Pediatric Pulmonology 14:201-205 (1992)
Original Articles -
Cilia in Children With Recurrent Upper Respiratory Tract Infections: Ultrastructural Observations Pier Luigi Giorgi, M D , ~Nicola Oggiano, MD,’ Pier Carlo Braga, phD,* Carlo Catassi, M D , ~ Orazio Gabrielli, MD,’ Giovanni Valentino Coppa, MD,‘ and Ahmad Kantar, MD’ Summary. We investigated the ultrastructureof nasal cilia in 27 children suffering from recurrent infections of the upper respiratory tract, during and after the onset of an acute respiratory infection, and after a convalescent period of 12 weeks. Our results demonstrated that in seven subjects after resolution of infection, the morphologyof a large proportionof the cilia (32%) was not back to normal. These findings suggest a long-term residual effect of infection, or the inability to reestablish normal ciliary structure during the convalescent period in some subjects with recurrent upper respiratory tract infection. Pediatr Pulmonol. 1992; 14:201-205. 0 1992 Wiley-Liss. Inc.
Key words: Ciliary motion analysis; electron microscopy; transmission electron microscopy; microbiology; acute, convalescent, post-recovery tests.
INTRODUCTION Purkinje and Valentin in the last century were the first to discover ciliary motion on the epithelia of the respiratory tract and the reproductive system. I For the last decades respiratory cilia have been of prime interest to investigators. Studies of the morphology, physiology, and biochemistry of the ciliaz4 laid the foundation for our present knowledge on the subject. Although these investigations were of general interest, they remained remote from medical attention until 1976, when Afzelius identified an ultrastructural lesion of cilia in patients with Kartagener’s syndrome.‘ Over the last 15 years a great deal of work has been focused on the study of cilia in relation to respiratory diseases including primary ciliary dyskinesia, cystic fibrosis, asthma, and recurrent respiratory infections.”” The mucociliary system of the respiratory tract constitutes a defense barrier that must be overcome by potential pathogens during their invasion. Alterations in ciliary function cause reduction in the mucociliary defense mechanism and predispose the respiratory tract to insults. Abnormalities of ciliary motility may be primary (primary ciliary dyskinesia) or secondary (induced by infection or injury). Recurrent upper respiratory infections (URI) in early childhood are common clinical problems facing pediatricians during the winter months. The majority of children with recurrent URI have no defects in 0 1992 Wiley-Liss, Inc.
host defense or detectable abnormalities predisposing to infection. l 3 In an attempt to determine the role of respiratory cilia in children with recurrent URI, we have evaluated the ultrastructure of the nasal ciliary apparatus in children at various stages of their illness. MATERIALS AND METHODS
Patients We studied 27 children with recurrent infection of the upper respiratory tract, all with more than six episodes annually. The study group consisted of 10 females and 17 males, between 2 and 6.2 years of age (mean, 3.2 & 0.6 years). No evidence of congenital respiratory or immunological abnormalities was found in these children, based on laboratory studies of the humoral and cellular defenses, phagocytic system, sweat electrolyte concentrations, and serum alpha- 1-antitrypsin concentration, as From the Pediatric Clinic, University of Ancona, Ancona‘ and the Department of Pharmacology, University of Milan, Milan,? Italy.
Receivcd July 3, 1990; (revision) accepted for publication June 16, 1992. Address correspondence and reprint requests to Dr. P.L. Ciorgi, Pediatric Clinic, University of Ancona, via Corridoni I 1.1-601 23 Ancona, Italy.
202
Giorgi et al.
