Cholesteatoma Tissue Research Articles

Up‐Regulation of Peroxidase Proliferator‐Activated Receptorγ in Cholesteatoma

To evaluate the localization and expression of peroxidase proliferator-activated receptor (PPAR)gamma in cholesteatoma epithelium. Experimental study. Reverse-transcription polymerase chain reaction was performed on cholesteatoma tissues from 10 adult patients undergoing tympanomastoid surgery for middle ear cholesteatoma and on 10 samples of normal external auditory canal skin tissue. The expression levels of PPARgamma to glyceraldehyde-3-phosphate dehydrogenase transcripts were semiquantified by densitometry. We also characterized the cellular localization of the PPARgamma protein immunohistochemically. Ki-67 was also localized to compare the proliferative activity of cells in cholesteatoma epithelium and in normal external auditory canal skin. PPARgamma mRNA and protein were detected in normal external auditory canal skin and in cholesteatoma epithelium. The expression level of PPARgamma mRNA in cholesteatoma was significantly increased compared with that in normal external auditory canal skin. PPARgamma protein was expressed in cells mainly in the granular and prickle cell layers. However, the intensity of its expression was generally decreased in the parabasal layer of the cholesteatoma epithelium. Ki-67 was expressed in the nuclei of cells in the basal and parabasal layers, and a greater number of cells were Ki-67 immunopositive in cholesteatoma epithelium. PPARgamma is up-regulated in the cholesteatoma epithelium compared with normal external auditory canal skin. These results suggest that PPARgamma may play an important role in the pathogenesis of cholesteatoma.

Expression of female sex hormone receptors in human middle-ear cholesteatomas

The purpose of this study was to establish the eventual presence of progesterone receptor (PGR) and oestrogen receptor (EGR) in human middle-ear cholesteatoma (MECh) tissues and to compare their expression between male and female patients. An immunohistochemical technique was employed for detection of PGR- and EGR-specific immunoreactivity in MECh samples using formalin-fixed paraffin-embedded tissue sections. The positive results were verified with reverse transcriptase polymerase chain reaction (RT-PCR). The morphological study revealed stable expression of PGR in suprabasal layers of all cholesteatoma samples. Weaker immunoreactivity for PGR was demonstrated in external auditory canal skin (EACS) samples in comparison with MECh, while PGR-specific staining was not observed in retroauricular skin (RAS) samples. EGR was detected only at mRNA levels. Stronger expression of EGR PCR products was disclosed in female cholesteatoma samples, while PGR mRNA was predominantly detected in male cholesteatoma specimens. Our preliminary experimental results give us ground to assume that female sex hormones may stimulate proliferation and affect differentiation of MECh keratinocytes.

Nucleoplasm staining patterns and cell cycle–associated expression of Ki-67 in middle ear cholesteatoma

Objectives To compare Ki-67 expression patterns in middle ear cholesteatoma with the corresponding retroauricular and external auditory canal skins, and to determine the cell cycle–dependent localization of Ki-67. Material and methods MIB-1 monoclonal antibody was used for comparative assessment of proliferative activity of middle ear cholesteatoma, external auditory canal skin, and retroauricular skin samples on formalin-fixed paraffin-embedded tissue sections. Primary keratinocytes from cholesteatoma tissue were isolated and subjected to kinetic analysis of the cell cycle. Results Higher proliferative activity was established in cholesteatoma in comparison with retroauricular and external auditory canal skins. Three different staining patterns have been described. Kinetic analysis revealed continuous expression of Ki-67 during all active phases of the cell cycle and remained “silent” in resting cells. Conclusion The established correlation between the staining patterns and cell cycle–associated expression of Ki-67 specifies Ki-67 as a reliable and stable marker of proliferation for middle ear cholesteatoma.

Photodynamic effect of argon and diode laser on cholesteatoma cell cultures after intravital staining with absorption enhancers

