Genetic and Immunologic Aspects of Cystic Fibrosis

Review article Supported by a grant from Zeneca Pharmaceuticals

Genetic and Immunologic Aspects of Cystic Fibrosis Bettina C Hilman, MD

Learning Objectives: Reading this article will enable the readers to reinforce their knowledge of the pathophysiology of cystic fibrosis (CF), the pathogenesis of the lung disease, the criteria for diagnosis, and CF genotype/phenotype relationships. The focus of this review is on the genetic and immunologic aspects of CF. Data Source: Relevant articles, current texts, data presented at the annual North American Cystic Fibrosis Conferences and distributed to the Directors of CF Centers by the CF Foundation were reviewed. A MEDLINE database using subject keywords was searched from 1987 to date. Background information derived from the author’s 33 years of clinical experience at three of the CF Foundation’s CF Care, Teaching and Resource Centers was also included. Study Selection: Since CF is an inherited disorder, the genetic aspects are emphasized. With the cloning of the CF gene, DNA analysis has assumed an important role in confirming the clinical diagnosis and in the improved understanding of the pathophysiology of this disorder. Although DNA testing is highly specific, it is not very sensitive. Results: Cystic fibrosis gene structure and function are described briefly. The pathophysiology of CF, as it relates to the CF gene defect, and the current knowledge of the pathogenesis of the lung disease are reviewed. The criteria for the diagnosis proposed by the Clinical Practice Guidelines for CF are discussed. Problems of establishing the diagnosis and the importance of correlations of laboratory and clinical findings in CF are emphasized. Conclusions: As a multisystem disorder, CF can masquerade as other disorders, including allergic respiratory disease. Primary care physicians often refer patients to allergists/immunologists because of recurrent respiratory problems. This review discusses the genetic heterogeneity of CF. Ann Allergy Asthma Immunol 1997;79:379–94.

INTRODUCTION Cystic fibrosis (CF), the most common life-threatening recessive genetic disease among Caucasians, can present a challenge to the allergist/immunologist in both diagnosis and management. The allergist is often the specialist to whom primary care physicians refer their patients with recurrent respiratory Professor of Pediatrics, Chief, Pulmonary/Allergy, Louisiana State University, Medical Center, Shreveport, Louisiana. Received for publication July 7, 1997. Accepted for publication in revised form October 3, 1997.

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infections. Cystic fibrosis can masquerade as allergic respiratory disease and result in a delay in the diagnosis. Interest among allergists and immunologists ranges from the challenge in the diagnosis because of the variable clinical symptoms and signs to a desire to understand the underlying immunologic mechanisms associated with the progressive bronchopulmonary disease. Cystic fibrosis is characterized by a broad spectrum of clinical features (phenotypes) that appear to be caused by defects in a single gene.1 Advances

in molecular biology contributed to the discovery and cloning of the gene for CF. The CF gene, cystic fibrosis transmembrane conductance regulator (CFTR), functions in ion transport as a chloride channel in the apical membranes of epithelial cells. The identification of CFTR has led to improvement in our understanding of the pathophysiology of this complex multisystem disorder and the pathogenesis of the sweat gland dysfunction and pancreatic disease.2–5 One of the current areas of CF research is the relationship of the abnormalities of CFTR function to the pathogenesis of the lung disease. It is hoped that this knowledge can be utilized to develop improved therapeutic interventions and possible correction of the defective gene through gene therapy. In individuals with two CF disease-producing mutations and one or more clinical characteristic CF phenotypes, CFTR is quantitatively or qualitatively reduced and the chloride permeability is decreased in the affected epithelial cells.6 –9 Patients with CF have diverse clinical features due to the abnormalities of ion transport on epithelial surfaces or in exocrine glands (pancreas, eccrine sweat glands, exocrine glands lining the respiratory, digestive and reproductive tracts). Characteristic clinical findings in CF include chronic sinopulmonary disease, gastrointestinal/ nutritional abnormalities, salt loss syndromes, and urogenital abnormalities resulting in obstructive azoospermia in most affected males.10,11 The specific CF phenotypic features are outlined in more detail in Table 1.12 The wide het-

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Table 1. Phenotypic Features Consistent with a Diagnosis of Cystic Fibrosis ● Chronic sinopulmonary disease a. Persistent endobronchial infection with typical CF pathogens including Staphylococcus aureus, mucoid and non-mucoid Pseudomonas aeruginosa, and Burkholderia cepacia. b. Chronic cough; sputum production. c. Abnormalities on chest radiographs, CT scans of chest (eg, bronchiectasis, atelectasis, infiltrates, and hyperinflation). d. Airway obstruction manifested by wheezing and air trapping. e. Nasal polyps; abnormalities of the paranasal sinuses on sinus radiographs or CT scans of sinuses. f. Digital clubbing. ● Gastrointestinal and nutritional abnormalities: a. Intestinal Meconium ileus Distal intestinal obstruction syndrome (DIOS) Rectal prolapse b. Pancreatic Pancreatic exocrine insufficiency Recurrent pancreatitis c. Hepatic Chronic hepatic disease manifested by clinical or histologic evidence of focal biliary cirrhosis or multi-lobular cirrhosis d. Nutritional Failure to thrive (protein-calorie malnutrition) Hypoproteinemia and edema Complications secondary to fat-soluble vitamin deficiency ● Salt loss syndromes a. Acute salt depletion b. Chronic metabolic alkalosis ● Male urogenital abnormalities resulting in obstructive azoospermia. Modified from: Boat TF, et al. The diagnosis of cystic fibrosis: consensus statement, consensus conferences: concepts of care. Vol. VII, Section 1. Clinical practice guidelines for CF. CF Foundation, Bethesda, MD, 1996.

erogeneity of clinical manifestations is due to the extent of the involvement of the various organ systems, the variability of gene expression, and the age of presentation. GENETIC ASPECTS AND PATHOPHYSIOLOGY Cystic Fibrosis Gene Structure The CF gene, a large gene with 250,000 base pairs and 27 exons located on chromosome 7, encodes for a transport protein, CFTR. The CFTR protein has two transmembrane spanning domains (TM1 and TM2), two nucleotide binding domains (NSD1 and NSD2) and an intracellular R or regulator domain8,13 (Fig 1). The nucleotide binding domains are the sites of adenosine tri-phosphate (ATP) binding/hydrolysis.