previously described. l4 Other pathological conditions such as allergy, asthma, and chronic gastroesophageal reflux were also excluded. A control group consisting of 20 healthy children, 7 females and 13 males, between 3 and 6.4 years old (mean, 3.8 ? 0.4 years) was selected from patients attending our clinic because of short stature. None of the children in the control group had a family or personal history of a chronic respiratory disease or had suffered from acute respiratory disease during the previous 3 months. Informed consent was obtained from the parents. Methods After having cleaned excess secretions from the nose and washed the turbinates with sterile isotonic saline solution, the nasal passage was examined, using an otoscope, to be sure it was clear of mucus. Tissue samples of ciliated epithelium were obtained from at least three distinct sites of the inferior turbinate and septum on each side by gentle brushing, as described by Rutland and Cole. Is A cytology brush (Microvasive, Milford, MA) was used. Cellular material adhering to the brush was dislodged by brisk agitation in a sterile tube containing 2 mL isotonic saline solution for ultrastructural evaluation and in a second sterile tube containing 2 mL of Medium 199 cell culture fluid (Flow Laboratories) for ciliary motion analysis and counting of beat frequency. The sample for ultrastructural study was immediately fixed after centrifugation in 2.5% glutaraldehyde in cacodylate buffer. Initial evaluation of the specimens was done by light microscopy of histological sections of the Epo-embedded material stained with Richardson’s blue stain. Ultrathin sections of the same sample were cut subsequently on an ultramicrotome (111 LKB) and later colored with lead citrate. These sections were studied with an electron microscope (Philips 301) and transmission electron microscope (Zeiss 902). In each sample a minimum of 150 clearly visualized cross sections of cilia were randomly selected and analyzed for each subject. Samples obtained for ciliary motion analysis were kept at 37”C, and transferred to a variable thickness culture chamber as previously described. l6 Cilia were observed under an optic microscope (Diaplan Leitz; Leica, MilanItaly) equipped with a Nomarsky differential interference contrast set and with a magnification of X 100 as previously described.” The microscope was connected to a high-resolution video camera head, equipped with a solid-state charge-coupled device and 6: 1 zoom power (JVC TK-870 E). The video camera was connected to a video recorder with a zoom power of 4:l (Sanyo VHRD56701) and a TV monitor (JVC TM-15OPSN). This system offers the possibility of observing the cilia on the TV monitor screen with a final magnification of X2,400. Ciliary motion was recorded to evaluate the motion pat-
TABLE 1-Infectious Agents Cultured During the Acute Episode of Infection No.
Agent Influenza virus, types A and B Respiratary syncytial virus Rhinovirus Parainfluenza virus I . 2. 3 Hacmophilus irrjluenza rhinovirus Sfuphylococcus aureus + influenza virus
+
tern and beat frequency, using a slow speed technique. In each sample an average of 50 individual cells or groups of cells was observed and mean ciliary beat frequency was calculated. Study Design
Samples of nasal epithelium of each patient were studied on threc separate occasions: first during the presentation of acutc URI symptoms (fever, nasal discharge, pharyngeal erythema, or cough); second, approximately 3 weeks after recovery from the acute episode; and third, after the summer season. During convalescence, all subjects were free of respiratory tract infection symptoms for at least 12 weeks. On each occasion microbiological studies were done by standard hospital laboratory techniques. The control group was sampled on two occasions. RESULTS
Analysis of ciliary motion and beat frequency counting demonstrated that in the control group no samples were without actively beating cilia. Ciliary motion was synchronous, coordinated, and metachronal. The mean k SD beat frequency was 12 % 1.6 Hz. Electron microscopic studies revealed normal ciliary ultrastructure in 94% of the examined cilia: a 9 2 microtubular pattern with prominent dynein arms and radial spokes and with the central pairs oriented in the same direction. In 6% of the cilia examined ultrastructural abnormalities were observed: multiple cilia (two or more axonemes enclosed by one cell membrane) and central or peripheral microtubular abnormalities (absence or addition of single microtubule) with a prevalence of peripheral defects and central translocation of peripheral cilia. The etiologic agents cultured from the studied group during the presentation of symptoms of acute URI are shown in Table 1. Evaluation of ciliary motion analysis and counting of nasal ciliary beat frequency during the acute infectious episode demonstrated reduced ciliary beat frequency compared to the control group; thc mean beat frequency was 7.8 & 1.8 Hz. Ciliary motion was synchronous, coordinated, and metachronous in 70% of the examined
+
Ciliary Structure in Recurrent Infections
cilia, whereas areas of uncoordinated and asynchronous motion, mainly on single cells, were observed in 30%of the examined cilia. Ultrastructural studies revealed that 30.8% of the cilia analyzed were abnormal; the main abnormalities were: multiple cilia, swollen cilia, peripheral and central microtubular abnormalities (absence or addition of single or multiple microtubules or doublets) with a prevalence of central abnormalities, and the presence of some microtubules of diverse axonemes lacking inner or outer dynein arms. Ciliary motion analysis and beat frequency counting in samples obtained 3 weeks after the onset of the acute episode demonstrated a normal ciliary motion in 84% of the observed cilia and a mean beat frequency of 9.6 1.2 Hz. By ultrastructural observations 39.0%of the examined cilia showed abnormalities such as multiple cilia, swollen cilia (a significant increase over the acute phase), peripheral and central microtubular abnormalities (absence of single or multiple microtubules or doublets), prevalence of peripheral abnormalities, and reduction of the number of microtubular misarrangements relative to the acute phase. Samples obtained on the third occasion (subjects free of symptoms of URI for at least 12 weeks) demonstrated that 20 subjects (9 females, 1 1 males) had normal ciliary motion and beat frequency and 10% of their cilia had abnormalities similar to those observed in the second sample. The cilia of the remaining seven subjects had reduced mean beat frequency of 10.8 & 0.6 Hz, and in 23% of the studied cilia uncoordinated and asynchronous motion was observed. Ultrastructurally32.0%of the cilia examined were abnormal. Compared with the preceding sample, multiple cilia and disarranged microtubules were reduced. Moreover, swollen cells were not present in these samples. However, we noticed the persistence of ultrastructural abnormalities characteristic of chronic infection: absence of single or multiple microtubules (central and/or peripheral) and some aspects of congenital alterations (absence and/or shortening of inner or outer dynein arms, dislocation of the central doublet, central translocation of a peripheral doublet, and absence of the radial spokes). Table 2 shows ultrastructural findings in the group of seven on the three sampling occasions. Table 3 gives the clinical and microbiological data for the seven subjects during the acute episode of infection. Figure 1 and 2 show the ultrastructure of some cilia examined.
