Cystic Fibrosis

Cystic fibrosis or mucoviscidosis is a serious and potentially fatal genetic disorder, characterized by a tendency to develop chronic lung infections combined with an inability to absorb fats and other nutrients from food. The main characteristic feature of cystic fibrosis (CF) is the secretion of sticky, viscous mucus in the nose, throat, airways, and intestines.

Causes

Cystic fibrosis is caused by an inherited defect in a gene. The defect is recessive, which means that one faulty gene must be inherited from each parent before any abnormality appears. People with only one defective gene have no symptoms but are “carriers” and can pass the gene on to their children.The defective gene causes a biochemical abnormality in which the faulty movement of ions across cell membranes affects mucus formation. As a result, the mucus-forming glands in several organs do not function properly. Most seriously, the glands in the lining of the bronchial tubes produce thick mucus, which predisposes the person to chronic lung infections. Another serious malfunction is poor or absent secretion of pancreatic enzymes, which are involved in the breakdown and absorption of fats in the intestine. The sweat glands are also affected and excrete excessive amounts of salt.

Symptoms and complications

The course and severity of cystic fibrosis vary. Typically, a child passes unformed, pale, oily, foul-smelling faeces and may fail to thrive. Often, growth is stunted and the child has recurrent respiratory infections. Without prompt treatment, pneumonia, bronchitis, and bronchiectasis may develop, causing lung damage. Most males and some females are infertile. Excessive salt loss from sweating may lead to heatstroke and collapse.

Diagnosis and treatment

Early diagnosis, confirmed by simple sweat and blood tests, improves the outlook for children with cystic fibrosis. Prompt treatment with intensive physiotherapy and antibiotics helps to reduce the severity and frequency of lung infections. In addition, lung function may be improved by treatment with dornase alfa, a genetically engineered version of a human enzyme, which is administered by nebulizer. Pancreatin and a diet rich in proteins and calories are given to bring about weight gain and encourage more normal faeces. Supervision of the treatment is best carried out from a special centre that is staffed by paediatricians, nurses, and physiotherapists who have particular knowledge of the disease.

Ivacaftor is a new oral drug, available for the 5% of cystic fibrosis patients with a G551D mutation. Ivacaftor is a potentiator of the CFTR channel that works by increasing the time the channel remains open after being activated; it has been found to improve lung function by 10% within 2 weeks of treatment, decrease pulmonary exacerbations by 55%, and decrease sweat chloride into the indeterminate range. CFTR corrector therapy for the most common mutation (DF508) is currently under trial.

Outlook

The highly specialized treatment now available for people with cystic fibrosis maximizes their chances of a reasonable quality of life. About 9 in 10 children survive into their teens; many live well into their 40s. Progressive respiratory failure is the usual cause of death, but in some cases a heart-lung transplant may be considered.

Cystic fibrosis in more detail - technical

Cystic fibrosis (CF) or mucoviscidosis is the most frequent autosomal recessive genetic disorder in people with European ancestry. The classic form of CF results from loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene is localized to the long arm of chromosome 7 and encodes a protein that acts both as a chloride channel and as a regulator of other transporters. Loss of CFTR activity results in a plethora of pathological consequences, suggesting a central role for CFTR in epithelial physiology.

CFTR is an ABC transporter-class ion channel that transports chloride and thiocyanate ions across epithelial cell membranes. Mutations of the CFTR gene affect functioning of the chloride ion channels in these cell membranes, leading to cystic fibrosis and congenital absence of the vas deferens.

The disease is characterized by viscous secretions of the exocrine glands in multiple organs and elevated levels of sweat chloride. The most life-threatening clinical features of CF include chronic bacterial infections of the upper and lower airways, airway obstruction, bronchiectasis, respiratory failure, and cor pulmonale.

The gastrointestinal manifestations are primarily caused by inefficient exocrine pancreatic function and include meconium ileus, maldigestion, and failure to thrive. Male infertility and reduced fertility in females are also important symptoms in adults with CF.

Current therapy focuses on ameliorating the symptoms of malnutrition, airway obstruction, and inflammation. As a result of these symptomatic treatments, the survival improved dramatically. The new therapeutic approaches fall into two broad categories, gene replacement and targeted drug development.

