Epithelial dynamics shed light on the mechanisms underlying ear canal defects.

Defects in ear canal development can cause severe hearing loss as sound waves fail to reach the middle ear. Here, we reveal new mechanisms that control human canal development and highlight for the first time the complex system of canal closure and reopening. These processes can be perturbed in mutant mice and in explant culture, mimicking the defects associated with canal atresia. The more superficial part of the canal forms from an open primary canal that closes and then reopens. In contrast, the deeper part of the canal forms from an extending solid meatal plate that opens later. Closure and fusion of the primary canal was linked to loss of periderm, with failure in periderm formation in Grhl3 mutant mice associated with premature closure of the canal. Conversely, inhibition of cell death in the periderm resulted in an arrest of closure. Once closed, re-opening of the canal occurred in a wave, triggered by terminal differentiation of the epithelium. Understanding these complex processes involved in canal development sheds light on the underlying causes of canal atresia.

Microsecretory adenocarcinoma of the external ear canal.

Microsecretory adenocarcinoma (MSA) is a recently described salivary gland tumor characterized by unique histomorphologic and immunohistochemical features as well as recurrent MEF2C::SS18 gene fusion. Since 2019, 24 cases have been reported in the literature, primarily arising in the oral cavity, with a single reported case arising in the parotid gland. Here, we present a case of MSA that arose in the external ear canal in an 89-year-old woman and was discovered during management of vertigo symptoms. Excisional biopsy of the lesion showed multiple fragments of squamous epithelium with hyperplastic changes and a distinct subepithelial infiltrating neoplasm composed of bland cells forming tubules and cords. Neoplastic cells expressed keratin, S100 protein, p63, and TLE1 and did not express p40, mammaglobin, pan-TRK, synaptophysin, or chromogranin by immunohistochemistry. SS18 gene rearrangement was shown with break-apart fluorescent in situ hybridization. Overall, the histomorphologic, immunohistochemical, and cytogenetic findings confirm a diagnosis of MSA arising in a unique extraoral location.

Expression of EGFR and p16 in Squamous Cell Carcinoma of External Auditory Canal.

The expression of EGFR and p16 in the external auditory canal squamous cell carcinoma (EACSCC) and their impacts on oncological outcomes were not well studied. Seventeen-one consecutive patients who were treated for EACSCC at Kobe University Hospital from 1995 to 2018 were enrolled in this study. The expression of EGFR, and p16 were evaluated and their impacts on oncological outcomes were statistically analyzed. Positive expression of EGFR was observed in 62 patients (87%). Strong positive expression of p16 were observed in 18 patients (32.4%), and weakly positive expression in 30 patients (42.3%), respectively. While the number of the patients with negative EGFR expression were limited, all the surgically treated patients with negative EGFR expression have been alive without disease. In the patients with T3 & T4a EACSCC, prognosis of the patients with positive p16 expression EACSCC tended to be better than those with negative p16 expression. These results suggest the clinical significance of EGFR and p16 expressions in the patients with advanced EACSCC to predict oncological outcomes.

The effect of BMP-2, BMP-4 and BMP-6 on bone destruction of cholesteatoma presence.

OBJECTIVE: The aim of our study was to investigate the relationship between the destruction of temporal bone structures, ossicular chain destruction, dissemination of cholesteatoma and the expressions of bone morphogenetic proteins (BMPs), BMP-2, BMP-4 and BMP-6 in patients with acquired cholesteatoma. MATERIAL AND METHODS: This study was performed in a total of 80 patients with cholesteatoma and without cholesteatoma who had undergone surgery due to chronic otitis media. The patients were grouped as the study and the control groups. The study group comprised patients with primary acquired cholesteatoma, and the control group consisted of chronic otitis media patients without cholesteatoma. The samples were obtained from cholesteatoma tissue and the external acoustic meatus skin in study group patients and they were obtained from the external acoustic meatus skin only in control group patients. The Reverse Transcriptase Polymerase Chain Reaction method was used for the measurements of BMPs, BMP-2, BMP-4 and BMP-6 expressions. Polymerase Chain Reaction was studied by isolation of Ribonucleic Acid from the tissue samples. RESULTS: When the expressions of BMP in the external acoustic meatus skin were compared between the study and the control groups, the BMPs, BMP-2 and BMP-6 were determined to have a statistically significant relation in the study group (p<0.05), but BMP-4 was not significant (p>0.05). When the expression of BMP in cholesteatoma tissue was investigated in the study group patients, the BMPs, BMP-2 and BMP-6 were determined with statistically significant positivity (p<0.05), but there was no significant positivity for BMP-4 (p>0.05). In the study group, there was no statistical significance between the expressions of BMPs, BMP-2, BMP-4 and BMP-6 in cholesteatoma tissue, in the external acoustic meatus skin, and temporal and ossicular chain destruction, and dissemination of cholesteatoma (p>0.05). A statistically significant positivity for BMPs expression in cholesteatoma tissue was determined in patients with destruction of the incus+malleus+stapes (p<0.05). CONCLUSION: The expressions of BMPs, BMP-2 and BMP-6, were elevated in cholesteatoma tissue. Furthermore, the positivity of BMPs expression was statistically significant in patients with destruction of all the ossicles, and we think that this marker can be used for evaluation of the aggressiveness of cholesteatoma.

