Ultrastructural abnormalities of cilia in the human respiratory tract

Ultrastructural Abnormalities of Cilia in the Human Respiratory Tract LESLEYA. SMALLMAN, MRCPATH, and J. GREGORY, FIMLS Primary ciliary dyskinesla is thought to be caused by a primary defect of ciliary ultrastructure and function. However, atypical cilia have also been described in humans with and without acquired respiratory tract disease. With few exceptions, these abnormalities have not been quantified. Ciliary ultrastructure was therefore studied in 21 specimens of bronchial mucosa from patients with a variety of respiratory problems and in five specimens of nasal mucosa from asymptomatic nonsmokers. The incidence of microtubular abnormalities and compound cilia was generally less than 10 per cent, and there was no correlation bet w e e n the incidence of these abnormalities and the presence of lung carcinoma or smoking habits. Transposition of ciliary microtubules and radial spoke defects, specific microtubular abnormalities thought to be pathognomonic for primary ciliary dyskinesia, were observed in a number of specimens, and visualization of dynein arms, particularly inner dynein arms, was extremely difficult. It is concluded that ultrastructural abnormalities of cilia should be carefully quantified in patients with primary ciliary dyskinesia and control subjects before it can be assumed they have pathologic significance. HUM PATItOL 17:.~48-855, 1986. In recent years it has b e e n shown that patients with K a r t a g e n e r ' s s y n d r o m e t (a h e r e d i t a r y disease c h a r a c t e r i z e d by situs i n v e r s u s v i s c e r u m , sinusitis, a n d bronchiectasis) as well as patients with similar s y m p t o m s but without situs inversns have defective m u c o c i l i a r y t r a n s p o r t , i m m o t i l e o r dyskinetic cilia, a n d a b n o r m a l c i l i a r y u l t r a s t r u c t u r e . 9-4 C o n s e -

Received from the Depart,nent of l'athology, The Medical School, University of Birmingham, England. Revision accepted for publication January 27, 1986. Supported by a grant from the Endowment Research Committee of the Central Birmingham tteahh District. Address correspondence and reprint requests to Dr. Smallman: Department of Pathology, The Medical School, University of Birmingham, Birmingham B 15 2TJ, Englaud.

quently, the s y n d r o m e is now r e f e r r e d to as the in]motile cilia s y n d r o m e 5 o r p r i m a r y ciliary dyskinesia. 6 T h e first u l t r a s t r u c t u r a l a b n o r m a l i t y r e p o r t e d was complete o r partial absence o f dynein a r m s , 7-9 a n d Sturgess a n d colleagues later described cilia with defective radial spokes t~ a n d transposition o f ciliary mic r o t u b u l e s . ~ M o r e recently, patients with p r i m a r y ciliary dyskinesia in w h o m ciliary u l t r a s t r u c t u r e is n o r m a l h a v e b e e n descl'ibed, t2-14 It is n o w clear, therefore, that t h e r e exists not a well-defined entity but r a t h e r a h e t e r o g e n e o u s g r o u p o f conditions it] which a wide r a n g e o f abnormalities o f u l t r a s t r u c t u r e and function o f cilia can be d e m o n s t r a t e d . A l t h o u g h a b n o r m a l i t i e s o f ciliary u l t r a s t r u c t u r e have received m u c h e m p h a s i s , little o r no a t t e m p t has been m a d e to q u a n t i f y the differences in ultrastructure, if an),, that o c c u r in patients with p r i m a r y ciliary dyskinesia a n d in suitable control subjects. It was t h e r e f o r e the aim o f this study to e x a m i n e cilia f r o m the m u c o s a o f the respirator)' tract o f patients with various acquired p u l m o n a r y conditions a n d o f n o r m a l subjects in o r d e r to d e t e r m i n e the incidence o f u h r a s t r u c t u r a l abnormalities and to a t t e m p t to assess their significance.

