Randomized controlled study of aerosolized hypertonic xylitol versus hypertonic saline in hospitalized patients with pulmonary exacerbation of cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by chronic bacterial infection and recurrent pulmonary exacerbations. Xylitol is a 5-carbon sugar that can lower the airway surface salt concentration and augment innate immunity. We examined the safety and efficacy of aerosolized xylitol use for 2 weeks in subjects hospitalized with a pulmonary exacerbation of CF. METHODS: In a 2-week study, 60 subjects with cystic fibrosis and FEV1 > 30% predicted were enrolled to receive aerosolized 7% hypertonic saline (4 ml) or 15% xylitol (5 ml) twice a day for 14 days. Outcomes assessed included change from baseline in FEV1% predicted, change in sputum microbial density, revised CF quality of life questionnaire including the respiratory symptom score, time to next hospitalization for a pulmonary exacerbation, and frequency of adverse events. RESULTS: 59 subjects completed the study (one subject in the saline group withdrew before any study product administration). No significant differences were noted between the 2 arms in mean changes in lung function, sputum microbial density for Pseudomonas aeruginosa and Staphylococcus aureus, body weight, quality of life, and frequency of adverse events. CONCLUSIONS: Aerosolized hypertonic xylitol was well-tolerated among subjects hospitalized for CF pulmonary exacerbation. Future studies examining efficacy for long term use in patients with CF lung disease would be worthwhile. The clinical trial registration number for this study is NCT00928135.

Impact of pharmacy services on cystic fibrosis transmembrane conductance regulator modulator prescribing at a pediatric cystic fibrosis center.

BACKGROUND: This study was undertaken to determine if the presence of a clinical pharmacy team impacted patients' access to cystic fibrosis transmembrane conductance regulator (CFTR) modulators. METHODS: A retrospective chart review of electronic medical records from the University of Iowa Hospitals and Clinics (UIHC) was conducted. Data were collected regarding the timing of prior authorization (PA) submissions and approvals from 2012 to 2018. The Wilcoxon rank-sum test was used to compare the meantime (days) between prescription and PA submission dates, and PA submission and approval date for all patients included in the analysis. Comparisons were made for pre- and postpharmacy services eras as well as the UIHC Specialty Pharmacy versus a non-UIHC Specialty Pharmacy. RESULTS: Sixty-three patients were included in the final analysis. The average time between prescription date and PA submission was 12.5 days (standard deviation [SD] = 17.4 days) in the preclinical pharmacy services era and 3.5 days (SD = 5.8 days; P = .028) in the postclinical pharmacy services era. The average time to PA submission significantly decreased from 9.8 days (SD = 13.1 days) to 1.3 days (SD = 4.2 days; P < .0001) when prescriptions were filled by the UIHC Specialty Pharmacy vs a non-UIHC Specialty Pharmacy. CONCLUSIONS: There was a significant benefit to CFTR modulator prescribing when clinical pharmacy services were incorporated in our cystic fibrosis (CF) care team, which will become increasingly important with the anticipation of new CF medications in the near future.

Impact of a cystic fibrosis transmembrane conductance regulator (CFTR) modulator on high-dose ibuprofen therapy in pediatric cystic fibrosis patients.

BACKGROUND: This study was undertaken to determine if a clinically relevant drug-drug interaction occurred between ibuprofen and lumacaftor/ivacaftor. METHODS: Peak ibuprofen plasma concentrations were measured prior to and after lumacaftor/ivacaftor initiation. A Wilcoxon signed rank sum test was used to compare the values. RESULTS: Nine patients were included in the final analysis. Peak ibuprofen plasma concentrations decreased an average of 36.4 mcg/mL after initiation of lumacaftor/ivacaftor with a relative reduction of 41.7%. The average peak plasma concentration was 84.2 mcg/mL (SD = 10.9) prior to lumacaftor/ivacaftor initiation and 47.9 mcg/mL (SD = 16.4) following initiation (P = 0.0039). Peak concentrations occurred at an average of 100 min (SD = 30) and 107 min (SD = 40) prior to and following lumacaftor/ivacaftor initiation, respectively. CONCLUSIONS: We suggest a clinically relevant drug-drug interaction exists between ibuprofen and lumacaftor/ivacaftor. Lumacaftor may cause subtherapeutic ibuprofen plasma concentrations due to the induction of CYP enzymes and increased metabolism of ibuprofen. Based on this analysis, we have modified our use of ibuprofen in several patients after evaluation of this drug-drug interaction.

