Cholesteatoma Samples Research Articles

Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium

Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.

The Role of p21 and p53 Proteins in Congenital Cholesteatoma

Immunoexpression analysis of p53 and p21 in congenital cholesteatoma with assessing their exact localization in cholesteatoma layers and the level of expression. P53 and p21 are apoptosis-related molecules that regulate cell cycle. These markers have been not completely evaluated in congenital cholesteatoma, and the role of apoptosis in congenital cholesteatoma is also not completely understood. Congenital cholesteatoma samples, a study group (n = 13) and normal auditory meatal skin, a control group (n = 12) from patients who underwent surgery for cholesteatoma were included in the study. Acquired cholesteatoma samples were used as a comparable group (n = 12). Tissue sections were investigated with the immunohistochemistry technique based on binding of biotinylated secondary antibody with the enzyme-labeled streptavidin with using appropriate primary antibodies. Cells with immunoexpression of analyzed antigens: p53 and p21 were defined as antigen positive. In each section, cells were counted, and the percentage of positive cells was determined. The level of significance was set at p < 0.05. The mean percentage of p21-positive was statistically significant higher in congenital cholesteatoma than in the control group (p < 0.05). There was no meaningful difference between congenital and acquired cholesteatoma with respect to p21 expression. There was significant difference between congenital and acquired cholesteatoma regarding p53. Up-regulation of p21 protein is expected to play a significant role in CC development. Apoptosis is an important process in the pathogenesis of congenital cholesteatoma. It seems reasonable to perform studies on apoptosis in congenital cholesteatoma in the prospective way taking into consideration more specimens.

Optical imaging with a high‐resolution microendoscope to identify cholesteatoma of the middle ear

High-resolution optical imaging is an imaging modality that allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation, and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Ex vivo imaging of histopathologically confirmed samples of cholesteatoma and uninvolved middle ear epithelium. Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin and eosin staining to allow histopathological correlation. Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, whereas nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin, allowing for discrimination of cholesteatoma from normal middle ear epithelium. This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. Laryngoscope, 2012.

Telomerase activity detection in cholesteatoma

BackgroundIn cholesteatoma, there is lack of control over cell proliferation that is why ectodermal tissue grows to be an epidermoid cyst. So cholesteatoma may be considered as cell growth disorder but still the cause of this uncontrolled proliferation is unknown. ObjectiveThe aim of this study is to detect telomerase activity in cholesteatoma and meatal skin (as a control) and its relation to the clinical findings and severity of the disease. Patients and methodsSamples of cholesteatoma and meatal skin were obtained from 31 patients during ear surgery. Telomerase activity was detected using polymerase chain reaction based on telomeric repeat amplification protocol assay then collected data were statistically analyzed. ResultsTelomerase activity was positive in 17/31 (54.8%) of cholesteatoma samples and was negative in all meatal skin samples. Significant high rates of intraoperative ossicular erosion and preoperative air bone gap more than 40dB were associated with positive telomerase activity. ConclusionsTelomerase activity may play a role in the hyperproliferative nature and uncontrolled aggressive growth pattern of cholesteatoma.

The role of EGFR/PI3K/Akt/cyclinD1 signaling pathway in acquired middle ear cholesteatoma.

Cholesteatoma is a benign keratinizing and hyper proliferative squamous epithelial lesion of the temporal bone. Epidermal growth factor (EGF) is one of the most important cytokines which has been shown to play a critical role in cholesteatoma. In this investigation, we studied the effects of EGF on the proliferation of keratinocytes and EGF-mediated signaling pathways underlying the pathogenesis of cholesteatoma. We examined the expressions of phosphorylated EGF receptor (p-EGFR), phosphorylated Akt (p-Akt), cyclinD1, and proliferating cell nuclear antigen (PCNA) in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium by immunohistochemical method. Furthermore, in vitro studies were performed to investigate EGF-induced downstream signaling pathways in primary external auditory canal keratinocytes (EACKs). The expressions of p-EGFR, p-Akt, cyclinD1, and PCNA in cholesteatoma epithelium were significantly increased when compared with those of control subjects. We also demonstrated that EGF led to the activation of the EGFR/PI3K/Akt/cyclinD1 signaling pathway, which played a critical role in EGF-induced cell proliferation and cell cycle progression of EACKs. Both EGFR inhibitor AG1478 and PI3K inhibitor wortmannin inhibited the EGF-induced EGFR/PI3K/Akt/cyclinD1 signaling pathway concomitantly with inhibition of cell proliferation and cell cycle progression of EACKs. Taken together, our data suggest that the EGFR/PI3K/Akt/cyclinD1 signaling pathway is active in cholesteatoma and may play a crucial role in cholesteatoma epithelial hyper-proliferation. This study will facilitate the development of potential therapeutic targets for intratympanic drug therapy for cholesteatoma.