Chronic epitympanic otitis media, or chronic suppurative osteitis, is a destructive form of chronic middle-ear inflammation. The therapy of choice is complete surgical removal of the squamous epithelium from the middle ear. It is often impossible to inspect all areas of the middle ear with the posterior canal wall intact. Not all recesses can be reliably monitored with the microscope, particularly in the area of the antrum and hypotympanum. Residual squamous epithelium here causes frequent recurrences following cholesteatoma surgery. This study examines the effect of argon and diode lasers on cholesteatoma tissue. The aim is to develop a laser treatment selectively directed against cholesteatoma cells that can be performed after cholesteatoma surgery to eliminate any residual squamous epithelium. Intraoperatively harvested monolayer-cultured cholesteatoma cells stained in vivo with various absorption enhancers served as the in vitro examination model. Argon (499 nm) and diode lasers (810 nm) were applied since their irradiation has an appropriate tissue penetration depth and is absorbed by various chromophores such as neutral red (475-500 nm), fluorescein (488 nm), and indocyanine green (790-810). Intracellular staining of cultured cells increased the optical density at the wavelength corresponding to the dye. Neutral red damaged 50-60% of cultured cells merely by intracellular accumulation at high concentrations. An additive cell destruction of about 30% was achieved by also applying argon laser irradiation. Fluorescein diacetate caused no appreciable stain-induced damage to cultured cholesteatoma cells. Argon laser irradiation destroyed up to 60% of the cultures. Indocyanine green resulted in only minor damage to cultured cells. The diode laser destroyed up to 60% of the irradiated cells. Selective staining of cholesteatoma cells was not achieved with any of the dyes examined. Thus, other stained tissue could be damaged. Staining and subsequent laser irradiation destroys up to 60% of cultured cholesteatoma cells. Unstained irradiated cells are not affected. Indocyanine green and fluorescein are nontoxic and may thus be used as absorption enhancers. The diode and argon lasers appear to be basically suitable. Cell staining is not selective, i.e., other tissues would also be stained and damaged. To avoid such unwanted damage, it would be desirable to couple the chromophore to a specific antibody that binds only to cholesteatoma cells.

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.

Suppressive Activity of Vitamin D3 on Matrix Metalloproteinase Production From Cholesteatoma Keratinocytes In Vitro

There is much evidence that degradation of the extracellular matrix is essential for the development of cholesteatomas and that this is induced by activation of matrix metalloproteinases (MMPs). Vitamin D3 (VD3) has several well-recognised biological activities, including suppression of MMP production. The present study, therefore, was undertaken to examine whether VD3 could suppress MMP production from cholesteatoma keratinocytes in vitro. Keratinocytes (2.5 × 105 cells/mL) induced from cholesteatoma tissue specimens were cultured with various concentrations of VD3. After one hour, lipopolysaccharide was added to the cell cultures at 100 μg/mL. The culture supernatants were then collected and assayed for MMP-1 and MMP-3 by ELISA. We also used ELISA to measure the levels of both TIMP (tissue inhibitor of metalloproteinase)-1 and TIMP-2 in culture supernatants. Addition of VD3 into keratinocyte cultures caused the suppression of MMP and TIMP production, which was increased by LPS stimulation. This was dose-dependent. The present results showing the suppressive activity of VD3 on the production of MMPs, which are responsible for tissue remodeling, strongly suggest that VD3 would be a good candidate for an agent in the medical treatment of, or prophylaxis for, cholesteatomas.

Micronucleus frequency in acquired middle ear cholesteatoma

Objective—To determine the micronucleus (MN) frequency of acquired cholesteatoma tissue using an MN assay.Material and Methods—Eighteen patients were diagnosed as having chronic otitis media with acquired cholesteatoma and were divided into primary and secondary acquired cholesteatoma groups. Cholesteatoma tissue and normal tissue specimens from the external ear canal skin were taken from the patients during surgical operations. MN frequencies of cholesteatoma and control samples were determined according to standard criteria.Results—The MN frequencies of the cholesteatoma and control tissues were 0.54%±0.31% and 0.24%±0.11%, respectively (p<0.01). MN frequencies for the primary and secondary acquired cholesteatoma groups were 0.63%±0.36% and 0.46%±0.26%, respectively (p>0.05). MN frequencies in cholesteatoma patients without and with complications were 0.42%±0.19% and 0.85%±0.37%, respectively (p<0.05).Conclusion—MN frequencies were found to be increased in cholesteatoma tissues when compared with external ear canal skin. The MN frequency in five cases with complications was higher than in cases without complications. These results indicate that there could be associations between MN frequency and acquired cholesteatoma and between MN frequency and complications.

