a lung biopsy.

Sensitivity, specificity, and predictive values of closed pleural biopsy.

To determine the clinical value of a nonspecific pleural biopsy specimen and fluid in malignant neoplasm and tuberculosis, we retrospectively reviewed records of all patients with pleural effusions undergoing the procedure at three community hospitals over six years. Two hundred eleven patients underwent biopsies. Adequate tissue was obtained in 207. The results were compared with the ultimate clinical and pathologic outcome by follow-up for 12 to 72 months. The initial procedure was diagnostic of malignant neoplasm in 54 patients and granulomatous disease in ten. A nonspecific or normal result was found in 143 (68%). Malignant neoplasms or tuberculosis was eventually established in 30 and excluded in 101 of the 143 patients. In 12 patients, no diagnosis was made. The procedure's sensitivities were 65% (malignant neoplasm) and 90% (tuberculosis). One false-positive result occurred in a patient with nontuberculous granulomatous pleuritis. The specificity and positive predictive value were 99% and 98%, respectively. The negative predictive value was 77%. Closed pleural biopsy with simultaneous fluid analysis is a valuable diagnostic procedure in community hospital patients, but a nonspecific result does not exclude malignant disease.

Pulmonary function and dysfunction in multiple sclerosis.

Pulmonary function was studied in 25 patients with clinically definite multiple sclerosis with a range of motor impairment. Forced vital capacity (FVC), maximal voluntary ventilation (MVV), and maximal expiratory pressure (MEP) were normal in the ambulatory patients (mean greater than or equal to 80% predicted) but reduced in bedridden patients (mean, 38.5%, 31.6%, and 36.3% predicted; FCV, MVV, and MEP, respectively) and wheelchair-bound patients with upper extremity involvement (mean, 69.4%, 50.4%, and 62.6% predicted; FVC, MVV, and MEP, respectively). Forced vital capacity, MVV, and MEP correlated with Kurtzke Expanded Disability Status scores (tau = -0.72, -0.70, and -0.65) and expiratory muscle weakness occurred most frequently. These findings demonstrate that marked expiratory weakness develops in severely paraparetic patients with multiple sclerosis and the weakness increases as the upper extremities become increasingly involved.

Evaluation of pulmonary function in neuromuscular disease.

Pulmonary function tests were performed on 40 patients with neuromuscular disease. The maximum expiratory pressure was the most sensitive indicator of weakness and was decreased in 87% of adult patients. A comparison of Stead-Wells, electronic (Vanguard), and bellows spirometry (Vitalor) indicated an excellent correlation between the Stead-Wells and electronic devices. The bellows spirometer consistently underestimated volumes, particularly in severely weak patients.

Raynaud's phenomenon of the lung.

To determine if pulmonary vessels develop vasospasm during Raynaud's phenomenon, digital vasospasm was induced by hand immersion in 15 degrees C water (cold pressor test) in 17 subjects, and pulmonary function was measured during the subsequent 120 minutes. Five healthy persons were control subjects, seven subjects had well documented systemic disorders associated with Raynaud's phenomenon (secondary Raynaud's), and five subjects had a history of Raynaud's phenomenon but no evidence of an associated disorder (primary Raynaud's). The only measure of pulmonary function that changed significantly following cold pressor testing was carbon monoxide diffusing capacity. Subjects with primary Raynaud's phenomenon had normal baseline carbon monoxide diffusing capacity (23.7 +/- 4.6 ml/minute/mm Hg) but demonstrated significant decreases (p less than 0.05) at 15 minutes (21.2 +/- 3.5 ml/minute/mm Hg), 45 minutes (19.5 +/- 3.7 ml/minute/mm Hg), and 120 minutes (17.1 +/- 2.1 ml/minute/mm Hg) after cold pressor testing. Subjects with secondary Raynaud's phenomenon had low baseline carbon monoxide diffusing capacity (71 percent predicted) and showed no significant change following cold pressor testing. These findings indicate that digital vasospasm in patients with primary Raynaud's phenomenon is part of a systemic vascular response that includes a decrease in the size of the pulmonary capillary bed.

Chemical breakdown of technetium-99m DTPA during nebulization.