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Proposed Function of Normal CFTR Cystic fibrosis transmembrane conductance regulator is a transport protein that is embedded in the apical cell membranes of epithelial cells that provides a passageway (channel) for charged ions such as chloride (Cl⫺) to be selectively transported to regulate fluid balance across the cell membranes. This transport protein is an adenosine 3⬘5⬘ monophosphate-(cAMP)dependent Cl⫺ channel that is regulated by protein kinase A (PKA) phosphorylation.14 –17 Cystic fibrosis transmembrane conductance regulator has other functions such as regulation of the activity of other channel proteins.18 The R domain of CFTR functions as a Cl⫺ channel inhibitor, until it is phosphorylated by PKA and undergoes conformational change to open the channel.19 Phosphorylation of any one

of the four main sites is sufficient for the Cl⫺ channel opening20,21; the closed state of the Cl⫺ channel is accomplished by dephosphorylation of the R domain.21 Function of Defective CFTR in CF The primary physiologic defect in CFTR in patients with CF is reduced chloride conductance at the surface of the apical epithelial membranes. It is this defect in the cAMP dependent Cl⫺ channel that results in the cell being impermeable to the Cl⫺, and in failure of the transport and secretion of chloride from the epithelial cells via this complex signal transduction pathway. The defect in ion transport in CF manifests itself differently in the various organs. Other proposed functions of CFTR include the acidification of intracellular organelles that result in the alteration of sulfation or sialylation of CF mucins and alteration of other cAMP-mediated functions such as endocytosis and exocytosis. Although the promoter for the CF gene that controls transcriptions has not yet been identified, it has some characteristics of the “housekeeping genes.”22 There is a complex regulatory process for CFTR transcription with apparently tissue-specific regulation. The primary transcript is also processed differentially, resulting in alternatively spliced variants.21 Some of these variants do result in in-frame deletions. A hierarchy of organ sensitivity has been proposed to the deficits in functional CFTR. The tissues of the vas deferens have the greatest requirement for normal functioning CFTR and the sweat ducts are the next most sensitive tissues.21 More functional CFTR is required for normal sweat duct function than for normal pancreatic tissue function. The patients with the most severe deficiency of CFTR have pulmonary disease, pancreatic exocrine dysfunction, and abnormal concentrations of sweat chlorides.21 In the ducts of eccrine sweat glands, CFTR is the only channel that has the capabilities of reabsorption of chloride from the sweat. In CF, malfunction of the CFTR results in alteration of reab-

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Figure 1. Cystic fibrosis transmembrane conductance regulator (CFTR) gene with 2 membrane spanning domains, 2 nucleotide (ATP) binding domains and the R domain with model of CFTR protein. Modified from Zielenski J and Tsui L-C. Ann Rev Genet 1995;29:780, used with permission.

sorption of Cl⫺ from the ductal cells as the sweat moves along the ducts to the skin surface. There is diminished permeability of the ductal cells of the sweat gland to the Cl⫺ in CF, but sodium transport across the sweat ducts is not increased. In CF, when there is diminished reabsorption of chloride by the ductal cells, fewer sodium ions are reabsorbed, resulting in elevation of chloride and sodium in the sweat. The abnormality of electrolytes in the sweat was first reported by diSant’ Agnese et al23 and is the basis for the most reliable diagnostic test for CF.24 Because of the increased sodium and chloride in the sweat, patients with CF may have signs and symptoms of salt depletion syndromes and infants may present with metabolic alkalosis.25,26 In CF, the CFTR defect in pancreatic duct cells results in reduced Cl⫺ permeability causing a decrease in bicarbonate ions that normally enter the pancreatic duct cells via the chloride ion/bicarbonate exchange. This defective CFTR function leads to insufficient alkalinization and hydration of pancreatic secretions. Both patients with pancreatic sufficiency and pancreatic insufficiency have a reduction of bicarbonate in pancreatic secretions.27–29 Most patients with CF are born with pancreatic insufficiency, others can develop pancreatic insufficiency due to autodigestion of the exocrine portion of the pancreas because their pancreatic enzymes cannot reach the small intestine. The autodigestion process in the pancreas may be associated with symptomatic pancreatitis.30 A few patients with CF remain pancre-

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atic sufficient all their lives. In the patients with pancreatic insufficiency, the defective CFTR in the pancreatic ductules and ducts is associated with reduced water content of the secretions. The dehydration of pancreatic secretions contributes to the obstruction of the pancreatic ducts and inspissation of secretions, leading to autolysis and fibrosis. With the fibrosis of the exocrine portion of the pancreas, pancreatic enzymes delivered to the small intestine are decreased. The fibrosis in the exocrine portion of the pancreas can result in loss of endocrine-secreting cells and glucose intolerance or cystic fibrosis related diabetes mellitus. In the intestinal tract, CFTR is the only apical Cl⫺ channel in most regions.21,31 In CF, defects in CFTR result in the inability of chloride secretion (with water following chloride) into the lumen of the intestine. Reduced water in the intestinal contents in CF is thought to contribute to the obstruction due to sludging of the intestinal contents associated with meconium ileus of the newborn and distal intestinal obstruction syndrome.21 Intestinal mucous glands undergo hypertrophy and hyperplasia early in the life of patients with CF.32 This altered pathophysiology may play a role in the gastrointestinal complications in CF. In the respiratory epithelium in CF, the defective CFTR results in failure of Cl⫺ secretion due to either a defect in the chloride channel per se or from defective regulatory proteins.21 In addition to the dysfunction of the cAMP dependent Cl⫺ channel, there is increased reabsorption of sodium by the

