Eosinophil cationic protein in mucosal biopsies from patients with allergy and otitis media with effusion DAVIDS. HURST,MD,and KJELDFREDENS,MD, Uppsala, Sweden, and Aarhus, Denmark
Nine patients with persistent middle ear effusion and allergy confirmed by skin testing were evaluated for eosinophUs by histochemical staining of middle ear mucosal biopsy specimens for eosinophil cationic protein. The study was designed to determine whether eosinophils were present in the middle ear mucosa of these patients and whether the elevated levels of eosinophil cationic protein reported in effusion from patients with chronic otitis media with effusion and allergy might originate within the mucosa itself. Seven of nine patients with otitis media with effusion had eosinophil cationic protein containing eosinophils (12 to 15 per high-power field) and degranulated eosinophil cationic protein material in the stroma of their mucosal biopsy specimens. Positive and negative biopsy findings correlated directly with respective high and low effusion levels of eosinophil cationic protein (p = 0.03), reflecting an intrinsic immune-mediated process occurring within the middle ear mucosa. (Otolaryngol Head Neck Surg 1997;117:42-8.)
F o r decades allergy has been proposed as a cause of chronic middle ear inflammation, ~-3 but research has been impeded by an absence of methods to verify that relationship. Immunologic studies of the effect of allergy on the respiratory system have been dominated by research on rhinitis and asthma because of the ease of access to the nose and lung. Clinical studies would suggest that the immunologic processes responsible for these classic models of allergy also take place in the middle ear. Perpetuation of inflammation, regardless of origin, is the crucial distinction between recurrent acute otitis and persistent otitis media with effusion (OME). It has been shown that chronic inflammation is a direct continuation of an acute episode in only half the cases and is not caused by inadequate therapy. 4 A different cause of inflammation must therefore be responsible for maintaining the disease, which progresses to OME in more than 20% of patients with otitis. This study was designed to extrapolate proven methods of immunolog-
From the Department of Clinical Chemistry (Dr. Hurst), Uppsala University; and the Department of Neurobiology (Dr. Fredens), Institute of Anatomy,Aarhus Universitet. Presented at the Annual Meeting of the American Academy of Otolaryngic Allergy, Phizer Symposium, San Diego, Calif., Sept. 24, 1994. Reprint requests: David S. Hurst, MD, c/o R.R. 4, Box 4548, Farmington, ME 04938. Copyright © 1997 by the American Academy of OtolaryngologyHead and Neck SurgeryFoundation, Inc. 0194-5998/97/$5.00 + 0 23/1/'74544 42
ic research and to apply them to the study of otitis media (OM) to determine whether eosinophils or eosinophil cationic protein (ECP) granules can be demonstrated in the mucosa of ears with elevated ECP levels. It is generally accepted that selective recruitment of eosinophils is important in the pathogenesis of allergic rhinitis and asthma. Similarly, most patients with OME and allergy have been shown to have elevated levels of ECP in their middle ear fluid, as distinguished from controls. 5 Earlier research introduced a method to investigate the inflammatory processes taking place in the middle ear 5 on the basis of the standard model for measuring levels of cell mediators in both nasal allergy6,7 and asthma. 8 The parameters of intradermal testing, RAST and IgE, have been used to confirm clinically that an effusion ECP level greater than 10 gg/L is also a marker with high sensitivity (92.1%) and positive predictive value (87.5%) for allergy in patients with OME. 5 Biopsy specimens from nine random ears of patients preselected to have both allergy and effusion for more than 2 months were examined by polyclonal antibody staining for ECP to determine whether their effusion ECP originated from the middle ear mucosa. Chronic inflammation is the prerequisite for the development of persistent OME. There is disagreement as to whether mediators identified in middle ear effusion originate from the plasma or from local tissue. Palva et al. 9 concluded that OME is a local process and that it "may be assumed that the fluid cytology and the
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HURST and FREDENS 43
Volume 117 Number 1
Fig. 1. Middle ear mucosal biopsy specimen showing intact surface epithelium
(top left) and
shedding. (Hematoxylin and eosin stain; original magnification x165.)
biopsy cell make-up give an accurate picture of the ongoing immune biological events." Using animal studies, Nakata et alJ ° found few eosinophils in the effusion of immunized chinchillas in the acute phase of inflammation but did conclude that "middle ear effusion is a local product of the middle ear mucosa rather than a transudate from plasma." Previous reports of high levels of ECP in effusion from OME patients with allergy suggest that chronic effusion represents a latephase allergic response, as seen in asthma)1 Elevated effusion ECP levels have prompted the question of whether this mediator is produced locally.