EGFR expression in acquired middle ear cholesteatoma in children and adults

Middle ear cholesteatomas are characterized by the presence of keratinized stratified squamous epithelium inside this cavity. It is considered to be more aggressive in childhood. In normal skin, the epidermal growth factor receptor (EGFR) is expressed in the cytoplasmic membrane of epithelial cells of the basal layer. In contrast, its expression in middle ear cholesteatoma extends to suprabasal layers. The objective of this study is to detect the presence of EGFR in cases of acquired cholesteatoma of the middle ear and correlate the expression of this receptor with patients' ages. In this cross-sectional study, cholesteatoma samples were collected from 50 patients (35 adults and 15 children) who underwent otological surgery, throughout 1 year of study. These samples were subjected to histological and immunohistochemical assays. Results were submitted to statistical analyses and main findings were: EGFR was present in the parabasal layers in 27 cases and EGFR expression was extended to all layers of the matrix in 17 cases. There were no statistically significant differences in what concerns age-related variances in EGFR expression. The intensity and location of EGFR expression in acquired cholesteatoma of the middle ear confirm the hyperproliferative capacity of keratinocytes.

Interleukin‐8 production in response to tumor necrosis factor‐alpha by cholesteatoma keratinocytes in cell culture

Keratinocytes harvested from acquired cholesteatoma and grown in cell culture will demonstrate increased interleukin-8 (IL-8) production in response to tumor necrosis factor (TNF)-alpha as compared with a control keratinocyte cell line. Immunohistochemical studies have identified IL-8 and TNF-alpha, mediators of bony destruction, in tissue samples of cholesteatoma. TNF-alpha stimulates IL-8 production in healthy epidermal keratinocyte cell lines. It is not known whether TNF-alpha stimulates IL-8 production in cultured cholesteatoma keratinocytes. Prospective controlled tissue culture experiment. Tissue from an acquired cholesteatoma was dissociated and grown in keratinocyte serum-free media for 8 weeks. Cholesteatoma keratinocytes and a control cell line of skin epidermal keratinocytes were treated with TNF-alpha. Conditioned media were harvested; production of IL-8 was measured by enzyme-linked immunosorbent assay, and cell counts were performed. At a zero concentration of TNF-alpha, mean production of IL-8 by cholesteatoma keratinocytes was 39,809 pg/mL/24hr/1 × 10(6) cells versus 1,907 pg/mL/24hr/1 × 10(6) cells from skin epidermal keratinocytes, a statistically significant difference (P < .05). The cholesteatoma keratinocytes showed a 2.1-fold increase in response to 2 pg/mL of TNF-alpha and a 2.44-fold increase in response to 20 pg/mL of TNF-alpha. The skin epidermal keratinocyte cell line demonstrated a 1.07- and 1.13-fold increase to respective concentrations of TNF-alpha. Cholesteatoma keratinocytes appear to retain cell signaling characteristics in vitro that distinguish them from skin epidermal keratinocytes. This finding may indicate that cholesteatoma keratinocytes undergo a change in behavior in vivo that is preserved after the cells are removed from the inflammatory environment of the middle ear.