MATERIALSAND METHODS T w e n t y - o n e s p e c i m e n s o f bronchial m u c o s a (17 biopsies, f o u r brushings) were obtained f r o m 21 patients (16 m e n , 48 to 78 )'ears o f age; five w o m e n , 17 to 72 years o f age) who were being investigated fox" various p u l m o n a r y diseases (lung carcinoma, 11 patients; u n r e s o l v e d p n e u m o n i a , t h r e e patients; hemoptysis, two patients; fibrosing alveolitis, two patients; m y c e t o m a , o n e patient; lipoma b r o n c h u s , o n e

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patient; and adenoma bronchus, one patient). None of the patients studied had primary ciliary dyskinesia or bronchiectasis. The specimens were taken from areas o f mucosa with normal appearances in the right or left main bronchus during diagnostic bronchoscopy under local anesthesia. An additional five specimens, consisting of nasal mucosa, were obtained from the inferior turbinate area under general or local anesthesia from five subj e c t s w h o were n o n s m o k e r s and had no nasal symptoms or signs; two of the subjects (a 39-year-old man and a 40-year-old woman) were patients tradergoing transsphenoidal hypophysectomy for pituitary tumors, and three were healthy vohmteers (two men, 30 and 73 ),ears of age; one woman, 18 )'ears of age).

Informed consent was obtained from the 26 patients, and the project was passed by tile Research Ethical Committee of tile Central Birmingham Health District. The 26 specimens were fixed in 2.5 per cent glutaraldehyde ill 0.1 ,~l cacodylate buffer to which 2 mmol of magnesium sulfate had been added in view of its reputed ability to cause the dynein arms to attach to adjacent microtubules and thereby make their identification easier. 15 Each specimen was postfixed in Caulfield's fixative for one hour, prestained in 0.1 per cent uranyl acetate in methanol for one hour, dehydrated in graded alcohols, cleared in propylene oxide, and embedded in Araldite. Brnshings were processed in the same manner as 849

HUM/~ PATHOLOGY

Volume '17,No. 8 [August'1986)

seen were included in the quantitative analysis, with cilia sectioned near the base or tip excluded. Each ciliary profile was classified as normal or abnormal, and the incidence o f abnormal cilia was expressed as a percentage o f the total n u m b e r counted (fig. 1). In addition, the n u m b e r o f inner and outer dynein arms was counted in 50 cilia in each specimen. More subtle defects, such as shortened dynein arms, shortened radial spokes, and absence o f spoke heads a n d central sheath, were not assessed in tiffs study.

RESULTS

Normal Ultrastructure of Cilia

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FIGURE7 (top]. Normal 9 + 2 arrangement [left cilium) and transposition of ciliary microtubules [right cilium]. The central singlets are missing, and an outer doublet has been displaced into the central region of the axoneme (arrow). (x 165,000.) [Reprinted with permission from Lancet 1:965, 19843 EIGUR[ 8 (boflom]. Cilium showing the radial spoke detect. Radial spokes cannot be distinguished, and an outer doublet has been displaced toward the central region [arrow]. ( x 155,0003

T h e internal structure o f the axoneme of cilia has the classic 9 + 2 microtubtflar pattern./9 T h e pair in the center is composed o f single microtubules (singlets). Those in the peripheral row o f nine are double-barelled (doublets), composed of subunits A a n d B. T w o s h o r t d i v e r g i n g arms ( d y n e i n arms) project clockwise from subunit A of each doublet toward the next doublet. In addition, radial spokes connect subunit A to a central sheath that surrotmds the central singlets (fig. 2).

