Receptor-mediated activation of CFTR via prostaglandin signaling pathways in the airway.

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone (a Food and Drug Administration-approved drug known to target prostaglandin receptors) and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary-cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to the levels comparable to forskolin (Fsk). Pretreatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pretreated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) cotreatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ∼60% compared with the treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that cotreatment with lubiprostone can further restore modulator-rescued CFTR.

Characterizing mucociliary clearance in young children with cystic fibrosis.

BACKGROUND: This research characterized mucociliary clearance (MCC) in young children with cystic fibrosis (CF). METHODS: Fourteen children (5-7 years old) with CF underwent: two baseline MCC measurements (Visits 1 and 2); one MCC measurement approximately 1 year later (Visit 3); and measurements of lung clearance index (LCI), a measure of ventilation inhomogeneity. RESULTS: Median (range) percent MCC through 60 min (MCC60) was similar on Visits 1 and 2 with 11.0 (0.9-33.7) and 12.8 (2.7-26.8), respectively (p = 0.95), and reproducible (Spearman Rho = 0.69; p = 0.007). Mucociliary clearance did not change significantly over 1 year with median percent MCC60 on Visit 3 [12.8 (3.7-17.6)] similar to Visit 2 (p = 0.58). Lower percent MCC60 on Visit 3 was significantly associated with higher LCI scores on Visit 3 (N = 14; Spearman Rho = -0.56; p = 0.04). CONCLUSIONS: Tests of MCC were reproducible and reliable over a 2-week period and stable over a 1-year period in 5-7-year-old children with CF. Lower MCC values were associated with increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF. IMPACT: This is the first study to characterize mucociliary clearance (MCC) in children with cystic fibrosis (CF) who were 5-7 years old. Measurements of mucociliary clearance were reproducible and reliable over a 2-week period and stable over a 1-year period. Variability in MCC between children was associated with differences in ventilation homogeneity, such that children with lower MCC values had increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF.

The Changing Face of Cystic Fibrosis: An Update for Anesthesiologists.

Cystic fibrosis (CF) is the most common fatal genetic disease in North America. While CF is more common among Whites, it is increasingly being recognized in other races and ethnicities. Although there is no cure, life expectancy has steadily improved, with the median survival exceeding 46 years in the United States. There are now more adults than children with CF in the United States. CF is caused by mutations in a gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, expressed in many epithelial cells. More than 2100 CFTR mutations have been linked to CF, and newer CFTR modulator drugs are being used to improve the production, intracellular processing, and function of the defective CFTR protein. CF is a multisystem disease that affects primarily the lungs, pancreas, hepatobiliary system, and reproductive organs. Anesthesiologists routinely encounter CF patients for various surgical and medical procedures, depending on the age group. This review article focuses on the changing epidemiology of CF, advances in the classification of CFTR mutations, the latest innovations in CFTR modulator therapies, the impact of the coronavirus disease pandemic, and perioperative considerations that anesthesiologists must know while caring for patients with CF.

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Clinical Phenotypes and Genotypic Spectrum of Cystic Fibrosis in Chinese Children.

OBJECTIVES: To characterize the clinical phenotypes and genotypic spectrum of cystic fibrosis (CF) in Chinese children. STUDY DESIGN: We recruited and characterized the phenotypes of 21 Chinese children with CF. All 27 exons and their flanking sequences of the CF transmembrane conductance regulator gene were amplified and sequenced to define the genotypes. RESULTS: Bronchiectasis (95.2%) and sinusitis (76.2%) were the most common clinical presentations among our patients. By contrast, pancreatic insufficiency was rare (14.3%). The predominant organism found in the airways was Pseudomonas aeruginosa (66.7%). There were obvious reductions of forced expiratory volume in the first second (mean ± SD: 71.8% ± 17.2% predicted) and forced expiratory flows at 75% of exhaled vital capacity (33.7% ± 20.4% predicted) in children with CF. Overall, we identified 22 different mutations, including 12 missense, 5 nonsense, 2 frameshift, 1 in-frame insertion, 1 splice site, and 1 3'untranslated region mutation. Of these, 7 were novel observations (W216X[780G→A], 1092insA, Q359X, D567Y, 2623-126T→C, 3439delA and 4575+110C→G), and the most common types were L88X and I556V. One de novo mutation (1092insA) was also revealed. Except for N1303K and R334W, none of them were present in the common Caucasian CF transmembrane conductance regulator mutation-screening panels. CONCLUSIONS: There was a 5.7-year delay between the first clinical presentation and the eventual CF diagnosis, suggesting that CF may be underdiagnosed in China. The clinical phenotypes and genotypic spectrum are different from that observed in Caucasians.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia.

