Janice L. Launspach

Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth.

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms. A Matter of Life and Breath The CafePress and Zazzle Web sites and most yoga-wear boutiques sport an array of teeshirts, bumper stickers, and water bottles prepared to offer simple advice to those living a harried life: “Just breathe.” Not so simple for a cystic fibrosis (CF) patient. Very early on, physicians recognized that difficulty breathing was the most ominous of the mosaic of symptoms that characterize this syndrome. Indeed, lung disease is the main cause of death in cystic fibrosis patients, but the lack of an animal model that mirrors the CF lung pathology seen in people has slowed translational cystic fibrosis research. Now, Stoltz et al. report findings in cystic fibrosis pigs that survive long enough to develop human-like lung disease. At the heart of this recessive genetic disease is the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride-ion channel. CF-causing mutations in the CFTR gene give rise to an aberrant channel that is defective in its ability to transport ions and water across cell membranes, resulting in a dizzying array of defects in the pancreas, intestines, reproductive system, liver, and lungs. It has been hypothesized that the impaired channel causes cells that line body cavities and passageways to become coated with thick mucus. In such an environment, bacteria thrive, leading to the chronic infections characteristic of this disease. However, the precise mechanisms by which CFTR mutations manifest as the complex phenotypes that constitute CF remain unclear, particularly with respect to the inflamed and infected airways of the CF lung. Despite substantial research efforts, scientists have been unable to achieve two crucial goals,to mold an animal model that mimics human CF lung disease and to pinpoint the trigger of CF lung pathology in pristine airways. Stoltz et al. tackled both of these obstacles by producing genetically modified CF pigs and analyzing their airways from birth to 6 months of age. Their studies revealed a spontaneously arising human-like lung disease that developed over time and had the CF hallmarks: multibacterial infections, inflammation, and mucus buildup. Although the lungs of the newborn CF piglets were not yet inflamed, they were less likely to be sterile and less able to eliminate bacteria that had been introduced into their lungs, relative to wild-type animals. Together, these findings suggest that bacterial infiltration spurs the pattern of lung inflammation and pathogenesis associated with CF. Having a clearer conception of CF lung disease can help clinicians devise preventive treatments that can be initiated early in the lives of CF patients. Such interventions may let CF suffers live and breath more fully. Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). Understanding the pathogenesis of this disease has been hindered, however, by the lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with mutated CFTR genes. We now report that, within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Our finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.

Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.

The effects of recombinant human hemoglobin on esophageal motor function in humans

Abstract Background & Aims: Nitric oxide controls lower esophageal sphincter (LES) relaxation and esophageal peristalsis in opossums, but its role in the control of esophageal motility in humans is not defined. Hemoglobin inactivates NO by binding it. Recombinant human hemoglobin (rHb1.1) was used to test the hypothesis that NO mediates esophageal motor functions in humans. Methods: rHb1.1 or human serum albumin was administered intravenously to fasting male volunteers. Esophageal manometric studies were performed before, during, and up to 6 hours after the infusion. Results: rHb1.1 increased the velocities of peristaltic contractions to produce simultaneous contractions in 6 of 9 subjects. It increased the amplitude and duration of contractile waves in the esophagus. There was no consistent effect on the resting tone of the LES, but LES relaxation was inhibited. Spontaneous, simultaneous high-pressure contractions occurred in 8 of 9 subjects. Lower retrosternal chest pain during swallowing was observed in 4 subjects. Conclusions: rHb1.1 interfered with esophageal peristalsis and LES relaxation. It precipitated esophageal spasm in some subjects. These data support the hypothesis that the timing of smooth muscle esophageal peristalsis and LES relaxation are mediated by NO. They suggest that some disorders of esophageal motor function may result from defects in NO neuromuscular communication.

Loss of Anion Transport without Increased Sodium Absorption Characterizes Newborn Porcine Cystic Fibrosis Airway Epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR⁻(/)⁻ pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR⁻(/)⁻ epithelia showed markedly reduced Cl⁻ and HCO₃⁻ transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR⁻(/)⁻ pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl⁻ conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl⁻ and HCO₃⁻ in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections

Rationale: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D‐CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. Objectives: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. Methods: We studied 12 subjects with G551D‐CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. Measurements and Main Results: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. Conclusions: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D‐CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR‐targeting treatments.