Expression of tumor necrosis factor-like weak inducer of apoptosis in human middle ear cholesteatoma.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) was recently identified as a member of the TNF superfamily of structurally related cytokines. It regulates TNF-α and numerous cellular responses. We investigated whether TWEAK is upregulated in human middle ear cholesteatoma compared to the skin of the normal external auditory canal (EAC). The expression of TWEAK was analyzed using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemical staining. TWEAK expression was correlated with that of TNF-α as determined by Western blotting. The expression of TWEAK and TNF-α protein was stronger in cholesteatoma tissue than in EAC skin. TWEAK was expressed to a greater degree in the suprabasal layer in the cholesteatoma than in EAC skin. The expression of TWEAK was correlated more closely with single-stranded DNA than Ki-67 immunohistochemically. These findings imply that TWEAK plays an important role in modulating TNF-α expression and apoptosis in cholesteatoma.

Isolation, culture, and identification of ceruminous gland cells.

External auditory canal (EAC) stenosis or atresia usually requires a skin graft to repair, but due to the lack of a graft containing functional glands, postoperative complications such as infection and eczema are common. The aim of this study was to isolate and characterize seed cells for the construction of tissue engineered EAC skin containing ceruminous gland by isolating and cultivating cells of ceruminous gland. In this study, EAC skin samples were harvested from adult goats for ceruminous gland cell isolation. Cell morphology and proliferation rates, expression of CK7, CK8, CK18, and CK19 (glandular cell specific-markers), and secretion of ß-defensin-1, lysozyme, and polysaccharides were evaluated at different passages to verify the presence of ceruminous gland cells and determine whether function and proliferation potential were maintained. Ceruminous glands were successfully isolated and extracted from goat EAC skin. Furthermore, the isolated glandular cells maintained robust proliferation potential, exhibited high expression of CK7, CK8, CK18, and CK19, and vigorously secreted ß-defensin-1, lysozyme, and polysaccharides in this culture system. However, expression of glandular cell specific-markers and secretory function gradually declined with increasing passage number, indicating dedifferentiation of the subcultured ceruminous gland cells after five passages. In conclusion, ceruminous glands were successfully isolated, cultured, and expanded from goat EAC skin using the serumcontaining culture system. Importantly, the isolated glandular cells retained robust proliferation potential and maintained their phenotype and function in early passages (P1-P3), indicating the method's potential application for ceruminous gland regeneration.

Comparison between auricular and standard rectal thermometers for the measurement of body temperature in dogs.

Although the rectal mucosa remains the traditional site for measuring body temperature in dogs, an increasing number of clinicians have been using auricular temperature to estimate core body temperature. In this study, 88 mature healthy dogs had body temperatures measured with auricular and rectal thermometers. The mean temperature and confidence intervals were similar for each method, but Bland-Altman plots showed high biases and limits of agreement unacceptable for clinical purposes. The results indicate that auricular and rectal temperatures should not be interpreted interchangeably.

ATP2A2 SINE Insertion in an Irish Terrier with Darier Disease and Associated Infundibular Cyst Formation.