Histologic Studies Normal tympanic mucosa is described as low cuboidal, devoid of lymphoid tissue and eosinophils. The submucosa is often just thick enough to transport vessels, nerves, and lymph channels) 2 Temporal bone sections are required for evaluation of normal ear mucosa, which is only two cell layers thick, making biopsy an impossibility. Eosinophils are not described in the few reports of temporal bone specimens from patients with serous OM, 13 nor did Karmody observe eosinophils on review of the histology of 100 temporal bones from normal children (Karmody C, Personal communication, February 1996). Conventional histology leads to varying conclusions regarding inflammatory Changes in the mucosa itself because degranulated eosinophils are not readily identifiable. 13-16 Histopathologic examination of effusion demonstrates that eosinophils and neutrophils are inte-
gral components in these secretions 13,17but are not necessarily present in the mucosal lining. Koch 18 initially described cytologic changes in chronic OM and found eosinophils in mucosa from 52 of 62 cases of allergic otitis. Mice ears with a chronic immune response show an extensive mucosal hyperplasia consisting of lymphocytes and neutrophils in the submucosa with neutrophils, macrophages, and diffuse eosinophilic material in the cellular effusions. This is accompanied by numerous interleukin-5+ cells in both the subepithelial tissue and effusion in the chronic stages. 19 Modern methods of identification of specific cells with monoclonal or polyclonal antibody stains have made histopathology more precise. These methods have been used to identify eosinophils in allergic inflammation involving tissue damage of skin, kidney, lung, and nasal mucosa. 2°-23 Inflammation involved in otitis can similarly be characterized by use of immunohistochemically stained biopsy specimens, which are more sensitive m~d specific than conventional histologic methods.
METHODS Subjects, Data Collection, and Analysis Subjects included nine children with allergies (aged 3 to l0 years) selected in a random, prospective manner. None was immunodeficient, exhibited congenital malformations, or had had an acute infection within the preceding 8 weeks. All had had documented hearing loss, flat tympanograms, and effusion unresponsive to antibiotics and/or decongestants for 3 to 18 months. Middle ear effusion was collected quantitatively in a
44 HURSTand FREDENS
OtolaryngologyHead and Neck Surgery July 1997
Fig. 2. Middle ear mucosal biopsy specimen stained for ECP immunoreactivity. Slide is a consecutive section to Fig. 1. (Peroxidase-anfiperoxidase procedure; original magnification x165.)
Juhn Tym-Tap and diluted with precisely 2 ml of normal saline solution at the time they underwent routine myringotomy for placement of tympanostomy tubes. Variation of the volume of middle ear fluid was previously considered by measuring 14 samples containing lithium chloride. 24 Effusion volumes were shown to be quite similar, ranging from 0.11 to 0.47 ml (mean, 0.28 + 2 SD; 0.12 ml). 5 The statistically significant differences observed between individual ECP levels in this study and normal means were thus unlikely to be explained by variation of volumes and dilution of sampies. Biopsy specimens of the mucosa from the promontory of the middle ear were taken after approval of the Hospital Committee on Ethics and Human Experimentation and with parental consent. Biopsy specimens were obtained with a microcup forceps through the myringotomy incision. The tissue was immersion-fixed in 4% formaldehyde in 0.1 mol/L phosphate buffer (pH 7.3) embedded in paraffin, sectioned at 20 ~m in a cryostat, and subjected to immunoperoxidase staining for the demonstration of ECE The ECP antiserum was a gift from E Venge, Uppsaia, Sweden. The endogenous peroxidase activity was blocked by 50 ml 3% hydrogen peroxide in 200 ml absolute methanol for 20 minutes. The sections were then gently rinsed in buffer. After being soaked for 30 minutes in Tris-buffered saline (pH 7.4) containing 1% Triton X-100 (Triton-TBS; Sigma), sections were exposed to a 1% solution of swine serum for 30 minutes, rinsed briefly in Triton-TBS, and
exposed to optimally diluted antisera for 20 hours at 4 ° C followed by 2 hours of reequilibration at room temperature. The site of antigen-antibody reaction was demonstrated by the peroxidase-antiperoxidase procedure of Stemberger. 25 Antibody dilution for ECP was 1:1200. Conventional staining controls as detailed by Stemberger and absorptional (specificity) controls were used. Consecutive sections were stained with hematoxylin and eosin (Fig. 1) to describe the tissue. Diagnosis of I m m e d i a t e - t y p e Hypersensitivity