*
203
TABLE 2-Occurrence of Ultrastructural Abnormalities (%) of the Nasal Epithelium in Seven Subjects at Three Occasions of SamDlina Sampling
I
11
111
9.4
10.8
~
Mean beat frequency (Hz) Abnormalities Abnormal cilia Multiple cilia Swollen cilia Extraperipheral microtubules Missing peripheral microtubules Extracentral microtubules Missing central micmtubules Translocated central microtubules Translocated peripheral microtubules Axoneme without innerlouter dynein arms Axoneme with short dynein Outer doublets misarranged Absence of radial sookes
7.6
32 I 0 0 8
36 4 3 1 3
40 8 6
6
2
7
1
0 4
3
I
8
1
9
6
4
3
8
1
2
4
4 0
I
2
0 4
5 8
TABLE 3-Clinical and Microbiological Description of Seven Subjects During the Acute Episode of Infection Patient no. 1
2 3 4
5 6
I
Clinical and microbiological findings Fever, nasal discharge, cough, and pharyngitis; influenza virus types A and B Fever, rhinorrhea, and sore throat; parainfluenza virus I , 2, 3 Fever, rhinorrhea, and sore throat; parainfluenza virus I , 2, 3 Fever, rhinorrhea, and pharyngitis; parainfluenza virus I , 2. 3 Fever, rhinorrhea, and otitis media; parainfluenza virus 1 , 2, 3 Fever, cough, pharyngitis, and cervical adenitis; Sraphylococcus aureus + influenza virus Fever, vomiting, myalgia, and pharyngitis; Sraphylococcus aureus + influenza virus
induced by such agents include damage to the ciliary membrane and to the internal structure. The activation of phagocytic cells is accompanied by the release of potentially toxic products such as proteinase, elastase, and oxygen metabolite^,^^ which contribute to cilia inMoreover, it has been demonstrated that several mediators involved in the inflammatory process can DISCUSSION influence mucociliary function, such as leukotrienes and A wide spectrum of ciliary defects and concomitant platelet-activating f a ~ t o r . ~ ' -Thus, ~' the cilia are caught mucociliary dysfunction has been documented in URI in an unfortunate cycle of microbial pathogens as well as caused by various infectious pathogens. Microbial agents toxic products released by the defense mechanism. and their products have been found to induce structural Return to normal ciliary structure may be essential for and functional ciliary defects. Ultrastructural changes normal mucociliary clearance after an insult. The process
204
Giorgi et al.
Fig. 2. Electron micrograph of transverse sections of multiple cilia. Magnlflcation, ~60,000.
9 [hl&OBIAL
/ - - - - - - - -
I
I
]*I
C- P
f
i
A
E
' :I
PATHOGENIC AGENTS]
I
] C-
I
INFLAMMATION
DEFENSE MECHANISM
Fig. 1. Electron micrographs of transverse sections of cilia in patients with recurrent URI. (Magnification, x 120,000). a: Normal cilium showing the 9 + 2 microtubule pattern, inner and outer dynein arms, and central spokes. b: Missing single peripheral microtubules and missing dynein arms. c: Two extra microtubules inside 9 + 2 arrays. d: Extra single central microtubule. e: Extra central mlcrotubule doublets inside 9 + 2 array and extra single peripheral microtubule. f: Mlsslng single peripheral microtubule doublets. g: Swollen cilia.
