Apoptosis in lung pathophysiology.

As recently as 1993, fewer than 10 manuscripts had been published on the topic of apoptosis specifically in the lung. Although that number is increasing, far fewer papers appear each year on apoptosis in the lung than in the other major organs. Therefore, our knowledge of this important aspect of lung cell physiology is relatively rudimentary. Recent literature is beginning to define important roles for apoptosis in normal lung cell turnover, lung development, and the pathogenesis of diseases such as interstitial pulmonary fibrosis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease. Although the involvement of lung cell apoptosis in each of these examples seems clear, the many factors comprising the normal and abnormal regulation of cell death remain to be elucidated and are likely to be different in each situation. The definition of those factors will be an exciting and challenging field of research for many years to come. In that context, the goal of this symposium was to discuss, from a physiological perspective, some of the most recent and exciting advances in the definition of signaling mechanisms involved in the regulation of apoptosis specifically in lung cell populations.

In-person vs telephone-administered multiple-pass 24-hour recalls in women: validation with doubly labeled water.

OBJECTIVE: To determine the accuracy of energy intakes estimated with the multiple-pass 24-hour recall method in women by conducting in-person and telephone interviews. Doubly labeled water measurements of total energy expenditure were used for validation. SUBJECTS: Thirty-five weight-stable women (mean age = 30 years, range = 19 to 46 years) participated. DESIGN: Total energy expenditure was measured over a 14-day period using the doubly labeled water method. During this time, 4 multiple-pass 24-hour recalls were obtained from the women (2 in-person, 2 by telephone) who were provided 2-dimensional food models to estimate portion sizes. The Food Intake Analysis System was used to analyze recall data. STATISTICAL ANALYSES: Paired t tests were conducted to examine differences between energy intake estimated from the telephone and in-person interviews. Agreement between the energy intake estimates from the telephone recalls and the in-person recalls was assessed using the technique of Bland and Altman. Paired t tests were used to compare energy intake estimated from the telephone and in-person recalls to total energy expenditure. RESULTS: No significant difference in mean daily energy intake was found between the telephone (2,253 +/- 688 kcal) and in-person (2,173 +/- 656 kcal) interviews (P = .36). However, the mean energy intake from each interview method was significantly lower than total energy expenditure (2,644 +/- 503 kcal) (P = .006 and .001, respectively). APPLICATIONS/CONCLUSIONS: Underreporting of energy intake was widespread in the sample. Although the multiple-pass 24-hour recall method did not generate a group measure of energy intake that was accurate or unbiased, the telephone-administered multiple-pass 24-hour recall was just as effective in estimating energy intake as the recall administered in-person. Dietetics professionals should be aware of the pervasive and serious problem of under-reporting of self-reported food intakes.

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.

Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma.

BACKGROUND: Recent evidence has shown that nitric oxide (NO) levels are increased in asthmatic airways. Although the role of NO in asthma is unknown, reactive metabolites of NO may lead to nitrotyrosine formation and promote airway dysfunction. OBJECTIVE: The aim of this study was to determine whether nitrotyrosine, as a marker of nitrating species, could be found in the airways and lung parenchyma of subjects with asthma who died of status asthmaticus or other nonrespiratory causes. METHODS: Lung tissue specimens were obtained from 5 patients who died of status asthmaticus, 2 asthmatic patients who died of nonrespiratory causes, and 6 nonasthmatic control subjects who died of nonrespiratory causes. Lung sections were stained for immunofluorescence with use of an antinitrotyrosine antibody, followed by a indiocarbocyanine (Cy5, Jackson Immunochemicals, Westgrove, Pa)-conjugated secondary antibody. RESULTS: Nonasthmatic lungs showed little or no nitrotyrosine staining, whereas asthmatic lungs demonstrated significantly more staining of nitrotyrosine residues distributed in both the airways and lung parenchyma. CONCLUSION: This study demonstrates the presence of nitrotyrosine, and hence evidence of formation of nitrating species, in the airways and lung parenchyma of patients with asthma who died of status asthmaticus or other nonrespiratory causes. This finding supports the concept that widespread airway and parenchymal inflammation occurs in asthma, and, more specifically, that NO and its reactive metabolites may play a pathophysiologic role in asthma.

Depletion of nitric oxide causes cell cycle alterations, apoptosis, and oxidative stress in pulmonary cells.