Introduction

Cystic fibrosis (CF) is a monogenic disorder with a complex phenotype and clinical variability. It is observed in approximately 1 in 2500 births among Caucasians, and has a carrier frequency of 1 in 25 individuals, making it the most common lethal monogenetic disease within this demographic group. CF is caused by mutations in a 180 kb gene localized to the long arm of chromosome 7. The gene encodes the 1480 amino acid cystic fibrosis transmembrane conductance regulator (CFTR) protein.

CF cases present with complex phenotypes and clinical variability. Defects in CFTR lead to altered exocrine secretion and pathological changes in multiple organ systems. Therefore, CF provides an excellent example of the ways in which a single gene defect confers a wide variety of clinical symptoms. The most commonly affected organs include the airways, gastrointestinal (GI) tract, pancreas, sweat glands, hepatobiliary system, and genital tract. The most life-threatening clinical features of CF are recurrent and persistent airway infections leading to bronchiectasis, respiratory failure, and cor pulmonale. Pancreatic insufficiency is present in 90% of patients due to obstruction of the pancreatic ducts and subsequent fibrosis. Exocrine pancreatic dysfunction begins in utero and causes postnatal steatorrhea and failure to thrive. Meconium ileus is present in B10% of newborns with CF. CF patients also present abnormally high levels of chloride (Cl) and sodium (Naþ) in the sweat, which is considered as a diagnostic standard of the disease before genetic testing. In 95% of adult CF males, infertility is caused by azoospermia as a result of the complete bilateral absence of vas deferens (CBAVD). In females, reduced fertility may result from abnormally viscous intrauterine mucus. Other manifestations of the disease include liver cirrhosis and diabetes mellitus.

Early references to CF describe a serious infant disorder characterized by unusual saltiness of skin. Detailed studies have been performed only since the beginning of the twentieth century, prior to which most infants died soon after birth. Landsteiner, who also defined the basic human blood groups, described cases of meconium ileus associated with defective pancreas function. However, CF was first recognized as a distinct pathological entity in the 1930s by pathologist Dorothy Anderson, who defined one of the most important signs of the disease, the ‘‘cystic fibrosis of the pancreas.’’ After this report, Farber described CF as ‘‘generalized state of thickened mucus’’ and named it mucoviscidosis, a term that is still widely used in Europe.

Anderson and Hodges discovered the autosomal recessive inheritance pattern of the disease in 1946, and suggested that CF was caused by a single gene defect. In the 1950s, Di Sant’Agnese and colleagues observed that levels of Cl and Naþ were increased in the sweat of children with CF. This led to the development (in 1959) of the first accurate diagnostic test, the sweat test for cystic fibrosis. In the 1980s, two critical observations led to a better understanding of the basic pathophysiology of the disease. First, Knowles and colleagues found that respiratory epithelium in CF has defective Naþ and Cl transport. Second, Paul Quinton described the sweat ducts of CF patients as impermeable to Cl.

The gene responsible for the disease was identified in 1989. This opened exciting possibilities for understanding the molecular basis of CF and provided new hope for potential therapies. Fifteen years later, this advance was further reinforced by the successful completion of the low resolution structure of the CFTR protein, along with the high-resolution structures of mouse and human wild-type and DF508 NBD1 domains. Although there is no cure for CF to date, the life expectancy of CF patients has increased significantly. In the 1950s, few children with CF lived to school age; whereas today, patient survival extends to the mid-30s.

Etiology

CF is caused by mutations of the CFTR gene encoding the CFTR protein. Mutations result in decreased levels or inefficient function of CFTR and subsequent pathological changes (Figure 1). CFTR is a multidomain, integral membrane glycoprotein consisting of two homologous halves. Each half contains six predicted transmembrane segments (TMD1 and TMD2) and a nucleotide-binding domain (NBD1 and NBD2). A regulatory domain (R domain) is interposed between the two halves of the protein. CFTR is part of a multiprotein assembly in the apical plasma membrane of epithelial cells.

Although more than 1400 mutations (listed in the CFTR Mutation Database, see ‘Relevant websites’) are known in CFTR, deletion of phenylalaline at the 508 position (DF508) is the most frequent cause of the disease and contributes to over 90% of CF cases. Other mutations are often linked to certain geographical regions or are unique. Only four of them (G542X, N1303K, G551D, and W1282X) have a frequency above 1%.