Aerosols of 99mTc diethylenetriaminepentaacetic acid ([99mTc]DTPA) used for measuring lung permeability and lung ventilation require a radioaerosol delivery system to produce an aerosol with reproducible size and radiochemical purity. To test how well nebulizers meet this requirement, radiochemical purity of aerosols produced with a jet and an ultrasonic nebulizer was evaluated. The activity median aerodynamic diameter (AMAD) and geometric standard deviation (sigma g) of radioaerosols were 0.46 micron (sigma g = 1.6) for the jet nebulizer and 0.70 micron (sigma g = 1.7) for the ultrasonic nebulizer. Paper and liquid chromatographic assays were obtained on the [99mTc]DTPA aerosol solute produced with each nebulizer. The results of these tests showed major differences in radiochemical purity. Aerosols produced in the jet nebulizer consistently showed greater than 90% of the radioactivity bound to the DTPA ligand whereas aerosols produced in the ultrasonic nebulizer showed less than 10% of the radioactivity bound to DTPA. The results support the need to test radiochemical purity of aerosols before using an aerosol nebulizer for pulmonary imaging and clearance studies.

Fatal acute bacterial myocarditis after dentoalveolar abscess.

A 19 year old woman presented with chest pain after a dental extraction for a dentoalveolar abscess. Electrocardiographic and serum isoenzyme changes were consistent with acute anterior myocardial infarction. At autopsy bacteria were demonstrated, within the myocardium in the absence of a myocardial abscess or endocarditis. This case illustrates the occurrence of isolated acute bacterial myocarditis after a dental extraction.

Effects of inhaled acid aerosols on lung mechanics: an analysis of human exposure studies.

There exist significant gaps in our understanding of human health effects from inhalation of pollutants associated with acid precipitation. Controlled clinical studies examine effects of criteria pollutants almost exclusively by assessing changes in lung mechanics. One constituent of acid precipitation, sulfuric acid aerosols, has been shown to induce bronchoconstriction in exercising extrinsic asthmatics at near ambient levels. These asthmatics may be an order of magnitude more sensitive to sulfuric acid aerosols than normal adults. More recently, a second component nitrogen dioxide has been observed to provoke changes in lung mechanics at progressively lower concentrations. To date, virtually no data exist from clinical exposures to acidic aerosols for subjects with chronic obstructive pulmonary disease.

Toxicologic methods: controlled human exposures.

The assessment of risk from exposure to environmental air pollutants is complex, and involves the disciplines of epidemiology, animal toxicology, and human inhalation studies. Controlled, quantitative studies of exposed humans help determine health-related effects that result from breathing the atmosphere. The major unique feature of the clinical study is the ability to select, control, and quantify pollutant exposures of subjects of known clinical status, and determine their effects under ideal experimental conditions. The choice of outcomes to be assessed in human clinical studies can be guided by both scientific and practical considerations, but the diversity of human responses and responsiveness must be considered. Subjects considered to be among the most susceptible include those with asthma, chronic obstructive lung disease, and cardiovascular disease. New experimental approaches include exposures to concentrated ambient air particles, diesel engine exhaust, combustion products from smoking machines, and experimental model particles. Future investigations of the health effects of air pollution will benefit from collaborative efforts among the disciplines of epidemiology, animal toxicology, and human clinical studies.

Environmental tobacco smoke exposure and asthma in adults.

Environmental tobacco smoke (ETS) contaminates indoor air in homes and workplaces. Although the adverse effects of active cigarette smoking on the respiratory tract have been extensively characterized, the effects of ETS exposure on adult asthma have not yet been investigated extensively and the available data are limited. This article examines the evidence for ETS exposure as a cause of asthma and asthma exacerbation in adults, and for ETS exposure in the workplace specifically as contributing to these health effects. It addresses methodological barriers that limit the available data and evaluates the adequacy of the data for risk assessment.

Human breathing and eye blink rate responses to airborne chemicals.