Paracrine Loops of Keratinocyte Stimulation in Cholesteatoma Tissue

In relation to the keratinocyte growth factor (KGF) expression in cholesteatoma tissue, the inflammatory infiltrate present in this disease could play a relevant role in the paracrine stimulation of keratinocytes. Cholesteatoma is a temporal bone pathologic disease characterized by active proliferation of epithelial cells, with progressive growth and involvement of the neighboring middle/inner ear structures. The pathogenetic mechanism underlying the hyperproliferation of keratinocytes is not yet fully clarified. It has been suggested that keratinocyte proliferation and migration could be mediated by several autocrine and paracrine growth factors and their receptors. A previous study has reported that the expression of KGF receptor is increased in more differentiated areas of the cholesteatoma tissue, whereas the expression of the epidermal growth factor receptor is associated with proliferative and migratory areas of the lesion. Fresh cholesteatoma samples were collected during surgical procedures. Serial cryosections were examined by conventional hematoxylin and eosin or by immunofluorescence with antipancytokeratin antibodies. The ultrastructural features were analyzed by transmission electron microscopy. The expression of KGF, K1, and filaggrin in the samples was evaluated by quantitative reverse transcription-polymerase chain reaction and correlated with the dermal degree of inflammatory infiltrate and the presence of stromal cells. Increased expression of KGF was associated with strong levels of the inflammatory infiltrate. These results would suggest that KGF up-modulation is a consequence of fibroblast stimulation by inflammatory cells and that this paracrine loop could be responsible not only for the hyperproliferation of keratinocytes in cholesteatoma tissue but also for the deregulation of epidermal differentiation.

In vitro cholesteatoma growth and secretion of cytokines

Conclusion: Our results show a significant difference between skin and cholesteatoma biology in vitro. Objectives: Cholesteatoma disease is a process of destruction characterized by uncontrolled growth of squamous epithelial cells in the middle ear or temporal bone. The pathophysiology behind the cholesteatoma development is controversial, and the mechanisms driving the cholesteatoma growth, migration and destructive properties is still unclear. We aimed to provide a method to study the effect of various compounds on cholesteatoma and skin tissue growth, as well as to further investigate the biological differences between normal skin and cholesteatoma tissue. Methods: We have established a method to study cholesteatoma biopsy tissue in vitro. Cholesteatoma tissues from patients undergoing surgery for chronic otitis were grown in culture medium and compared to growth patterns and behaviour of normal retroauricular skin. Conditioned medium was analysed for various secreted cytokines. Results: We found a radial outgrowth of keratinocyte epithelium from the circular biopsies. After 5 days of culture we found a significant growth of both cholesteatoma and skin-derived cells. Cholesteatoma samples showed higher growth rate as compared with skin control cultures from the same patient. Moreover, the cholesteatoma cells showed higher production of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6 as compared with normal skin.

Cholesteatoma growth and proliferation: posttranscriptional regulation by microRNA-21.

The goal of this study was to identify novel regulatory mechanisms controlling the growth and proliferation of cholesteatoma. Specifically, the potential role of microRNAs, regulators of protein translation, was studied in cholesteatoma. This study represents a molecular biologic investigation characterizing and comparing microRNA and protein expression in cholesteatoma and normal postauricular skin. Cholesteatoma and normal skin were taken from patients at the time of surgery. Tissue was processed for RNA and protein extraction. Real-time reverse-transcriptase-polymerase chain reaction was used to assess levels of human microRNAs, reverse-transcriptase-polymerase chain reaction was used to confirm the presence of upstream regulators, and Western blot analyses were used to assess levels of downstream target proteins. Among the microRNAs investigated, human microRNA-21 (hsa-miR-21) showed a 4.4-fold higher expression in cholesteatoma as compared with normal skin (p = 0.0011). The downstream targets of hsa-miR-21, PTEN and programmed cell death 4, were found to be greatly reduced in 3 of 4 cholesteatoma samples. Proposed upstream regulators of hsa-miR-21 expression (CD14, interleukin 6R, gp130, and signal transducer and activator of transcription 3) were present in all cholesteatoma tissues. MicroRNAs represent powerful regulators of protein translation, and their dysregulation has been implicated in many neoplastic diseases. This study specifically identified up-regulation of hsa-miR-21 concurrent with down-regulation of potent tumor suppressor proteins PTEN and programmed cell death 4. These proteins control aspects of apoptosis, proliferation, invasion, and migration. The results of this study were used to develop a model for cholesteatoma proliferation through microRNA dysregulation. This model can serve as a template for further study into potential RNA-based therapies for the treatment of cholesteatoma.