CFTR gene mutations are classified into six broad groups based on their impact on CFTR synthesis and intracellular trafficking. Class I, nonsense, and frameshift mutations abolish CFTR synthesis. Examples include the G542X and R1162X nonsense mutations that encode premature stopcodons. These mutations result in truncated mRNA transcripts that are unstable. Mutations that result in low CFTR levels (Class V) include missense defects such as A455E, and promoter and splicing abnormalities. Class I and V mutations result in little or no protein product.

Class II mutations include the most frequent mutation, DF508. Deletion of phenylalanine at the 508 position leads to the synthesis of a protein that is recognized by the ER quality control system as misfolded and degraded by the proteasome after retrotranslocation from the ER. Class III mutations lead to the production of CFTR that reaches the plasma membrane, but has defective regulation and function. G551D, a missense mutation within NBD1, is an example of this class and the third most common disease-associated mutation.

The G551D mutant is unable to conduct chloride in response to cAMP. Other mutants in this category exhibit ineffective nucleotide binding or hydrolysis (S1255P, G551S, G1244E, and G1349D). Class IV mutants, such as the R117H and R347P mutants, result in the synthesis of a CFTR protein with defective chloride conductance. The most recently identified class of CFTR mutants (Class VI) maintain normal biosynthetic processing and chloride channel function of the truncated CFTR, but the biological stability of the fully processed protein is dramatically reduced.

Pathology

Respiratory Tract

Histological signs of CF lung disease have been observed as early as the third trimester, with the obstruction of submucosal gland ducts by thick mucus. Recurring pulmonary exacerbations, due to repeated cycles of infection and inflammation, result in lung damage and compromise pulmonary function. Inflammation is present at an early age and remains active even in clinically stable patients. As a result, pulmonary fibrosis, bronchiectasis, respiratory insufficiency, and cor pulmonale remain the hallmarks of CF pathology and the primary cause of morbidity.

Gastrointestinal Tract

The pathological features of the gastrointestinal tract are linked to reduced exocrine pancreatic functions and inadequate intestinal absorption. Neonatal meconium ileus, a diagnostic feature of CF, occurs only in 10% of cases. Distal intestinal obstruction syndrome (DIOS), constipation, megacolon, rectal prolapse, and pancreatic fibrosis develop later during adolescence. Pancreatic insufficiency is present in approximately 90% of CF subjects. Pancreatic fibrosis leads to an increased incidence of diabetes mellitus. Diabetes affects B1% of children and 15% of adults with CF. Hepatobiliary System Hepatic dysfunction increases with age and 10–15% of adolescents and adults with CF have liver cirrhosis. Genital Tract Azoospermia is present in 95% of adult CF males as a result of CBAVD and consequential dilated or absent seminal vesicles. In women, there is reduced fertility that is caused by abnormally viscous intrauterine mucus.

Hepatobiliary System

Hepatic dysfunction increases with age and 10–15% of adolescents and adults with CF have liver cirrhosis.

Genital Tract

Azoospermia is present in 95% of adult CF males as a result of CBAVD and consequential dilated or absent seminal vesicles. In women, there is reduced fertility that is caused by abnormally viscous intrauterine mucus.

Clinical Features

Diagnosis

CF cases represent a great variety of clinical symptoms that vary from patient to patient and by age groups. In the majority of cases, CF is diagnosed before adolescence, but some remain asymptomatic until adult age. In 10% of newborns with CF, meconium ileus is the first diagnostic sign. Later, the symptoms are related mainly to respiratory infections and intestinal malabsorption. The classical diagnostic sweat test for CF involves the biochemical detection of high levels (4100 mg ml1) of sodium and chloride in a sweat sample collected following pilocarpine iontophoresis. The excessive loss of electrolytes may result in heat exhaustion in hot climates. Diagnosis can be confirmed by genetic testing of blood or buccal cells. The classic clinical symptoms of CF are summarized in Table 1.