Nitric oxide (NO.) is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate how endogenous NO. regulates the function of airway epithelial cells, we used carboxy-PTIO, a hydrophilic, negatively charged NO. trap, to scavenge NO. from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and to determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of NO. with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO. formation. ESR spectroscopy revealed the production of NO. in RLE or RPM cells over a period from 1 to 24 h of exposure, indicating scavenging of NO. by PTIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefold increases in S and G2/M phases after exposure to 100-200 microM PTIO as well as increases in the fraction of cells undergoing apoptosis. Direct addition of PTI to cells failed to elicit cell cycle perturbations or apoptosis. The guanylyl cyclase inhibitor ODQ mimicked the effects of PTIO. 8-Bromo-cGMP but not 8-bromo-cAMP ameliorated the PTIO- or ODQ-mediated cell cycle perturbations and apoptosis, suggesting that cGMP-dependent pathways are involved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cells treated at confluence. Assessment of 5-bromo-2'-deoxyuridine incorporation to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G2/M phase. Last, incubation with dichlorofluorescin diacetate revealed oxidative stress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO. induces oxidative stress, cell cycle perturbations, and apoptosis. Our findings illustrate the importance of endogenous NO. in the control of cell cycle progression and survival of pulmonary and pleural cells and that a critical balance between NO. and superoxide may be necessary for these physiological events.

Literacy and body fatness are associated with underreporting of energy intake in US low-income women using the multiple-pass 24-hour recall: a doubly labeled water study.

OBJECTIVE: The accuracy of the multiple-pass 24-hour recall method for estimating energy intake in low-income women in the United States was ascertained by comparing the method with measurements of total energy expenditure. The multiple-pass 24-hour recall is designed to provide respondents with multiple cues and opportunities to report their food intake. It consists of 3 distinct passes: the quick list, detailed description, and review. Predictors of energy intake misreporting (energy intake--total energy expenditure) in the sample were determined. DESIGN: Four multiple-pass 24-hour recalls (2 in person, 2 by telephone) were obtained over a 14-day period to estimate energy intake. Total energy expenditure was measured over the same 14-day period using the doubly labeled water method. Body composition was measured using dual energy x-ray absorptiometry, and literacy was measured by the Wide Range Achievement Test (WRAT) for reading and spelling. SUBJECTS/SETTINGS: Thirty-five low-income women between the ages of 19 and 46 years were tested at the General Clinical Research Center at the University of Vermont, Burlington. Low income was defined as a household income at or below 130% of the federal poverty level. STATISTICAL ANALYSIS: Pearson product moment correlation coefficients, t tests, paired t tests, and stepwise multiple regression analysis were used to test the relationships among study variables. RESULTS: Mean energy intake was significantly lower than mean total energy expenditure (2,197 +/- 607 vs 2,644 +/- 503 kcal, P = .001) and the correlation between the 2 measures was poor (r = .22, P = .20). Percentage body fat and the combined age-adjusted reading and spelling WRAT scores were the best predictors of misreporting of energy intake (R = .52, P = .006). CONCLUSIONS: The multiple-pass 24-hour recall did not generate a group measure of energy intake that was accurate or unbiased in this sample. Underreporting was strongly associated with increased body fatness. The ability to read and spell as measured by the WRAT improved the accuracy of the women's recall of their food intake. APPLICATIONS: Dietetics professionals should take into consideration the problem of underreporting whenever conclusions are made about associations between diet and health and/or when evaluating the impact of food assistance programs on dietary intake.

Factors predicting a successful outcome after pharmacologic bowel compensation.

OBJECTIVES: The authors determined those factors that predict a successful outcome in patients who receive pharmacologic agents to promote bowel absorption after massive intestinal resection. SUMMARY BACKGROUND DATA: Patients with the short bowel syndrome are maintained on long-term total parenteral nutrition (TPN) or more frequently considered for intestinal transplantation as part of their treatment program. The authors have administered a combination of trophic agents and a specialized diet to further enhance intestinal compensation and optimize nutrient absorption in patients with intestinal failure. METHODS: Forty-five TPN-dependent adults with a jejunal-ileal remnant < or = 50 cm and a portion of colon in continuity were treated with growth hormone, glutamine, and a modified diet for 4 weeks and observed for an average of 1.8 years. RESULTS: The average age of the patients was 43 years, the average jejunal-ileal length was 23 cm, and the average length of time the patient received TPN was 4.3 years. After 4 weeks of therapy, 26 (58%) were free of TPN support. Predictors of a favorable response included greater bowel length, lower body weight, and greater bowel length-body weight ratio. At follow-up, the percentage of patients who were not receiving TPN had fallen to 40%. CONCLUSIONS: Approximately half of a group of patients, thought to have absorptive surface area inadequate to be independent of TPN support, can maintain themselves on enteral feedings after this intestinal rehabilitation program. Because of the risk, costs, and alterations in lifestyle associated with long-term TPN or intestinal transplantation or both, it seems prudent to consider a program of bowel rehabilitation with an individual patient before embarking on another therapeutic plan.