A 4-month-old female Irish Terrier presented with a well demarcated ulcerative and crusting lesion in the right ear canal. Histological analysis revealed epidermal hyperplasia with severe acantholysis affecting all suprabasal layers of the epidermis, which prompted a presumptive diagnosis of canine Darier disease. The lesion was successfully treated by repeated laser ablation of the affected epidermis. Over the course of three years, the dog additionally developed three dermal nodules of up to 4 cm in diameter that were excised and healed without complications. Histology of the excised tissue revealed multiple infundibular cysts extending from the upper dermis to the subcutis. The cysts were lined by squamous epithelium, which presented with abundant acantholysis of suprabasal keratinocytes. Infundibular cysts represent a novel finding not previously reported in Darier patients. Whole genome sequencing of the affected dog was performed, and the functional candidate genes for Darier disease (ATP2A2) and Hailey-Hailey disease (ATP2C1) were investigated. The analysis revealed a heterozygous SINE insertion into the ATP2A2 gene, at the end of intron 14, close to the boundary of exon 15. Analysis of the ATP2A2 mRNA from skin of the affected dog demonstrated a splicing defect and marked allelic imbalance, suggesting nonsense-mediated decay of the resulting aberrant transcripts. As Darier disease in humans is caused by haploinsufficiency of ATP2A2, our genetic findings are in agreement with the clinical and histopathological data and support the diagnosis of canine Darier disease.

Sensory neurons in the human jugular ganglion.

The jugular ganglion (JG) contains sensory neurons of the vagus nerve which innervate somatic and visceral structures in cranial and cervical regions. In this study, the number of sensory neurons in the human JG was investigated. And, the morphology of sensory neurons in the human JG and nodose ganglion (NG) was compared. The estimated number of JG neurons was 2721.8-9301.1 (average number of sensory neurons ±â€¯S.D. = 7975.1 ±â€¯3312.8). There was no significant difference in sizes of the neuronal cell body and nucleus within the JG (cell body, 1128.8 ±â€¯99.7 µâ€¯m2; nucleus, 127.7 ±â€¯20.8 µâ€¯m2) and NG (cell body, 963.8 ±â€¯225.7 µâ€¯m2; nucleus, 123.2 ±â€¯32.3 µâ€¯m2). These findings indicate that most of sensory neurons show the similar morphology in the JG and NG. Our immunohistochemical method also demonstrated the distribution of ion channels, neurotransmitter agents and calcium-binding proteins in the human JG. Numerous JG neurons were immunoreactive for transient receptor potential cation channel subfamily V member 1 (TRPV1, mean ±â€¯SD = 19.9 ±â€¯11.5 %) and calcitonin gene-related peptide (CGRP, 28.4 ±â€¯6.7 %). A moderate number of JG neurons contained TRPV2 (12.0 ±â€¯4.7 %), substance P (SP, 15.7 ±â€¯6.9 %) and secreted protein, acidic and rich in cysteine-like 1 (SPARCL1, 14.6 ±â€¯7.4 %). A few JG neurons had vesicular glutamate transporter 2 (VGLUT2, 5.6 ±â€¯2.9 %) and parvalbumin (PV, 2.3 ±â€¯1.4 %). SP- and TRPV2-containing JG neurons had mainly small and medium-sized cell bodies, respectively. TRPV1- and VGLUT2- containing JG neurons were small to medium-sized. CGRP- and SPARCL1-containing JG neurons were of various cell body sizes. Sensory neurons in the human JG were mostly free of vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY). In the external auditory canal skin, subepithelial nerve fibers contained TRPV1, TRPV2, SP, CGRP and VGLUT2. Perivascular nerve fibers also had TRPV1, TRPV2, SP, CGRP, VIP, NPY and TH. However, PV- and SPARCL1-containing nerve endings could not be seen in the external auditory canal. It is likely that sensory neurons in the human JG can transduce nociceptive and mechanoreceptive information from the external auditory canal. Theses neurons may be also associated with neurogenic inflammation in the external auditory canal and ear-cough reflex through the vagus nerve.

Localisation of basic fibroblast growth factor in cholesteatoma matrix: an immunochemical study.

OBJECTIVE: To investigate the expression of basic fibroblast growth factor in the matrix of human acquired cholesteatoma compared to the deep meatal skin. This topic does not appear to have been fully investigated before. METHODS: An immunochemical study was conducted. Cholesteatoma tissues from adult patients were collected during surgery (n = 19). Control specimens were taken from the deep meatal skin (n = 8) and compared. RESULTS: A highly significant difference in basic fibroblast growth factor expression was identified between cholesteatoma and skin (mean ± standard error = 58.53 ± 3.6 per cent in cholesteatoma vs 40.6 ± 3.5 per cent in skin; p = 0.005). Both basal and parabasal keratinocytes were stained positive with basic fibroblast growth factor. Additionally, there was specific staining in the basal columnar middle-ear epithelium and mast cell membrane. CONCLUSION: Basic fibroblast growth factor plays an active role in proliferative activity of cholesteatoma through its overexpression in basal and parabasal layers of cholesteatoma matrix. Moreover, its expression in the mast cell membrane supports its role in bone resorption activity.