Table 1: Clinical features of cystic fibrosis

Respiratory tract
- Chronic respiratory tract infection
- Recurrent wheeze
- Bronchiectasis
- Nasal polyposis
- Chronic sinusitis
- Cor pulmonale
Hepatobiliary system
- Liver cirrhosis
Genital tract
- Male infertility (90%, CBAVD, azoospermia)
- Reduced fertility in females (20%)
Gastrointestinal tract
- Meconium ileus
- Steatorrhea
- Distal intestinal obstruction syndrome
- Vitamin deficiency
- Failure to thrive
- Rectal prolapse
- Diabetes mellitus
Electrolyte imbalance
- High sweat sodium and chloride
- Excessive salt loss in sweat
- Systemic electrolyte imbalance

Airway Infection and Inflammation

At birth, typically there are no significant signs of lung disorder in infants with CF. During early childhood, however, cough and thick mucus production, impaired mucociliary clearance, and high susceptibility to infections begin to manifest themselves. Chronic upper airway infections and the production of thick, viscous mucus result in sinus obstruction and nasal polyposis. Pulmonary infection and inflammation in the CF lung are caused by surprisingly few bacterial pathogens. Pseudomonas aeruginosa is the most common isolate (80%), followed by Staphylococcus aureus (51%), Haemophilus influenzae (17%), Methicillin-resistant Staphylococcusaureus (MRSA) (12%), and Stenotrophomonas maltophilia (11%). Viral infections with respiratory syncytial virus (RSV),influenza, and adenovirus are also frequent in children with CF. Therefore, CF should be considered in any young child with persistent symptoms following infection with these pathogens. Chronic lower airway infections leading to fibrosis, bronchiectasis, respiratory insufficiency, and cor pulmonale remain the hallmarks of morbidity in CF.

Gastrointestinal and Pancreatic Manifestations

CF patients suffer from gastrointestinal problems related to inadequately controlled intestinal absorption and pancreatic insufficiency. These include meconium ileus in newborns, steatorrhea, DIOS, constipation and acquired megacolon, rectal prolapse, pancreatitis, and failure to thrive during adolescence.

Genital Manifestations

Fertility Ninety-five per cent of adult CF males are infertile as a result of CBAVD and consequential dilated or absent seminal vesicles. Although about 80% of adult female CF patients are fertile, reduced fertility may result from abnormally viscous intrauterine mucus, that is, o80% water in contrast to the normal 93– 96% hydration that appears to be necessary for sperm migration.

Pathogenesis

Ion channels selectively expressed in the apical or basolateral membrane domains of epithelial cells regulate ion composition and hydration of secreted material. Discrepancies in the expression of these ion transporters engender impaired salt composition and aberrant hydration of the glandular fluid. CFTR is a cAMP-regulated, bidirectional Cl channel primarily expressed in the apical membranes of epithelial cells in a variety of organs including the respiratory tract, gastrointestinal tract, exocrine secretory glands, kidneys, and bile ducts. Tissue specificity is known to be acquired both at the level of transcription and by expression of CFTR splice variants. Interestingly, CFTR mRNA, protein, and functional activity have also been demonstrated in T and B lymphocytes, red blood cells, and cardiomyocytes. Based on its broad expression pattern, it is apparent that CFTR has a central role in electrolyte transport regulation in a number of tissues and cell types. The current concepts regarding CF pathogenesis can be summarized based on the primary functions of CFTR.

CFTR as Ion Transporter

Pathogenesis of sweat abnormalities in CF The direction of chloride movement through CFTR depends on the function of the epithelia in which CFTR is expressed. In the apical surface of the secretory coils of sweat glands, CFTR controls Cl efflux. Movement of Cl promotes H2O movement and hydration of the secretum. In contrast, in the ducts of the sweat glands, Cl is reabsorbed through CFTR. Because the secretory coil cells express either CFTR or other (non-CFTR, Ca2þ-activated) anion channels, in the absence of CFTR, Cl can still be secreted in the secretory coils. However, Cl (and consequently Naþ) reabsorption are deficient because in the duct cells, CFTR is the only chloride channel able to reabsorb Cl.

Pathogenesis of airway abnormalities in CF

Because of the multiple physiological functions of CFTR, several models have been proposed to link CFTR mutations to the development of airway infection. Results from CF mice, raised under germfree conditions and from infants with CF studied by polymerase chain reaction (PCR) or other sensitive pathogen detection methods, suggest that inflammation precedes infection. Not all studies, however, are in agreement with this hypothesis and support the idea that infection precedes inflammation.

