Human Middle Ear Cholesteatoma Research Articles

Super-enhancer Acquisition Drives FOXC2 Expression in Middle Ear Cholesteatoma.

Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in middle ear cholesteatoma. It is known that histone acetylation destabilizes the nucleosome and chromatin structure and induces gene activation. The association of histone acetylation with chronic inflammatory diseases has been indicated in recent studies. In this study, we examined the localization of variously modified histone H3 acetylation at lysine 9, 14, 18, 23, and 27 in paraffin-embedded sections of human middle ear cholesteatoma (cholesteatoma) tissues and the temporal bones of an animal model of cholesteatoma immunohistochemically. As a result, we found that there was a significant increase of the expression levels of H3K27ac both in human cholesteatoma tissues and the animal model. In genetics, super-enhancers are clusters of enhancers that drive the transcription of genes involved in cell identity. Super-enhancers were originally defined using the H3K27ac signal, and then we used H3K27ac chromatin immunoprecipitation followed by sequencing to map the active cis-regulatory landscape in human cholesteatoma. Based on the results, we identified increased H3K27ac signals as super-enhancers of the FOXC2 loci, as well as increased protein of FOXC2 in cholesteatoma. Recent studies have indicated that menin-MLL inhibitor could suppress tumor growth through the control of histone H3 modification. In this study, we demonstrated that the expression of FOXC2 was inhibited by menin-MLL inhibitor in vivo. These findings indicate that FOXC2 expression under histone modifications promoted the pathogenesis of cholesteatoma and suggest that it may be a therapeutic target of cholesteatoma.

Evaluation of YAP signaling in a rat tympanic membrane under a continuous negative pressure load and in human middle ear cholesteatoma

Objectives: Mechanotransduction plays an important role in cell-proliferative activities. Negative pressure in the middle ear is thought to be an important factor related to the etiology of acquired middle ear cholesteatoma. However, the correlation between negative pressure in the middle ear and the mechanism of middle ear cholesteatoma formation remains unclear. In this study, we investigated the expression of key molecules for mechanotransduction immunohistochemically.Methods: An immunohistochemical analysis was performed using anti-Wnt5a (a marker of alternative Wnt signaling), -Yes-associated protein (YAP) (a marker of mechanosensing) and -pYAP (phosphorylated YAP at Ser 127: inactivated YAP) antibody in the tympanic membrane (TM) under a negative pressure load and in human middle ear cholesteatoma tissues.Results: The number of Wnt5a-positive cells had increased and YAP nuclear translocation was observed in epithelial and mesenchymal cells in the pars flaccida (PF) of the TM under a negative-pressure load and in human middle ear cholesteatoma tissues.Conclusions: We demonstrated that negative pressure in the middle ear might possibly induce cell proliferation PF of TM in response to mechanical force (mechanotransduction) through YAP nuclear translocation mediated by alternative Wnt signaling, thus affecting human middle ear cholesteatoma formation.

The expression and significance of DDR2 and MMP-13in human middle ear cholesteatoma

Objective:To ascertain the expression level of discoidin domain receptors 2(DDR2) and matrix metalloproteinase-13(MMP-13) in human middle ear cholesteatoma tissues and further investigate their roles in bone destruction and correlation.Method:Using immunohistochemical S-P method to detect the expression level of DDR2 ,MMP-13 in 30 specimens with middle ear cholesteatoma epithelial tissue and 10 specimens with normal ear epithelial tissues．At the same time,the computer image analysis system was used to detect the expression of the two indexes by the quantitative analysis,analyzing their expression in middle ear cholesteatoma epithelial tissue and the correlation between the extent of bone destruction．Result:The expression of DDR2 and MMP-13 were confirmed in human middle ear cholesteatoma epithelial tissues and normal ear epithelial tissues． The mean optical density of DDR2 and MMP13 in human middle ear cholesteatoma epithelial tissues which were tested by the computer image quantitative analysis system were higher than those in normal ear epithelial tissues(P<0．05).The expression of DDR2 and MMP-13 in middle ear cholesteatoma epithelial tissues were positively correlated(r=0.738,P<0.01).In addition,the two indexes were associated and relative to the extent of bone destruction,the wider the extent of bone destruction was,the higher the expression of both indexes(P<0．05)．Conclusion:DDR2 and MMP-13 may play important roles in bone destruction of human middle ear cholesteatoma．.

