Is there a relationship between myeloperoxidase activity and conductive hearing loss in chronic otitis media complicated by cholesteatoma?

We conducted a prospective, controlled study of patients with chronic otitis media and cholesteatoma (1) to examine the expression of myeloperoxidase (MPO) using immunohistochemical staining techniques and (2) to investigate the relationship between MPO activity and the degree of conductive hearing loss in these patients. Our study population included 51 adults-26 men and 25 women, aged 18 to 58 years (mean: 37.5)-who had been diagnosed with chronic otitis media and cholesteatoma by physical examination and computed tomography (study group). Another 30 patients-13 men and 17 women, aged 18 to 52 years (mean: 32.7)-who had chronic otitis media without cholesteatoma served as the control group. Following audiometric evaluations, all patients underwent appropriate surgery. Postoperatively, cholesteatoma samples were analyzed by immunostaining for MPO positivity as a marker for acute inflammation. We found that MPO activity was present in all 51 study patients (100%) but in only 10 controls (33.3%); the difference was statistically significant (p< 0.01). In the study group, the degree of MPO activity was slight in 6 patients (11.8%), moderate in 24 patients (47.1%), and intense in 21 patients (41.2%), while in the control group, all 10 MPO-positive cases showed only a slight degree of activity. We also found a statistically significant association in the study group between the degree of MPO activity and the degree of conductive hearing loss (χ(2) = 13.518; p < 0.001). We encourage further study of all steps in the process of cholesteatoma formation.

Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma.

PURPOSE: Jak-Stat signaling pathway is one of the major signal transduction cascades which regulates most of the cellular events such as cell proliferation, differentiation, cell migration and apoptosis. This study aims to determine the activity of Jak-Stat signaling pathway in the pathogenesis of cholesteatoma. MATERIALS AND METHODS: Cholesteatoma and skin samples were obtained from 10 patients who underwent tympanomastoidectomy for chronic otitis media with cholesteatoma. Immunohistochemical analysis of cholesteatoma and skin was performed using anti-Jak1, anti-Jak2, anti-Jak3, anti-Stat1, anti-Stat2, anti-Stat3, anti-Stat4 and anti-Stat5 antibodies. The immunoreactivities in cholesteatoma and skin were quantified using H-score measurement and statistical comparison was performed. RESULTS: Jak1, Jak2, Jak3, Stat1 and Stat3 immunoreactivities were not detected in cholesteatoma; in contrast to the skin (129.8; 226.7; 33.0; 66.4;115.9). In addition, when H-score measurements of Stat2, Stat4 and Stat5 immunoreactivities were compared between cholesteatoma (172.8; 166.7; 120.0) and skin (400.0; 284.9; 292.0), statistically significant differences were found (p<0.0001, p<0.0001, p<0.0001). CONCLUSIONS: A remarkable deficiency in the family members of Jak-Stat signaling pathway was demonstrated in cholesteatoma. Therefore, perturbations in Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma.

Cholesteatoma gene expression of matrix metalloproteinases and their inhibitors by RT-PCR.

UNLABELLED: Acquired middle ear cholesteatoma is a benign keratinizing hyperproliferative squamous epithelial lesion that may result in the destruction of the bone structures surrounding the temporal bone. Recent studies show that variations in cellular production of matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) contribute to the pathophysiology of cholesteatoma. OBJECTIVE: This study aims to analyze the use of RNA amplification tests to evaluate the expression of MMP and TIMP isoforms in cholesteatomas and their correlation with disease severity. MATERIALS AND METHODS: This is a prospective study. Nineteen cholesteatoma cases at different stages were selected. RNA collected from biopsy specimens was submitted to reverse transcription polymerase chain reaction (RT-PCR) for semiquantitative amplification of MMP2, MMP3, MMP9, MMP13 and TIMP1. RESULTS: Six cholesteatomas were positive for at least one of the studied genes. Four samples amplified a single gene (MMP2 or MMP13) and two samples amplified three genes (MMP2, TIMP1 and MMP3 or MMP13). No sample amplified MMP9. CONCLUSION: RT-PCR can be used to assess MMP and TIMP gene expression in cholesteatomas despite technical difficulties. Gene expression profiles could not be related to disease severity.