Alterations in salt and fluid secretion

The development of infection in the CF lung may result from alterations in salt and fluid secretion. Two models have been put forward to explain how fluid and electrolyte secretion alterations may contribute to lung infections. Under physiological conditions, the function of CFTR in the apical membrane of airway epithelial cells and serous secretory cells is to secrete or reabsorb Cl (Figure 5(a)). Intracellular ion conditions are maintained mainly through the action of the basolateral sodium–potassium pump and other ion transporters. Under basal conditions, both serous acinus cells and surface epithelial cells secrete Cl, Naþ, and H2O to hydrate the airway surface liquid (ASL). Physiological ASL volume and salt concentration is maintained through regulated Naþ and Cl reabsorption through ENaC and CFTR.

The first hypothesis, referred to as the ‘low-volume hypothesis’, suggests that in the absence of CFTR, ENaC is hyperactive, resulting in increased Naþ absorption from the periciliary fluid layer. This causes increased water absorption from the airways and leads to isotonic, but diminished airway surface fluid. As a result, the airway fluid is poorly hydrated. Infections follow because the bacteria are not cleared and become trapped in the viscous surface fluid.

A second model termed the ‘high-salt hypothesis’ is based on the assumption that under normal conditions, airway and submucosal gland epithelial cells behave in a similar way to sweat glands and absorb more Cl and Naþ than water, resulting in hypotonic airway surface liquid. In CF, CFTR is missing or functionally defective, and therefore Cl absorption is decreased, resulting in higher luminal Cl and Naþ concentrations than in healthy individuals (Figure 5(c)). This model is supported by the finding that defensins are inactivated under high salt conditions, and loss of defensin activity may predispose patients to bacterial infections. Distinguishing between these two possible models continues as an ongoing debate. CFTR as Receptor Loss of the CFTR protein has also been linked to altered cell surface protein sialysation, resulting in increased asialo-GM1 molecule expression. This result has significance because asialo-GM1 is a receptor for a number of respiratory pathogens including P. aeruginosa and S. aureus. CFTR itself has been proposed as receptor for P. aeruginosa, suggesting that epithelial cells without CFTR are unable to bind, internalize, and clear this pathogen.

CFTR as Regulator of Other Transporters

CFTR plays a central role not only in secretion of chloride and bicarbonate ions, but also regulates a number of other ion channels. These regulatory functions appear to be tissue-and cell type-specific, and reflect the ion channel expression profiles of the specific cells. CFTR has been shown to regulate Naþ reabsorption by inhibiting the activity of the epithelial Naþ channel, ENaC, suggesting that CFTR defects may lead to disturbances of both Naþ secretion and absorption. It has been proposed that the lung pathology in CF is primarily due to disregulation of sodium transport through ENaC. Supporting this hypothesis, transgenic mice with airway-specific overexpression of ENaC (i.e., increased Naþ reabsorption), but normal CFTR levels, have recently been shown to develop mucus obstruction, goblet cell metaplasia, neutrophilic infiltration, and poor bacterial clearance, that are the hallmarks of CF lung disease.

In addition to the evidence that CFTR conducts HCO3 , it also regulates HCO3 transport through the HCO3 /Cl exchanger. In the intestinal lumen and pancreatic ducts, secretion of fluid containing high concentrations (100–140mM) of HCO3 is necessary to maintain the solubility of mucins and the inactive state of digestive enzymes. The majority of bicarbonate transport in these organs is achieved through an epithelial Cl/HCO3 exchanger, regulated by CFTR. This particular CFTR function is therefore likely to have important physiological implications, and may help explain the severely impaired HCO3 secretion in CF.

Chloride channels other than CFTR, including Ca2þ, activated and outwardly rectified anion channels require the presence of CFTR in the plasma membrane. Loss of CFTR has profound effects on chloride transport both directly and indirectly through these pathways, compounding the defects in epithelial chloride transport that result from loss of CFTR.