Intradermal skin testing for 12 inhalants (Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae l, timothy and Bermuda grass, giant and short ragweed, birch and oak trees, cat, dog, Alternaria, and Hormodendrum) and 12 foods26 (milk, wheat, soy, corn, baker's yeast, chicken, potato, egg, coconut, sugar, beef, and pork) was performed on all patients. The diagnosis of allergy was biased so that a patient so classified had to demonstrate either (1) a positive RAST reaction to two allergens, and/or (2) intradermal skin testing with positive wheal response to at least two allergens at a concentration of 1:500 or less for inhalants or 1:50 for foods. Food skin testing was confirmed by positive oral challenge test results. Histamine diphosphate (1.0 mg/ml) was the positive control, and glycerol/phosphate-buffered saline (1:1) was the negative control (Table 1). Antigens were obtained through Hollister-Stier (Miles Laboratories). All skin reactions were evaluated at 10 minutes, and a mean wheal diam-
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HURSTand FREDENS 45
Fig. 3. Enlargement of right side, of Fig. 2 showing infiltration of ECP-positive cells below basal layer. (Peroxidase-antiperoxidose procedure; original magnification ×340.)
eter 2 mm greater than the glycerol control was considered a positive reaction. Titration of Mediators
ECP in effusion Was measured by a double-antibody radioimmunoassay (Kabi Pharmacia Diagnostic AB) with a polyclonal rabbit antibody as previously described. The ECP standards are calibrated against a pure ECP according to the method of Venge et al. ~7 The technique was carried out according to the package insert. The interassay coefficient of variation was 3% to 8%, and levels under 1 tag/L were undetectable. The effusion was considered to be related to allergic inflammation if the ECP level was greater than 10 pg/L (control mean n 2 SD) in a nonpurulent ear. 5 RESULTS
Intradermal testing demonstrated that all nine patients had allergies. Immunostaining clearly showed ECP containing eosinophils and degranulated ECP material in the epithelial and subepithelial layers of the mucosa (Figs. 2 and 3). All seven of the biopsy specimens from patients with elevated effusion levels of ECP (>10 I~g/L)28 (Table 1) showed shedding of the surface epithelium and infiltration of ECP-positive cells in the tissue. Cell infiltration occurred on the surface of the epithelium, within the epithelium, and especially below the basal layer, in the connective tissue. There was no crush artifact in the specimens. In addition to 12 to 15 ECP-positive cells (eosinophils) per high-power field,
extracellular granutar deposits were seen at many places. These are the findings of active degranulation of eosinophils indicating release of cytotoxic proteins with resultant nonselective damage to the mucosa in all seven cases. These were compared with the two biopsy specimens (patients 8 and 9) with low-effusion ECP levels (5.4 and 0.8 pg/L, respectively). The mucosa from these two patients stained negative for ECP but were not normal. Both specimens demonstrated significant fibrosis indicating the sequelae of a more longlasting, chronic inflammatory process. Retrospective review of these two case histories revealed that patients 8 and 9 (aged 7 and 8 years, respectively) had effusion present for 8 and 18 months before placement of their tubes. These low-effusion ECP levels correlated with the biopsy specimens, which demonstrated that an eosinophil response was not present. Thus the biopsy specimens showed that elevated middle ear effusion ECP directly correlates to the presence of degranulafing eosinophils in the local mucosa (Fischer's exact test, p = 0.03). DISCUSSION
Eosinophils represent key cells of the allergeninduced, IgE-mediated, late-phase mucosal inflammation seen in rhinitis, asthma, 29,3° and atopic dermatitis. 31 Only allergens related to patient symptoms and eliciting positive skin test results and RAST induce extracellular release of eosinophil mediators. 32 It has never been possible to differentiate allergic and nonal-
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46 HURSTand FREDENS
Table I. Effusion ECP a n d positive skin test allergens in nine patients with OME Patient no.