Implementation of cystic fibrosis clinical pathways improved physician adherence to care guidelines.

INTRODUCTION: There is significant variability in clinical outcomes, including growth and lung function, between the various cystic fibrosis (CF) centers. No specific or unique therapeutic practices have been identified to account for these differences. However, more uniform care within centers was associated with better outcomes. The objective of this study was to implement clinical pathways for diagnosis and treatment of nutritional failure and lung inflammation in order to achieve better health care provider adherence to center-specific, agreed-on practices. METHODS: Agreed-on clinical pathway treatment plans for both nutrition and lower airway inflammation were implemented on January 1, 2010. The primary outcome measure was to evaluate if patients' diagnoses and treatments were consistent with the agreed-on clinical pathways. RESULTS: The proportion of clinic visits from baseline to 18 months post-intervention where the provider completely followed nutrition clinical pathway increased from 57.72% to 79.49% (P = 0.049) and the proportion for lower airway inflammation clinical pathway increased from 65.85% to 86.32% (P = 0.035). The use of nutritional diagnosis and documentation of associated clinical pathway in the clinical plan increased from 16.26% to 61.54% and 56.10% to 94.87%, respectively. Similarly, diagnosis of lower airway inflammation and documentation related to their treatment plans increased from 1.63% to 43.59% and 30.08% to 87.18%, respectively. CONCLUSION: Implementation of clinical pathways for nutrition and lower airway inflammation issues resulted in more uniform care of CF patients. Having objective criteria for diagnoses and agreed-on treatment plans for each of those diagnoses allowed for monitoring and individual feedback. Increases in utilization of correct diagnoses and discussion of specific therapeutic interventions in the clinic notes were associated with increased adherence to clinical pathways. Pediatr Pulmonol. 2017;52:175-181. © 2016 Wiley Periodicals, Inc.

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co-culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air-liquid interface to allow an in-depth evaluation of a simulated colonization state. Exposure to wild-type, S. aureus bacteria or conditioned media killed airway epithelial cells within 1 day, while mutant S. aureus strains lacking alpha-toxin (hla) persisted on viable cells for at least 2 days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real-time polymerase chain reaction of known virulence factors showed the expression profile of S. aureus grown in co-culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co-culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.

Highly differentiated human airway epithelial cells: a model to study host cell-parasite interactions in pertussis.

BACKGROUND: Bordetella pertussis colonizes the human respiratory mucosa. Most studies on B. pertussis adherence have relied on cultured mammalian cells that lack key features present in differentiated human airway cells or on animal models that are not natural hosts of B. pertussis. The objectives of this work were to evaluate B. pertussis infection in highly differentiated human airway cells in vitro and to show the role of B. pertussis fimbriae in cell adherence. METHODS: Primary human airway epithelial (PHAE) cells from human bronchi and a human bronchial epithelial (HBE) cell line were grown in vitro under air-liquid interface conditions. RESULTS: PHAE and HBE cells infected with B. pertussis wild-type strain revealed bacterial adherence to the apical surface of cells, bacteria-induced cytoskeleton changes, and cell detachment. Mutations in the major fimbrial subunits Fim2/3 or in the minor fimbrial adhesin subunit FimD affected B. pertussis adherence to predominantly HBE cells. This cell model recapitulates the morphologic features of the human airway infected by B. pertussis and confirms the role of fimbriae in B. pertussis adherence. Furthermore, HBE cells show that fimbrial subunits, and specifically FimD adhesin, are critical in B. pertussis adherence to airway cells. CONCLUSIONS: The relevance of this model to study host-parasite interaction in pertussis lies in the striking physiologic and morphologic similarity between the PHAE and HBE cells and the human airway ciliated and goblet cells in vivo. These cells can proliferate in vitro, differentiate, and express the same genetic profile as human respiratory cells in vivo.

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.

Tracheomalacia is associated with lower FEV1 and Pseudomonas acquisition in children with CF.