Another important cellular effect of CFTR is volume regulation through the Ca2þ-dependent potassium channel, KCNN4. During regulatory volume decrease (RVD), anion and cation channels are activated, permitting the passive loss of inorganic ions and osmotically obliged water. In a CFTR knockout mouse model, cell volume regulation in jejunal crypts is deficient as a consequence of dysfunctional KCNN4. The activity of other Kþ channels such as ROMK2 and KvLQT1 have also been shown to be modulated by CFTR expression. To make matters worse, proper cell volume through aquaporin 3 also appears to be dependent on CFTR. Finally, the activity of gap junction channels is affected by functional CFTR expression, indicating that CFTR plays a significant role in maintaining electrolyte transport not only across individual cells, but also across the epithelial monolayer as a whole.

CFTR and Gene Expression Regulation

Because of the difficulties that have been experienced in attempting to correlate CF genotype and pulmonary disease severity, alternative functions of CFTR remain of considerable interest. One of the hallmarks of CF is enhanced expression of proinflammatory mediators in CF lungs. For example, reduced Smad3 expression has been reported to selectively alter TGFb1- mediated signaling in CF epithelium, and CF epithelial cells may be functionally deficient in IL- 10, a key anti-inflammatory cytokine that controls expression of the inhibitory subunit, IkB. The presence of cell surface CFTR has also been shown to be necessary for RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) expression, a chemokine that selectively influences the migration of monocytes, CD45 ROþ memory T lymphocytes, and eosinophils. Furthermore, CF airway epithelial cells fail to express RANTES in response to P. aeruginosa, the predominant bacterium observed in CF patients. Although the complete list of inflammatory genes affected by CFTR expression in different tissues remains incomplete, this area of research may explain the multifaceted nature of CF lung disease and will remains an important aspect for future studies.

Animal Models

Important structural and functional characteristics of CFTR have been discovered using cell culture models. Despite the progress made, animal models are necessary to investigate the pathogenesis of CF and evaluate novel therapeutic strategies. After cloning the CF gene in 1989, a novel approach was applied to generate mouse strains with mutations in the mouse Cftr gene. Mutations to specific sites in the mouse genome were targeted using homologous recombination in mouse embryonic stem cells. The mutant embryonic stem cells were then injected into blastocysts to create chimeric mice. Chimeric mice were bred to obtain individuals homozygous for the null mutation. Several different CF mouse strains were developed using this approach. A comprehensive list of models can be found in the Virtual Repository of Cystic Fibrosis European Network.

Gene Targeted (‘Knockout’ or KO) and Residual Function CF Mouse Models

Gene targeted (KO) mouse models were created using a method called ‘replacement strategy’. An interruption that was introduced to the mouse Cftr gene resulted in complete loss of CFTR mRNA and protein. Residual function models were created using the ‘insertion strategy’. These models, despite the interruption in the Cftr genome may produce some CFTR mRNA and demonstrate minimal residual CFTR activity. Although these models represented an important step in CF research, it was apparent that the artificial mutations would not accurately model naturally occurring mutations.

Mouse Models with Naturally Occurring, Disease- Causing Mutations in the Mouse Cftr Genome

New mouse strains with naturally occurring mutations, most importantly those with DF508, G480C, (Class II), and G551D (Class III) mutations, have been developed. These models were created either by the ‘exon 10 replacement’ strategy or by the ‘hit and run’ procedure. The ‘exon replacement’ strategy uses a selection marker inserted into one of the introns that regulates the activity of the transcription of the mutant gene. The ‘hit and run’ method results in a mutant exon only and the transcription and activity of the mutant allele is therefore identical to the normal allele. It has been shown that the DF508 mutation in the mouse Cftr sequence caused a similar processing defect as in the human protein.

Transgenic Mouse Models Expressing Human CFTR

Transgenic mouse models were constructed on a ‘knockout’ background with a null mutation in the endogenous CFTR locus (Cftr/). First, the human wild-type CFTR transgene was expressed in the intestinal tract under the control of the fatty-acidbinding protein (FABP) promoter from rat. These mice have an improved survival rate compared to the null mutants. Using a similar approach, a second transgenic mouse model was developed expressing human CFTR carrying the G542X mutation. These mice have been used to study the effects of different compounds on the suppression of this premature stop mutation.