Fig. 3. Schematic representation of the presumed association among infection, ciliogenesis, and the mucociliary defense mechanism.
subjects, although there was no difference in the etiology of the initial infection between this group and the group of 20. These alterations may be viewed as long-term residual effects of infection. The mechanisms leading to the persistence of ciliary abnormalities may be similar to those proposed by Carson et al.''9'9 in acute viral upper respiratory tract infections, in which conditions for producing errors in ciliogenesis exist. The data obtained demonstrate that some time after of ciliogenesis in the normal nasal epithelium is continuclinical resolution of infection there is still evidence that a ous and rdpid.32 Various congenital and environmental large portion of cilia has not returned to normal morpholfactors have been shown to influence the process of ciliog e n e ~ i s . ~Altered ~ - ~ ~ ciliogenesis following continuous ogy in some children with URI. It is not known whether insults can lead to additional impairment of the mucocili- the reported abnormalities have an effect on mucociliary ary defense mechanism with consequent increase in se- defense mechanism. The role of these ciliary abnormaliverity and chronicity of the infection. This situation may ties in predisposing to recurrent infection needs to be investigated. Further studies are in progress to assess the create a vicious circle (Fig. 3). In our study group we observed the persistence of mucociliary transport system in children with recurrent ciliary abnormalities after the convalescent period in 7 URI.
Ciliary Structure in Recurrent Infections
ACKNOWLEDGMENTS
We are indebted to Prof. S. Cinti of the Institute of Human Morphology, University of Ancona, for giving us access to electron microscopy facilities.
18. Chandrer DKF, Barile MF. Ciliostatic, hemagglutinating and pro-
19. 20.
REFERENCES I . Purkinje JE, Valentin GG. Entdeckung continuerlicher durch wimperhaare einzeugter flimmer der bewegungen, als eines allgemeine phanomens in der klassen der amphibien, Vogel und Saugethiere. Mullers Arch Anat Physiol. 1834391400. 2. Warner FD. The fine structure of the ciliary and flagellar axoneme. In: Sleigh MA, ed. Cilia and Flagella. London: Academic Press, 1974:ll-37. 3. Iravani J. Physiologie und pathophysiologie der cilientatigkeit und des schleimtransportes in tracheobronchialbum (Untersuchungen an ratten). Pneumonologie. 1971; 144:93-112. 4. Gibbons IR. Chemical dissection of cilia. Arch Biol (Liege). 1965; 76~317-352. 5 . Afzelius BA. A human syndrome caused by immotile cilia. Science. 1976; 193:317-319. 6. Wanner A. Alteration of tracheal mucociliary transport in airway disease. Effect of pharmacologic agents. Chest. 1981; 80(Suppl):867-870. 7. Chao J, Turner JAP, Sturgess JM. Genetic heterogeneity of dynein-deficiency in cilia from patients with respiratory disease. Am Rev Respir Dis. 1982; 126:302-305. 8. Rossman CM, Lee RMKW, Forrest JB, Newhouse MT. Nasal ciliary ultrastructure and function in patients with primary ciliary dyskinesia compared with that in normal subjects and in subjects with various respiratory disease. Am Rev Respir Dis. 1984; 129:161-167. 9. Pysher TJ, Neustein HB. Ciliary dysmorphology. Perspect Pediatr Pathol. 1984; 8:101-131. 10. Rossman CM, Newhouse MT. Primary ciliary dyskinesia: Evaluation and management. Pediatr Pulmonol. 1988; 5:36-50. 1 1 . Carson JL, Collier AM. Ciliary defects: Cell biology and clinical perspectives. Adv Pediatr. 1988; 35:139-166. 12. Buchdal RM, Reiser J, Ingram D, Rutman A, Cole PJ, Warner JO. Ciliary abnormalities in respiratory disease. Arch Dis Child. 1988; 63238-243. 13. Rubin BK. The evaluation of the child with recurrent chest infection. Pediatr Infect Dis J. 1985; 4:88-98. 14. Kantar A, Oggiano N, Romagnoni GG, Giorgi PL. Effect of oral administration of bacterial extracts on the bactericidal capacity of polymorphonuclear leukocytes in children with recurrent respiratory infections. J Intern Med Res. 1991; 19:451456. 15. Rutland J, Cole PJ. Non-invasive sampling of nasal cilia for measurement of beat frequency and study of ultrastructure. Lancet. 1980; 11564-565. 16. Braga PC.A variable-thickness, multipurpose culture chamber for high-magnification observation. J Microsc. 1990; 159:285-288. 17. Braga PC,Bossi R, Allerga L. A method for maintaining, in vitro, TV-monitoring and counting ciliary beat frequency of samples from human ciliated respiratory epithelium brushing. Methods Find Exp Clin Pharmacol. 1986; 8:321-326.
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21. 22.
23. 24. 25. 26. 27.
28.
29. 30.
31.
32.