Clinical practice guideline: cerumen impaction.

OBJECTIVE: This guideline provides evidence-based recommendations on managing cerumen impaction, defined as an accumulation of cerumen that causes symptoms, prevents assessment of the ear, or both. We recognize that the term "impaction" suggests that the ear canal is completely obstructed with cerumen and that our definition of cerumen impaction does not require a complete obstruction. However, cerumen impaction is the preferred term since it is consistently used in clinical practice and in the published literature to describe symptomatic cerumen or cerumen that prevents assessment of the ear. This guideline is intended for all clinicians who are likely to diagnose and manage patients with cerumen impaction. PURPOSE: The primary purpose of this guideline is to improve diagnostic accuracy for cerumen impaction, promote appropriate intervention in patients with cerumen impaction, highlight the need for evaluation and intervention in special populations, promote appropriate therapeutic options with outcomes assessment, and improve counseling and education for prevention of cerumen impaction. In creating this guideline the American Academy of Otolaryngology-Head and Neck Surgery Foundation selected a panel representing the fields of audiology, family medicine, geriatrics, internal medicine, nursing, otolaryngology-head and neck surgery, and pediatrics. RESULTS: The panel made a strong recommendation that 1) clinicians should treat cerumen impaction that causes symptoms expressed by the patient or prevents clinical examination when warranted. The panel made recommendations that 1) clinicians should diagnose cerumen impaction when an accumulation of cerumen is associated with symptoms, or prevents needed assessment of the ear (the external auditory canal or tympanic membrane), or both; 2) clinicians should assess the patient with cerumen impaction by history and/or physical examination for factors that modify management, such as one or more of the following: nonintact tympanic membrane, ear canal stenosis, exostoses, diabetes mellitus, immunocompromised state, or anticoagulant therapy; 3) the clinician should examine patients with hearing aids for the presence of cerumen impaction during a healthcare encounter (examination more frequently than every three months, however, is not deemed necessary); 4) clinicians should treat the patient with cerumen impaction with an appropriate intervention, which may include one or more of the following: cerumenolytic agents, irrigation, or manual removal other than irrigation; and 5) clinicians should assess patients at the conclusion of in-office treatment of cerumen impaction and document the resolution of impaction. If the impaction is not resolved, the clinician should prescribe additional treatment. If full or partial symptoms persist despite resolution of impaction, alternative diagnoses should be considered. The panel offered as an option that 1) clinicians may observe patients with nonimpacted cerumen that is asymptomatic and does not prevent the clinician from adequately assessing the patient when an evaluation is needed; 2) clinicians may distinguish and promptly evaluate the need for intervention in the patient who may not be able to express symptoms but presents with cerumen obstructing the ear canal; 3) the clinician may treat the patient with cerumen impaction with cerumenolytic agents, irrigation, or manual removal other than irrigation; and 4) clinicians may educate/counsel patients with cerumen impaction/excessive cerumen regarding control measures. DISCLAIMER: This clinical practice guideline is not intended as a sole source of guidance in managing cerumen impaction. Rather, it is designed to assist clinicians by providing an evidence-based framework for decision-making strategies. It is not intended to replace clinical judgment or establish a protocol for all individuals with this condition, and may not provide the only appropriate approach to diagnosing and managing this problem.

Presence of hBD-1 and hBD-2 in human cerumen and external auditory canal skin.

CONCLUSION: Human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) antimicrobial peptides present in the cerumen, which is composed of exfoliated epithelial keratin and gland secretion, might provide the first line of defense against microbes in external auditory canal (EAC) skin. OBJECTIVES: Cerumen (earwax) plays a primary role in protecting the EAC skin and tympanic membrane. Even though the protection by antimicrobial peptides present in the skin secretion has been well established, little is known about the intrinsic role of the peptides in the EAC skin and cerumen. The aim of this study was to examine the presence of important antimicrobial peptides, hBD-1 and hBD-2, in the cerumen and EAC skin. MATERIALS AND METHODS: Cerumen was collected from 20 healthy adults, and the EAC skins were obtained from 12 patients who underwent middle ear surgery with canaloplasty. The presence of hBD-1 and hBD-2 was analyzed using immunohistochemistry and Western blotting. RESULTS: In the immunohistochemical study of the EAC skin, expression of hBD-1 and hBD-2 was observed in both the epithelium and the glands. The presence of hBD-1 and hBD-2 peptides in the cerumen was confirmed by Western blotting.