Age (yr)
ECP
+ Skin test allergens
4.9 9.8 4.9 3.4 4.5 7.8 4.4 7.2 8.0
46 289 16 97 145 12.6 10.4 5.4 0.8
Gr, Dg, Dt, Md Dt, Rg, Tr, Md, Eg, Sy, Mk, Ck Dt, Md, Gr, Rg, Sy Cn, Eg, Mk Dt, Ct Mk, Md Ct, Dg, Dt, Rg, Gr Eg, Wt, Po, Dg, Dt, Rg, Md Dt, Gr, Rg, Md
I
1 2 3 4 5 6 7 8 9
Inhalants: Ot, Dust (Der p 1 and Der f 1); Gr, grass (2); Tr, trees (birch, pine); Rg, ragweed; Wd, weeds; Md, mold (Alternaria, Cladosporium); Ct, cat; Dg, dog, Foods: M/<, Milk; Yt, yeast; Eg, egg; Wt, wheat; Cn, corn; Sy, soy; Ck, chicken; Po, potato; Co, coconut; Sg, sugar; Bf, beef; Pk, pork.
lergic subjects on the basis of eosinophils or their granule proteins alone. That distinction requires intradermal skin testing or mucosal biopsy. 7 Thus the association of allergy with chronic otitis requires the presence of eosinophils in a patient with positive skin test results, as demonstrated in this study. Eosinophilic inflammation of the airway correlates with the severity of allergic symptoms. 33,34Pathologically, small amounts of ECP produce both disruption of the epithelial lining and destruction of pulmonary epithelium. 22 Clinically, ECP induces bronchoconstriction, increases mucosal permeability,35 inhibits tracheal ciliary function,22 increases mucus secretion,36 and reduces mucociliary clearance.37 These pathologic changes also characterize the inflammation seen in chronic OM. Eosinophils and allergy are integral to the histopathophysiology of OME. 15,38 It has been reported that 89.6% of patients selected randomly with nonacute OME had elevated levels of effusion ECE 28 This elevation has been shown to occur in those patients in whom allergy is a significant disease; ECP is not present in the effusion from either persons without allergies or those whose allergy season does not correlate with the season of their otitis. 5,u Levels of ECP in serum and effusion do not correlate. 28 It is rightly questioned whether effusion ECP arises from the middle ear mucosa and reflects localized middle ear eosinophil activation. Mature eosinophils survive only 13 to 18 hours in blood and perhaps several days in tissue, but ECP has a half-life of only 45 minutes. 39 Cell kinetics dictate that elevated levels of ECP reflect an active inflammatory process involving the continual recruitment and degranulation of eosinophils into the middle ear space. In studies of nasal allergy the combined assessment of eosinophil count and ECP concentration in nasal secretions has been shown to be "a good indicator of mucosal inflammation in allergic
patients." Furthermore, ECP levels in nasal secretions are considered to "reflect the state of the activation of the eosinophils present. ''34 In all middle ears various mediators are produced by leukocytes during an acute infection. It has been hypothesized that the allergic subject is more prone to OME developing after an episode of acute otitis for reasons similar to those causing the hyperreactivity of an asthmatic lung during times of infection.4° Progenitors of mast cells and eosinophils are primed by allergen challenge and circulate in increased numbers in the blood of patients with various forms of airway inflammation and are more prone to react to any stimulus. 41 Allergic reactions, infectious inflammation, and local immunologic response associated with persistence of pathogenic bacteria, bacterial components, or viruses have all been considered factors responsible for the preemptive dysfunction of the eustachian tube leading to OME. 42 Repeated antigen exposure may alter inflammatory reactivity to strengthen eosinophil participation in the late-phase reaction while neutrophil involvement diminishes. 31 Various mediators, including prostaglandins, cytokines, and interleukins, persist for some time in the middle ear after the acute episode has subsidedfl 9,43 In normal subjects this does not matter, but in subjects who have eosinophils primed by allergy circulating in their blood, cytokines and ligands in the middle ear may attract and activate those cells, with the resultant chronic release of ECR A fundamental problem in investigating the pathogenesis of respiratory tract disease has been to explain why eosinophils preferentially accumulate in inflamed mucosa. The middle ear is capable of immune responses including the local generation of antibodies. The middle ear normally has a very weak response to antigen challenge, but a previously sensitized middle ear responds with a vigorous immune response when antigen is presented to it. 17,44 On appropriate antigenic
OtolaryngologyHead and Neck Surgery Volume 117 N u m b e r 1