airway epithelium.4 –5,33–35 Since CFTR plays a role in the regulation of other chloride channels and serves as a regulatory “switch” that allows cAMP to inhibit the absorption of sodium (Na⫹), all these functions are altered in CF. The amiloride-sensitive sodium channel in the airways has been cloned recently and identified as the epithelial sodium channel (ENaC) in the airways. When expressed with CFTR, the ENaC channel activity is reduced and its regulation by cAMP is inhibited.35,36 In CF, the defective CFTR results in reduced water content of the periciliary fluid with dehydration of the airway secretions, leading to inspissation of secretions and airway obstruction. The vicious cycle of airway inflammation/infection and obstruction impair the clearance of airway secretions and lead to persistence of endobronchial infectious agents. Although the systemic immunity in CF is intact, local airway host defenses are impaired due to several factors. The airway obstruction due to increased respiratory secretions and the persistence of infection/inflammation results in impairment of the local host defenses in CF, especially in regard to phagocytic activity. The role of the neutrophil will be described further under the section on the “Pathogenesis of Lung Disease and Immunologic Aspects of CF.” Recently investigators have identified human beta defensin-1 (HBD-1); the HBD-1 may be the link between the basic CF gene defect and the pathogenesis of the chronic bronchopulmonary infections.37 Mode of Inheritance The CF gene is inherited as an autosomal recessive disorder. In the presence of two CF disease-producing mutations, there is impairment of ion transport in exocrine gland cells and epithelial tissues due to quantitative and qualitative reduction in function of CFTR. CF Mutations There are now more than 600 putative mutations of the CF gene that have

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Table 2. World Frequency of CFTR Mutation Chromosomes Mutation ⌬F508 G542X G551D N1303K W1282X R553X 621 ⫹ 1G3T 1717⫺1G3A R117H R1162X ⌬1507 R347P R560T 1078delT G85E R334W A455E 3659delC 3849⫹10kbC3T 3905insT 711⫹1G3T S549N Y122X 2184delA Y1092X S549R(T3C) 1898⫹1G3A V520F 2789⫹5G3A Q493X 3849⫹4A3G R347H

Found

Screened (%)

20,153 29,983 (67.2) 674 20,1998 (3.4) 492 20,827 (2.4) 306 16,739 (1.8) 300 14,408 (2.1) 248 19,600 (1.3) 186 14,056 (1.3) 151 13,715 (1.1) 82 10,460 (0.8) 74 8,699 (0.9) 58 12,465 (0.5) 54 10,307 (0.5) 47 11,527 (0.4) 34 3,192 (1.1) 32 4,801 (0.7) 31 8,733 (0.4) 31 7,048 (0.4) 29 3,634 (0.8) 28 1,955 (1.4) 27 1,313 (2.1) 25 2,860 (0.9) 21 12,516 (0.2) 19 6,535 (0.3) 18 2,598 (0.7) 15 3,118 (0.5) 15 5,616 (0.3) 14 1,589 (0.9) 14 6,890 (0.2) 14 1,305 (1.1) 12 4,317 (0.3) 11 1,120 (1.0) 10 15,060 (0.1)

Data from Cystic Fibrosis Genetic Analysis Consortium, May 1992. Modified from Hilman BC, Lewiston NJ. Clinical aspects of cystic fibrosis. In: Pediatric respiratory disease, ed. Hilman BC, Philadelphia, PA: W.B. Saunders, 1993; 661–73.

been reported to the CF Genetic Analysis Consortium.38 Table 2 lists the 32 most common mutations.39 There are available resources for analyzing DNA for up to 70 CF mutations, including the 32 more common CF mutations. With screening of the 70 CF mutations, the detection rates are reported to be 90% for Caucasians of Northern European ancestry and 61% for African-Americans.40 Few of the CF mutations occur on CF alleles at a fre-

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quency ⬎1%. The relative frequency of specific mutations is higher in certain geographic areas and ethnic groups (eg, among Ashkenazi Jews the mutation W1282X is 60%).41 Because the CF gene is so large and codes for a large protein, the CFTR, different defects in the gene cause a variety of abnormalities in function. At the molecular level, the CF mutations include several different types of mutations: missense mutations, nonsense mutations, frameshift mutations, splice mutations, null mutations, and mutations causing deletions or premature stop codons.21,42 The missense mutations are those in which one base is substituted for another, changing the meaning of the codon containing that base (eg, G551D). Nonsense mutations cause a change in a base resulting in an inappropriate termination of signal and short fragments of protein with reduced function (eg, G542X). Premature stop codons give rise to a truncated CFTR and some also reduce CFTR mRNA levels43 (eg, G542X and W1282X). Deletion mutations result from a loss of the part of the DNA sequence such as the deletion of phenylalanine in the most common CF mutation, the deletion at the ⌬F508 codon. Frameshift mutations are caused by small insertions or deletions of 1, 2, or 3 nucleotides. Deletions and insertions shift the reading frame and usually lead to a downstream, stop codon and a truncated CFTR. 3905 insT is a single nucleotide insertion that alters the reading frame of the mRNA. Null mutations are due to a change in the area of the gene that regulates expressions and halts protein production. Splice-site mutations are due to abnormal exon splicing and give rise to a range of variable CFTR expressions.44 These mutations alter the efficiency of normal mRNA processing and the quality of the CFTR. There is a variety of mechanisms by which mutations in CFTR achieve quantitative or qualitative (or both) reductions in its function in affected individuals. Zelenski and Tsui propose five general functional classes of CF mutations45 (Fig 2). These classes are

not mutually exclusive. In the most common CF mutation, ⌬F508, there is deletion of an amino acid, phenylalanine; this mutant CFTR is not only misprocessed, but fails to respond normally to activation signals.21 In class I mutations there is defective protein production with premature termination of CFTR mRNA translation caused by either base substitutions that create stop codons (eg, G542X) or by frameshift mutations such as the 3905 insT mutation due to insertion of a single nucleotide. Mutations in class I usually produce few or no functioning CFTR chloride channels. Class II mutations are due to defective processing or trafficking of CFTR so that it does not reach its intended site at the apical surface membrane eg, ⌬F508, (a deletion mutation) and N1303K, (a missense mutation). Class III mutations have defective regulation of CFTR, even though the mutant protein reaches the cell surface eg, G551D (a missense mutation). In class IV mutations, CFTR reaches the apical surface membrane, but conduction is defective due to altered conduction or gating channel properties eg, R117H or R347P (missense mutations). Class V mutations are associated with reduced synthesis.45 This class of mutations may include promoter mutations that reduce transcription, nucleotide alterations that promote alternative splicing of the CFTR transcript, and amino acid substitutions that cause inefficient protein maturation, all reducing the amount of functional CFTR. The missense mutation A455E or alternative splicing mutation 3849 ⫹10Kb C⫺⬎T belong to this class of mutations. Genotype-Phenotype Relationships The genotype-phenotype relationships are complicated due to many factors such as the large number of CF mutations, other genetic modifiers, nutritional status, frequency and severity of respiratory tract infections, presence of complications, and environmental factors. The ⌬F508 CF mutation is associated with variable clinical manifestations of disease from mild to severe.