Differences in the Phenotypic Properties of CF Mouse Models and Human CF

Because a number of laboratories have created CF mouse models, it is important to note that although the phenotypic hallmarks are the same, important differences between models with the same mutation have been observed. These variations relate to the genetic background and a number of other factors.

In general, intestinal disease is the most prominent feature of CF mice and the symptoms are comparable to CF in humans. Because of the severity of intestinal obstruction, CF mice are kept on a liquid diet to improve survival. The pancreatic involvement in CF mice is less severe than in humans.

The most striking differences regarding the respiratory defect between the mouse models and human CF were described in studies. Initial characterization of CF mice showed no significant lung disease. Later, pulmonary abnormalities were described in mice bred on a different genetic background, suggesting the presence of genetic modifiers. However, despite extensive studies, an appropriate model for CF lung disease in CF mice has proved challenging. Pathological changes in the lungs of CF mice resemble pulmonary fibrosis rather than abnormalities seen in human patients with CF. It appears that there are additional Cl transporters in mouse lungs that make modeling of the human disease incomplete. Interestingly, infection of CF mice with Pseudomonas promotes some changes consistent with CF. Although the suitability of mouse models for CF lung disease remains controversial, some of the models have already been used to test new therapeutic interventions.

Other Animal Models

Sheep, pig, and ferret models are being considered because the structure of their lungs resembles the human lung more. These models may become more important in the future for testing new therapeutic interventions.

Therapy

Traditionally, cystic fibrosis treatments have focused on ameliorating symptoms of intestinal obstruction, malnutrition, airway obstruction, infection, and inflammation. Pancreatic enzyme replacement decreased the severity of these symptoms significantly, resulting in improved survival, and increased the importance of ambulatory care. Increased understanding of CF pathophysiology and genetic defects provide a conceptual basis for targeted drug development. New therapeutic approaches fall into two broad categories: gene replacement and drug development (CFTR repair). In theory, the gene replacement therapies could be applied to all mutations, whereas drugs target specific mutations. Compounds intended for CFTR repair are being screened to rescue DF508 from the ER quality control (Class II mutants), to stimulate defective channels at the cell surface such as G551D (Class III mutants), or to promote readthrough of premature stopcodons for mutants such as W1282X (Class I mutants).

Preventive and Symptomatic Treatments

The increased life expectancy of CF patients is a direct result of the improved preventive care and antibiotic treatments against respiratory pathogens. A variety of general and transmission-based, CFspecific infection control guidelines are now being implemented. General precautions focus on the potentially infectious nature of body fluids, particularly respiratory tract secretions. Transmission-based precautions, on the other hand, prevent transmission of multidrug-resistant organisms such as Burkholderia cepacia, P. aeruginosa, or viruses such as RSV, influenza, and adenovirus by employing procedures such as maintaining sterilized respiratory equipment, segregating patients, and vaccinating young CF patients. These measures have decreased the severity of lung disease and resulted in a dramatic improvement in the patients’ health status.

Antibiotic Treatments

S. aureus and H. influenzae are often the first agents to infect patients with CF. In the short term, treatment of these pathogens with oral antibiotics is typically effective. Later in the course of the disease, P. aeruginosa (particularly the mucoid strains) become predominant. Mucoid P. aeruginosa is remarkably resistant to even intensive antibiotic regimens in part because antibiotics fail to penetrate bacterial biofilms. A specifically formulated tobramycin (TOBI) solution developed for use in inhalers provides a high-dose aminoglycoside antibiotic in the lungs that is effective against P. aeruginosa. TOBI has been shown to increase lung function in CF patients, with less toxicity compared to systemic aminoglycoside treatments.

Macrolide antibiotics are well tolerated by CF patients. One member of this class, azithromycin, exhibits broad antibacterial activity and has also been shown to decrease neutrophil chemotaxis, inhibit the expression of proinflammatory cytokines, and interfere with biofilm formation by P. aeruginosa.

It is clear that the use of combined antibiotic therapy during acute exacerbations significantly reduces the development of resistant organisms and chronic airway infections. To date, however, no consensus has been reached regarding the possible benefit of periodic antibiotic administration in the absence of overt pulmonary exacerbation. No significant advantage of this type of treatment (compared to symptomatic antibiotic therapy) has been shown for this type of therapy.