Avaliação da expressão gênica de metaloproteinases de matriz e seus inibidores em colesteatomas por amplificação de ácidos nucleicos / Cholesteatoma gene expression of matrix metalloproteinases and their inhibitors by RT-PCR

O colesteatoma adquirido da orelha média é uma lesão epitelial escamosa queratinizante e hiperproliferativa benigna que pode resultar na destruição das estruturas ósseas circunvizinhas do osso temporal. Estudos recentes demonstram que alterações na produção celular de metaloproteinases de matriz (MMPs) e seus inibidores específicos (TIMPs) contribuem para a fisiopatologia do colesteatoma. OBJETIVO: Verificar a aplicabilidade da amplificação de RNA para avaliação da expressão de isoformas de MMPs e TIMPS em colesteatomas para correlação com a agressividade da doença. MATERIAIS E MÉTODOS: Estudo prospectivo. Dezenove casos de colesteatomas em diferentes estágios de evolução foram selecionados. RNA extraído das biópsias foi submetido à transcrição reversa - reação da polimerase em cadeia (RT-PCR) para amplificação semiquantitativa de MMP2, MMP3, MMP9, MMP13 e TIMP1. Resultados: Seis colesteatomas apresentaram reação positiva para pelo menos um dos genes estudados. Quatro amostras amplificaram apenas um gene (MMP2 ou MM13) e duas amostras amplificaram três genes (MMP2, TIMP1 e MMP3 ou MMP13). Nenhuma amostra amplificou MMP9. CONCLUSÃO: A avaliação da expressão gênica de MMPs e TIMPs em colesteatomas pode ser realizada por RT-PCR, apesar de dificuldades técnicas. Não foi possível realizar associação entre o perfil de expressão gênica e a agressividade da doença.

Acquired middle ear cholesteatoma is a benign keratinizing hyperproliferative squamous epithelial lesion that may result in the destruction of the bone structures surrounding the temporal bone. Recent studies show that variations in cellular production of matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) contribute to the pathophysiology of cholesteatoma. OBJECTIVE: This study aims to analyze the use of RNA amplification tests to evaluate the expression of MMP and TIMP isoforms in cholesteatomas and their correlation with disease severity. MATERIALS AND METHODS: This is a prospective study. Nineteen cholesteatoma cases at different stages were selected. RNA collected from biopsy specimens was submitted to reverse transcription polymerase chain reaction (RT-PCR) for semiquantitative amplification of MMP2, MMP3, MMP9, MMP13 and TIMP1. RESULTS: Six cholesteatomas were positive for at least one of the studied genes. Four samples amplified a single gene (MMP2 or MMP13) and two samples amplified three genes (MMP2, TIMP1 and MMP3 or MMP13). No sample amplified MMP9. CONCLUSION: RT-PCR can be used to assess MMP and TIMP gene expression in cholesteatomas despite technical difficulties. Gene expression profiles could not be related to disease severity.

Cholesteatoma-associated pathogenicity: potential role of lysosomal exoglycosidases.

UNLABELLED: The enzymatic profile of lysosomal exoglycosidases in middle ear cholesteatoma has not been well known. The assessment of glycoconjugate catabolism may contribute to a better understanding of cholesteatoma pathogenesis. OBJECTIVE: The study aim was to evaluate catabolic processes of glycoproteins, glycolipids, and proteoglycans in cholesteatoma through outlining the concentration of N-acetyl-ß-hexosaminidase (HEX), ß-glucuronidase (GLUC), and ß-galactosidase (GAL) activity as well as in serum of cholesteatoma patients and healthy volunteers. STUDY DESIGN: Acquired cholesteatomas (n = 25) and normal retroauricular skin specimens (n = 25) were taken during surgery as well as serum from cholesteatoma patients and healthy volunteers. HEX, GAL, and GLUC activity was assessed on basis of p-nitrophenol release from derivatives of the substrate (HEX: N-acetylglucosamine i N-acetylgalactosamine, GAL from galactose, and GLUC from glucuronide). RESULTS: The mean concentration of activity of HEX 1142.39 pKat/ml, GAL 8.90 pKat/ml, and GLUC 14.06 pKat/ml was significantly higher compared with the concentration of enzyme activity in normal tissue: HEX 267.65 pKat/ml, GAL 3.44 pKat/ml, and GLUC 3.90 pKat/ml. In the serum of cholesteatoma patients, the mean concentration of enzyme activities were as follows: HEX 641.62 pKat/ml, GAL 4.55 pKat/ml, and GLUC 12.80 pKat/ml and were significantly higher compared with the concentration of HEX activity (215.75 pKat/ml), GAL (1.89 pKat/ml), and GLUC (5.51 pKat/ml) in the serum of the healthy control group. In cholesteatoma compared with the normal tissue, there is an increase of the glycoconjugate catabolism due to significantly higher concentration of HEX, GAL, and GLUC activity in cholesteatoma. Cholesteatoma causes systemic reaction due to the increase of HEX, GAL, and GLUC activity in patient serum.