Localization of osteopontin at calcification sites of cholesteatoma: possible role as a regulator of deposition of calcium phosphate in the middle ear

Objective: Recently, we showed that osteopontin (OPN), a major acidic phosphorylated glycoprotein of bone, participates in the pathological calcification that occurs as a result of chronic otitis media. To investigate the possibility of OPN as a common regulator of the pathological calcification in the middle ear, we here examined whether or not OPN is localized at the calcification sites of cholesteatoma, which is clearly different from chronic otitis media in the pathogenesis. Methods: Middle ear tissues including cholesteatoma were obtained from 32 cases who underwent tympanoplasty. The tissues of 29 cases were used for the immunohistochemistry of OPN and CD68, the marker of macrophages, and in situ hybridization of OPN mRNA. And those of the other three for reverse transcriptase polymerase chain reaction (RT-PCR). Results: Middle ear tissues including cholesteatoma were histologically classified as to the degree of calcification. In hyalinized tissues with macroscopic calcification, OPN was immunohistochemically found at the calcification sites. In inflammatory tissues with microscopic calcification, OPN was also found in the calcifying foci, and many OPN mRNA-expressing cells, as determined by in situ hybridization, were located near the foci. Moreover, immunohistochemical double staining for OPN and CD68 showed that the OPN-expressing cells were CD68-positive, indicating the cells were macrophages. In inflammatory tissues without calcification, immunohistochemistry of CD68 and in situ hybridization of OPN mRNA revealed that most OPN mRNA-expressing cells were CD68-positive. The expression of OPN mRNA in cholesteatoma tissues was also demonstrated by means of RT-PCR. Conclusion: OPN secreted by exudate macrophages was localized in the pathological calcification that occurs in association with cholesteatoma. These results are consistent with the observations in chronic otitis media. Therefore, it is suggested the possibility that OPN might contribute as a common regulator of the deposition of calcium phosphate in the middle ear.

Expression of hypoxia inducible factor 1 alpha and Von Hippel Lindau protein in human middle ear cholesteatoma.

To evaluate the role of hypoxia in cholesteatoma progression. Immunohistochemical analysis of paraffin-embedded human specimens. Thirteen middle ear cholesteatomas and 10 samples of normal human external ear canal skin were stained immunohistochemically for the presence of hypoxia inducible factor 1 alpha (HIF 1 alpha) and Von Hippel Lindau protein. Specimens were then analyzed semiquantitatively. Staining for both antibodies could be detected in all cholesteatomas (perimatrix and matrix), as well as in the samples of normal human ear canal skin. Cholesteatoma specimens showed statistically significant increased staining when compared with normal human skin and mucosa. The age of patients and relapse surgery affected immunohistochemical staining of HIF 1 alpha and Von Hippel Lindau protein. Elevated staining intensities for HIF 1 alpha and Von Hippel Lindau protein in cholesteatoma tissue indicated that cholesteatoma may be hypoxic. In addition, our data indicated that relapse cholesteatomas yield a higher degree of hypoxia than ears without surgery.

Detection of Chlamydia pneumoniae in cholesteatoma tissue: any pathogenetic role?

Acquired cholesteatoma is a complication of chronic otitis media that is usually associated with an intense local inflammatory reaction. Cholesteatoma probably arises from epithelial migration close to an ongoing host inflammatory response attributable to a chronic bacterial infection. Chlamydia pneumoniae is an intracellular microorganism associated with several pathologic conditions originally considered noninflammatory, including asthma, atherosclerosis, and Alzheimer disease. To investigate a possible relationship between C. pneumoniae and the development of cholesteatoma, tissue was studied in three different layers by polymerase chain reaction analysis. The results were compared with those relative to other two common middle-ear pathogens, Mycoplasma pneumoniae and Haemophilus influenzae. Cholesteatoma specimens were collected from 32 patients undergoing middle ear surgery. A series of 5 microm-thick specimens were obtained at three different tissue levels, internal (matrix), intermediate (perimatrix), and external (granulation tissue), and processed by polymerase chain reaction for detection of C. pneumoniae, H. influenzae, and M. pneumoniae. Fragmentation and polymerase chain reaction amplification were carried out using two substantially different techniques. C. pneumoniae was detected with either polymerase chain reaction techniques in the internal layers in 16 of the 32 cholesteatomas (50%), associated with a positive finding in the intermediate layer in two cases and in the external layer in one case. Four specimens contained H. influenzae, always in the external layer, whereas none contained M. pneumoniae. The close relationship between cholesteatoma and C. pneumoniae demonstrated by the findings of this study could suggest a direct cause and effect link between the pathogen action and the clinical manifestations. Otherwise, a facilitated colonization by C. pneumoniae and chronic pathology of the ear could both take origin from a peculiar immunologic background of the host.