Multiple sodium and chloride channels on the apical surface of nasal epithelial cells contribute to periciliary fluid homeostasis, a function that is disrupted in patients with cystic fibrosis (CF). Among these channels is the chloride channel CLCN2, which has been studied as a potential alternative chloride efflux pathway in the absence of CFTR. The object of the present study was to use the nasal potential difference test (NPD) to quantify CLCN2 function in an epithelial-directed TetOn CLCN2 transgenic mouse model (TGN-K18rtTA-hCLCN2) by using the putative CLCN2 pharmacological agonist lubiprostone and peptide inhibitor GaTx2. Lubiprostone significantly increased chloride transport in the CLCN2-overexpressing mice following activation of the transgene by doxycycline. This response to lubiprostone was significantly inhibited by GaTx2 after CLCN2 activation in TGN-CLCN2 mice. Cftr(-/-) and Clc2(-/-) mice showed hyperpolarization indicative of chloride efflux in response to lubiprostone, which was fully inhibited by GaTx2 and CFTR inhibitor 172 + GlyH-101, respectively. Our study reveals lubiprostone as a pharmacological activator of both CFTR and CLCN2. Overexpression and activation of CLCN2 leads to improved mouse NPD readings, suggesting it is available as an alternative pathway for epithelial chloride secretion in murine airways. The utilization of CLCN2 as an alternative chloride efflux channel could provide clinical benefit to patients with CF, especially if the pharmacological activator is administered as an aerosol.

N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier.

Protective mucus coatings typically trap and rapidly remove foreign particles from the eyes, gastrointestinal tract, airways, nasopharynx, and female reproductive tract, thereby strongly limiting opportunities for controlled drug delivery at mucosal surfaces. No synthetic drug delivery system composed of biodegradable polymers has been shown to penetrate highly viscoelastic human mucus, such as non-ovulatory cervicovaginal mucus, at a significant rate. We prepared nanoparticles composed of a biodegradable diblock copolymer of poly(sebacic acid) and poly(ethylene glycol) (PSA-PEG), both of which are routinely used in humans. In fresh undiluted human cervicovaginal mucus (CVM), which has a bulk viscosity approximately 1,800-fold higher than water at low shear, PSA-PEG nanoparticles diffused at an average speed only 12-fold lower than the same particles in pure water. In contrast, similarly sized biodegradable nanoparticles composed of PSA or poly(lactic-co-glycolic acid) (PLGA) diffused at least 3,300-fold slower in CVM than in water. PSA-PEG particles also rapidly penetrated sputum expectorated from the lungs of patients with cystic fibrosis, a disease characterized by hyperviscoelastic mucus secretions. Rapid nanoparticle transport in mucus is made possible by the efficient partitioning of PEG to the particle surface during formulation. Biodegradable polymeric nanoparticles capable of overcoming human mucus barriers and providing sustained drug release open significant opportunities for improved drug and gene delivery at mucosal surfaces.

Net benefit of ivacaftor during prolonged tezacaftor/elexacaftor exposure in vitro.

BACKGROUND: A decrease in the lumacaftor-mediated increase in F508del-CFTR function and expression upon prolonged exposure to ivacaftor (VX-770) has previously been described. However, the efficacy observed with ivacaftor-containing CFTR modulator therapies in vivo is in conflict with these reports. We hypothesized that a portion of the apparent decrease in CFTR function observed after prolonged ivacaftor exposure in vitro was due to an increase in constitutive CFTR-mediated ion transport. METHODS: Human nasal epithelial (HNE) cells were obtained by brushings from three CF individuals homozygous for the F508del CFTR mutation. Differentiated epithelia were pre-treated with prolonged (24 h) exposure to either lumacaftor (VX-809; 3 µM), tezacaftor (VX-661; 3 µM), elexacaftor (VX-445; 3 µM), and/or ivacaftor (0.1-6.4 µM) or DMSO (vehicle control), and CFTR function was assayed by Ussing chamber electrophysiology. RESULTS: In cells treated with lumacaftor, constitutive CFTR activity was not increased at any concentration of co-treatment with ivacaftor. Constitutive CFTR activity was also unchanged in cells treated with the combination of tezacaftor and elexacaftor. An increase in constitutive CFTR activity above the DMSO controls was only observed in cells treated with the combination of tezacaftor and elexacaftor and co-treated with at least 0.1 µM ivacaftor. CONCLUSIONS: These results demonstrate that ivacaftor is a critical component in the triple combination therapy along with tezacaftor and elexacaftor to increase constitutive CFTR function. This work further elucidates the mechanism of action of the effective triple combination therapeutic that is now the primary clinical tool in treating CF.

IL-13-programmed airway tuft cells produce PGE2, which promotes CFTR-dependent mucociliary function.

Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single-cell RNA sequencing of airway epithelium and mouse modeling, we found that IL-13 expanded and programmed airway tuft cells toward eicosanoid metabolism and that tuft cell deficiency led to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bore a signature of PGE2 activation, and PGE2 activation led to cystic fibrosis transmembrane receptor-dependent ion and fluid secretion and accelerated mucociliary transport. These data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.

Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

Cystic fibrosis and estrogens: a perfect storm.