Bronchial Specimens

T h e m e a n n u m b e r o f cilia counted in each bronchial specimen was 797 (range, 238 to 1,072). M i c r o t u b u l a r a b n o r m a l i t i e s are d e f i n e d as failure to c o n f o r m to the 9 + 2 a r r a n g e m e n t or to the structure characteristic o f the base or tip of the cilium. Abnormalities o f m a n y different types were the biopsy specimens up to the dehydration stage. If seen; t h e y i n c l u d e d i n c r e a s e or d e c r e a s e in the settling of the fragments was incomplete in any of the number of doublets; extra microtubules (either stages o f processing, the specimen was centrifi~ged at singlets or doublets inside and/or outside the periph1,400 rprn for 5 m i n u t e s . A f t e r p r e s t a i n i n g with eral ring); increase or decrease in the n u m b e r o f uranyl acetate, a d r o p of liqtfid 2 per cent agar (42~ was a d d e d to the specimen; after it had settled for 3 singlets; and totally disorganized cilia (figs. 3 to 6). T h e mean incidence o f microtubular abnormalities minutes, it was solidified at 4~ Excess agar was was 2.68 per cent. However, in one unique specimen t r i m m e d from the block, which was cut into two or three pieces and then d e h y d r a t e d and processed in 73.25 per cent o f the microtubules were abnormal; the same way as a tissue specimen. 16 T h e r e was no 54.35 per cent of the abnormalities involved transposition of ciliary microtubules (fig. 7), and 1.74 per morphologic d i f f e r e n c e between tissue specimens cent were radial spoke defects (fig. 8). and brushings, except that the tissues yielded greater T h e incidence o f c o m p o u n d cilia, i.e., cilia connumbers of cilia. Sections were cut at 1 p.m with a microtome a n d taining more than one 9 + 2 microtubular arrangestained with equal parts o f 0.1 per cent thionin and m e n t within a single a x o n e m e , was 1.81 per cent. 0.1 per cent borax. U h r a t h i n sections (exhibiting T h e n u m b e r o f 9 + 2 a r r a n g e m e n t s within each gold-silver interference) were then cut and picked up c o m p o u n d cilium varied f r o m two to 60, and in some on 3-mm 100-mesh copper grids, double stained with the microtubular pattern was incomplete. C o m p o u n d saturated uranyl acetate and 0.1 to 0.4 per cent lead cilia o c c u r r e d in two m a i n f o r m s , a d h e s i v e a n d citratefl 7:s carbon-coated, a n d examined at 80 kv in bulging. T h e more c o m m o n type (the adhesive type) an electron microscope equipped with a goniometer was characterized by closely packed parallel 9 + 2 stage. arrangements, whereas in the bulging type the 9 + 2 Electron micrographs were taken of the whole a r r a n g e m e n t s lacked a r e g u l a r directional p a t t e r n section at a magnification o f x 18,000 and printed at and were dispersed in a b u n d a n t matrix (figs. 9 to a final magnification of x54,000. T h e goniometer 14). T h e c o m p o t m d cilia tended to occur in clusters, stage was used to obtain the m a x i m u m n u m b e r of with none in some photomicrographs and up to 17 in transverse sections. Only transversely sectioned cilia others from the same specimen. in which the microtubular a r r a n g e m e n t was clearly T h e distribution pattern of microtubular abnor850

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realities differed from that of c o m p o u n d cilia (fig. I 15). A scatter diagram o f the incidence of microtubular abnormalities against the incidence o f comp o u n d cilia divided into four quadrants by arbitrary lines showed that the greater the incidence o f microtubular abnormalities, the greater was the incidence o f c o m p o u n d cilia, and vice versa (fig. 16, table 1). A 9 similar scatter diagram for patients with and without lung carcinoma showed no correlation between the incidence of these abnormalities and the presence or absence o f t u m o r (fig. 17, table 2); a scatter diagram 85t

for patients with various smoking habits showed that a h h o u g h the incidence o f these abnormalities tended to be higher in smokers than in nonsmokers or exsmokers, tile difference did not reach statistical significance ( M a n n - W h i m e y U-test) (fig. 18, table 3). I n n e r dynein arms were much more difficult to see than outer dynein arms. T h e mean n u m b e r o f outer dynein arms was 8.96 (normal, nine), and tile mean n u m b e r o f inner dynein arms was 4.76. No instances o f absence o f all microtubules or o f aplasia of cilia were seen.

HUMAN PATHOLOGY

Volume 'IL No. 8 [August 1986]

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Nasal Specimens

The mean number of cilia counted ill eacll specimen was 831 (range, 389 to 1,851). The incidence of microtubular abnormalities was 2.51 per cent, and the incidence of compound cilia was 0.27 per cent. The mean number of outer dynein arms was 8.94, and the mean n u m b e r o f inner dynein arms was 5.26. DISCUSSION

