Unique Cilia Ultrastructure Findings in a 9/11 Rescue Worker

Rizwan Aslam, DO, Hamot Medical Center, Erie, PAJames McMahon, PhD, Cleveland Clinic Foundation, Cleveland, OH

Stephen Schell, MD, Hamot Medical Center, Erie, PA

AbstractOBJECTIVES: 1) Describe unique transmission electron microscopy (TEM) findings of cilia ultrastructure changes in a World Trade Center 9/11 rescue worker. 2) Compare normal and pathologic cilia ultrastructure changes using TEM micrograph images. 3) Understand primary and acquired ciliary dyskenesia. METHODS: A case report and literature review was performed. A 42 years old previously healthy female presented to the outpatient clinic with a several month history of odynophagia and hoarseness that developed while participating in relief efforts at “Ground Zero” immediately following the events of 9/11. Initial fiberoptic laryngoscopy demonstrated erythema of the laryngeal surface of the epiglottis, arytenoids, and interarytenoid mucosa. Following failed medical therapy and persistence of symptoms, panendoscopy was performed and specimens of the epiglottis, anterior tracheal wall, and nasal floor were acquired. Biopsy results were analyzed using TEM. RESULTS: TEM of ciliated cells of the anterior trachea wall demonstrated consistent absence of dynein arms normally associated with peripheral doublets. Another consistent finding was the displacement of peripheral microtubules. Numerous compound cilia were also seen. There were no reported cases of 9/11 patients having cilia ultrastructure changes in the otolaryngology literature. CONCLUSIONS: Ultrastructuredefects of cilia can lead to impaired cilia motion, causing decreased mucociliary clearance and significant morbidity. This is believed to be first reported case of cilia ultrastructurechanges in a patient exposed to the Ground Zero environment. We describe unique cilia changes in a patient with no previous manifestations of primary or previously acquired ciliay dyskinesia.

peripheral doublet is slightly tilted such that the microtubular subunit (A) bearing the dyneinarms is, in general, slightly closer to the center of the axoneme than the subunit (B) not having dynein arms. This allowed us to identify each of the peripheral doublets and describe the disarray despite the absence of dynein arms. Consistently evident were axonemes having a similar pattern of disarray. Doublet number 9 was uniformly out of line and was closer to the central pair than the others (Figure 2). This pattern was present in all of the cilia showing significant disarray but also to some degree in cilia less affected. There was no visualization of spoke structures, nexin links, or central sheaths surrounding the central pair of microtubules. On 2/20/2007 additional ciliary specimens from the nasal inferior turbinate and trachea were obtained in order to assess persistence of the motile quality and structural changes seen in the initial specimen. A wet mount preparation of living cells obtained by brushings of the turbinate showed moderately rapid ciliary motility with recognizable metachronic wave activity. Electron microscopy of the biopsy specimen of the trachea showed normal ciliary ultrastructure. There was no microtubular disarray as was seen in the initial biopsy specimen. Radial spokes and the sheath surrounding the central pair of microtubules were clearly present, as were the dynein arms; none of which were evident in the initial biopsy. It was apparent that the abnormalities seen in the initial specimen were no longer present in the follow-up specimen nearly 4 ½ months later.

1. Kokayi, K. Findings of and treatment for high levels of mercury and lad toxicity in ground zero rescue and recovery workers and lower Manhattan residents. Explore 2006;2:400-7.2. Wolf, R.K. Effects of Airborne Pollutants on Mucociliary Clearance. Environmental Health Perspectives 1986;66: 223-237. 3. Herbert R, et al. The World Trade Center disaster and the healthcare of workers: Five-year assessment of a unique medical screening program. Environ Health Perspect 2006;

114: 18534. Watanabe, Y. Ultrastructural study of immotile cilia syndrome. Rhinology 1984;22:193-199.5. Ho, J.C. The Effect of Aging on Nasal Mucociliary Clearance, Beat Frequency, and Ultrastructure of Respiratory Cilia. Am J Respir Crit Care Med 2001;163:983-988.6. Niewoehner, DE. Morphologic basis of pulmonary resistance in the human lung and effects of aging. J Appl Physiol 1974;36: 412-418.7. Agius, A.M. Age, smoking and nasal ciliary beat frequency. Clin Otolaryngol 1998;23:227-230.8. Rutland, J. Random ciliary orientation. N Engl J Med 1990;323:1681-1684.9. Houtmeyers, S.E. Regulation of mucociliary clearance in health and disease. Eur Respir J 199;13:1177-1188.10. Yager, J. Measurement of frequency of ciliary beats of human respiratory epithelium. Chest 1978;73:627-633.11. Mortensen, J. Lung mucociliary clearance. Eur J Nucl Med 1994;21:953-961.12. www.microscopy.fsu.edu