33. 34. 35.
teolytic activity in a cell extract of Mycoplasma pneumonia. Infect Immun. 1980; 29:1111-1116. Carson JL, Collier AM, Hu SS. Acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections. N Engl J Med. 1985; 312:463468. Wilson R, Roberts D, Cole P. Effect of bacterial products on human ciliary function in vitro. Thorax. 1985; 40:125-131. Hingley ST, Hastie AT, Kueppers F, Higgins ML. Disruption of respiratory cilia by proteases including those of Pseudoinonus aeruginosa. Infect Immun. 1986; 54:379-385. Luzzatto E, Kopernik G, Sarvo I, Priel Z. Effect of Chlamydia frachomurisinfection on ciliary activity in single cells from cultures of human nasal polyps. J Gen Microbiol. 1989; 135:105109. Thomas EL, Lehrer RI,Rest RF. Human neutrophil antimicrobial activity. Rev Infect Dis. 1988; lO(Suppl):45&456. Tegner H, Ohlsson K, Toremalm NG, Mecklenburg C. Effect of human leukocyte enzymes on tracheal mucosa and its mucociliary activity. Rhinology. 1979; 17:199-206. Smallman LA, Hill SL, Stockely RA. Reduction of ciliary beat frequency in vitro by sputum from patients with bronchiectasis: A serine proteinase effect. Thorax. 1984; 39:663-667. Hasite AT, Loegering DA, Gleich GJ, Kueppers F. The effect of purified human eosinophil major protein on mammalian ciliary activity. Am Rev Respir Dis. 1987; 135:848-853. Sykes DA, Wilson R, Greenstone M, Cume DC, Steinfort C, Cole PJ. Deleterious effects of purulent sputum sol on human ciliary function in vitro: At least two factors identified. Thorax. 1987; 42256-261. Bisgaard H, Pedersen M. SRS-a leukotrienes decreaqe the activity of human respiratory cilia. Clin Allergy. 1987; 17:95-103. Seybold ZV, Mariassy AT, Stroh S, Kim CS, Cazeroglu H, Wanner A. Mucociliary interaction in vitro: Effects of physiological and inflammatory stimuli. J Appl Physiol. 1990,68:1421-1426. Weisman Z, Fink A, Alon A, Poliak Z, Tabachnik E, Priscu L, Bentwich Z. Leukotriene C, decreases the activity of respiratory cilia in vitro. Clin Exp Allergy. 1990; 20:389-393. Nieminen MM, Moilanen EK, Nyholm JEJ, Koskinen MO, Karvonen JI, Metsa-Ketela TJ, Vapaatalo H. Platelet-activating factor impairs mucociliary transport and increases plasma leukotriene 9, in man. Eur Respir J. 1991; 4551-560. Carson JL, Collier AM, Knowles MR, Boucher RC, Rose JG. Morphometric aspects of ciliary distribution and ciliogenesis in human nasal epithelium. Proc Natl Acad Sci USA. 1981; 78:6996-6999. Richard S,Nezelof C, Pfister A, de Blic J. Scheinmann P, Paupe J. Congenital ciliary apalasia in two siblings. A primitive disregulation of ciliogenesis? Pathol Res Pract. 1989; 185: 18 1-1 83. Stockinger L, Sellner W, Ellinger A, Hofler H. Pathophysiology of the ciliated epithelium of the respiratory mucosa in humans. Disorders of ciliogenesis. Exp Lung Res. 1989; 15:925-941. Kawakami M, Yasui S, Yamawaki I, Katayama M, Nagai A, Takizawa T. Structural changes in airways of rats exposed to nitrogen dioxide intermittently for 7 days. Comparison between major bronchi and terminal bronchioles. Am Rev Respir Dis. 1989; 140:1754-1762.
Original Articles -
Cilia in Children With Recurrent Upper Respiratory Tract Infections: Ultrastructural Observations Pier Luigi Giorgi, M D , ~Nicola Oggiano, MD,’ Pier Carlo Braga, phD,* Carlo Catassi, M D , ~ Orazio Gabrielli, MD,’ Giovanni Valentino Coppa, MD,‘ and Ahmad Kantar, MD’ Summary. We investigated the ultrastructureof nasal cilia in 27 children suffering from recurrent infections of the upper respiratory tract, during and after the onset of an acute respiratory infection, and after a convalescent period of 12 weeks. Our results demonstrated that in seven subjects after resolution of infection, the morphologyof a large proportionof the cilia (32%) was not back to normal. These findings suggest a long-term residual effect of infection, or the inability to reestablish normal ciliary structure during the convalescent period in some subjects with recurrent upper respiratory tract infection. Pediatr Pulmonol. 1992; 14:201-205. 0 1992 Wiley-Liss. Inc.