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.

Increased expression of p63 and survivin in cholesteatomas

Conclusion. This study showed increased expression of p63 and survivin in cholesteatoma. Our finding indicates a putative role of p63 and survivin in the development of certain cholesteatomas. Objectives. Keratinocytes in cholesteatoma demonstrate uncoordinated hyperproliferation, migration, and invasion properties. p63 is a p53 homologue and a marker expressed in replicating keratinocytes. Survivin is an inhibitor of apoptosis protein that is abundantly expressed in most solid and hematologic malignancies. The purpose of this study was to investigate the differential expression of p63 and survivin in human middle ear cholesteatoma epithelium. Materials and methods. The expression levels of p63 and survivin protein were examined by immunohistochemical analysis of 40 cholesteatomas and 5 skin tissues obtained from patients during ear surgery. Results. Expression of p63 protein was diffusely observed in entire samples of cholesteatoma, especially in acquired cholesteatoma, compared with the control group. Congenital cholesteatoma showed variable p63 reactivity in a basal cell-like pattern. Primary and recurrent cholesteatomas showed no significant difference in p63 expression. Survivin was detected in 31 of 40 cholesteatoma samples. Acquired cholesteatomas showed especially increased survivin expression compared with congenital cases. The expression of p63 was correlated with survivin expression.

Chromosomal imbalances are associated with increased proliferation and might contribute to bone destruction in cholesteatoma

Our aim was to evaluate the copy number alterations of chromosomes 3, 7, 8, and 17 in middle ear cholesteatomas and define the association between the rate of cell proliferation and chromosome number changes. Tissues were obtained from 16 patients. Fluorescence in situ hybridization was performed on tumor imprint preparations. Cell proliferation was characterized with Ki-67 monoclonal antibody on cholesteatoma samples and on postauricular skins as control. Different degrees of aneusomy were found for all chromosomes except for chromosome 3. Chromosome copy number alterations were associated with elevated proliferative rate and related also with the aggressiveness of the lesions. Based on our results, we assume that aneusomy of chromosomes 7, 8, and 17 might play an important role during invasion of the adjacent bony structures of cholesteatoma, as well as associate with increased cell proliferation activity, which might lead to the aggressive behavior of the tissue.

R445 – Expression of Proliferative Markers in Cholesteatoma

Problem Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium believed to have hyperproliferative properties. Inflammatory stimulation of the underlying connective tissue, as well as an autocrine mechanism, may be responsible for the dysregulation and abnormal proliferative feature of the keratinocytes in cholesteatoma. Methods Comparative investigations were performed to assess the epithelial cell kinetics of cholesteatoma and auditory meatal skin. Monoclonal antibody PCNA and Ki-67 immunohistochemical staining were applied. Results Specimens of cholesteatoma samples (n=30) showed an average PCNA score of 26.6% and an average Ki-67 score of 15.9%. Auditory meatal skin(n=8) revealed an average PCNA score of 8.2% and average Ki-67 score of 4.9%. The results of this study confirm a highly increase in the proliferation rate of cholesteatoma keratinocytes, which had an PCNA score that was 3.24 times higher than the score for keratinocyte of auditory meatal skin and Ki-67 score of cholesteatoma was 3.24 times higher than auditory meatal skin. Conclusion We conclude that cholesteatoma is a hyperproliferative activity and that PCNA and Ki-67 immunohistochemical staining are valuable tool for assessing cell kinetics in cholesteatoma. Significance The cholesteatoma possesses a variable degree of proliferative activities depending on its histologic composition. The formation and accumulation of the keratin due to the continuous epithelial growth probably are important elements in inducing the inflammation.

Melanocyte localization and distribution in human cholesteatoma.