Irreversible destruction and widening of the airways due to acquired infections or genetic mutations as well as those of unknown cause are more severe in females. Differences between male and female anatomy, behavior, and hormonal state have been proposed to explain the increased incidence and severity in females with airway disease such as cystic fibrosis (CF); however, a mechanism to explain a sex-related difference has remained elusive. In this issue of the JCI, Coakley et al. report that elevations in the major estrogen hormone in humans--17beta-estradiol--reduce Ca2+-activated Cl- secretion by airway epithelial cells in culture, thereby disrupting ion and water balance (see the related article beginning on page 4025). They measure a similar diminution of nasal epithelial Ca2+-activated Cl- secretion in women with CF during the menstrual cycle phase at which 17beta-estradiol level is at its highest. These data suggest that for about one week of a four-week menstrual cycle, women with CF will have a reduced ability to efficiently clear airway secretions, the buildup of which is a hallmark of CF. The authors suggest that these data warrant the testing of antiestrogen therapy in females with CF and propose an alternative avenue for CF therapeutic development.

Elexacaftor is a CFTR potentiator and acts synergistically with ivacaftor during acute and chronic treatment.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding ("correctors") and/or increase channel function ("potentiators") have proven to be highly effective in the therapeutic treatment of CF. Notably, incorporation of the next-generation CFTR corrector, elexacaftor, into a triple combination therapeutic (marketed as Trikafta) has shown tremendous clinical promise in treating CF caused by F508del-CFTR. Here, we report on a newly-described role of elexacaftor as a CFTR potentiator. We explore the acute and chronic actions, pharmacology, and efficacy of elexacaftor as a CFTR potentiator in restoring function to multiple classes of CFTR mutations. We demonstrate that the potentiating action of elexacaftor exhibits multiplicative synergy with the established CFTR potentiator ivacaftor in rescuing multiple CFTR class defects, indicating that a new combination therapeutic of ivacaftor and elexacaftor could have broad impact on CF therapies.

Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure.

Quantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.

Downregulation of epithelial sodium channel (ENaC) activity in human airway epithelia after low temperature incubation.

INTRODUCTION: The incubation of airway epithelia cells at low temperatures is a common in vitro experimental approach used in the field of cystic fibrosis (CF) research to thermo-stabilise F508del-CFTR and increase its functional expression. Given that the airway epithelium includes numerous ion transporters other than CFTR, we hypothesised that there was an impact of low temperature incubation on CFTR-independent ionoregulatory mechanisms in airway epithelia derived from individuals with and without CF. METHODS: After differentiation at the air-liquid interface, nasal epithelia were incubated at either 37°C or 29°C (low temperature) for 48 hours prior to analysis in an Ussing chamber. RESULTS: While F508del-CFTR activity was increased after low temperature incubation, activity of CFTR in non-CF epithelia was unchanged. Importantly, cultures incubated at 29°C demonstrated decreased transepithelial potential difference (TEPD) and short-circuit currents (Isc) at baseline. The predominant factor contributing to the reduced baseline TEPD and Isc in 29°C cultures was the reduced activity of the epithelial sodium channel (ENaC), evidenced by a reduced responsiveness to amiloride. This effect was observed in cells derived from both non-CF and CF donors. DISCUSSION: Significant transcriptional downregulation of ENaC subunits ß and γ were observed, which may partially explain the decreased ENaC activity. We speculate that low temperature incubation may be a useful experimental paradigm to reduce ENaC activity in in vitro epithelial cultures.

Applications of proteomic technologies for understanding the premature proteolysis of CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-dependent anion channel. Disease-causing mutations can affect channel biogenesis, trafficking or function, and result in reduced ion transport at the apical surface of many tissues. The most common CFTR mutation is a deletion of phenylalanine at position 508 (DeltaF508), which results in a misfolded protein that is prematurely targeted for degradation. This article focuses on how proteomic approaches have been utilized to explore the mechanisms of premature proteolysis in CF. Additionally, we emphasize the potential for proteomic-based technologies in expanding our understanding of CF pathophysiology and therapeutic approaches.

Elevated serum IL-8 levels in ataxia telangiectasia.

Serum interleukin (IL)-8 levels were measured in 50 patients with ataxia telangiectasia (A-T) and 22 without A-T. In a cross-sectional study, the geometric mean of IL-8 level was significantly higher in the patients with A-T (P <.0001). Elevated serum IL-8 levels in patients with A-T suggest that systemic inflammation may contribute to the disease phenotype.

Description of a standardized nutrition classification plan and its relation to nutritional outcomes in children with cystic fibrosis.

OBJECTIVE: Better nutrition enhances lung function and increases survival for children with cystic fibrosis (CF). Therefore, we developed a standardized strategy to evaluate nutritional status and create individualized treatment plans to ensure that all patients received the same high-quality care in a busy CF Center. METHODS: A quality improvement approach was undertaken to develop a novel nutrition classification strategy to identify and treat children with subtle manifestations of nutritional deficits in addition to those with obvious nutritional issues. RESULTS: During the 15-month study period, the median body mass index (BMI) percentile increased from 35.2 (0-95.9) to 42.0 (0-97.7), p < .005. Additionally, the number of children with a BMI >or=50th percentile increased by 11.8%. CONCLUSIONS: Adoption of a standardized approach to nutritional assessment and treatment led to significant improvement in nutritional outcomes of CF patients, demonstrating that systematic changes in clinical practice can improve clinical outcomes substantially over a short period of time.