Anti-Inflammatory Therapy

Both steroid and nonsteroid anti-inflammatory drugs have been used in CF treatments with limited benefits. Although treatment with steroids may improve function, they also have a number of deleterious side effects (growth retardation, glucose intolerance, and cataract development). High doses of nonsteroidal anti-inflammatory drugs such as ibuprofen have also been used to treat CF lung disease and have been shown to reduce the decline in lung function. Based on altered pharmacokinetics of ibuprofen in CF patients and the potential adverse effects, however, the widespread use of anti-inflammatory agents of this sort has been limited.

Physiotherapy and Mucolytic Treatment

A major goal regarding CF therapy is to prevent the development of pulmonary hypertension and consequent cor pulmonale. Daily chest physiotherapy and aerosol inhalation are often used to improve oxygen delivery. A typical physiotherapy session involves either manual chest percussion (pounding), or use of a device such as the ThAIRapy vest or the intrapulmonary percussive ventilator (IPV) to help loosen thick, mucous plugs within the airways. Similarly, aerosolized medicines such as albuterol, ipratropium bromide, and Pulmozymes (dornase alfa) are used to solubilize and break up CF airway secretions.

Lung Transplantation

Lung or heart–lung transplantation is an important option for CF patients with severe lung disease. Based on present criteria, patients are considered for transplant waiting lists when the forced expiratory volume in s (forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC)) falls below 30%. While precedence should be given to patients with the most severe symptoms, due to the long waiting period for a transplant, patients should be evaluated well before FEV1/FVC falls below 30%. Contraindications for transplantation include ventilator dependence, drugresistant bacterial infection, and poor overall health/ nutritional status. Based on recent reports, this treatment increases the 5-year survival rate from 30% to 66% and perhaps more importantly, dramatically improves quality of life.

Treating the Fundamental CF Defect – Prospects for CF Interventions

Gene Therapy

Since the discovery of the CFTR gene in 1989, gene replacement strategies have been sought using both nonviral and viral vectors. Proof-of-principle in the lung has been established and progress achieved in understanding the barriers preventing efficient gene transfer to surface airway epithelium. A number of gene therapy clinical trials have been completed, although none with positive, long-term effects. Newly developed adeno-associated and lenti-viral vectors provide continuing hope for gene therapy-based CF interventions in the future.

Translational Readthrough, Rescue Agents, and Channel Modulators

Treatment of premature stop mutations with aminoglycoside antibiotics is based on the finding that these agents promote translational readthrough. Although clinical results from using this strategy have been promising, mutations that are treatable with this approach (such as W1284X) are rare. Agents that ‘rescue’ the common DF508 mutant from the ER quality control are of particular interest. A number of laboratories and pharmaceutical and academic centers have launched high throughput screening programs to identify compounds that correct CFTR folding. Similar studies concentrating on modulators of dysfunctional CFTR mutants such as G551D are being tested in preclinical and clinical trials. It is likely that a combination of several of these approaches will be required to overcome the CFTR defects. For example, treatment of patients with DF508 CFTR may require both, rescue of the protein from ERAD and methods or agents that activate the channel and stabilize the protein at the apical surface.

The past few years have provided significant advancements in our understanding of the CFTR protein, and future prospects for alleviating many of the symptoms of CF are at hand. Establishing new interventions based on molecular understanding of CF defects will require further insights regarding CFTR regulation, processing, and protein–protein interactions.&Ivacaftor is a new oral drug, available for the 5% of cystic fibrosis patients with a G551D mutation.

Ivacaftor is a potentiator of the CFTR channel that works by increasing the time the channel remains open after being activated; it has been found to improve lung function by 10% within 2 weeks of treatment, decrease pulmonary exacerbations by 55%, and decrease sweat chloride into the indeterminate range. CFTR corrector therapy for the most common mutation (DF508) is currently under trial.

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Relevant Websites

http://www.genet.sickkids.on.ca – This is a database of mutations in the CFTR gene and is currently maintained by the laboratory of Lap-Chee Tsui.

http://www.cff.org – This is a patient registry that was started by the CF Foundation to track the condition of patients with CF in the US. The registry is updated yearly by the CF Foundation.