The Role of Rho/Rho-Kinase Pathway in the Pathogenesis of Cholesteatoma.

OBJECTIVE: To assess the role of Rho/Rho-kinase pathway in the pathogenesis of cholesteatoma. MATERIALS AND METHODS: Thirty-eight patients with cholesteatoma, who had gone mastoidectomies were enrolled in this prospective study. Cholesteatomas matrix (CM) and a piece of the external ear canal skin (EECS as control) were taken and transferred to the liquid nitrogen and kept at -86 °C for Rho A and Rho-kinase (ROCK) analysis with Western blotting and commercial ELISA kits (Cell Biolabs Inc., San Diego, CA). The tissues were homogenized by an appropriate ice-cold lysis buffer. Following centrifugation, the supernatant was taken and total protein amount was detected by the Bradford method. Thereafter, tissue homogenates were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis electrophoresis then transferred to nitrocellulose membrane where it was treated with specific monoclonal primary antibody against to ROCK-2 and HRP-conjugated seconder antibody, respectively. The protein blots were visualized with commercial x-ray film and dansitometrically analyzed by the Scion Image Program (Cell Biolabs Inc., San Diego, CA). In another series of experiments, Rho-kinase activities were assessed by ROCK-2 ELISA kits. RESULTS: There were no statistical differences in Rho A translocation between CM and EECS. However, ROCK activity was found to be lower in CM than EECS as detected by ELISA kits. Furthermore, ROCK protein expression was also significantly lower in CM than EECS as demonstrated by Western blotting. CONCLUSION: Given Rho-kinase could take essential roles in cell differentiation, the results of this study implicate that down-regulated Rho-kinase could be responsible for the keratinocyte undifferentiation seen in cholesteatoma pathogenesis.

Human ceruminous gland: ultrastructure and histochemical analysis of antimicrobial and cytoskeletal components.

The ceruminous glands in the skin of the human external auditory canal are modified apocrine glands, which, together with sebaceous glands, produce the cerumen, the ear wax. Cerumen plays an important role in the protection of the ear canal against physical damage and microbial invasion. We studied the morphology of the glandular cells by light and electronmicroscopy. Antimicrobial and cytoskeletal components of the ceruminous glands were investigated by immunohistochemical methods. Numerous antimicrobial proteins and peptides are present in the ceruminous glandular cells: beta-defensin-1, beta-defensin-2, cathelicidin, lysozyme, lactoferrin, MUC1, secretory component of IgA. These data indicate a crucial role in the innate host defense against diverse pathogens. The apocrine secretion mechanism is a special mode of secretion by which the apical part of the cell cytoplasm surrounded by a membrane is pinched off. We could show that the presence of actin filaments, CK 19 and CK 7, seems to play a role in the pinching-off mechanism. Finally, we showed the secretion of lipid vesicles from the ceruminous gland. We could extend the number of detected antimicrobial peptides and proteins in human ceruminous glandular cells that protect the surface of the external auditory meatus. In addition, we detected proteins involved in the apocrine secretion mode of the ceruminous gland.

The role of S100A1 in external auditory canal cholesteatoma.