Key words: Ciliary motion analysis; electron microscopy; transmission electron microscopy; microbiology; acute, convalescent, post-recovery tests.
INTRODUCTION Purkinje and Valentin in the last century were the first to discover ciliary motion on the epithelia of the respiratory tract and the reproductive system. I For the last decades respiratory cilia have been of prime interest to investigators. Studies of the morphology, physiology, and biochemistry of the ciliaz4 laid the foundation for our present knowledge on the subject. Although these investigations were of general interest, they remained remote from medical attention until 1976, when Afzelius identified an ultrastructural lesion of cilia in patients with Kartagener’s syndrome.‘ Over the last 15 years a great deal of work has been focused on the study of cilia in relation to respiratory diseases including primary ciliary dyskinesia, cystic fibrosis, asthma, and recurrent respiratory infections.”” The mucociliary system of the respiratory tract constitutes a defense barrier that must be overcome by potential pathogens during their invasion. Alterations in ciliary function cause reduction in the mucociliary defense mechanism and predispose the respiratory tract to insults. Abnormalities of ciliary motility may be primary (primary ciliary dyskinesia) or secondary (induced by infection or injury). Recurrent upper respiratory infections (URI) in early childhood are common clinical problems facing pediatricians during the winter months. The majority of children with recurrent URI have no defects in 0 1992 Wiley-Liss, Inc.
host defense or detectable abnormalities predisposing to infection. l 3 In an attempt to determine the role of respiratory cilia in children with recurrent URI, we have evaluated the ultrastructure of the nasal ciliary apparatus in children at various stages of their illness. MATERIALS AND METHODS
Patients We studied 27 children with recurrent infection of the upper respiratory tract, all with more than six episodes annually. The study group consisted of 10 females and 17 males, between 2 and 6.2 years of age (mean, 3.2 & 0.6 years). No evidence of congenital respiratory or immunological abnormalities was found in these children, based on laboratory studies of the humoral and cellular defenses, phagocytic system, sweat electrolyte concentrations, and serum alpha- 1-antitrypsin concentration, as From the Pediatric Clinic, University of Ancona, Ancona‘ and the Department of Pharmacology, University of Milan, Milan,? Italy.
Receivcd July 3, 1990; (revision) accepted for publication June 16, 1992. Address correspondence and reprint requests to Dr. P.L. Ciorgi, Pediatric Clinic, University of Ancona, via Corridoni I 1.1-601 23 Ancona, Italy.
202
Giorgi et al.
previously described. l4 Other pathological conditions such as allergy, asthma, and chronic gastroesophageal reflux were also excluded. A control group consisting of 20 healthy children, 7 females and 13 males, between 3 and 6.4 years old (mean, 3.8 ? 0.4 years) was selected from patients attending our clinic because of short stature. None of the children in the control group had a family or personal history of a chronic respiratory disease or had suffered from acute respiratory disease during the previous 3 months. Informed consent was obtained from the parents. Methods After having cleaned excess secretions from the nose and washed the turbinates with sterile isotonic saline solution, the nasal passage was examined, using an otoscope, to be sure it was clear of mucus. Tissue samples of ciliated epithelium were obtained from at least three distinct sites of the inferior turbinate and septum on each side by gentle brushing, as described by Rutland and Cole. Is A cytology brush (Microvasive, Milford, MA) was used. Cellular material adhering to the brush was dislodged by brisk agitation in a sterile tube containing 2 mL isotonic saline solution for ultrastructural evaluation and in a second sterile tube containing 2 mL of Medium 199 cell culture fluid (Flow Laboratories) for ciliary motion analysis and counting of beat frequency. The sample for ultrastructural study was immediately fixed after centrifugation in 2.5% glutaraldehyde in cacodylate buffer. Initial evaluation of the specimens was done by light microscopy of histological sections of the Epo-embedded material stained with Richardson’s blue stain. Ultrathin sections of the same sample were cut subsequently on an ultramicrotome (111 LKB) and later colored with lead citrate. These sections were studied with an electron microscope (Philips 301) and transmission electron microscope (Zeiss 902). In each sample a minimum of 150 clearly visualized cross sections of cilia were randomly selected and analyzed for each subject. Samples obtained for ciliary motion analysis were kept at 37”C, and transferred to a variable thickness culture chamber as previously described. l6 Cilia were observed under an optic microscope (Diaplan Leitz; Leica, MilanItaly) equipped with a Nomarsky differential interference contrast set and with a magnification of X 100 as previously described.” The microscope was connected to a high-resolution video camera head, equipped with a solid-state charge-coupled device and 6: 1 zoom power (JVC TK-870 E). The video camera was connected to a video recorder with a zoom power of 4:l (Sanyo VHRD56701) and a TV monitor (JVC TM-15OPSN). This system offers the possibility of observing the cilia on the TV monitor screen with a final magnification of X2,400. Ciliary motion was recorded to evaluate the motion pat-
TABLE 1-Infectious Agents Cultured During the Acute Episode of Infection No.