stimulation the middle ear becomes infiltrated by lymphocytes too rapidly to be accounted for by proliferation of the few resident lymphocytes that occur in the normal mucosa. 45 A more likely source is the circulating pool of systemic lymphocytes. Lymphocytes are known to enter the middle ear in a nonspecific response to immune-mediated inflammation.46 Ryan et al.45 have shown that lymphocytes from i m m u n i z e d donors appear to possess more surface determinants for the recognition of endothelial cell surface molecules than those from unimmunized donors and has demonstrated increased homing to the middle ear. There has been substantial work showing that respiratory syncytial virus enhances synthesis of proinflammatoiy cytokines (IL-1 ~3, TNF-cq IL-6) and cell adhesion molecules (ICAM-1, ELAM-1, VCAM-1) in the middle ears of infected individuals.4° This viral sensitization may contribute to the initial inflammatory process leading to OME. It may be that both mechanisms participate in the eventual formation of effusion. Ichimiya et al. 44 suggest that the middle ear mucosa is an immunoreactive site only after it has been activated with pathogens. The viral hypothesis is not contradictory to the allergy hypothesis. Perhaps only in the allergic individual does viral or bacterial inflammation lead to the chronic recruitment of inflammatory cells with the resultant release of their potent inflammatory mediators and the resultant production and maintenance of effusion in a sterile ear. The mechanism underlying the attraction of eosinophils and release of ECP in the middle ear of patients with OME is not understood and should be a major focus of future studies.
CONCLUSION The eosinophil activity of allergic otitis mimics the inflammatory response seen in late-phase allergic asthma and rhinitis. Heretofore allergy has not been considered a direct cause of OME, but rather was thought to be responsible only for interference of eustachian tube clearance of middle ear effusion. 47 The inflammation we observed in the promontory mucosa of patients with OME who were proved by skin tests to have allergy appears to be accompanied by the continual recruitment and activation of eosinophils. This is demonstrated by the high numbers of eosinophils (12 to 15 per high-power field) and the high density of ECP-staining granule protein deposits found within the tissue from seven of nine mucosal biopsy specimens. Positive and negative biopsy findings correlated directly with respective high and low effusion levels of ECP (p = 0.03). These biopsy findings support the hypothesis that abnormally elevated levels of ECP in effusion reflect
HURST and FREDENS 47
an intrinsic i m m u n e - m e d i a t e d process involving eosinophils activated within the middle ear itself.
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22. Pipkom U, Karlsson G, Enerback L. The cellular response of the human allergic mucosa to natural allergen exposure. J Allergy Clin Immunol 1988;82:1046-54. 23. Demoly P, Vachier I, Pene J, Michel F, Godard P, Damon M. IgE produces monocyte superoxide anion release: correlation with CD23 expression: comparison of patients with asthma, patients with rhinitis, and normal subjects. J Allergy Clin Immunol 1994;93:108-16. 24. Linder A, Ronquist G, Deuschl H. Random distribution of exogenous lithium in nasal secretion and its application in substance determination. Acta Otolaryngol 1983;96:287-93. 25. Sternberger LA. Immunocytochemistry. New York: John Wiley and Sons, 1979. 26. Bock AA, Atkins FM. Patterns of food hypersensitivity during sixteen years of double-blind, placebo-controlled food challenges. J Pediatr 1990;117:561-7. 27. Venge P, Roxin LE, Olsson I. Radioimmunoassay of human eosinophil cationic protein. Br J Heamatol 1977;37:331-5. 28. Hurst DS, Venge P. The presence of eosinophil cationic protein in middle ear effusion. Otolaryngol Head Neck Surg 1993; 108:711-22. 29. Gleich G. The late phase of the immunoglobnlin-E mediated reaction: a link between anaphylaxis-and common allergic disease? J Allergy Clin Immunol 1982;70:160-9. 30. Weller PE Immunoglobulin E-dependent inflammation: eosinophils and other inflammatory cells. In: Levinson AI, Paterson Y, editors. Molecular and cellular biology of the allergic response. New York: Marcel Dekker Inc., 1994:361-410. 31. Ott N, Gleich G, Peterson E, Fujisawa T, Sur S, Leiferman K. Assessment of eosinophil and neutrophil participation in atopic dermatitis: comparison with the IgE-mediated late-phase reaction. J Allergy Clin Immunol 1994;94:120-8. 32. Tomassini M, Tsicopoulos A, Tai PC, et al. Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation. J Allergy Clin Immunol 1991;88:365-75. 33. Bousquet J, Chanez P, Lascoste JY, et al. Eosinophilic inflammation in asthma. N Engl J Med 1990;323:1033-9. 34. Wang D, Clement P, Smitz J, de Waelle M, Derde M-P. Monitoring nasal allergic inflammation by measuring the concentration of eosinophil cationic protein and eosinophils in nasal secretions, Allergy 1995;50:147-51.