rial in a single basal body 22,3~ and that c o m p o u n d cilia could arise by fnsion of axonemes or by the extension of more than one 9 + 2 arrangement into a cytoplasmic protusion. 2~ More complex mechanisms may be invoh, ed, however, becanse in no specimen did we observe tile filsion of more than two axonemes together or the association of more than two basal bodies with one compound cilium. Since micro~ tubular abnormalities and compound cilia have different distribution patterns, it is possible that the factors causing them to form are also different. Fox et al. 23 originally found a low incidence of ultrastructural abnormalities (microtubular abnormalities, 5.48 per cent; c o m p o u n d cilia, 0.27 per cent), with no significant differences between patients with and without lung carcinoma or between s m o k e r s a n d n o n s m o k e r s . H o w e v e r , the m e a n number of cilia counted in each specimen was only 39. More recently, these workers 24 re-examined the specimens and counted more than 500 cilia; they concentrated on microtubular abnormalities and found an incidence of 2.88 per cent, with a statistically significant difference in nticrotubular abnormalities between groups. The unique specimen in our series was from a patient who had smoked 100 cigarettes/day until three months prior to his admission to the hospital, when he reduced the number of cigarettes smoked to 40/day. It is tempting to suggest that the remarkably high incidence of microtubular abnormalities in the cilia from this patient was related to his very heavy smoking habits and, possibly, the presence of a lung carcinoma, but for all cases in our study, no significant correlation could be demonstrated between tile incidence of ciliary abnormalities and these two factors. Sturgess and colleagues described two o t h e r major abnormalities in primary ciliary dyskinesia: transposition of ciliary microtubules and cilia with defective radial spokes. When ciliary microtubules are transposed, tile central singlets are missing, and one of the outer doublets is displaced into the central region of the axoneme. Radial spokes cannot be distingnished in cilia with the radial spoke defect; the central singlets are slightly eccentric in the axoneme, and one of the outer doublets is displaced into the central region. In the case with the high incidence of microtnbular abnormalities discussed earlier, transposition of ciliary microtubules was common, with the radial spoke defect seen much less frequently, but there was no evidence of a hereditary disease or

Abnormal cilia have been described in the bronchial mucosa in patients with carcinoma o f the lung, 2~ chronic bronchitis, 25 asthma, 26 cystic fibrosis, 27 bronchiectasis and recurrent infections,22'z8 tuberculosis, 23,24,29 and various other conditions. 22-24 To explain the apparent association, it has been postulated that abnormalities of ciliary ultrastructure would cause diminished ciliary activity and, hence, lead to "impairment of the vital cleansing mechanism that rids the respiratory tract of irritants and carcinogens. ''2~ It is clear from the present study, however, TABLE 1. Incidence of Microtubular Abnormalities versus that the incidence of ultrastructural abnormalities of Incidence of Compound Cilia cilia is, with one exception, so low in patients with Compound acquired pulmonary diseases tlmt ciliary dysfimction Cilia (%) produced in this way is very unlikely to play a part in the causation of the disease. <0.45 >0.45 Very little is known about the mode of formation Microtubular abnormalities (%) of microtubular abnormalities and/or of compotmd >1.85 2 9 cilia. It has been suggested that microtubular abnor<1.85 9 I malities result from the mutation of the genetic mate852

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FIGURE 17 (fop right]. Similar scarier d i a g r a m for patients with and without lung carcinoma. There is no correlation b e t w e e n the incidence of these ciliary abnormalities a n d the presence or absence of tumor. FIGURE t 8 [bo#om right]. Scarier d i a g r a m for patients with various smoking habits. The incidence of these ciliary abnormalities tends to b e higher in smokers t h a n in nonsmokers or exsmokers, but the difference does not reach statistical significance.

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of bronchiectasis. Tile two microtubular abnormalities were also seen occasionally in several other specimens. It would appear, therefore, that these abnormalities are not pathognomonic for primary ciliary dyskinesia. No cilia showed complete absence o f dynein arms. However, it was very difficuh to demonstrate dynein arms and radial spokes in their entirety, and the identification o f inner dynein arms was particularly difficult. Much depends on fixation and on section quality and thickness. Fox et al. 24 obtained pieces of bronchial wall with normal appearances from a lobectomy specimen and used them to stud)' the effect of six standard fixatives; variation in the shape of the cilia, internal structure, and the number of dynein arms visualized were assessed. It was shown that dif853

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ferent fixatives had a marked effect on the ultrastructural appearances o f cilia, ahhough there was often variation even within the same specimen. However, it was not possible to relate the composition of the fixative to the apperances of the cilia. Modified Karnovsky fixative was considered to be the best fixative, but we obtained similar resuhs with 2.5 per cent glutaraldehyde in cacodylate buffer to which 2 mmol of magnesium sulfate had been added. Other m e t h o d s for e n h a n c i n g d y n e i n arms a n d radial spokes include a demembranization technique with Triton-X 100 ~l and tile Markham technique. ~l.~z There are obvious difficuhies in obtaining specimens of bronchial mucosa from healthy subjects. Wisseman et al. 33 s t u d i e d bronchial cilia in two " n o r m a l " children at necropsy immediately after

HUMAN PATHOLOGY TABLE 2.

