Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia.

Complexes of DNA and cationic lipid offer potential advantages for gene transfer to airway epithelia. However, we found that application of DNA-lipid (DMRIE-DOPE) complexes to primary cultures of human ciliated airway epithelia or explants of rabbit trachea generated only low levels of gene transfer. In contrast, when we applied the DNA-lipid to immature human epithelia shortly after seeding, transgene expression was substantially higher. We identified two barriers that limit gene transfer. First, uptake of the DNA-lipid complexes into airway cells across the apical membrane decreased rapidly with time after seeding and paralleled the decrease in transgene expression. Second, cell division decreased with time after seeding, and we found that cells in mitosis (labeled with BrdU) were much more likely to express transgene than BrdU-negative cells. These data suggest that the entry step across the apical membrane and the low rate of cell division are important barriers for cationic lipid-mediated gene transfer to airway epithelia. Attempts to modify these two processes may yield improvements in the efficiency of gene transfer to the airways in cystic fibrosis.

Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.

Pathogenesis of respiratory infections and host defenses.

At the cellular level, the respiratory tract has a variety of defense mechanisms to prevent bacterial infection. Recent data have demonstrated that the respiratory epithelium plays a very active role in host defense. In this review we start by examining the respiratory epithelia and its function in mucociliary clearance, and extend our review to include its role in the secretion and regulation of inflammatory cytokines and production of antimicrobial factors. Furthermore, we examine how recent advances in understanding cystic fibrosis have provided useful insights into the pathogenesis of lower respiratory tract infection. In addition, we examine how two common respiratory pathogens, Streptococcus pneumoniae and Pseudomonas aeruginosa, subvert the defense mechanisms at the cellular level. Finally, we attempt to identify new or potential therapeutic approaches that have arisen from some of the insights into the pathogenesis of lower respiratory tract infections.

Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.

Liddle's syndrome is an inherited form of hypertension caused by mutations that truncate the C-terminus of human epithelial Na+ channel (hENaC) subunits. Expression of truncated beta and gamma hENaC subunits increased Na+ current. However, truncation did not alter single-channel conductance or open state probability, suggesting there were more channels in the plasma membrane. Moreover, truncation of the C-terminus of the beta subunit increased apical cell-surface expression of hENaC in a renal epithelium. We identified a conserved motif in the C-terminus of all three subunits that, when mutated, reproduced the effect of Liddle's truncations. Further, both truncation of the C-terminus and mutation of the conserved C-terminal motif increased surface expression of chimeric proteins containing the C-terminus of beta hENaC. Thus, by deleting a conserved motif, Liddle's mutations increase the number of Na+ channels in the apical membrane, which increases renal Na+ absorption and creates a predisposition to hypertension.

A mouse model for the delta F508 allele of cystic fibrosis.

The most common cause of cystic fibrosis is a mutation that deletes phenylalanine 508 in cystic fibrosis transmembrane conductance regulator (CFTR). The delta F508 protein is misprocessed and degraded rather than traveling to the apical membrane. We used a novel strategy to introduce the delta F508 mutation into the mouse CFTR gene. Affected epithelia from homozygous delta F508 mice lacked CFTR in the apical membrane and were Cl-impermeable. These abnormalities are the same as those observed in patients with delta F508 and suggest that these mice have the same cellular defect. 40% of homozygous delta F508 animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. These animals should provide an excellent model to investigate pathogenesis and to examine therapies directed at correcting the delta F508 defect.

Predicting postoperative pulmonary function in patients undergoing lung resection.

OBJECTIVE: Our aim was to determine the effect of lung resection on spirometric lung function and to evaluate the accuracy of simple calculation in predicting postoperative pulmonary function in patients undergoing lung resection. DESIGN: We reviewed preoperative and postoperative pulmonary function test results on patients who were followed in the multidisciplinary lung cancer clinic between July 1991 and March 1994 and who underwent lung resection. The predicted postoperative FEV1 and FVC were calculated based on the number of segments resected and were compared with the actual postoperative FEV1 and FVC. SETTING: This study was conducted at a university, tertiary referral hospital. PATIENTS: All patients were evaluated at a multidisciplinary lung cancer clinic and underwent lung resection by one surgeon (L.A.L.). MEASUREMENTS AND MAIN RESULTS: Sixty patients undergoing 62 pulmonary resections were reviewed. The predicted postoperative FEV1 and FVC were calculated using the following formula: predicted postoperative FEV1 (or FVC) = preoperative FEV1 (or FVC) x (1-(S x 0.0526)); where S = number of segments resected. The actual postoperative FEV1 and FVC correlated well with the predicted postoperative FEV1 and FVC for patients undergoing lobectomy (r = 0.867 and r = 0.832, respectively); however, the predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by approximately 250 mL. For patients undergoing pneumonectomy, the actual postoperative FEV1 and FVC did not correlate as well with the predicted postoperative FEV1 and FVC (r = 0.677 and r = 0.741, respectively). Although there was considerable variability, the predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by nearly 500 mL. Of the patients undergoing lobectomy, eight also received postoperative radiation therapy. When analyzed separately, patients receiving combined therapy lost an average of 5.47% of FEV1 per segment resected. This contrasts with a 2.84% per segment reduction in FEV1 for patients who did not receive radiation therapy. CONCLUSIONS: This simple calculation of predicted postoperative FEV1 and FVC correlates well with the actual postoperative FEV1 and FVC in patients undergoing lobectomy. The predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by approximately 250 mL. The postoperative FEV1 and FVC for patients undergoing pneumonectomy is not accurately predicted using this equation. The predicted postoperative FEV1 for patients undergoing pneumonectomy was underestimated by an average of 500 mL and by greater than 250 mL in 12 of our 13 patients. Thus, by adding 250 mL to the above calculation of predicted postoperative FEV1, we improve our ability to we identify a minimal postoperative FEV1 for patients undergoing pneumonectomy. Finally, combined modality treatment with surgery followed by radiation therapy may result in additive lung function loss.