Expression and significance of PDK1 and NF-κB in the middle ear cholesteatoma

Objective:To detect the expression of 3-phosphoinositide-dependent kinase-1(PDK1) and nuclear factor-κB(NF-κB) in human middle ear cholesteatoma tissue and to analyze their correlation.Method:Immunohistochemical method was taken to detect the expression and location of PDK1 and NF-κB in 60 cases of cholesteatoma tissue and 30 cases of normal ear skin specimens.SPSS 19.0 software was used to analyze the data. Result:Immunohistochemistry revealed PDK1 was positive in cytoplasm and the positive expression rate in cholesteatoma was significantly higher than normal skin (P <0.05); NF-κB expressed in the nucleus of cholesteatoma and the positive expression rate in cholesteatoma significantly higher than normal skin(P <0.05);In cholesteatoma specimens,there was a significant positive correlation between protein PDK1 and NF-κB(P <0.05). Conclusion:Abnormal expression of PDK1 and NF-κB may be related to the proliferation of cholesteatoma epithelium and they reinforce each other.

In vivo over-expression of KGF mimic human middle ear cholesteatoma.

We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.

Expression of the epidermal growth factor system in human middle ear cholesteatoma

Conclusion: The detection of the HER4 receptor in 50% of cholesteatomas but never in the reference tissue, and the increased expression of its activating ligand EPI, suggest that EPI-mediated activation of HER4 might play a role in cholesteatoma growth. Objective: To investigate the expression of the epidermal growth factor (EGF) system in human middle ear cholesteatoma. Methods: Forty-seven patients referred for surgery due to cholesteatoma were included in the study. Clinical data were collected. Biopsies of cholesteatoma and skin from the external ear canal were obtained during surgery. mRNA expression was quantified with real-time PCR. The corresponding proteins were visualized using immunohistochemistry. Results: A systematic investigation of all four receptors, HER1, HER2, HER3, and HER4, and the ligands EGF, transforming growth factor (TGF)-α, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin (EPI) of the EGF system is presented. At the mRNA level, the study demonstrates an up-regulation of mRNA encoding EPI and AR. In contrast HER1 and EGF were down-regulated. HER4 mRNA could be detected in 50% of cholesteatoma and 20% of reference tissues, and the HER4 protein was detectable only in cholesteatoma tissue. HER1 and HER2 were also visualized by immunohistochemistry, whereas the ligands EPI, AR, and EGF were undetectable with our methods.

Retraction: Expression of PLC-1 in Human Middle Ear Cholesteatoma

Retraction: Expression of PLC-1 in Human Middle Ear Cholesteatoma

Elevated RANKL expression is associated with increased epithelial proliferation and submucosal inflammation in human middle ear cholesteatomas

Elevated RANKL expression is associated with increased epithelial proliferation and submucosal inflammation in human middle ear cholesteatomas

Retraction: Expression Pattern of Gap Junction Protein, Connexin 26 and 43 in Human Middle Ear Cholesteatomas

Retraction: Expression Pattern of Gap Junction Protein, Connexin 26 and 43 in Human Middle Ear Cholesteatomas

Expression of IL-17 and Its Role in Bone Destruction in Human Middle Ear Cholesteatoma

Purpose: The present study was designed to elucidate the immunoreactivity and protein level of IL-17 in human cholesteatomas. Procedures: The expression and localization of IL-17 and receptor activator of nuclear factor ĸB ligand (RANKL) were examined by immunohistochemistry in tissue specimens collected from 24 patients with cholesteatomas. The cellular sources of IL-17 were assessed by double staining with CD4. The level of IL-17 protein was determined using an enzyme-linked immunosorbent assay. The degree of bone destruction was compared with the IL-17 immunoreactivity. Results: IL-17 immunoreactivity was detected in the inflammatory cells in the granulation tissues and there were increased levels of IL-17 protein. The localization of IL-17 expression coincided with CD4-positive lymphocytes. IL-17 was correlated with the cells positive for RANKL. The degree of bone destruction was dependent on the number of infiltrated IL-17-positive cells. IL-17-driven pathology was observed in the congenital type as compared with the acquired type. Conclusions: The present study suggests that IL-17 is related to the pathogenesis of the disease.