S100 proteins were reported to be involved in different biological activities such as transduction of intracellular calcium signalling. It has been reported that each member of the S100 protein family exhibits a distinct tissue-specific pattern of expression. Furthermore, altered S100 protein expression and function correlate with many diseases. The expression of S100A1 was reported to be increased in different tissue with hyperplasia. The external auditory canal cholesteatoma (EACC) is a benign hyperplasia of the auditory meatal skin. However, without treatment, EACC destroys adjacent tissue. Seventeen EACC specimens were collected and investigated immunohistochemically against S100A1. Normal auditory meatal skin served as control. In the EACC, S100A1 showed a more homogeneous staining pattern, positively expressed throughout the epithelial layers. The keratin debris showed no detectable expression of S100A1. In the auditory meatal skin (control), S100A1 was only expressed in the basal layer of the epithelium. We showed that there are different staining patterns in normal auditory meatal skin, middle ear cholesteatoma and external auditory canal cholesteatomas. The immunoreactivity increased with the stage of the disease. Contrary to that reported previously in the middle ear cholesteatomas, the reactivity was strongest in the upper epithelial layers. In our collective, the epithelial matrix of the EACC showed strong reactivity throughout all the layers. Surprisingly, there is no study regarding the connection between growth factors and S100A1. In previous experiments we showed significant increase of growth factors in EACC. This correlates with our new data concerning S100A1. This is the first study to show the different reactivity pattern of S100A1 in the external auditory canal cholesteatoma.

Expression of the gap junction proteins connexin 26 and connexin 43 in human middle ear cholesteatoma.

CONCLUSION: The results of this study showed upregulated expression and a change in localization of both connexin 43 (Cx43) and Cx26 in human middle ear cholesteatoma compared to those in normal retroauricular skins (RASs) and ear canal skins (ECSs). This suggests that perturbations of intercellular communication through gap junctions may be associated with the pathology of human cholesteatomas. OBJECTIVE: Cholesteatomas in the middle ear require intercellular signal exchange through gap junctions as well as intracellular signal pathways for the hyperproliferation and differentiation of epithelial cells. Cx is a gap junction protein involved in intercellular communication. The objective of this study was to analyze the expression and possible roles of Cx43 and Cx26 in human cholesteatoma compared to normal epithelium. MATERIAL AND METHODS: Ten RASs, 10 ECSs and 10 cholesteatomas were obtained during middle ear operations. Immunohistochemical staining, Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect Cx43 and Cx26. The expression patterns of Cx43 and Cx26 were also compared with that of the proliferation marker Ki67. RESULTS: In human cholesteatomas, Cx43 was expressed in whole suprabasal layers, except in the basal layer, and Cx26 was usually expressed in the suprabasal and basal layers. However, normal RASs showed weak expression of Cx43 in the upper spinosal and granular layers (with no expression in the basal layers) and restricted localization of Cx26 in the basal layer. The expression of Cx43 and Cx26 in ECSs was weak but showed similar patterns to that of cholesteatoma. RT-PCR and Western blotting showed that the expression of Cx43 and Cx26 was higher in cholesteatoma than in RASs. Epithelial cells expressing Cx43 and Cx26 in cholesteatoma were not exactly identical to Ki67-expressing cells on immunohistochemical staining.

Do tissue spears used to clear ear canal pus improve hearing? A case series study of hearing in remote Australian Aboriginal children with chronic suppurative otitis media before and after dry mopping with tissue spears.

OBJECTIVE: To determine whether the use of tissue spears to remove otorrhoea from Aboriginal children's ear canals improves hearing in the affected ear. DESIGN: Case series study with controls. METHODS: The study comprised 61 Aboriginal children from communities in the remote arid zone of South Australia who had otorrhoea obscuring the tympanic membrane in 1 or both ears. Eighty ears were treated with tissue spears, and hearing was assessed before and after treatment. RESULTS: Forty-two children had unilateral and 19 had bilateral active disease. An additional 13 ears without otorrhoea served as controls. Improvement by 5 dB HL or greater in a four-frequency pure tone average occurred in 41.3 per cent of treated ears. Subsequently, blinded audiologists made qualitative judgements that a functional improvement in hearing had occurred after tissue spear use in 28.4 per cent of ears. CONCLUSION: Tissue spears can improve hearing thresholds in a significant proportion of children with otorrhoea. However, the duration of the effect is unknown.

[The significance of keratinocyte in hyperproliferation of middle ear cholesteatoma].