The imbalance of enzymatic antioxidants in cholesteatoma.

CONCLUSION: Depletion of enzymatic antioxidants was observed in cholesteatoma. However, a relationship between activity of enzymatic antioxidants and the extent of bone erosion was not found. OBJECTIVES: To measure the level of major enzymatic antioxidants in cholesteatoma, and to investigate the relationship between the level of enzymatic antioxidants and the extent of bone erosion. PATIENTS AND METHODS: The cholesteatoma and skin samples were obtained during otologic surgeries. All cases were grouped according to the number of bone erosion sites. Samples were examined biochemically and the levels of enzymatic antioxidants were measured. The results were analyzed statistically. RESULTS: Thirteen patients were included in the study. The mean level of superoxide dismutase in cholesteatoma and skin was 45.87 U/mg and 71.04 U/mg, respectively. When the catalase level was evaluated, the mean level was 5.04 U/g in cholesteatoma and 11.62 U/g in skin. The mean level of glutathione peroxidase in cholesteatoma and skin was 12.13 IU/g and 236.74 IU/g, respectively. All the results of cholesteatoma and skin samples were compared through non-parametric tests and statistically significant differences were found. However, a statistically significant difference between the levels of enzymatic antioxidants and the extent of bone erosion was not observed.

Expressions of caspase-14 in human middle ear cholesteatoma.

OBJECTIVES: Cholesteatoma is characterized by an excessive proliferation and differentiation of keratinocytes with a progressive accumulation of keratin debris. Caspase-14 is a novel regulator of keratinocyte terminal differentiation. The purpose of this study was to investigate the expression patterns and localizations of caspase- 14 in cholesteatoma and in normal external auditory canal (EAC) epithelium. METHODS: The expression levels of caspase-14 mRNA were evaluated through real-time polymerase chain reaction (PCR) and Western blotting. Cholesteatoma and normal EAC epithelium were immunostained with a monoclonal antibody to caspase-14. The localizations of immunoreactivity to the caspase-14 antibody were compared between cholesteatoma and normal EAC epithelium through immunohistochemical staining. RESULTS: As shown by real-time reverse-transcription PCR, the expression level of caspase-14 mRNA in cholesteatoma epithelium was significantly higher than in normal EAC epithelium. Caspase-14 protein was detected in both normal EAC and cholesteatoma, but its expression was shown to be greater in cholesteatoma on Western blot analysis. As shown with immunohistochemical staining, caspase-14 protein was primarily expressed in the granular layer and the upper parts of the spinous layer in cholesteatoma epithelium and in the superficial layer of normal EAC epithelium. The expression of caspase-14 was more intense in cholesteatoma tissues than in normal EAC epithelium. CONCLUSION: The increased level of caspase-14 expression in cholesteatoma tissues may play a role in terminal differentiation of epithelium and accumulation of keratin debris from external matrix.

Gelatinolytic activity of matrix metalloproteinases 2 and 9 in middle ear cholesteatoma.