Volume 1L No. 8 [August 1986}

Incidence of Ciliary Abnormalities versus Incidence of Lung Carcinoma

Compound Cilia (%}

Lung carcinoma Microtubular abnormalities (%) >1.85 <1.85 Nonlung carcinoma Microtubular abnormalities (%) >1.85 <1.85

TABLE 3.

<0.45

>0.43

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Incidence of Ciliary Abnormalities versus Smoking History

Compound Cilia (%) <0.45 Smokers Microtubular abnormalities (%) > 1.85 < 1.85 Nonsmokers and ex-smokers Microtubular abnormalities (%) >1.85 <1.85

>11.45

tory o f sinusitis a n d bronchiectasis, with o r without situs inverstis viscerum; 2) functional studies o f cilia; and 3) u l t r a s t r u c t u r e o f cilia. T h e absence o f d y n e i n arms, particularly o u t e r d y n e i n a r m s , may be a real f e a t u r e o f this s y n d r o m e , b u t the fact that m i c r o t u bular abnormalities a n d c o m p o u n d cilia are f o u n d in various p u h n o n a r y diseases a n d u n d e r " n o r m a l " conditions suggest ttmt these abnormalities are n o n s p e cific and, probably, acquired. T h e y do not, however, a p p e a r to be related to the p r e s e n c e o f h m g carcin o m a or to cigarette s m o k i n g . T r a n s p o s i t i o n o f ciliary m i c r o t u b u l e s a n d t h e radial s p o k e d e f e c t a r e merely f u r t h e r e x a m p l e s o f nficrotubular a b n o r m a l i ties a n d not p a t h o g n o m o n i c for p r i m a r y ciliary dyskinesia, as s u g g e s t e d p r e v i o u s l y . Patients with the latter c o n d i t i o n a r e said to be p r o n e to r e c u r r e n t u p p e r a n d lower r e s p i r a t o r y tract infections, a n d it is possible that the infections are not a c o n s e q u e n c e o f the a b n o r m a l i t i e s o f ciliary u l t r a s t r u c t u r e (such as m i c r o t u b u l a r a b n o r m a l i t i e s a n d c o m p o t t n d cilia) but that infection, i n f l a m m a t i o n , a n d r e g e n e r a t i o n o f the mucosa give rise to these structural abnorntalities.

1 3

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Acknowledgments. The authors thank Professor R. C. Curran for advice and encouragement, and Dr. J. M. Hopkin for obtaining the bronchial biopsy specimens and brushings.

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REFERENCES

!. Kartagener M: Zur Pathogenese der Bronchiektasien. I: Bronchiektasien bei situs viscerum inversus. Beitr Kiln Tuberk 83:489, 1933 2. Canmer P, Mossberg B, Afzelius BA: Evidence of congenital non-functioning cilia in tile tracheobronchial tract of two subjects. Am Rev Respir Dis 112:807, 1975 3. Pedersen M, Morkassel E, Nielsen MH, et al: Kartagener's syndrome. Preliminary report on cilia structure function and upper airway symptoms. Chest 80(suppl):858, 1981 4. Rossman CM, Forrest JM Lee FMFW, et al: The dyskinetic cilia syndrome. Abnormal ciliary motility in association with abnormal nhrastructure. Chest 80(suppl):860, 1981 5. Eliasson R, Mossberg B, Canmer P, et al: The immotile-cilia syndrome. A congenital abnormality as an aetiologic factor in chronic airway infections and male sterility. N EnglJ Med 297:!, 1977 6. Sleigh MA: Primary ciliary dyskinesia. Lancet 2:476, 1981 7. Pedersen H, Rebbe H: Absence of arlns in tile axoneme of immotile human spermatozoa. Biol Reprod 12:541, 1975 8. Afzelius BA, Eliasson R, Johnsen O, et al: Lack ofdynein arms in immotile human spermatozoa. J Cell Biol 66:225, 1975 9. Pedersen H, Mygind N: Absence of axonemal arms in nasal mucosal cilia in Kartagener's syndrome. Nature 262:494, 1976 10. Sturgess JM, Chao J, Wong J, et ah Cilia with defective radial spokes. A cause of lnmaan respiratory disease. N EnglJ Med 300:53, 1979 11. SturgessJM, ChaoJ, TurnerJAP: Transposition of ciliary nilcrotubules. Another cause of impaired ciliary motility. N EnglJ Med 303:318, 1980 12. tterzon FS, Murphy S: Normal ciliary uhrastructure in children with Kartagener's syndrome. Ann Otol Rhinol Laryngol 89:81, 1980 13. Schidlow DV, Katz SM: Immotile cilia syndrome. N Engl J Med 308:595, 1982 14. Greenstone MA, Dewar A, Cole PJ: Ciliary dyskinesia with normal ultrastru6ture. Thorax 38:875, 1983