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associated with CF only when the allele also contains the 5 T variant.45,57

Figure 2. General classes of CFTR mutations based on proposed functional abnormalities of CFTR channel function (molecular basis of CFTR chloride channel dysfunction). Modified from Zielenski J and Tsui L-C. Ann Rev Genet 1995;29:792, used with permission.

Pancreatic function can be related to some specific CF mutations. Pancreatic sufficiency is closely related to those CF mutations associated with detectable chloride channel activity eg, R117H and A455E.46 – 47 The mutations with little chloride channel activity (eg, ⌬F508) or those that result in severe quantitative reductions of CFTR (eg, W1282X) are associated with pancreatic insufficiency.21,45 In the respiratory tract involvement, the relationship between genotype and phenotype is less clear.21 The extent of pulmonary involvement varies considerably among patients with the same CF gene mutations, including members of the same family.42,45 Patients with pancreatic sufficiency, in general, have milder pulmonary disease than those with pancreatic insufficiency.48 One of the mutations associated with an atypical presentation of CF is the 3849 ⫹ 10 kb C3 T mutation. Highsmith et al were the first to report the 3849 ⫹ 10kb C3 T mutation in a patient with mild lung disease and normal sweat chlorides.49 Augarten et al described 15 patients with compound heterozygotic 3849 ⫹ 10kb C3 T mutations, including five with ⌬F508/ 3849 ⫹ 10kb C3 T.50 Dreyfus et al recently reported a 40-year-old Hispanic male homozygous for this mutation with severe pulmonary disease, normal sweat chlorides, male fertility, and pancreatic sufficiency.51 Patients with the 3849 ⫹ 10kb C3 T mutation have variable severity of disease.

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Approximately 95% of males with CF are azoospermic due to congenital bilateral absence of the vas deferens (CBAVD).21 Congenital bilateral absence of the vas deferens has also been reported in otherwise healthy males with no other organ involvement.45 The carrier frequency of the ⌬F508 mutation in males with CBAVD is approximately 0.5 in contrast to a carrier frequency of .03 in general population.52 A number of the males with CBAVD carry a second CF mutation on their non-⌬ F508 chromosome that is often a mutation with some residual chloride channel activity (eg, R117H).21 It has recently been reported that many males with CBAVD who have only one detectable CF mutation are also carriers of the 5T Exon 9 splice variant on the other chromosome.53 Coexistence of two independent DNA alterations in the same CFTR allele have been reported (ie, secondsite mutations).45 These second site mutations can modulate the effect of the principal mutation.45 The mutation R117H, a missense mutation, can be modulated by the length of a polythymidine tract (T-tract) upstream of the 3⬘ splice site in intron 8, the intron preceding exon 9.45,54 Of the three Ttract variants, the 5T variant shows the lowest splicing efficiency with over 90% of mRNA CFTR missing exon 9.55 Mutant CFTR missing the region corresponding to exon 9 is not functional as a cAMP-regulated chloride channel.56 The CF mutation R 117H is

DIAGNOSIS OF CYSTIC FIBROSIS A high index of clinical suspicion is essential to the early diagnosis of CF. For the accurate diagnosis of CF, the clinician has to have access to laboratories that offer reliable quantitative analysis of sweat electrolytes by experienced personnel utilizing standardized methodologies, consistent with the guidelines of the National Committee for Clinical Laboratory Standards.58 Reliable quantitative sweat electrolyte testing requires technical precision and quality control measures.59 LeGrys has outlined practical considerations in the use of the sweat test for the diagnosis of CF.60 According to the current Clinical Practice Guidelines and the Consensus Statement for the Diagnosis of Cystic Fibrosis by the CF Foundation, the only acceptable method for diagnostic sweat testing is the quantitative analysis of sweat electrolytes, after stimulation of sweating by pilocarpine iontophoresis.12 A sweat chloride concentration ⬎60 mmol/L is consistent with the diagnosis of CF; however, the results must be interpreted in context of the patient’s age and clinical features characteristic of CF by a physician knowledgeable about CF.12 Sweat chlorides of 40 to 60 mmol/L are considered borderline. Sweat chloride values ⬍40 mmol/L have been documented in genetically proven patients with CF. Some recent data suggests that a sweat chloride of ⬎40 mmol/L is highly suggestive of CF in infants ⬍3 months of age.61 Sweat tests should be done in duplicate and should be repeated on a separate day, if the test is borderline or positive. Although confirming the clinical diagnosis of CF based on the presence of two CF-disease producing mutations is highly specific, it is not very sensitive. Even when the test sensitivity approaches 95%, there will be a substantial fraction of patients with CF who will carry an unidentified CF mutation.