BACKGROUND: Cholesteatoma of the middle ear or mastoid is a hyperproliferative disorder of keratinocytes characterized by a progressive bone erosion. Matrix metalloproteinase (MMP)-2 and MMP-9 gelatinases are endopeptidases targeting extracellular protein. Several studies examined the role of gelatinases in the pathogenesis of cholesteatoma, but the biologic mechanism by which cholesteatoma destroys the bone tissue remains unclear. OBJECTIVES: The aim of this study was to characterize the activity of MMP-2 and MMP-9 in human cholesteatoma and external auditory canal skin. METHODS: In the study, specimens of cholesteatoma and middle ear canal skin from 14 patients treated surgically at the Department of Otolaryngology were used. After two-step extraction of MMP-2 and MMP-9 from tissue samples, gelatinolytic activity was assessed with zymography. RESULTS: We noticed the augmentation of MMP-9 (p = .0001) and MMP-2 (p = .046) activity obtained from cholesteatoma in comparison with control skin. The MMP-9 active to latent ratio was significantly higher in cholesteatoma samples versus normal skin. CONCLUSION: The present study indicates that MMP-9 and, to a lesser degree, MMP-2 overexpression may be implicated in the molecular mechanisms of cholesteatoma invasion and bone destruction.

Role of N-acetyl-beta-d-hexosaminidase in cholesteatoma tissue.

Cholesteatoma is a destructive disease characterized by the progressive expansion of keratinizing squamous epithelium in the middle ear and mastoid, and chronic inflammatory reaction of the subepithelial connective tissue. N-Acetyl-beta-d-hexosaminidase (HEX) catalyzes the release of terminal non-reducing N-acetyl-d-hexosamine residues acting on glucosides and galactosides in glycoproteins, G(M2)-gangliosides and glycosaminoglycans (GAGs). In this study the activities of HEX were measured in cholesteatoma tissue and in normal skin to demonstrate a possible role of HEX in bone resorption in the area adjacent to cholesteatoma. Cholesteatomas (n = 21) and normal adult retroauricular skin (controls, n = 21), were collected from patients during surgery due to chronic otitis media. In 20 of 21 specimens a significantly higher activity of HEX was observed in cholesteatoma tissue compared with that in normal skin. Mean release of HEX from the activated cells was 68.55 +/- 30.77 nkat/g wet tissue in cholesteatoma and 31.79 +/- 10.02 nkat/g wet tissue in skin specimens. It may explain the process of bone resorption in the area adjacent to cholesteatoma, i.e. ossicles or temporal bone. This study suggests that drugs inhibiting HEX activity, such as iminocyclitols, may be useful in cholesteatoma treatment.

Matrix metalloproteinase 2: an important genetic marker for cholesteatomas.

AIM: This study is to determine the MMP2s presence in cholesteatomas and whether complicating cholesteatomas show a higher immunohistochemical expression of matrix metalloproteinase 2. Cholesteatoma produces enzymes that cause bone erosion like matrix metalloproteinase 2 (MMP2). MATERIAL AND METHODS: We analyzed the expression of MMP2 in invasive (causing complications) compared to latent cholesteatomas (not causing complications). A cross-sectional study with nineteen slides and paraffin blocks of cholesteatomas derived from mastoidectomies were located and processed, including 8 invasive and 11 latent cholesteatomas. Immunohistochemical technique was empregated to MMP2. RESULTS: The results are expressed as 0, + (to low), ++ and +++(high) according to the quantity and color of the immunohistochemical staining of MMP2. Higher expression of MMP2 was observed in 7 (87.5%) of the 8 invasive cholesteatomas. With respect to latent cholesteatomas, higher expression of MMP2 was observed in 27.3% (3 cases), with Fishers exact test indicating a significant difference (p=0.015). CONCLUSIONS: Cholesteatoamas express MMP2 and Invasive cholesteatomas had high MMP2 compared to latent cholesteatomas.

The localization of matrix metalloproteinases-8 and -13 in cholesteatoma, deep-meatal and post-auricular skin: a comparative analysis.