d e a t h a n d r e p o r t e d the incidence o f m i c r o t u b u l a r abnormalities as 2 to 4 p e r cent a n d that o f c o m p o u n d cilia as 1.6 to 3.2 p e r cent. Wilsman et al. 31 studied c i r c u l a r cuffs o f m u c o s a f r o m the tracltea o f d o g s a n d f o u n d incidences o f m i c r o t u b u l a r abnormalities a n d c o m p o u n d cilia o f 2.2 a n d 0.4 p e r cent, respectively. A l t h o u g h o u r c o n t r o l s , which c o n s i s t e d o f nasal s p e c i m e n s f r o m n o r m a l subjects, are not strictly c o m p a r a b l e to bronchial specimens, the incidence o f ultrastructural abnormalities in t h e m is similar to the f i g u r e s t h a t we a n d o t h e r s ( W i s s e m a n et al. a n d Wilsman et al.) o b t a i n e d f o r bronchial mucosa. A1thoug.h the incidence o f c o m p o u n d cilia was 1.81 p e r cent i n b r o n c h i a l s p e c i m e n s a n d 0.27 p e r cent in nasal specimens, this d i f f e r e n c e was not statistically significant ( M a n n - W h i t n e y U-test). M o r e o v e r , within this c o n t r o l g r o u p small n u m b e r s o f cilia s h o w e d transposition o f ciliary m i c r o t u b u l e s and the radial s p o k e defect, a n d visnalization o f the i n n e r d y n e i n a r m s was difficult. It is clear f r o m the resuhs o f this study that ultrastructural abnormalities o f cilia front patients with p r i m a r y ciliary dyskinesia a n d front control subjects m u s t be carefully quantified b e f o r e the abnormalities can be a s s u m e d to h a v e p a t h o l o g i c significance. A diagnosis o f p r i m a r y ciliary dyskinesia can be m a d e only a f t e r t h r e e factors h a v e b e e n considered: 1) his854

CILIARYULTRASTRUCTURE[Smallman& Gregory) 15. Warner FD: Cation-induced attachment of ciliary dynein cross bridges. J Cell Biol 77:R19, 1978 16. Rutland J, Dewar A, Cox T, et al: Nasal brushing for the stud) of ciliary nhrastructure. J Clin Pathol 35:357, 1982 17. Reynolds ES: The nse of lead citrate at high pH as an electroq opaque stain for electron microscop). J Cell Biol 17:208, 1983 18. Fasca JM, Parks VR: A routine teclmiqne for double-staining ultrathin sections using uranyl and lead salts. J Cell Biol 65:157, 1965 19. Fawcett DW, Porter KR: A stud)" of the fine structnre of ciliated epithelia. J Morphol 94:221, 1954 20. Ailsb)" RL, Ghadiall)' FN: Atypical cilia in human bronchial mucosa. J Pathol 109:75, 1973 21. Torikarta C, Takeuchi H, Yamaguchi tt, et al: Abnormal cilia in the bronchial mucosa. Case reports of non-smoking women with bronchogenic carcinoma and an experimental model in guinea pigs. Virchows Arch [A] 371:121, 1976 22. McDowell EM, Barrett LA, ttarris CC, et al: Abnormal cilia in h u m a n bronchial epithelium. Arch Pathol Lab Med 100:429, 1976 23. Fox B, Bull TB, Make)' AR, et al: The significance of ult r a s t r u c t u r a l a b n o r m a l i t i e s o f h u m a n cilia. C h e s t 80(suppl):796, 1981 24. Fox B, Btdl TB, Oliver TN: The distribution and assessment

25. 26. 27. 28.

29. 30. 31. 32. 33.

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