OBJECTIVE: In order to investigate the interaction between the cytokines and keratinocyte and determine the role of cytokines in hyperproliferative of chronic otitis media with cholesteatoma, we observe the expression of matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and its receptor (KGFR) in middle ear cholesteatoma. METHOD: We examined the expression of MMP9, VEGF, KGF, KGFR and Ki-67 by immunohistochemistry in 50 specimens from chronic otitis media with cholesteatoma and 15 specimens from the normal skin of external auditory meatus. Ki-67 as an evaluation of cholesteatoma proliferation markers were used to detect the keratinocyte proliferative activity. RESULT: (1) The expression of VEGF and MMP9 in cholesteatoma specimens was higher than normal skin, and the difference was statistically significant (t = 4.914, P < 0.01; t = 3.284, P < 0.01). (2) The expression of KGF and KGFR in middle ear tissues was higher than normal skin, and the difference was statistically significant (t = 4.814, P < 0.01; t = 3.104, P < 0.01); The expression of KGF and KGFR increased, and the expression of Ki-67 also correspondly increased in the cholesteatoma. (3) In the tissue MMP9 and VEGF were positive. Mean optical density increased as well. KGF expression also increased accordingly. CONCLUSION: MMP9, VEGF, KGF and KGFR proteins played an important role in hyperproliferation of cholesteatoma tissues. VEGF, MMP9 and KGF had a synergistic effect in hyperproliferation of cholesteatoma tissues.

Targeting TGF-beta1 increases hepatocyte growth factor (HGF/SF) levels in external auditory canal cholesteatoma (EACC) epithelial cell culture.

OBJECTIVE: The transforming growth factor (TGF)-beta 1 is known to have pro- and anti-angiogenic actions. Hepatocyte growth factor/scatter factor (HGF/SF) antagonizes TGF-beta1 by stabilizing SMAD transcriptional co-repressor TGIF. HGF/SF is a multifunctional polypeptide with morphogenic, motogenic, angiogenic, and proliferative capabilities. We assume HGF to be a pivotal factor during the pathogenesis of the external auditory canal cholesteatoma (EACC). In this study, we investigate the effect of antisense targeting TGF-beta1 on HGF/SF levels in the epithelial EACC-culture. MATERIALS: For 48 h, epithelial EACC cell culture was incubated with 3 and 6 mumol antisense targeting TGF-beta1, respectively. Levels of HGF/SF were determined and normalized to cellular protein. Untreated EACC cell culture and scrambled TGF-beta1-antisense served as control. In the second experiment, EACC cells were incubated with rh TGF-beta1 (2 and 4 ng/ml) for 48 h and HGF/SF was determined. RESULTS: After incubation with 3 mumol TGF-beta1-antisense, the average level of HGF/SF was measured at 43.68 pg/ml. Incubation with 6 micromol TGF-beta1-antisense showed 64.95 pg/ml. In untreated EACC (control), the average level of HGF/SF after 48 was 34.55 pg/ml. Incubation with scrambled TGF-beta1 oligonucleotide showed an average HGF/SF level of 34.41 (3 micromol) and 35.66 (6 micromol), respectively. The difference between the scrambled antisense and the targeting antisense TGF-beta1 was significant (p<0.05). After incubation with 2 ng/ml TGF-beta1, the HGF/SF levels were at 22.16 pg/ml. TGF-beta1, 4 ng/ml, resulted in 15.33 pg/ml of HGF/SF. The difference of the levels of HGF/SF after incubation with exogenous TGF-beta1 was significant (p<0.05). CONCLUSION: In this study, levels of HGF/SF increased in the epithelial EACC cell culture after incubation with 3 and 6 mumol antisense TGF-beta1 oligonucleotides depending on the concentration of the antisense. In reverse, TGF-beta1 acted as inhibiting cytokine on HGF/SF levels. In conclusion, TGF-beta1 may be a useful therapeutic agent for managing EACC.

Up-regulation of beta-catenin in external auditory canal cholesteatoma.

The external auditory canal cholesteatoma (EACC) is a rare disease with hyperproliferation and destructive growth in the adjacent structures. Down-regulation of beta-catenin (key component of the zonula adherens) is a pivotal factor for loose tissue integrity and invasiveness. Transforming growth factor beta1 (TGF-beta1) was reported to decrease beta-catenin in mammary epithelium. We investigated the abrogation of TGF-beta1 and beta-catenin expression in EACC culture cells. Cultured EACC-specimens were incubated with 6 micromol TGF-beta1 antisense. After 48 h, expression of beta-catenin was determined by means of immunohistochemistry. The cells showed an increased mural reactivity to beta-catenin, and intracellular reactivity was unchanged. The untreated cells showed a loss of beta-catenin expression at the membranes. The predominant membranous location after treatment with TGF-beta1 antisense suggests increased tendency of the cells for tissue formation and strong cell-cell adhesion rather than migratory and invasive character, and thus TGF-beta1 antisense application is a useful therapeutical strategy.