CONCLUSION: The presence of matrix metalloproteinase (MMP)-8 and MMP-13 was found to be significantly higher in cholesteatoma compared with post-auricular skin. The results show that the control group used has implications for further studies. OBJECTIVES: To compare the presence of MMP-8 and MMP-13 in cholesteatoma, deep meatal and post-auricular skin. Our null hypothesis was that there was no difference in expressions of MMP-8 and MMP-13 in the three groups. MATERIALS AND METHODS: The study was carried out in a secondary care specialist centre and used prospective retrieval of specimens for immunohistological localization of MMP-8 and MMP-13. Eleven patients undergoing cholesteatoma surgery were recruited for the study. Eleven cholesteatoma specimens, 10 deep meatal skin specimens and 10 post-auricular skin specimens were analysed. Specimens were analysed by immunohistochemistry using monoclonal antibodies to MMP-8 and MMP-13. Two observers scored the slides independently in a blind fashion. RESULTS: The presence of MMP-8 and MMP-13 was found to be significantly higher in cholesteatoma compared to post-auricular skin (p=0.02, p=0.03, respectively). There were no significant differences in expression of MMP-8 and MMP-13 between cholesteatoma and deep meatal skin (p=0.08, p=0.09, respectively). There were no significant differences in the control groups.

Matriz Metaloproteinase 2: um importante marcador genético para colesteatomas / Matrix Metalloproteinase 2: an important genetic marker for cholesteatomas

Este estudo foi desenvolvido para determinar a presença de MMP2 em colesteatomas humanos e observar se colesteatomas que complicam (invasivos) apresentam uma maior expressão imunohistoquímica de Matriz Metaloproteinase 2 (MMP2). Colesteatomas produzem enzimas que causam erosão óssea, como a MMP2. MATERIAL E MÉTODO: Analisamos a expressão imunohistoquímica de MMP2 em colesteatomas invasivos, comparando-os aos latentes. Um estudo de corte transversal com dezenove lâminas e blocos parafinados de colesteatoma, derivados de mastoidectomias, foram desparafinados e submetidos à técnica imunohistoquímica com anticorpos anti-MMP2. RESULTADOS: Os resultados foram expressos em 0 (tênue), + (leve), ++ (moderado) e +++ (intenso), de acordo com a intensidade da expressão de MMP2. As expressões 0 e + foram denominadas Fraca e as expressões ++ e +++, Forte. Dos 8 colesteatomas invasivos, 7 apresentaram Forte expressão de MMP2 (87,5 por cento). Com relação aos colesteatomas latentes (11), apenas 3 apresentaram Forte expressão de MMP2 (27,3 por cento), com um teste exato de Fisher significante (p= 0,015). CONCLUSÃO: Colesteatomas expressam MMP2 e colesteatomas invasivos expressam MMP2 com maior intensidade, em relação aos latentes.

AIM: This study is to determine the MMP2Æs presence in cholesteatomas and whether complicating cholesteatomas show a higher immunohistochemical expression of matrix metalloproteinase 2. Cholesteatoma produces enzymesthat cause bone erosion like Matrixmetalloproteinase 2 (MMP2). MATERIAL AND METHODS: We analyzed the expression of MMP2 in invasive (causing complications) compared to latent cholesteatomas (not causing complications). A crosssectional study with nineteen slides and paraffin blocks of cholesteatomas derived from mastoidectomies were located and processed, including 8 invasive and 11 latent cholesteatomas. Immunohistochemical thecnique was empregated to MMP2. RESULTS: The results are expressed as 0, + (to low), ++ and +++(high) according to the quantity and color of the immunohistochemical staining of MMP2. Higher expression of MMP2 was observed in 7 (87.5 percent) of the 8 invasive cholesteatomas. With respect to latent cholesteatomas, higher expression of MMP2 was observed in 27.3 percent (3 cases), with FisherÆs exact test indicating a significant difference (p=0.015). CONCLUSIONS: Cholesteatoamas express MMP2 and Invasive cholesteatomas had high MMP2 compared to latent cholesteatomas.

Expression patterns of Ki-67 and telomerase activity in middle ear cholesteatoma.