Increased levels of air pollution have been linked with morbidity and mortality, but mechanisms linking physiologic responses to quality of life and productivity issues remain largely unknown. Individuals often report irritation of the nose and/or eyes upon exposures to environmental contaminants. Evaluation of these self-reports would be greatly aided by the development of valid physiological markers. Chamber studies (unencumbered exposures) of nonsmoker responses to environmental tobacco smoke offer two candidate end points: (a) Tidal volume increases and breathing frequency declines with stimuli that elicit only moderate irritation. (b) Eye blink rate increases only with a concentration sufficiently high to cause progressive worsening of eye irritation with prolonged exposure. Experiments with very brief nasal-only presentations also suggest the value of breathing changes as sensitive markers of irritation: (a) Tidal volume is inversely related to perceived nasal irritation (NI) intensity in both normal and anosmic (lacking olfactory input) individuals, although normals exhibit greater NI sensitivity. (b) Inhalation duration, in both groups, declines only with trigeminal activation sufficient to cause readily perceptible NI in anosmics. Changes in eye blink rate and breathing may be useful in the investigation of irritation and other effects of air pollution, and could be quite useful in investigations of mixtures of volatile organic compounds.

Interferon-alpha therapy associated with the development of sarcoidosis.

Interferons (IFNs) have been implicated in the pathogenesis of sarcoidosis. In particular, IFN-gamma has been linked to pulmonary macrophage activation, a characteristic feature of sarcoidosis. IFN-alpha is now being administered therapeutically in a variety of conditions. To date, IFN-alpha has not been implicated in the pathogenesis of sarcoidosis. We report the case of a 50-year-old woman who developed sarcoidosis while being treated with IFN-alpha for chronic myelogenous leukemia. Her disease activity correlated with the dosage of IFN-alpha. We speculate that the immunomodulatory effects of IFN-alpha triggered clinical manifestations of sarcoidosis in this patient.

Inhalation challenge with carbachol and isoproterenol to predict bronchospastic response to propranolol in COPD.

Disabling propranolol-induced bronchospasm occasionally prevents use of this drug in patients with chronic obstructive pulmonary disease (COPD). A means was developed to identify patients who have high risk for this adverse effect using bronchial challenge by inhaling the parasympathomimetic drug, carbachol, and isoproterenol. After baseline pulmonary function tests, 12 patients with varying degrees of COPD and 13 control patients underwent maximal beta-blockade using intravenously administered propranolol during cardiac catheterization followed by repeat pulmonary function testing. Seven of the patients with COPD and ten of the control subjects were restudied while taking propranolol orally for at least three weeks. The results indicated that the bronchodilator response to inhaled isoproterenol does not reliably identify patients who develop bronchoconstriction with propranolol, but bronchoconstriction after inhaling carbachol is indicative of the high risk of developing bronchoconstriction from propranolol.

Measurement of lung gas volume and regional density by computed tomography in dogs.

To determine if computed tomography (CT) can accurately measure lung volume, we compared lung gas volume measured by helium dilution with the equivalent volume calculated from CT total lung volume and density in 13 supine dogs. CT lung gas volume underestimated helium volume by 34% (range: -63 to 0%). Studies of wooden lung phantoms varying in density from 0.082g/cc to 0.776g/cc showed that only 15% of this error could be mimicked by the phantoms. The rest of the discrepancy is attributed to the lung's irregular borders, and the sharp density gradients surrounding and within the lung that result in x-ray beam hardening, sampling limitations, and partial volume measurement errors. Serial biweekly measurements in three dogs for 14 weeks showed CT gas volume to be highly reproducible with less scatter than seen in the helium measurements. Density in the lungs of all dogs showed a uniform gradual decrease from approximately 0.60g/cc at the dependent surface to 0.20g/cc at the superior surface with relatively constant density at any horizontal level. These studies show that whereas CT underestimates gas volume in the lungs, serial measurements are highly reproducible in experimental studies and are a promising technique to monitor diseases or response to therapy. Density gradients in the lungs were sufficiently uniform so that disruption of the normal gradient may be an indicator of early lung disease.

Effect of the rebreathing pattern on pulmonary tissue volume and capillary blood flow.