진주종 유래 섬유아세포에서의 Insulin-Like Growth Factor I과 Binding Protein의 역할

Background and Objectives: Insulin like growth factor-I is an important growth regulator for epidermis and its activity is regulated by specific insulin like growth factor binding protein. Authors observed the expression of mRNA of Insulin-like Growth FactorI (IGF-I) and mRNA of Insulin-like Growth Factor Binding Protein (IGFBP) in fibroblast of patient with cholesteatoma to find out the function of fibroblast in the progress of cholesteatoma. Materials and Method: During the operation in middle ears, the cholesteatoma and normal skin tissue were extracted from 10 patients with Cholesteatoma. Some cytokines were added such as Interferon β, Interferon γ and TNF α to those extraced fibroblast. We analyzed the expression of IGF-I, IGFBP-3, and IGFBP-5 by semi-quantative RT-PCR. Results: After stimulation by inflammatory cytokine, the expression of IGF-I from cholesteatoma was significantly increased ,while the expression of IGFBP-5 was significantly reduced. Conclusion: These results indicate that cholesteatoma fibroblast could play an important role in the proliferation of cholesteatoma tissue. (J Clinical Otolaryngol 2003;14:113-117)

Osteoclast stimulating and differentiating factors in human cholesteatoma.

To investigate the expression of osteoclast-activating and differentiating factors and to study the occurrence of osteoclast precursor cells and osteoclasts in acquired human cholesteatoma tissue. We examined 21 cholesteatoma samples versus 18 normal auditory canal skin specimens for the expression of osteoprotegerin ligand (OPGL), osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Immunohistochemistry and computer-assisted microscopy using markers CD4, CD11a, CD11b, CD14, CD51, CD68, and TRAP obtained the detection of osteoclast cell lineage. An increased expression of the investigated cytokines M-CSF, OPG, and OPGL was demonstrated by immunohistochemistry and RT-PCR in cholesteatoma tissue compared with normal external meatal skin. Several CD4-positive cells exhibited a co-expression for OPGL within the perimatrix of cholesteatoma. The presence of osteoclast precursor cells was confirmed in all samples of cholesteatoma tissue. This study reveals that the number of osteoclast precursor cells is markedly increased in the perimatrix of cholesteatoma tissue. Our results support a concept described for inflammatory arthritis: the inflammation related to cholesteatoma induces bone resorption by release of OPGL from activated T-cells and triggers osteoclastogenesis. This could be a major target for drugs to inhibit osteoclast formation and bone resorption and may be an adjunct in cholesteatoma management.

Expression of a cathelicidin antimicrobial peptide is augmented in cholesteatoma.

Antimicrobial peptides are active defense components of innate immunity. Their importance was confirmed at epithelial surfaces as immediate barrier effectors in preventing infection. Cathelicidins are peptide antibiotics that are receiving increasing attention. Several studies have shown that overexpression of cathelicidin results in augmented protection against bacterial infection and prevention of local infection and systemic invasion of microbes. The goal of the study was to investigate whether cathelicidin is upregulated in cholesteatoma epithelium compared with normal skin. Twenty patients from a prospective study of cholesteatoma tissues and normal skins were enrolled in the study. The specimens were divided into two portions. One portion was used for subsequent RNA studies; the other was used for immunohistochemical staining. Reverse transcriptase-polymerase chain reaction was used to assess the expression levels of cathelicidin messenger RNA (mRNA) both in cholesteatoma and in normal skin. Presumptive concentration of cathelicidin mRNA and beta2-microglobulin mRNA was evaluated. Ratio of beta2-microglobulin to cathelicidin was analyzed in each group. The expressions of cathelicidin in cholesteatoma and normal skin epithelium were investigated by an immunohistochemical technique. Cathelicidin mRNA in cholesteatoma epithelium was increased 5.5-fold compared with normal skin of the ear canal. In cholesteatoma epithelium, cathelicidin was located in all the layers, but in the normal skin it was expressed only in the granular and prickle cell layers. Cathelicidin is augmented in cholesteatoma epithelium, and the data in the present study are in agreement with the hypothesis that cathelicidin is likely to act as a key component in the first line of defense at the surface epithelium.

Cycline-dependent kinase inhibitor, p27 (KIP1), is associated with cholesteatoma.