BACKGROUND: Keratinocytes in cholesteatoma demonstrate uncoordinated hyperproliferation, migration, and invasion properties. There is a controversy regarding the impact of Ki-67 and telomerase activities on cellular proliferation in cholesteatoma. We studied expression of Ki-67 protein and telomerase activity in cholesteatoma and its relationship with clinical findings. METHODS: The expression level of Ki-67 protein was examined by immunohistochemical analysis of 51 cholesteatomas and 6 skin tissues obtained from patients during ear surgery. Telomerase activity was determined in 23 samples of cholesteatomas and 6 skin samples by polymerase chain reaction-based telomeric-repeat amplification protocol assay. RESULTS: The presence of Ki-67 protein was observed in 21 (41.2%) of 51 samples of acquired cholesteatoma. The average Ki-67 labeling index in the cholesteatoma group was 28.9 +/- 9.2 and was higher than that in the skin group (18.2 +/- 6.1). Telomerase activity was detected in 2 (8.7%) of 23 samples of cholesteatoma (21 of them were Ki-67 staining positive and 2, negative) and in 3 (50%) of 6 of control skin samples (p < 0.05). CONCLUSION: This study showed increased expression of Ki-67 in cholesteatoma, whereas there was no significant difference in rate of Ki-67 positive staining between skin and cholesteatoma (p = 0.066). Telomerase activation is a rare event in cholesteatoma. We assume that the absence of telomerase may lead to generation dysfunctional telomeres what in turn may impair the proliferative capacity of cholesteatoma.

Characterization of patterns of expression of protein kinase C-alpha, -delta, -eta, -gamma and -zeta and their correlations to p53, galectin-3, the retinoic acid receptor-beta and the macrophage migration inhibitory factor (MIF) in human cholesteatomas.

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin in the middle ear cavity. Due to roles in cell proliferation, apoptosis and differentiation members of the protein kinase C (PKC) family could be involved in disease progression. This study focuses on the expression of protein kinase C-alpha, -delta, -eta, -gamma and -zeta in the epithelial tissues of 56 human cholesteatomas and their correlations with those of previously characterized distributions of p53, galectin-3, retinoic acid receptor-beta (RARbeta) and macrophage migration inhibitory factor (MIF). We have previously reported this marker set to be correlated with keratinocyte differentiation in human cholesteatomas. Our present data clearly show that the percentage of PKC-alpha (but not PKC-delta, -gamma, -eta and -zeta)-immunopositive cells in epithelial tissue fro recurrent cholesteatomas was significantly higher than in non-recurrent cases. Correlations between the PKC isoenzymes and the biological markers were non-uniform. PKC-alpha (but not PKC-delta, -gamma, -eta and -zeta) expression in epithelial cholesteatoma cells correlated significantly and positively with the percentages of p53-immunopositive cells. The patterns of PKC-alpha and -delta expression, but not of PKC-gamma, -eta and -zeta, correlated significantly and positively with galectin-3 expression. In addition, the correlation levels between the expression of PKC-alpha and -delta and that of galectin-3 varied depending on the infection and recurrence status. Presence of RARbeta correlated significantly (and positively) with the expression of PKC-gamma and -zeta and also in relation to the infection and recurrence status. MIF correlated presence significantly (and positively) with that of the five PKCs under study, depending on whether the cholesteatomas were non-infected or infected as well as non-recurrent or recurrent. In conclusion, the present study suggests that modifications occurring at the level of keratinocyte differentiation in human cholesteatomas involve distinct effectors, to which the activation of PKC-alpha, -delta, -eta, -gamma and -zeta can be added.

Telomerase activity and cell proliferation index in cholesteatoma.

CONCLUSIONS: Telomerase activity was expressed in cholesteatomas, and cellular proliferation was significantly higher in cases where the telomerase activity was positive. Telomerase activity was also closely related with cellular proliferation in chronic hyperproliferating tissues such as cholesteatomas. OBJECTIVE: Telomerase activity is detected in most malignant tumors and is also known to have a close relationship with cell proliferation. Cholesteatoma shows cellular hyperproliferation. We studied telomerase activity in cholesteatoma and its relationship with cellular proliferation and clinical findings. MATERIAL AND METHODS: Cholesteatoma tissue was obtained from 40 patients during middle ear surgery. Telomerase activity was measured using a telomeric repeat amplification protocol method. As a cellular proliferation index, expression of Ki-67 was measured by means of immunohistochemical staining. Posterior auricular skin was used as a control. Telomerase activity was compared with Ki-67 expression. Clinical features such as hearing loss, the extension of cholesteatoma, the degree of bone destruction and the cause of cholesteatomas were compared with telomerase activity and the cellular proliferation index. RESULTS: Telomerase activity was positive in 21/40 cholesteatomas (52.5%), but absent in the control group. The average Ki-67 labeling index in the cholesteatoma group was 32.84+/-10.13, higher than that in the control group (21.83+/-7.76) (p<0.05). The average Ki-67 labeling indices of the 21 telomerase activity-positive and 19 telomerase activity-negative cholesteatomas were 37.76+/-8.53 and 27.39+/-9.06, respectively. The Ki-67 labeling index was significantly higher in telomerase-positive cholesteatomas (p<0.05). The clinical features did not show a relationship with either telomerase activity or the cellular proliferation index.