Bibliography

We performed a case review and a literature review. We searched MEDLINE and PubMed using the search words “cilia,” “cilia ultrastructure,” “ground zero,” “transmission electron microscopy,” and “9/11”. Transmission Electron Microscopy (TEM) and observation of living preparations were used to analyze cilia specimens from a single Lpatient on two different occasions. We describe in this report the case of LD a 42 year old previously healthy female who presented with a several month history of odynophagia and hoarseness that developed while she participated in rescue and relief efforts Ground Zero 3 weeks after 9/11. Her medical history also included upper respiratory congestion, headaches, dyspnea, cough, dizziness, chest pain, and paresthesias in all four extremities. She was treated with omeprazole and gaviscon for findings consistent with laryngopharyngeal reflux and a history of toxic exposure. The patient’s symptoms failed to improve with medical treatment and her respiratory complaints persisted. Biopsies were performed for staging suspicious synchronous upper aerodigestive tract lesions. Panendoscopy was performed and specimens of the epiglottis, anterior tracheal wall, and nasal floor were acquired. Specimens for evaluation of ciliary motility were received in RPMI. They consisted either of brushings of the posterior nasal turbinate or biopsies of the trachea. Specimens from the nasal turbinate were stripped from bronchoscopybrushes into a puddle of RPMI on a glass slide while specimens from the trachea were teased apart under RPMI using two pairs of watchmaker forceps. Both specimens were coverslipped with the addition of more RPMI to float the coverslip. Preparations were observed at room temperature using a standard compound microscope with the condenser lens lowered in order to produce a phase contrast-like effect. Motility was judged to be absent, slow, or normal based on the experience of one of the observers (JTM). Specimens judged to be immotile were tested for viability using the trypan blue exclusion test. Only sites consisting of strips or clusters of numerous intact ciliated cells were evaluated for motility. Single cells or small clusters of cells were not evaluated since they may have suffered trauma when separated from the epithelium and may be likely to show reduced motility or no motility at all. Specimens were fixed in 3.75% glutaraldehyde, 0.1M sodium cacodylate, 6% sucrose, pH 7.2-7.4 for a period of 4 hours to overnight. Specimens were postfixed in 1% osmium tetroxide, 0.1 M cacodylate, pH 7.2 –7.4 for 1 hour. Specimens were dehydrated and embedded in EM Bed 812. Thin sections were cut at 60 nm, stained in uranyl acetate and lead citrate, and examined using a Philips CM-12 electron microscope.

Methods and Materials

Figure 1: Normal Cilia Figure 2: Notice abnormal displacement of MT 9

In 2006, the Mount Sinai Medical Center released the findings of the World Trade Center and Volunteer Medical Screening Program, the largest multi-center clinical program providing medical screening examinations for the workers and volunteers who worked at Ground Zero and other sites following the 9/11 terrorist attacks. The screening program included close to 12,000 subjects from diverse professions aiding in the WTC rescue, recovery, and clean-up operations. 9,500 agreed to be included in the report. Most of the subjects suffered upper respiratory illnesses (84%), lower respiratory disorders (47%), psychological disorders (37%), and musculoskeletal problems (31%) suffered from injuries at the site.3