Noninvasive rebreathing measurements of pulmonary tissue volume (Vt) and pulmonary capillary blood flow (Qc) theoretically and experimentally vary with the rebreathing maneuver. To determine the cause of these variations and identify ways to minimize them, we examined the consequences of varying the volume inspired (VI), rebreathing rate (f), volume rebreathed (Vreb), and alveolar volume (VA) on the observed Vt and Qc in six normal sitting subjects. When VA was increased by progressively larger VI and Vreb, Vt increased 50 ml/l of VA. Increasing VA while keeping VI and Vreb constant did not significantly alter Vt. Diminishing Vreb while VA and VI constant caused Vt to fall 108 ml/l decrease in Vreb. Therefore the observed Vt is not simply a function of VA but increased with greater penetration of the inspired gas into the lungs. Diminishing f from 40 to 12 breaths/min caused the observed Vt to rise 27%, indicating time allowed for alveolar mixing is an important determinant of Vt. The observed Qc, in contrast, was essentially independent of the same variations in rebreathing. The above findings were similar regardless of solubility of the tracer gas (dimethyl ether instead of acetylene) or changing to the supine position. A two-compartment series lung model derived from the anatomy and rates of gas mixing in normal human pulmonary lobules produced similar changes in Vt. Thus the degree of uneven distribution between ventilation, VA, Vt, and Qc within the normal lung lobule can account for variations in the observed Vt with different ventilatory maneuvers. Slow deep breathing maneuvers tended to reduce variations in Vt. Unlike Qc, the observed value of Vt can be expected to vary substantially with pathological processes that alter pulmonary gas distribution.

Lung weight in vivo measured with computed tomography and rebreathing of soluble gases.

In nine anesthetized dogs, accuracy of noninvasive measurements of lung weight (W) and gas volume in vivo was determined from volume and density determined by computed tomography (CT) and by rebreathing helium and the soluble gases dimethyl ether (WDME) and acetylene (WC2H2). Reference standards were obtained from the postmortem scale weight of the frozen lungs (Wscale) and compared with the CT lung weights measured in the living dog (WCT-38) and the frozen carcass (WCT-cold). WCT-cold did not significantly differ from Wscale [-2 +/- 9% (SD), P = 0.7]. WCT-cold was 10% greater than WCT-38 (0.10 greater than P greater than 0.05), suggesting an increase in lung weight despite immediately commencing freezing after death. WDME measured 64 +/- 6% and WC2H2 56 +/- 12% of WCT-38. Serial multiple measurements in three dogs over 14 wk showed a coefficient of variation (CV) of 10 +/- 2% for WDME, 18 +/- 2% for WC2H2, 4.1 +/- 0.9% for WCT, 2.6 +/- 0.8% for CT density, and 3.5 +/- 1.6% for functional residual capacity (FRC) by CT. FRC calculated from CT consistently underestimated FRC measured by rebreathing helium by 18 +/- 8% (P less than 0.005). This error, despite good agreement between WCT and Wscale, was explained by underestimation of CT total lung volume and overestimation of lung density by factors known to affect CT readings, such as partial volume effects, beam hardening, and limited number of input signals. These data show that CT scanning can provide serial measurement of the mass, density, and volume of the lungs with a CV in the order of 5%, but the rebreathing of soluble gases gives more than double this variability. Measurements of WDME performed on the same day had a CV of 3 +/- 1%, so that WDME provides a precise noninvasive means to measure lung weight in acute studies.

Determination of production of nitric oxide by lower airways of humans--theory.

Exercise and inflammatory lung disorders such as asthma and acute lung injury increase exhaled nitric oxide (NO). This finding is interpreted as a rise in production of NO by the lungs (VNO) but fails to take into account the diffusing capacity for NO (DNO) that carries NO into the pulmonary capillary blood. We have derived equations to measure VNO from the following rates, which determine NO tension in the lungs (PL) at any moment from 1) production (VNO); 2) diffusion, where DNO(PL) = rate of removal by lung capillary blood; and 3) ventilation, where V A(PL)/(PB - 47) = the rate of NO removal by alveolar ventilation (V A) and PB is barometric pressure. During open-circuit breathing when PL is not in equilibrium, d/dt PL[V(L)/ (PB - 47)] (where V(L) is volume of NO in the lower airways) = VNO - DNO(PL) - V A(PL)/(PB - 47). When PL reaches a steady state so that d/dt = 0 and V A is eliminated by rebreathing or breath holding, then PL = VNO/DNO. PL can be interpreted as NO production per unit of DNO. This equation predicts that diseases that diminish DNO but do not alter VNO will increase expired NO levels. These equations permit precise measurements of VNO that can be applied to determining factors controlling NO production by the lungs.