BACKGROUND: Tracheomalacia (TM) occurs in approximately 1 in 2,100 children. Because the trachea develops abnormally in animal models of cystic fibrosis (CF), we hypothesized this may also occur in children with CF, increasing their risk of TM. PURPOSE: To examine the prevalence and clinical consequences of TM in children with CF. METHODS: We studied children with CF born between 1995 and 2012. TM was defined as dynamic collapse of the trachea, and the severity was recorded as described in the chart. The effect of TM on patient outcomes, including FEV1 , CT changes, and acquisition of CF pathogens, was assessed using a longitudinal patient dataset. RESULTS: Eighty-nine percent of children with CF had at least one bronchoscopy (n = 97/109). Fifteen percent of these children had TM described in any bronchoscopy report (n = 15/97). Of the patients with TM, eight had meconium ileus (P = 0.003) and all were pancreatic insufficient. Pseudomonas aeruginosa infection occurred 1.3 years earlier among children with TM (P = 0.01). Starting FEV1 values by age 8 were diminished by over 18% of predicted for patients with TM. Life-threatening episodes of airway obstruction occurred in 3 of 15 patients with CF and TM, including one leading to death. Gender, prematurity, and hepatic disease were not associated with TM. No difference was observed in the frequency of bronchiectasis. CONCLUSIONS: TM is significantly more common in infants and children with CF than in the general population and is associated with airway obstruction and earlier Pseudomonas acquisition.

Growth of preschool age children receiving daily inhaled corticosteroids.

Data on the effects of inhaled corticosteroid (ICS) on linear growth in children <5 years old are limited with conflicting results from existing studies. This study was designed to investigate growth effects of inhaled corticosteroid use in children <5 years of age treated for asthma with ICS administered through a valved holding chamber (VHC). A retrospective cohort study was performed of 145 children in three treatment groups: (1) metered-dose inhaler (MDI) containing ultrafine beclomethasone dipropionate (n = 62), (2) MDI containing fluticasone propionate (n = 32), and (3) oral montelukast sodium (n = 51). Children <5 years of age between 2000 and 2009 treated for asthma with one of the three drugs were included in the study. Linear mixed model analysis was used to examine and compare growth during sustained treatment with each of the three medications. The three treatment groups did not differ significantly in their effect on growth rates (p = 0.64). However, female subjects had significantly slower growth than male subjects (p = 0.017), and the addition of intranasal corticosteroids (INS; p = 0.013) and the presence of atopy (p = 0.015) had a significant negative effect on growth. In children <5 years of age receiving maintenance therapy for chronic asthma, low-to-medium doses of ultrafine beclomethasone or fluticasone administered through a VHC were not associated with growth inhibition compared with children receiving oral montelukast. A small but statistically significant decrease in growth was seen in subjects with positive skin testing to inhalant allergens in female subjects and in subjects receiving INS.

Multicenter intestinal current measurements in rectal biopsies from CF and non-CF subjects to monitor CFTR function.

Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm(2); 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P<0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.

Assessment of clinical response to ivacaftor with lung clearance index in cystic fibrosis patients with a G551D-CFTR mutation and preserved spirometry: a randomised controlled trial.