Agent Influenza virus, types A and B Respiratary syncytial virus Rhinovirus Parainfluenza virus I . 2. 3 Hacmophilus irrjluenza rhinovirus Sfuphylococcus aureus + influenza virus
+
tern and beat frequency, using a slow speed technique. In each sample an average of 50 individual cells or groups of cells was observed and mean ciliary beat frequency was calculated. Study Design
Samples of nasal epithelium of each patient were studied on threc separate occasions: first during the presentation of acutc URI symptoms (fever, nasal discharge, pharyngeal erythema, or cough); second, approximately 3 weeks after recovery from the acute episode; and third, after the summer season. During convalescence, all subjects were free of respiratory tract infection symptoms for at least 12 weeks. On each occasion microbiological studies were done by standard hospital laboratory techniques. The control group was sampled on two occasions. RESULTS
Analysis of ciliary motion and beat frequency counting demonstrated that in the control group no samples were without actively beating cilia. Ciliary motion was synchronous, coordinated, and metachronal. The mean k SD beat frequency was 12 % 1.6 Hz. Electron microscopic studies revealed normal ciliary ultrastructure in 94% of the examined cilia: a 9 2 microtubular pattern with prominent dynein arms and radial spokes and with the central pairs oriented in the same direction. In 6% of the cilia examined ultrastructural abnormalities were observed: multiple cilia (two or more axonemes enclosed by one cell membrane) and central or peripheral microtubular abnormalities (absence or addition of single microtubule) with a prevalence of peripheral defects and central translocation of peripheral cilia. The etiologic agents cultured from the studied group during the presentation of symptoms of acute URI are shown in Table 1. Evaluation of ciliary motion analysis and counting of nasal ciliary beat frequency during the acute infectious episode demonstrated reduced ciliary beat frequency compared to the control group; thc mean beat frequency was 7.8 & 1.8 Hz. Ciliary motion was synchronous, coordinated, and metachronous in 70% of the examined
+
Ciliary Structure in Recurrent Infections
cilia, whereas areas of uncoordinated and asynchronous motion, mainly on single cells, were observed in 30%of the examined cilia. Ultrastructural studies revealed that 30.8% of the cilia analyzed were abnormal; the main abnormalities were: multiple cilia, swollen cilia, peripheral and central microtubular abnormalities (absence or addition of single or multiple microtubules or doublets) with a prevalence of central abnormalities, and the presence of some microtubules of diverse axonemes lacking inner or outer dynein arms. Ciliary motion analysis and beat frequency counting in samples obtained 3 weeks after the onset of the acute episode demonstrated a normal ciliary motion in 84% of the observed cilia and a mean beat frequency of 9.6 1.2 Hz. By ultrastructural observations 39.0%of the examined cilia showed abnormalities such as multiple cilia, swollen cilia (a significant increase over the acute phase), peripheral and central microtubular abnormalities (absence of single or multiple microtubules or doublets), prevalence of peripheral abnormalities, and reduction of the number of microtubular misarrangements relative to the acute phase. Samples obtained on the third occasion (subjects free of symptoms of URI for at least 12 weeks) demonstrated that 20 subjects (9 females, 1 1 males) had normal ciliary motion and beat frequency and 10% of their cilia had abnormalities similar to those observed in the second sample. The cilia of the remaining seven subjects had reduced mean beat frequency of 10.8 & 0.6 Hz, and in 23% of the studied cilia uncoordinated and asynchronous motion was observed. Ultrastructurally32.0%of the cilia examined were abnormal. Compared with the preceding sample, multiple cilia and disarranged microtubules were reduced. Moreover, swollen cells were not present in these samples. However, we noticed the persistence of ultrastructural abnormalities characteristic of chronic infection: absence of single or multiple microtubules (central and/or peripheral) and some aspects of congenital alterations (absence and/or shortening of inner or outer dynein arms, dislocation of the central doublet, central translocation of a peripheral doublet, and absence of the radial spokes). Table 2 shows ultrastructural findings in the group of seven on the three sampling occasions. Table 3 gives the clinical and microbiological data for the seven subjects during the acute episode of infection. Figure 1 and 2 show the ultrastructure of some cilia examined.