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Lack of detection of CF mutations does not exclude a diagnosis of CF. The majority of cases where CF is suspected on clinical phenotypic features will be confirmed by positive sweat electrolyte tests. DNA analysis can be very helpful when the sweat electrolyte test is normal or equivocal, or only one of the CF-disease-producing mutations are identified. The active transport of ions such as sodium and chloride generates a transepithelial electrical potential difference (PD) that can be measured in vivo.8,62 Abnormalities in ion transport in the nasal respiratory epithelium of individuals with CF are associated with a different pattern of PD across nasal epithelium compared to normal epithelia.8,63,64 For patients with normal or borderline sweat tests in whom two disease-producing CF mutations are not identified, an abnormal nasal PD measurement, recorded on two separate days, may be used as a diagnostic aid, if methodologies are validated and reproducible.12 Interpretation of nasal PD measurements must be done with caution by those knowledgeable in ion transport characteristics of nasal epithelia. PATHOGENESIS OF LUNG DISEASE AND IMMUNOLOGIC ASPECTS IN CF Because lung disease is the major cause of morbidity and mortality in CF, understanding the pathogenesis of the lung disease is essential to the development of management strategies for the chronic progressive bronchopulmonary disease. The lung differs from most of the other affected organs in CF in that it appears normal in utero, but becomes involved shortly after birth. Cystic fibrosis transmembrane conductance regulator is expressed in the fetal lung and has been demonstrated by in situ hybridization.65,66 Sturgess et al reported dilated submucosal gland ducts in airways of infants with CF compared with control infants.67 Early markers of inflammation have been demonstrated in the airways of

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very young patients diagnosed by neonatal screening or newly diagnosed patients with CF within the first 3 months of age by analysis of bronchoalveolar lavage (BAL).68,69 Sequential BAL studies on young children with CF demonstrated evidence of inflammation in the absence of the characteristic CF bacterial pathogens in some patients.70 The evidence of acute inflammation found in BAL fluid in these studies included elevated levels of neutrophils, neutrophil elastase, and IL-8. Polymerase chain reaction (PCR) on BAL fluid detected low levels of P. aeruginosa DNA in 44% of children ⬍5 years of age; 88% of the PCRpositive BAL fluid were previously reported to be culture negative.71 Konstan et al reported BAL findings in patients with CF ⱖ12 years of age who were stable with clinically mild lung disease (FEV1 of 79% ⫾ 4% of predicted) who showed evidence of ongoing inflammation (elevated inflammatory cells, predominately neutrophils, and neutrophil elastase).72 Bonfield et al demonstrated that proinflammatory cytokines (TNF-␣, IL-1␤, and IL-8) are increased and the cytokine IL-10 is decreased in the airway secretions of patients with CF compared to healthy controls.73 Additional studies of Bonfield et al demonstrated that IL-10, a potent regulatory cytokine that decreases inflammatory responses and T cell stimulation, is down regulated in patients with CF.74 The precise sequence of pathophysiologic abnormalities that result from the defective CFTR and lead to chronic progressive bronchopulmonary disease are not clearly defined. The defective CFTR resulting in reduced chloride secretion through the cAMP Cl⫺ channel and the outward rectifying chloride channel (ORCC),75 along with the increased reabsorption of sodium by the airways34 in CF contribute to the dehydration of the periciliary fluid. Desiccated respiratory secretions in CF can alter the clearance of airway secretions and entrap bacteria or fungi and contribute to the persistence of endobronchial infections. The inspissation of

airway secretions and persistence of chronic endobronchial bacterial infection cause impairment of the host’s phagocytic defense mechanisms. No defects in specific primary systemic immune defenses in CF have been demonstrated to account for the recurrence and persistence of the sinobronchopulmonary infections. Alterations in local defenses in the airways have been proposed with the neutrophil playing a role in the self-perpetuation and amplification of the airway inflammation. The neutrophil is the major inflammatory cell in the airways of patients with CF. Neutrophils are recruited to the airways by a variety of mechanisms and can cause oxidant damage and can release enzymes such as neutrophil elastase (NE). This enzyme stimulates the airway epithelial cells to produce the cytokine interleukin 8 (IL-8) which is a chemoattractant for neutrophils, perpetuating the inflammatory response.76 LTB4 is a chemoattractant for neutrophils and serves as a neutrophil aggregator. Pseudomonas species release chemotactic factors such as lipopolysaccharide, contributing to recruitment of neutrophils. Immune complexes and direct action of neutrophils with bacteria or proteolytic cleavage by NE can release complement activation products such as C5a that serve as chemotactic factors for neutrophils. As a proteolytic enzyme, NE has capabilities of digesting the structural proteins of the lung.77 In addition, this enzyme has a spectrum of harmful effects on the control of pulmonary inflammation. The Fc part of immunoglobulin (IgG) can be digested by NE. Since the Fc portion of IgG is required for the binding of IgG to macrophages, the host defenses for protection against infections with Pseudomonas aeruginosa are impaired by NE.78 NE cleaves CR1, the receptor for the opsonic C3 fragment, on neutrophils.79 In addition, neutrophil elastase cleaves C3bi bound on opsonized Pseudomonas species.80 Neutrophils rely on complement-mediated opsonization of bacteria for optimal phagocytosis. The detrimental effects of NE create an “opsonin-

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receptor mismatch” and disarm the complement-mediated opsonization and phagocytosis of Pseudomonas aeruginosa in CF. Neutrophil elastase also promotes hyperplasia of mucous cells with hypersecretion of mucus in patients with CF, contributing to the obstruction of the airways. When the large numbers of neutrophils recruited to the airways disintegrate, they release large amounts of DNA and actin that increase the viscoelasticity of airway secretions. The retention of these secretions with increased viscoelasticity contributes to airway obstruction. The neutrophil-dominated inflammation in the CF airways is ineffective in eradicating the chronic endobronchial infection and overwhelms the protective pulmonary host defenses such as secretory leukoprotease inhibitor and alpha1 antitrypsin. The endobronchial infection, once established, is self-sustaining. The neutrophildriven inflammation leads to the destruction of airway and lung architecture, impairment of pulmonary gas exchange, and respiratory failure. Smith et al81 recently reported a defect in host defenses in CF lung. These investigators used primary cultures of human airway epithelial cells and showed that airway surface fluid from CF cells was not capable of killing pathogens such as P. aeruginosa. When the airway surface fluid from cells from patients with CF was changed to hypotonic solution, the killing of pathogens such as Pseudomonas aeruginosa was increased. Human beta defensin-1 (HBD-1), a naturally occurring antibiotic peptide has been recently identified.37 It is proposed as a possible link between the basic CF gene defect and the chronic endobronchial infections in CF.37 When functioning normally, this molecule appears to be a primary mechanism for the protection of the lung against bacterial infections (ie, provides innate immunity). This important peptide is inactivated by the high salt concentrations that are found in the airways and lungs of patients with CF.