[Detection of the activity of MMP2 and MMP9 in middle ear cholesteatoma].

OBJECTIVE: To study the relationship between the activity of MMP2, and bone resorption in middle ear cholesteatoma. METHOD: Specimens from 41 cases of middle ear cholesteatoma and 20 cases of external auditory canal skin were analysed for molecular corresponding to MMP2 and MMP9 by using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Zymography. RESULT: The active levels of MMP2 and MMP9 in the cholesteatoma, which were (0.954+/-0.411) and (2.676+/-0.734) respectively, were obviously higher than that in the external auditory canal skins, which were (0.355+/-0.160) and (1.166+/-0.443) gray area x mg(-1) x ml(-1) respectively, and showed close relationship (P < 0.01). The active levels of MMP2 and MMP9 in extensive cholesteatoma, which were (1.062+/-0.401) and (2.946+/-0.134) respectively, were significantly increased in comparison with in localized cholesteatoma which were (0.701+/-0.318) and (2.193+/-0.601) gray area x mg(-1) x ml(-1) respectively. No significant difference was observed between recurrent cases and first cases (P > 0.05). CONCLUSION: The increase of activity of MMP2 and MMP9 in cholesteatoma indicated that MMP2 and MMP9 may play a vital role in the bone destruction associated with cholesteatoma.

Matrix metalloproteinases in a gerbil cholesteatoma model.

OBJECTIVE: The goal of this study was to determine whether expression of matrix metalloproteinases (MMP) and tumor necrosis factor-alpha (TNF-alpha) occurs in a gerbil cholesteatoma model, which may be involved early in the pathogenesis of cholesteatoma. Study design Atelectasis was induced by transpalatal, bilateral Eustachian tube obstruction on gerbil ears (n = 60). Tympanic membranes were removed at days 0, 3, 7, 14, 28, 56, and 84, and quantitative analysis of TNF-alpha and MMPs was performed. RESULTS: Atelectasis and middle ear inflammation increased with time. TNF-alpha and MMP levels increased (P < 0.0083) with time, inflammation, and atelectatic stage with some variability seen between different MMPs. CONCLUSION: Elevation of TNF-alpha and MMPs associated with progressive tympanic membrane atelectasis suggests a possible role for these inflammatory mediators in the pathogenesis of cholesteatoma.

Telomerase activity, telomere length, and apoptosis: a comparison between acquired cholesteatoma and squamous cell carcinoma.

BACKGROUND: Cholesteatoma disease is characterized by accumulation of keratinizing epithelium. Several molecular markers of tumor formation have been found in cholesteatoma (e.g. upregulation of matrix metalloproteinases, c and activation of angiogenesis). Other molecular findings clearly distinguish between cholesteatoma and malignant tumors (e.g., lack of chromosomal instability, intact checkpoint responses). To further distinguish the molecular mechanisms in cholesteatoma from malignant tumors, the authors determined telomerase activity and telomere length in both tissue types. METHODS: To evaluate the role of telomerase activation and telomere length in cholesteatoma, 29 cholesteatoma samples and 9 squamous cell carcinomas were analyzed for telomerase activity and telomere length. In addition, the rate of apoptosis was determined in both groups, using the TdT-mediated dUTP nick end labeling technique. RESULTS: As previously described, a high proportion of squamous cell carcinoma exhibited telomerase activity (6/9, 66%). By contrast, a significantly lower rate of telomerase activity was found in cholesteatoma samples (1/29, 3.4%, p = 0.0002). Despite the differences in telomerase activity, the telomere length was similar in cholesteatoma (mean length 7.43 kb) and in squamous cell carcinoma (mean length 7.99 kb; difference not significant, p = 0.1364). The low rate of telomerase activity in cholesteatoma was accompanied by significantly higher rates of apoptosis in cholesteatoma (mean 30%) compared with squamous cell carcinoma tissue (mean 3%, p = 0.0031). CONCLUSIONS: Taken together, these data show that telomerase activation is a rare event in cholesteatoma and that the absence of telomerase activity is accompanied by high rates of apoptosis in cholesteatoma. It is proposed that the absence of telomerase limits the proliferative capacity of cholesteatoma by induction of apoptosis, whereas the presence of telomerase allows immortal growth of squamous cell carcinoma.