*
203
TABLE 2-Occurrence of Ultrastructural Abnormalities (%) of the Nasal Epithelium in Seven Subjects at Three Occasions of SamDlina Sampling
I
11
111
9.4
10.8
~
Mean beat frequency (Hz) Abnormalities Abnormal cilia Multiple cilia Swollen cilia Extraperipheral microtubules Missing peripheral microtubules Extracentral microtubules Missing central micmtubules Translocated central microtubules Translocated peripheral microtubules Axoneme without innerlouter dynein arms Axoneme with short dynein Outer doublets misarranged Absence of radial sookes
7.6
32 I 0 0 8
36 4 3 1 3
40 8 6
6
2
7
1
0 4
3
I
8
1
9
6
4
3
8
1
2
4
4 0
I
2
0 4
5 8
TABLE 3-Clinical and Microbiological Description of Seven Subjects During the Acute Episode of Infection Patient no. 1
2 3 4
5 6
I
Clinical and microbiological findings Fever, nasal discharge, cough, and pharyngitis; influenza virus types A and B Fever, rhinorrhea, and sore throat; parainfluenza virus I , 2, 3 Fever, rhinorrhea, and sore throat; parainfluenza virus I , 2, 3 Fever, rhinorrhea, and pharyngitis; parainfluenza virus I , 2. 3 Fever, rhinorrhea, and otitis media; parainfluenza virus 1 , 2, 3 Fever, cough, pharyngitis, and cervical adenitis; Sraphylococcus aureus + influenza virus Fever, vomiting, myalgia, and pharyngitis; Sraphylococcus aureus + influenza virus
induced by such agents include damage to the ciliary membrane and to the internal structure. The activation of phagocytic cells is accompanied by the release of potentially toxic products such as proteinase, elastase, and oxygen metabolite^,^^ which contribute to cilia inMoreover, it has been demonstrated that several mediators involved in the inflammatory process can DISCUSSION influence mucociliary function, such as leukotrienes and A wide spectrum of ciliary defects and concomitant platelet-activating f a ~ t o r . ~ ' -Thus, ~' the cilia are caught mucociliary dysfunction has been documented in URI in an unfortunate cycle of microbial pathogens as well as caused by various infectious pathogens. Microbial agents toxic products released by the defense mechanism. and their products have been found to induce structural Return to normal ciliary structure may be essential for and functional ciliary defects. Ultrastructural changes normal mucociliary clearance after an insult. The process
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Fig. 2. Electron micrograph of transverse sections of multiple cilia. Magnlflcation, ~60,000.
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Fig. 1. Electron micrographs of transverse sections of cilia in patients with recurrent URI. (Magnification, x 120,000). a: Normal cilium showing the 9 + 2 microtubule pattern, inner and outer dynein arms, and central spokes. b: Missing single peripheral microtubules and missing dynein arms. c: Two extra microtubules inside 9 + 2 arrays. d: Extra single central microtubule. e: Extra central mlcrotubule doublets inside 9 + 2 array and extra single peripheral microtubule. f: Mlsslng single peripheral microtubule doublets. g: Swollen cilia.
Fig. 3. Schematic representation of the presumed association among infection, ciliogenesis, and the mucociliary defense mechanism.
subjects, although there was no difference in the etiology of the initial infection between this group and the group of 20. These alterations may be viewed as long-term residual effects of infection. The mechanisms leading to the persistence of ciliary abnormalities may be similar to those proposed by Carson et al.''9'9 in acute viral upper respiratory tract infections, in which conditions for producing errors in ciliogenesis exist. The data obtained demonstrate that some time after of ciliogenesis in the normal nasal epithelium is continuclinical resolution of infection there is still evidence that a ous and rdpid.32 Various congenital and environmental large portion of cilia has not returned to normal morpholfactors have been shown to influence the process of ciliog e n e ~ i s . ~Altered ~ - ~ ~ ciliogenesis following continuous ogy in some children with URI. It is not known whether insults can lead to additional impairment of the mucocili- the reported abnormalities have an effect on mucociliary ary defense mechanism with consequent increase in se- defense mechanism. The role of these ciliary abnormaliverity and chronicity of the infection. This situation may ties in predisposing to recurrent infection needs to be investigated. Further studies are in progress to assess the create a vicious circle (Fig. 3). In our study group we observed the persistence of mucociliary transport system in children with recurrent ciliary abnormalities after the convalescent period in 7 URI.
Ciliary Structure in Recurrent Infections
ACKNOWLEDGMENTS
We are indebted to Prof. S. Cinti of the Institute of Human Morphology, University of Ancona, for giving us access to electron microscopy facilities.
18. Chandrer DKF, Barile MF. Ciliostatic, hemagglutinating and pro-
19. 20.
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