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Allergic Bronchopulmonary Aspergillosis Allergic bronchopulmonary aspergillosis (ABPA), first reported as a complication of CF in 1965, is a hypersensitivity reaction to the hyphae of Aspergillus fumigatus. It is hypothesized that the spores of A. fumigatus are inhaled, trapped, and grow saprophytically in the bronchial mucus of patients with CF and asthma.83 The immunologically mediated hypersensitivity reaction is associated with a rise in total IgE, the appearance of IgG and IgE antibodies specific for A. fumigatus, peripheral blood eosinophilia, and pulmonary infiltrates. The prevalence of ABPA reported in CF varies from 0% to 22%.83– 89 Untreated ABPA can result in exacerbations of airway inflammation and proximal or central bronchiectasis. Greenberger and Patterson90 proposed two subcategories of ABPA: (1) ABPA-CB with central bronchiectasis and proximal airway obstruction and (2) ABPA-S or seropositivity associated with radiographic findings limited to infiltrates.91 Criteria that are common to both of the subcategories of ABPA include: immediate cutaneous test reactivity to A. fumigatus (Af) antigens, Af specific antibodies (IgE, IgG, and precipitins), elevated total serum IgE, peripheral blood eosinophilia, and recurrent infiltrates on chest roentgenograms.90 Total serum IgE ⬎ 500 IU is more likely to be associated with ABPA. In patients with CF there is difficulty in documenting the diagnosis of ABPA and in subdividing them into ABPA-CB or ABPA-S. A 12-year longitudinal study of sensitivity to Aspergillus in patients with CF reported spontaneous diminution and loss of immune parameters in non-ABPA CF patients which prevented them from defining a sensitivity profile suggestive of ABPA.83 These authors noted the importance of follow-up studies and correlation with clinical symptoms, in patients with CF.83 Two recent articles reported the use of recombinant A. fumigatus allergen

I/a used to determine specific serological response92 and skin test reactivity93 in the diagnosis of ABPA in patients with CF. In the future a larger panel of recombinant allergens might be able to detect sensitivity to allergens to A. fumigatus better than using a single A. fumigatus allergen.93 CURRENT AND PROPOSED NEW APPROACHES TO THERAPY Since the lung disease in CF is responsible for most of the morbidity and is the predominant cause of death from CF, most of the therapeutic approaches have been directed toward control of the progressive bronchopulmonary disease. Current treatment goals are aimed at reducing airway obstruction, controlling the endobronchial infection, and improving the nutritional status. In addition to these basic therapeutic strategies, prompt detection and treatment of the complications have contributed to the increased survival and improved quality of life of individuals with CF. In the past 40 years, there has been a dramatic increase in the median age of survival, with the median age of 31.3 years reported in 1996 by the CF Foundation’s Patient Data Registry.94 Bronchodilators, usually beta-adrenergic agonists, have been recommended prior to segmental bronchial drainage and chest physical therapy in patients with CF to dilate airways and facilitate clearance of the respiratory secretions. The short-term response to bronchodilators can vary among patients and within the same patient at different times of testing.90 –98 In a given patient with CF, bronchodilators may be beneficial, of no use, or harmful (paradoxical effect with deterioration of pulmonary function). Patients in this latter group usually have floppy airways that depend on smooth muscle tone to prevent expiratory collapse.21 Long-term effects of bronchodilators in patients with CF have been addressed recently in a preliminary report by Ko¨nig et al.104 The results of this study suggested that maintenance aerosol albuterol reversed the progressive

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decline in lung function in the CF treatment group.104 This result is of interest in view of the evidence that long-term regular use of inhaled beta agonists may be associated with worsening of asthma.105 A large double-blind, placebo-controlled clinical trial of recombinant human DNase (dornase alfa) demonstrated efficacy in stabilizing the decline in lung function of patients with CF ⬎5 years of age with mild to moderate lung disease.106 Twice-a-day administration of inhaled dornase alfa demonstrated no therapeutic advantage over once-a-day treatments.106 Another multicenter study evaluated the safety and aerosol deposition of rhDNase as assessed by BAL in patients with CF ⬍5 years compared with those ⬎5 years.107 This latter study compared patients with CF 3 months to 4 years of age with patients 5 to 9 years of age; rhDNase was reported to be safe for the patients with CF ⬍5 years of age.107 Based on our current understanding of the pathophysiology resulting from the defective CF gene, both pharmacologic approaches and gene replacement therapy have been areas of intensive research. Pharmacologic approaches have been directed to improvement of ion transport. Alternative chloride conductance can be activated by pharmacologic agents, including ATP and uridine triphosphate (UTP).108 The very high basal potential difference in CF airways can be reduced by amiloride, a sodium channel blocker.8 A pilot study reported amiloride aerosol to be successful in slowing the rate of decline of pulmonary function after intensive antibiotic therapy.109 A larger multicenter study, however, did not confirm the beneficial results of nebulized amiloride in patients with CF.110 Gene replacement therapy directed at the basic defect is the ultimate goal of CF therapeutic approaches, since it offers the hope of a cure. There have been, however, formidable practical barriers identified in gene therapy trials. Both viral and non-viral (liposome) vectors have been utilized in the feasibility and safety of gene therapy