Neonates with cystic fibrosis have a reduced nasal liquid pH; A small pilot study

BACKGROUND Disrupted HCO3(-) transport and reduced airway surface liquid (ASL) pH in cystic fibrosis (CF) may initiate airway disease. We hypothesized that ASL pH is reduced in neonates with CF. METHODS In neonates with and without CF, we measured pH of nasal ASL. We also measured nasal pH in older children and adults. RESULTS In neonates with CF, nasal ASL (pH5.2 ± 0.3) was more acidic than in non-CF neonates (pH6.4 ± 0.2). In contrast, nasal pH of CF children and adults was similar to values measured in people without CF. CONCLUSIONS At an age when infection, inflammation and airway wall remodeling are minimal, neonates with CF had an acidic nasal ASL compared to babies without CF. The CF:non-CF pH difference disappeared in older individuals, perhaps because secondary manifestations of disease increase ASL pH. These results aid understanding of CF pathogenesis and suggest opportunities for therapeutic intervention and monitoring of disease.

Enhancement of Respiratory Mucosal Antiviral Defenses by the Oxidation of Iodide

Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.

Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions

A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN(-)) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN(-)). Airway epithelial cultures have been shown to secrete SCN(-) in a CFTR-dependent manner. Thus, reduced SCN(-) availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN(-) concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN(-) concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN(-) concentration was somewhat reduced. Among human subjects, SCN(-) concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN(-) levels had better lung function than those with low SCN(-) levels. Thus, although CFTR can contribute to SCN(-) transport, it is not indispensable for the high SCN(-) concentration in ASL. The correlation between lung function and SCN(-) concentration in CF patients may reflect a beneficial role for SCN(-).

Safety assessment of inhaled xylitol in mice and healthy volunteers

BackgroundXylitol is a 5-carbon sugar that can lower the airway surface salt concentration, thus enhancing innate immunity. We tested the safety and tolerability of aerosolized iso-osmotic xylitol in mice and human volunteers.MethodsThis was a prospective cohort study of C57Bl/6 mice in an animal laboratory and healthy human volunteers at the clinical research center of a university hospital. Mice underwent a baseline methacholine challenge, exposure to either aerosolized saline or xylitol (5% solution) for 150 minutes and then a follow-up methacholine challenge. The saline and xylitol exposures were repeated after eosinophilic airway inflammation was induced by sensitization and inhalational challenge to ovalbumin. Normal human volunteers underwent exposures to aerosolized saline (10 ml) and xylitol, with spirometry performed at baseline and after inhalation of 1, 5, and 10 ml. Serum osmolarity and electrolytes were measured at baseline and after the last exposure. A respiratory symptom questionnaire was administered at baseline, after the last exposure, and five days after exposure. In another group of normal volunteers, bronchoalveolar lavage (BAL) was done 20 minutes and 3 hours after aerosolized xylitol exposure for levels of inflammatory markers.ResultsIn naïve mice, methacholine responsiveness was unchanged after exposures to xylitol compared to inhaled saline (p = 0.49). There was no significant increase in Penh in antigen-challenged mice after xylitol exposure (p = 0.38). There was no change in airway cellular response after xylitol exposure in naïve and antigen-challenged mice. In normal volunteers, there was no change in FEV1 after xylitol exposures compared with baseline as well as normal saline exposure (p = 0.19). Safety laboratory values were also unchanged. The only adverse effect reported was stuffy nose by half of the subjects during the 10 ml xylitol exposure, which promptly resolved after exposure completion. BAL cytokine levels were below the detection limits after xylitol exposure in normal volunteers.ConclusionsInhalation of aerosolized iso-osmotic xylitol was well-tolerated by naïve and atopic mice, and by healthy human volunteers.

Absence of amiloride-sensitive sodium absorption in the airway of an infant with pseudohypoaldosteronism

We report the measurement of transepithelial voltage across the nasal epithelium in a neonate with pseudohypoaldosteronism (PHA) type 1. A 5-day-old infant was seen with hyponatremia, hyperkalemia, and elevated plasma renin and aldosterone levels. Sweat Cl(-) concentration was also increased. Measurements of voltage showed a basal value of zero and the absence of an amiloride-sensitive voltage. However, voltage changed as expected for normal cyclic adenosine monophosphate-stimulated Cl(-) transport. These data demonstrate the absence of amiloride-sensitive Na(+) transport across airway epithelia in a neonate with PHA. The findings suggest that measurements of voltage could be of value in the diagnosis of PHA.