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35. Hogg JC, Eglesston PA. Is asthma an epithelial disease? Am Rev Respir Dis 1984;129:207-8. 36. Lundgren JD, Davey RT Jr, Lundgren B, et al. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucous secretion. J Allergy Clin Immunol 1991;87:689-98. 37. Hastie AJ, Loegering DA, Gleich GJ, Kueppers E The effect of purified human eosinophil MBP on mammalian ciliary activity. Am Rev Respir Dis 1987;135:848-53. 38. Pelikan Z. Nasal response to food ingestion challenge. Arch Otolaryngol Head Neck Surg 1988;114:525-30. 39. Venge P, Bergstrand H, Hakansson L. Neutrophils and Eosinophils. In: Kelley W, Harris E Jr, Ruddy S, Sledge C, editors. Textbook of rheumatology. 4th ed. Philadelphia: WB Saunders Co., 1992:269-82. 40. Okamoto Y, Kudo K, Ishikawa K, et al. Presence of respiratory syncytial virus genomic sequences in middle ear fluid and its relationship to expression of cytokines and cell adhesion molecules. J Infect Dis 1993; 168:1277-81. 41. Denburg JA. Microenvironmental influences on inflammatory cell differentiation. Allergy 1995;50(suppl 25):25-8. 42. Miller M, Koltai P, Hetherington S. Bacterial antigens and neutrophil granule proteins in middle ear effusions. Arch Otolaryngol Head Neck Surg 1990; 116:335-7. 43. Bernstein JM. Applying today's concepts on pathogenesis ofotitis media. J Respir Dis 1990;11:402-18. 44. Ichimiya I, Kawanchi H, Mogi G. Analysis of immunocompetent cells in the middle ear mucosa. Arch Otolaryngol Head Neck Surg 1990;116:324-30. 45. Ryan AF, Sharp PA, Harris JP. Homing of mucosal and nonmucosal lymphocytes to the tympanic cavity. In: Sade J, editor. The eustachian tube, clinical aspects. Selected papers from a Conference on the Eustachian Tube and Middle Ear Disease, Geneva, Switzerland. New York: Kugler & Ghedini Publishers, 1989:33-7. 46. Bemstein JM, Tomasi TB, Ogra PL. The immunochemistry of middle ear effusions. Acta Otolaryngol 1974;99:320-6. 47. Mogi G. Immunologic and allergic aspects of otitis media. In: Lira D, Bluestone C, Klein J, Nelson J, Ogra P, editors. Recent advances in otitis media. Ontario, Canada: BC Decker Inc., t993:145-51.
Temporal Bone Histopathology This 1-day workshop, presented and sponsored by the Department of Otolaryngology, University of Minnesota, and the NIDCD National Temporal Bone, Heating and Balance Pathology Resource Registry, will be held Sept. 26, 1997, at the University of Minnesota, Minneapolis, Minn. It will provide hands-on instruction in the techniques of study of the human temporal bone and associated brain tissue. For further information, contact Patricia Schachern, Lions Research Building, Room 226, 2001 Sixth St., SE, Minneapolis, MN 55455; phone, (612)626-9876; fax, (612)626-9871; e-mail,
[email protected].