Expression of matrix-degrading cysteine proteinase cathepsin K in cholesteatoma.

Cholesteatoma is a nonneoplastic lesion of the middle ear space or mastoid that is histologically characterized by a progressive bone erosion of the ossicles and surrounding bone. Several matrix-degrading enzymes have been implicated as mediators of this bone erosion. Because the novel cysteine proteinase cathepsin K has been shown to play a central role in bone resorption, we examined the expression of this enzyme in tissue specimens of cholesteatoma. Tissue specimens of 9 patients with cholesteatoma were obtained during middle-ear surgery. Expression of cathepsin K mRNA was determined by RT-PCR using specific primers. Immunohistochemical analysis of cathepsin K protein expression in tissue sections was performed by using the streptavidin-alkaline phosphatase technique. Expression of both cathepsin K mRNA and protein was detected in areas affected by cholesteatoma, whereas specimens of nonaffected ear cartilage and surrounding tissue were not positive. In addition, cathepsin K was detected in numerous multinucleated giant cells, particularly osteoclasts at the site of bone degradation. In contrast, keratinized squamous epithelium was negative for cathepsin K. These data demonstrate that the matrix-degrading cysteine proteinase cathepsin K may be involved in bone erosion in cholesteatoma. Strong expression of this collagenolytic enzyme in osteoclasts suggests that these cells are mainly involved in cathepsin K-mediated bone destruction.

Detection of macrophage migration inhibitory factor (MIF) in human cholesteatomas and functional implications of correlations to recurrence status and to expression of matrix metalloproteinases-3/9, retinoic acid receptor-beta, and anti-apoptotic galectin-3.

OBJECTIVES: To investigate whether the expression of the macrophage migration inhibitory factor (MIF) 1) is detectable, 2) changes in relation to recurrence and infection status, and 3) relates to the levels of expression of growth regulators/differentiation markers, including galectin-1, -3, and -8, retinoid acid receptors (RAR)]-alpha, -beta, and -gamma, binding sites for sarcolectin, and invasion markers (cathepsins -B and -D, and matrix metalloproteinases [MMP]-2, -3, and -9) in human cholesteatomas. STUDY DESIGN: An analysis of 56 cholesteatomas resected by the same surgeon using canal wall up and canal wall down surgical procedures. METHODS: The immunohistochemical levels of expression of MIF and the proteases were quantitatively determined (using computer-assisted microscopy) on routine histologic slides by specific antibodies, and statistically correlated to parameters of the other markers determined previously in conjunction with data on apoptosis/proliferation. RESULTS: MIF expression was detected. It was significantly higher in the epithelium (P =.002) and vessels (P =.04) of the connective tissues (but not in the connective tissue itself) of recurrent as opposed to non-recurrent cholesteatomas. The MIF expression is significantly correlated (P =.006) to the RAR beta expression in non-infected cholesteatomas, and to MMP-3 (P <.01) and anti-apoptotic galectin-3 (P =.01) in infected cholesteatomas. The level of MIF expression was also correlated significantly to MMP-9 (P = 0.003), RAR beta (P <.001), and galectin-8 (P =.003) expression in the cholesteatomas regardless of their infection status. CONCLUSIONS: MIF expression in human cholesteatomas is related to the levels of biologic aggressiveness reflected in their recurrence status and MMP expression, and to the differentiation status reflected in their galactin and RAR beta expressions. Together with galectin-3, it could cooperate to form an anti-apoptotic feedback loop.