Cyclin-dependent kinases (CDKis) can arrest the cell cycle, which in turn inhibits the cell proliferation. P27 (KIP1) is a CDKi and acts as a tumor suppressor gene. In this study, we aimed to investigate the role of p27 CDKi in cholesteatoma, a disease characterized by the presence of hyperproliferative squamous epithelium. Immunohistochemical staining of 15 cholesteatoma and 18 control ear canal skin samples, which were taken intraoperatively, was performed for p27 positivity. The monoclonal antibodies to p27 were used for immunohistochemical staining of the sections. The streptavidin-biotin horseradish method was used. The number of cells staining positive for p27 was calculated, and the intensity of p27 positivity was graded. P27 positivity was obtained in 9 (50%) of 18 skin tissues. In the cholesteatoma tissues, p27 positivity was found only in 2 (13.3%) tissue samples. The difference between the groups were statistically significant (P =.03). The mean numbers of p27 positivity were 11.8 +/- 15.5 and 1.4 +/- 3.8 (mean +/- standard deviation) in the skin and cholesteatoma samples, respectively. This difference was also statistically significant (P =.02). The p27 results of primary and secondary cholesteatoma samples were not significantly different (P =.3). The results of p27 were not related to the gender of the patients (P =.8). P27 is involved directly or indirectly in the occurrence of cholesteatoma. Alterations of p27 levels in keratinocytes can influence the proliferative state of the keratinocytes. Altered p27 levels in cholesteatoma may suggest a molecular pathology in cholesteatoma. The search for significance of CDKis seems promising to better understand the pathogenesis of cholesteatoma.

Invasiveness of fibroblasts from experimental cholesteatomas.

Cultured fibroblasts derived from experimental gerbil cholesteatoma tissue exhibit an invasive phenotype in comparison with normal fibroblasts. Aural cholesteatomas are enlarging accumulations of keratin debris caused by keratinizing squamous epithelium in the middle ear. They characteristically result in the destruction of adjacent tissues, specifically bone erosion. The mechanisms by which cholesteatomas relentlessly invade the structures of the temporal bone are varied, but it has been suggested that one factor contributing to the aggressive nature of cholesteatomas is the transformation of resident fibroblasts into an invasive phenotype. The ability of cultured normal and cholesteatoma fibroblasts to invade a basement membrane matrix in a Boyden chamber assay was examined. Less than 1% of gerbil fibroblasts invaded the matrix, compared with almost 10% of the invasive HT-1080 fibrosarcoma cells. Normal and cholesteatoma fibroblasts did not differ from each other in their invasive potential. Normal fibroblasts and fibroblasts from induced cholesteatomas do not exhibit the invasive phenotype characteristic of true neoplastic cells.

중이 진주종에서 형질전환성장인자 α 및 β의 발현에 관한 연구

Background and Objectives: The cholesteatoma consists of keratinizing squamous epithelium in middle ear cavity. These abnormal behavior of cholesteatoma epithelium seems to be induced by presence of a heavy immune cell infiltrate releasing different cytokines and growth factor in high amount. Materials and Method: This study investigated the presence of transforming growth factor-alpha (TGF- α), beta (TGF- β) in the mucosa of cholesteatoma specimens of human middle ear cholesteatoma tissue (N =17) and external auditory canal skin were obtained from patients during ear surgery as a control group. Results: Immunostaining for TGF- α showed a cytoplasmic staining pattern in epithelial cells of normal skin and cholesteatoma. In normal skin samples the expression of TGF- α was restricted to epithelial cells in the basal layer and parabasal layer but all epithelial cell layers in cholesteatoma were positive with prominent staining of the basal cells. A number of infiltrated cells in cholesteatoma matrix also expressed TGF- α immunostaining. Cholesteatoma tissue showed a strongly enhanced expression of TGF- β in lymphocytes and fibroblasts of stroma, particularly in an area of heavy inflammatory infiltration. Conclusion: According to the well known roles of the TGF- α and β, these results suggest that TGF- α is an important factor for the hyperproliferative behavior of cholesteatoma epithelium and TGF- β protect the infiltration into the matrix of cholesteatoma. (J Clinical Otolaryngol 1999;10:178–183)

Role of Bcl-xL protein in differentiation and apoptosis of human middle ear cholesteatoma epithelium.