Melanocytes in skin are derived from the neural crest and colonize the epidermis in the first trimester of gestation. Melanocytes have been observed in the nasopharyngeal, inner ear and oral mucosa and should therefore be present in the middle ear mucosa. To identify and determine the distribution of melanocytes in human cholesteatoma and normal meatal skin in Caucasian adults. Human cholesteatoma (n=18) and normal meatal skin samples (n=10) were investigated immunohistochemically with anti-HMB-45 and MART-1 antibodies. Localization and distribution of melanocytes were assessed in the epidermis and cholesteatoma using an automatic analyzing system. Regular skin exhibited melanocytes within the epidermis and accounted for 10% of the total cell number. They occurred partly as membrane-bound clusters. Cholesteatoma matrix melanocytes were observed in the basal layer and exhibited an oval or roundmorphology. Decreased numbers of melanocytes in the basal layer correlated with keratinization within cholesteatoma samples. Melanocytes revealed monomorphous nuclei, abundant cytoplasm containing particles of melanin. Found adjacent to glands and blood vessels, melanocytes were also scattered among the mesenchymal cells. Accounting for 2-6% of the total cell number within the squamous epithelium, melanocyte density was significantly lower in cholesteatoma tissue than in skin. The melanocyte distribution pattern was different when comparing the epithelia of skin and cholesteatoma. The presence of melanocytes in cholesteatoma may be due to an ingrowth, consequently controlled by keratinocyte-derived signals. In terms of the pathogenesis of cholesteatoma, neither squamous metaplasia nor melanocyte metaplasia can be excluded by our data.

Pathogenesis of sinus cholesteatoma

The aim of the present study was to provide evidence for the establishment of sinus cholesteatoma, defined as postero-superior pars tensa retraction extending into the posterior tympanum and tympanic sinuses. There is clinical evidence for formation of a retraction, but there is a lack of explanation for the transition from a retraction pocket to an active and expanding sinus cholesteatoma. Epidemiological studies on incidence of postero-superior retractions of pars tensa and follow-up studies on patients with similar pars tensa retractions were performed. Additionally, expression of proliferation marker and analysis of basement membrane were studied in samples of sinus cholesteatoma. The prevalence of pars tensa pathology was between 9.2 and 24% of investigated ears. In children with manifest secretory otitis there were some sinus cholesteatomas and 5-6% severe retractions, some of those became pre-cholesteatomas, requiring treatment and controls. Immunohistochemistry of sinus cholesteatomas showed that proliferating keratinocytes were very often found within epithelial cones growing towards the underlying stroma. These growth cones exhibit focal discontinuities of the basement membrane especially in areas of intense subepithelial inflammation. As a possible explanation based on clinical and immunohistochemical findings, we propose a four-step concept for pathogenesis of sinus cholesteatoma combining the retraction and proliferation theory: (1) The retraction pocket stage. (2) The proliferation stage of the retraction pocket, subdivided in (a) Cone formation, (b) Cone fusion. (3) Expansion stage of attic cholesteatoma. (4) Bone resorption.

Expression Patterns of Ki-67 and Telomerase Activity in Middle Ear Cholesteatoma

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. 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. 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). 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.

Survival signaling and terminal differentiation in cholesteatoma epithelium

Conclusion: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. Objectives: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. Materials and methods: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. Results: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p≤0.05).

Cell Cycle Inhibitory Protein p27 in Human Middle Ear Cholesteatoma

Aim: To evaluate the immunohistochemical and molecular presentation of protein p27 in cholesteatoma. Methods: 42 cholesteatoma samples and 6 external ear canal skin (EECS) specimens were investigated and analyzed taking into consideration congenital, acquired, recurrent cholesteatoma, and EECS. Results: The expression of p27 was found in 16 (38.1%) out of 42 specimens of cholesteatoma and in 5 (83.3%) out of 6 specimens of EECS. There was a significant difference in p27-positive staining rate between EECS and cholesteatoma epithelium (p < 0.008). The presence of p27 was detected in 10 cases of acquired cholesteatoma, 2 cases of congenital and 3 cases of recurrent cholesteatoma. There was no significant difference between the presence of p27 in cholesteatoma and EECS (p = 0.01). Conclusion: The down-regulation of p27 is a key player in cell cycle control and plays an undefined role in the pathogenesis of all types 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.