BACKGROUND: Ivacaftor has shown a clinical benefit in patients with cystic fibrosis who have the G551D-CFTR mutation and reduced lung function. Lung clearance index (LCI) using multiple-breath washout might be an alternative to and more sensitive method than forced expiratory volume in 1 s (FEV1) to assess treatment response in the growing number of children and young adults with cystic fibrosis who have normal spirometry. The aim of the study was to assess the treatment effects of ivacaftor on LCI in patients with cystic fibrosis, a G551D-CFTR mutation, and an FEV1 >90% predicted. METHODS: This phase 2, multicentre, placebo-controlled, double-blind 2×2 crossover study of ivacaftor treatment was conducted in patients with cystic fibrosis, at least one G551D-CFTR allele, and an FEV1 >90% predicted. Patients also had to have an LCI higher than 7·4 at screening, age of 6 years or older, and a weight higher than or equal to 15 kg. Eligible patients were randomly allocated to receive one of two treatment sequences (placebo first followed by ivacaftor 150 mg twice daily [sequence 1] or ivacaftor 150 mg twice daily first followed by placebo [sequence 2]) of 28 days' treatment in each period, with a 28-day washout between the two treatment periods. Randomisation (ratio 1:1) was done with block sizes of 4, and all site personnel including the investigator, the study monitor, and the Vertex study team were masked to treatment assignment. The primary outcome measure was change from baseline in LCI. The study is registered at ClinicalTrials.gov, NCT01262352. FINDINGS: Between February and November, 2011, 21 patients were enrolled, of which 11 were assigned to the sequence 1 group, and 10 to the sequence 2 group. 20 of these patients received treatment and 17 completed the trial (eight in sequence 1 group and 9 in sequence 2 group). Treatment with ivacaftor led to significant improvements compared with placebo in LCI (difference between groups in the average of mean changes from baseline at days 15 and 29 was -2·16 [95% CI -2·88 to -1·44]; p<0·0001). Adverse events experienced by study participants were similar between treatment groups; at least one adverse event was reported by 15 (79%) of 19 patients who received placebo and 13 (72%) of 18 patients who received ivacaftor. No deaths occurred during study period. INTERPRETATION: In patients with cystic fibrosis aged 6 years or older who have at least one G551D-CFTR allele, ivacaftor led to improvements in LCI. LCI might be a more sensitive alternative to FEV1 in detecting response to intervention in these patients with mild lung disease. FUNDING: Vertex Pharmaceuticals Incorporated.

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

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

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.

Pseudomonas aeruginosa rugose small-colony variants have adaptations that likely promote persistence in the cystic fibrosis lung.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs.

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

Subinhibitory concentrations of azithromycin decrease nontypeable Haemophilus influenzae biofilm formation and Diminish established biofilms.

Nontypeable Haemophilus influenzae (NTHi) commonly causes otitis media, chronic bronchitis in emphysema, and early airway infections in cystic fibrosis. Long-term, low-dose azithromycin has been shown to improve clinical outcomes in chronic lung diseases, although the mechanism of action remains unclear. The inhibition of bacterial biofilms by azithromycin has been postulated to be one mechanism mediating these effects. We hypothesized that subinhibitory concentrations of azithromycin would affect NTHi biofilm formation. Laboratory strains of NTHi expressing green fluorescent protein and azithromycin-resistant clinical isolates were grown in flow-cell and static-culture biofilm models. Using a range of concentrations of azithromycin and gentamicin, we measured the degree to which these antibiotics inhibited biofilm formation and persistence. Large biofilms formed over 2 to 4 days in a flow cell, displaying complex structures, including towers and channels. Subinhibitory concentrations of azithromycin significantly decreased biomass and maximal thickness in both forming and established NTHi biofilms. In contrast, subinhibitory concentrations of gentamicin had no effect on biofilm formation. Furthermore, established NTHi biofilms became resistant to gentamicin at concentrations far above the MIC. Biofilm formation of highly resistant clinical NTHi isolates (azithromycin MIC of > 64 microg/ml) was similarly decreased at subinhibitory azithromycin concentrations. Clinically obtainable azithromycin concentrations inhibited biofilms in all but the most highly resistant isolates. These data show that subinhibitory concentrations of azithromycin have antibiofilm properties, provide mechanistic insights, and supply an additional rationale for the use of azithromycin in chronic biofilm infections involving H. influenzae.

Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis.

RATIONALE: Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF. OBJECTIVES: This study investigated ability of NTHi to form biofilms and its impact on airway epithelia using in vivo and in vitro analyses. METHODS: We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we developed a novel in vitro coculture model of NTHi biofilm formation on polarized human airway epithelial cells grown at the air-liquid interface. MEASUREMENTS AND MAIN RESULTS: In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF, we found morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms on plastic surfaces. NTHi formed biofilms on the apical surface of cultured airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial cells remained viable throughout 4 d of coculture, and responded to NTHi with nuclear factor-kappaB signaling, and increased chemokine and cytokine secretion. CONCLUSIONS: NTHi formed adherent biofilms on the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory cells exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data presented here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H. influenzae.

TGF-alpha regulates TLR expression and function on epidermal keratinocytes.

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-alpha, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-alpha regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human beta-defensins. These findings provide evidence that TGF-alpha up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.