To compare the mechanisms of proliferation, differentiation, and apoptosis in middle ear cholesteatoma epithelium with those of normal external ear canal epithelium. The localizations of the expression of Bcl-xL protein and involucrin and the presence of apoptotic cells were determined for tissue slices of middle ear cholesteatoma epithelium and compared with the findings for normal external ear canal epithelium. In addition, SCC-25/bcl-xL transfectants showing the overexpression of Bcl-xL were used to investigate the effect of this protein on the expression of involucrin, which is a marker of epithelial cell differentiation. Cholesteatoma tissue specimens were surgically excised from 10 patients. Normal skin specimens collected from the external ear canal of the 10 patients were used as control specimens. The expression of Bcl-xL was detected in the vicinity of the basal cell layer of both the cholesteatoma epithelium and the normal external ear canal epithelium. Conversely, the expression of involucrin (ie, a marker of epithelial cell differentiation) increased in proportion to the shallowness of the epithelial layer. In situ labeling detected apoptotic cells in the spinous and granular cell layers of cholesteatoma tissue sections and similar findings in the normal external skin specimens. Western blot analysis confirmed that the expression of involucrin protein was the same in both wild-type SCC-25 cells and the SCC-25/bcl-xL transfectants. In both the cholesteatoma epithelium and the normal external ear canal epithelium, differentiation and apoptosis begin when the epithelial cells separate from the basal cells. The mechanisms behind these changes, at least in apoptosis, appear to be controlled by the expression of the Bcl-xL protein.

Expression of transforming growth factor-alpha (TGF-alpha) in cholesteatoma.

This study aims at elucidating the role of cytokines in the mechanism of proliferation of cholesteatoma epithelium by investigating the mode of expression of epidermal growth factors, such as TGF-alpha. The subjects of this study were patients who had undergone operation for middle ear cholesteatoma. Skins of the bone region of the external ear canal (normal skin) of the same patients were used as the negative control. The mode of expression of TGF-alpha was studied by immunohistochemistry and in situ hybridization. In the immunohistochemical study, there were no conspicuous differences observed between cholesteatoma tissues and normal skin. After in situ hybridization, expression of TGF-alpha mRNA was mainly observed in the epidermal basal cell layer in the normal skin, while in the cholesteatoma epidermis with severe inflammatory cell infiltration, expression of TGF-alpha mRNA was observed up to layers superior to the basal cell layer. The expression of TGF-alpha mRNA is greatly affected by subepithelial connective tissue, strongly suggesting involvement of paracrine regulation in proliferation of cholesteatoma epithelium.

Expression of annexin II in human middle ear cholesteatoma

Annexin II has previously been discovered to have involvements in DNA replication and metabolism, bone resorption, and osteoclast formation. In our work, Western blotting and immunohistostaining studies revealed the presence of annexin II in human cholesteatoma tissue. With monoclonal mouse antiannexin II antibody, a 36,000 dalton protein (annexin II) was identified in the cholesteatoma protein extract. Immunoalkaline-phosphatase staining selectively localized annexin II to the keratinocytes in the basal and spinous layers of the cholesteatoma tissue. In normal human skin, annexin II is expressed mainly in the cytoplasmic membrane of its keratinocytes in the basal layer without significant staining in its nucleus. However, annexin II is expressed in both the cytoplasmic membrane and the nucleus of the keratinocytes in basal and spinous layers of human cholesteatomas. Our findings indicate a possible physiologic role of annexin II in keratinocyte cell hyperproliferation during development of human cholesteatoma. (Otolaryngol Head Neck Surg 1998;118:324-8.)

Cell proliferation and apoptosis in human middle ear cholesteatoma.

To compare the pattern of proliferation and apoptotic cell death in cholesteatoma tissues with that in normal skin. The cholesteatoma tissue samples were excised from 10 patients during surgery. Normal skin specimens collected from the external ear canal of 6 of the 10 patients were used as controls. In all cholesteatoma tissue samples, apoptotic cells were not seen in the basal cell layer, but they were observed in the suprabasal, prickle, and granular cell layers. In skin specimens obtained from normal external ear canal skin, in which the suprabasal cell layer was comparatively small, similar kinetics of apoptotic cell death were observed. Immunohistochemical analysis using a monoclonal antibody to proliferation cell nuclear antigen demonstrated the presence of proliferating cells in the basal and suprabasal cell layers of the normal external ear canal skin, whereas in the cholesteatoma tissue samples, large numbers of proliferation cell nuclear antigen-positive cells were also observed in the prickle and granular cell layers. Proliferation in cholesteatoma epidermal cells is not uncontrolled, as it is in malignant tumors. Our results demonstrate an increase in the rate of proliferation and apoptotic cell death in cholesteatoma epidermis.