Proper ciliary motility is key to efficient mucociliary transport along the respiratory tree. For cilia to perform properly there must be a correct combination of structural and physiological factors. When in their proper normal operating state, these factors allow cilia to move mucus containing entrapped bacteria, toxins, and environmental pollutants along cell surfaces lining the respiratory tract with escalation of particles towards the nasopharynx where they may be swallowed or expectorated. Genetic abnormalities, respiratory infections, environmental stresses, and locally applied drugs may cause absence or failure of any of these factors, which may lead to loss of mucociliary transport and the occurrence of respiratory disease. Ciliary defects that are genetic in origin comprise a complex set of diseases collectively called Primary Ciliary Dyskinesia (PCD) that have a variety of abnormalities that may be identified by electron microscopy. Primary ciliary defects include absent or partial inner and/or outer dynein arms, absent or disoriented central pair of microtubules, absent radial spokes, and translocation of microtubular doublets. Any of these defects may significantly alter the normal ciliary beat pattern and produce clinical symptoms including recurrent upper and lower respiratory tract infections, chronic sinusitis, chronic bronchitis, and bronchiectasis. Afzelius et al. (1979) were the first to report impairment of ciliary movement secondary to the absence of dynein arms is the main cause of Immotile Cilia Syndrome (ICS).4 Abnormal ciliaryfunction, which could result from abnormal cilia ultrastructure or de novo, can lead to recurrent sepsis of the respiratory tract.

While genetic abnormalities of PCD are permanent and can be treated only in a supportive manner, Secondary Ciliary Dyskinesia (SCD)is a transient form of ciliarydisease in which kinetic and/or structural abnormalities have non-genetic etiologies including infection, pollutants, and drugs that may be limited in duration or may be treated with subsequent recovery of normal ciliary structure and function over time. Patient LD had no prior symptoms of ciliary dismotility. Her TEM findings were consistent with ICS, but interestingly returned to normal function 4 ½ months later. This is believed to be the first reported case of such findings. The question of whether these findings were acquired is of significance given her exposure history. TEM can be a useful tool in diagnosing underlying microscopic ultrastructure changes in 9/11 patients suffering from respiratory illnesses. It can help us understand the disease process and offer a better understanding of how to appropriately manage patients with ciliary defects and mucociliary impairment.

IntroductionWhen the World Trade Center buildings burned and collapsed following terrorists

attacks on September 11, 2001, America suffered a human and environmental disaster of unprecedented proportions. Pulverized and vaporized, along with thousands of people, were an estimated 1.2 million tons of concrete, metal, asbestos, glass and other construction materials creating massive rolling clouds of toxic dust and debris that swept through lower Manhattan. The alkalinity of the toxic cloud approached the caustic level of aerosolized drain cleaner capable of searing the soft membrane linings of the lungs and respiratory tract. At the time of the attack, combustion of 20,000 gallons of fuel from two jet planes spewed out dense plumes of black smoke. Approximately 40,000 men and women participated in the rescue, recovery, and clean-up at the Ground Zero site. Since then several individuals who were exposed to those pollutants have suffered a myriad of illnesses including respiratory problems, digestive problems, skin rashes, sleeplessness, anxiety, depression, weight gains, hypertension, lethargy, and headaches.1 The mucocilliary clearance system is a first line of defense against inhaled agents, and so its compromise can adversely affect health.2 The focus of this report is to illustrate the significance of the respiratory cilia insult and its microstructural abnormalities of patients exposed to the environment of WTCGZ.

ResultsThe first cilia biopsy was taken 10/09/2006 from the trachea and prepared as a wet

mount preparation and for electron microscopy. Follow-up specimens from the nasal posterior turbinate and the trachea were taken on 2/20/2007 and similarly prepared. In the wet mount prep of the 10/09/2006 specimen, an intact strip of ciliated epithelium with no apparent motility was identified at the edge of the small biopsy specimen. Results of electron microscopy were striking. All well oriented cross sections of the cilia showed some degree of perturbation of the axoneme. Universally present were nine peripheral microtubular doublets and a central pair of singlet microtubules. However, dynein arms were not evident and the positions of the microtubules appeared disordered but, oddly enough, in a disordered pattern that was repeated from cilia to cilia. Since we were interested in describing the pattern of disarray, we used a standard convention for numerically identifying each of the peripheral microtubular doublets in the axoneme. The custom is to draw a perpendicular line through the central pair such that the line passes between two doublets at one end of the line and intersects a doublet at the other end. The intersected doublet is the number 1 doublet and the numbering proceeds from 1 to 9 in a procession indicated by the direction of the dynein arms. However, numbering cues also came from the orientation of each of the doublets. In normal cilia (Figure 1) each

11

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3344 55

66

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99

Figure 3: Cilia Ultrastructure

Discussion