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trials. Adenovirus-associated virus (AAV) is one of the favored vectors for gene transfer because it can infect non-dividing cells and is non-pathogenic. Safety studies using AAVCFTR gene transfer in adults with CF have shown that AAV-CFTR gene transfer can safely be used in humans.111 New approaches to CF therapy are directed at the problem that only small amounts of CFTR reach the cell membrane of individuals affected with CF and some of the CFTR at the cell membrane does not function normally. One strategy being investigated to solve these problems include the use of chemical chaperones to help the mutant CFTR fold properly.112 A number of low molecular weight compounds that stabilize proteins have shown promise in correcting the folding defect associated with the ⌬F508 CFTR mutation.112 To enhance delivery of the mutant CFTR to the apical surface of the cell for activation, other therapeutic strategies are addressing the problem of the mutant CFTR chloride channel staying open. Several potential “channel openers” are being investigated in CF cells, including milrinone (a phosphodiesterase inhibitor), genistein (a tyrosine kinase inhibitor), and CPX (cyclopentyl1,3, dipropylxanthine).113–115 Several approaches in vitro appear to activate chloride secretion via CFTR, both wild type and mutant CFTR, including CPX, class III phosphodiesterase inhibitors such as amrinone and milrinone.21,113–117 Lung transplantation is an option that is being sought by an increasing number of CF patients with progressive lung disease. Technical advances in surgery now indicate operative mortality below 5% and primary graft failure is rare.118 The major obstacles to long-term survival include infection and graft rejection. As more CF patients seek this option, the waiting lists for lung transplantation become longer. Mortality can exceed 20% for patients with CF and the average length of waiting can exceed 2 years in many transplant centers.118 Exclusion-

ary criteria vary with each transplant center. One of the recent alternatives to bilateral lung transplantation from cadaveric transplants are the lobar/livingrelated donor transplants (one lung lobe from each of two donors). The survival rates after bilateral living-related donor transplants are comparable to those with cadaveric bilateral lung transplants.118,119 During 1996, 130 patients with CF had bilateral lung transplantation, five had heart-lung transplants and 11 had lobar/living-related donors.94 SUMMARY Because of the complex effects of the CF gene on epithelial surfaces and cell membrane, involvement of multiple organ systems, and heterogeneity in clinical manifestations, CF presents a major challenge for molecular biological and clinical medicine. Despite the remarkable progress in CF research, there are many unanswered questions and the options for therapy are continually being assessed. This complex multisystem disorder with genetic heterogeneity poses a challenge to the clinician in both diagnosis and management. REFERENCES 1. Dean M, Santis G. Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations. Hum Genet 1994;93: 364 – 8. 2. Tsui LC, Buchwald M, Barker D, et al. Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 1985; 230:1054 –7. 3. Rommens JM, Iannuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245:1059 – 65. 4. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245: 1066 –73. 5. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989;245:

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al. Effect of genistein on chloride secretions in CF tissues in vivo and in vitro. Pediatr Pulmonol 1996;(Suppl)13:281A. 117. Middleton PG, Chadwick SL, Pollard KA, et al. Milrinone induces chloride secretion in normal but not CF airways in vivo. Pediatric Pulmonol 1996; (Suppl)13:282A. 118. Mallory GB Jr. Lung transplantation for cystic fibrosis: state of the art. Pediatr Pulmonol 1996; (Suppl)13:121. 119. Cohen RG, Barr ML, Schenkel FA, et al. Living-related donor lobectomy for bilateral lobar transplantation in patients with cystic fibrosis. Ann Thorac Surg 1994; 57:1473– 8. Request for reprints should be addressed to: Bettina C Hilman, MD Professor of Pediatrics Chief, Pulmonary/Allergy 1501 Kings Highway P.O. Box 33932 Shreveport, LA 71130-3932

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CME Examination No. 007-011 Questions 1–30: Hilman BC. Ann Allergy Asthma Immunol 1997;79:379 –93. CME Test Questions 1. The CF gene is: a. A chloride ion (Cl⫺) channel b. Activated by cAMP c. Dependent on protein kinase A phosphorylation d. All of the above e. None of the above 2. The following are true statements about the CF gene (CFTR) function: a. Regulates fluid balance across apical epithelial membranes b. More functional CFTR required for normal pancreatic function than sweat duct function c. Regulates activities of other chloride channels d. Only a and c are correct e. All are correct 3. In CF, the defect in CFTR causes all of the following except: a. Altered reabsorption of chloride from ductal cells in sweat glands. b. Increased sodium transport across ductal cells in sweat glands. c. Reduced chloride permeability in pancreatic duct cells. d. Reduction of bicarbonate in pancreatic secretions. e. Failure of chloride secretion by airway epithelium 4. Inheritance of CF: a. Autosomal recessive mode of inheritance b. CF Gene located chromosome 7 c. More than 600 mutations of CF gene d. a, b, and c are correct e. Only a and c are correct 5. Which of the following types of mutations are associated with CF?

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7.

8.

9.

a. Missense and nonsense mutations b. Frameshift and deletion mutations c. Splice-site mutations d. a and b are correct. e. All of the above The following are true statements about the most common CF mutation: a. Due to deletion of an amino acid, phenylalanine b. Misprocessed and fails to respond to activation signals. c. Due to defective processing or trafficking of CFTR so it does not reach the apical membrane d. a, b, and c are correct e. Only b and c are correct. Genotype-Phenotype Relationships: a. Pancreatic sufficiency (PS) is closely related to CF mutations with detectable chloride channel activity. b. Relationship between genotype and phenotype is related to extent of pulmonary involvement. c. Delta F 508 associated with variable severity of disease d. a, b, and c are correct e. Only a and c are correct Atypical cystic fibrosis a. May have normal or borderline sweat test b. Patients with 3849 ⫹ 10kb C⫺ ⬎ T mutation have variable severity of pulmonary disease c. Pancreatic sufficiency d. a, b, and c are correct e. Only a and c are correct. Azoospermia: a. About 95% of males with CF are azoospermic because of congenital bilat-

eral absence of the vas deferences (CBAVD). b. The carrier rate for delta F 508 mutation is increased in CBAVD without other organ involvement. c. Some of the males with only 1 delta F 508 mutation may also be carriers of the 5T Exon 9 splice variant on the non-delta F 508 chromosome. d. a and c are correct e. All of the above 10. The following are criteria for the diagnosis of CF: a. 1 or more CF phenotypes and elevated sweat chlorides ⬎ 60 mM/L b. 1 or more CF phenotypes, normal sweat chlorides, and in vivo elevated transepithelial nasal potential differences on 2 separate days c. Two CF disease-producing CF mutations on DNA analysis and 1 or more clinical CF phenotypes d. a, b, and c are correct e. only a and c are correct. 11. Role of DNA Testing for CF a. DNA Analysis for CF is very specific b. Inability to detect 2 CFdisease producing CF mutations does not rule out diagnosis of CF c. Not very sensitive, due to large number of CF mutations d. Only a is correct e. All of above correct 12. The CF mutations can be separated into classes of dysfunction of CFTR which include some or all of the following: a. Defective CFTR production with premature termination of CFTR

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mRNA translation caused by base substitutions that create stop codons or by frameshift mutations b. CFTR that reaches the cell surface, but have defective regulation due to missense mutations. c. Reduced functional CFTR due to amino acid substitutions d. a, b and c are correct e. Only a, and b are correct 13–15. Identify as true (a) or false (b) the following statements about pancreatic function in CF: 13. Most patients with CF are born with pancreatic insufficiency (PI) 14. Patients born with pancreatic sufficiency (PS) can develop PI and are subject to episodes of symptomatic pancreatitis. 15. Patients with CF who are PS generally have milder pulmonary disease than those with PI. 16. The following are true statements about sweat electrolyte analysis for confirming the clinical diagnosis of CF: a. Quantitative analysis of chlorides in sweat after pilocarpine iontophoresis is the most reliable laboratory diagnostic test to confirm CF. b. Samples of sweat ⬍75 mg are insufficient for quantitative analysis of electrolytes. c. All positive and borderline sweat chloride tests should be repeated on separate days. d. a and c are correct e. a, b, and c are correct 17–19. Specify if true (a) or false (b) statements about bronchopulmonary disease in CF: 17. Accounts for most of the morbidity 18. CFTR is expressed in fetal lung and has been

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demonstrated by in situ hybridization. 19. The lungs appear to be normal in utero, but become involved shortly after birth in affected individuals with CF. 20. The following statements are correct about the pathogenesis of the lung disease in CF: a. Early markers of inflammation can be found in the airways of infants with CF, detected by neonatal screening. b. Elevated levels of neutrophils, neutrophil elastase, and IL 8 have been demonstrated in bronchoalveolar lavage (BAL) fluid in children with CF with mild stable pulmonary disease. c. Proinflammatory cytokines (TNF-1, IL 1-b, and IL-8) are increased and IL-10 decreased in BAL from children with CF compared to healthy normal controls. d. Only b and c are correct e. all are correct 21. The following are true statements about host defenses in CF: a. No defects in specific primary immune function b. The function of human beta-defensin-1 is altered in patients with CF. c. The neutrophil plays a significant role in the self-perpetuation and amplification of the airway inflammation. d. a, b, and c are correct e. Only a and c are correct 22. The following are true statements about neutrophil elastase: a. Disarms complementmediated opsonization and phagocytosis. b. Stimulates airway epithelial cells to produce the cytokine IL-8 and pro-

motes hyperplasia of mucous cells. c. A proteolytic enzyme capable of digesting structural proteins of the lung. d. Only a and b are correct e. a, b, and c are correct

23–25. Identify as true (a) or false (b) statements about alterations in local host defenses in CF: 23. Neutrophil dominated inflammation overwhelms the host’s protective pulmonary defenses such as alpha1 antitrypsin and secretory leukoprotease inhibitor 24. Beta defensin-1 is inhibited by low salt concentrations found in airway surface fluid of patients with CF 25. Bacteria such as Pseudomonas aeruginosa release chemotactic factors for neutrophils. 26. The following are correct statements about Allergic Bronchopulmonary Aspergillosis (ABPA) in CF: a. Prevalence in CF varies up to 10% to 12%. b. May be difficult to confirm in CF and must be correlated with clinical symptoms and course. c. Untreated ABPA can result in exacerbations of airway inflammation due to central bronchiectasis. d. Only a and c are correct e. All are correct

27–30. Identify as true (a) or false (b) the following statements about gene therapy: 27. Both viral and non-viral vectors (e.g. liposomes) are being used in gene therapy trials.

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28. Adenovirus associated viruses is one of the current favored vectors since it can infect non-dividing cells

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and is non pathogenic. 29. Current gene therapy trials are mainly safety and feasibility trials.

30. The main problem with replacement of mutant CFTR is the rapid clearance by the immune system

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Instructions for Category I CME Credit Certification. As an organization accredited for continuing medical education, the American College of Allergy, Asthma, & Immunology (ACAAI) certifies that when the CME material is used as directed it meets the criteria for two hours’ credit in Category I of the American College of Allergy, Asthma, & Immunology CME Award and the Physician’s Recognition Award of the American Medical Association. Instructions. Category I credit can be earned by reading the text material, taking the CME examination and recording the answers on the perforated answer sheet entitled, “Continuing Medical

Education,” which can be found after the examination. Please record your ACAAI identification number and the quiz identification number in the spaces and scanning targets provided on the answer sheet. Your ACAAI identification number can be found on your ACAAI membership card, nonmembers of the College will be assigned an ACAAI identification number and this should be left blank on the answer sheet. The quiz identification number can be found at the beginning of the CME examination. Use a No. 2 or soft lead pencil for marking the answer sheet. You may erase but

do so completely in order to prevent computer reading errors. Your ACAAI identification number and quiz identification number will be used to record your credit hours earned on the CME transcript system. No records of individual performance will be maintained. Tear out the perforated answer sheet and print your name and address in the spaces provided. Return it within one month after the Annals is received to the American College of Allergy, Asthma, & Immunology, 85 West Algonquin Rd, Suite 550, Arlington Heights, IL 60005. Answers will be published in the next issue of the Annals of Allergy, Asthma, & Immunology.

Answers to CME examination—Annals of Allergy, Asthma, & Immunology, October 1997 (Identification No 007-010) Bardana EJ. Sick building syndrome—a wolf in sheep’s clothing. Ann Allergy Asthma Immunol 1997;79: 283–94. 1. e 6. e 11. b 16. a 2. c 7. e 12. d 17. e 3. c 8. e 13. a 18. e 4. e 9. c 14. d 19. c 5. a 10. c 15. e 20. e

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