Psoriasin (S100A7), an antimicrobial peptide, is increased in human middle ear cholesteatoma

Conclusion. Increased psoriasin in cholesteatoma epithelium may play a role in epithelial inflammatory response and differentiation. Objectives. Cholesteatoma is characterized by excessive keratinocyte differentiation leading to inflammation, granulation tissue, and osteolytic activity. Moreover, psoriasin may act as an antimicrobial peptide, stimulate granulocytes, and control keratinocyte differentiation. The purpose of this study was to investigate the differential expression patterns and the localization of psoriasin in cholesteatoma and in normal external auditory canal skin. Materials and methods. Expression levels of psoriasin mRNA were evaluated by real-time PCR and Western blotting. Cholesteatoma-affected and normal external auditory canal skin samples were immunostained with monoclonal antibody to psoriasin. Localization of immunoreactivity to psoriasin antibody was then compared. Results. By real-time PCR, expression levels of psoriasin mRNA in cholesteatoma were significantly higher than in normal external auditory canal skin, and this was confirmed by Western blotting. Immunohistochemical staining revealed that psoriasin protein is mainly expressed in the granular layer and in the upper parts of the spinous layer in cholesteatoma epithelium, but that it is expressed in the superficial layer of normal external auditory canal skin.

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.

Differential Expression of Human Beta Defensin 2 and Human Beta Defensin 3 in Human Middle Ear Cholesteatoma

The purpose of this study was to investigate the differential expressions of human beta defensin (hBD) 2 and hBD-3 in human middle ear cholesteatoma epithelium. The expressions of hBD-2 and hBD-3 were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining. Samples were obtained from 10 patients who underwent middle ear surgery for middle ear cholesteatoma. Real-time RT-PCR and Western blot analysis showed that the messenger RNAs and proteins of hBD-2 and hBD-3 were higher in the cholesteatoma epithelium than in normal external auditory canal skin. In cholesteatoma epithelium, hBD-2 and hBD-3 activities were present in the upper granular layer and in the prickle cell layer, but in the normal skin they were poorly expressed in all layers. Increased expressions of hBD-2 and hBD-3 in cholesteatoma epithelium suggest that cholesteatoma, a chronic inflammatory state of middle ear keratinocytes, may induce an innate immune response. That the induction of hBD-2 was found to be more intense than that of hBD-3 in cholesteatoma epithelium implies that hBD-2 is the major effector in terms of chronic epithelial inflammatory responses.

Expression of secretory leukocyte protease inhibitor in middle ear cholesteatoma

Secretory leukocyte protease inhibitor (SLPI) is a cationic protein and a member of the innate immunity-associated protein family. The main function of SLPI is to protect local tissue against the detrimental consequences of inflammation. We undertook this study to investigate the expression of SLPI in human middle ear cholesteatoma tissue as compared with normal external auditory canal skin. Cholesteatoma tissues and external auditory canal skin samples were obtained from eight patients during middle ear surgery. The expression levels of SLPI mRNA and protein were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Immunohistochemical staining was performed in both tissue groups using anti-SLPI antibody. Real-time RT-PCR showed that SLPI mRNA levels in cholesteatoma tissues were increased 7.8-fold on average as compared with normal auditory canal skin. Western blotting analysis showed that SLPI protein expression in cholesteatoma epithelium is up-regulated versus external auditory canal skin epithelium. Immunohistochemical staining for SLPI showed that SLPI is expressed mainly in the stratum granulosum and in subepithelial inflammatory cells. These findings imply that SLPI contributes to host protection against inflammatory cell and destructive enzymes in the chronic inflammatory state of cholesteatoma by affecting the innate immune system.

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

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

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.

Overexpression of ErbB‐2 Protein in Human Middle Ear Cholesteatomas

The purpose of this study is to verify the hypothesis that ErbB-2 protein is overexpressed in human middle ear cholesteatomas and to elucidate the relationship between overexpression of ErbB-2 protein, cell proliferation, and apoptosis. Prospective review of 20 patients between 2001 and 2003 with middle ear cholesteatoma. Middle ear cholesteatoma matrix and retroauricular skin were immunostained with anti-ErbB-2, Ki-67, and single-stranded DNA (ssDNA) antibody. The distribution of immunoreactivity to these antibodies and labeling indices were compared between cholesteatoma and retroauricular skin. In matrix of middle ear cholesteatoma, ErbB-2 and ssDNA were expressed in the keratinocytes of all layers and Ki-67 was expressed in the keratinocytes of the basal, lower spinous, and occasionally granular layer. In retroauricular skin, ErbB-2 and Ki-67 were expressed in the keratinocytes of the basal and occasionally lower spinous layer and ssDNA was expressed in the keratinocytes of all layers. Labeling indices against anti-ErbB-2, Ki-67, and ssDNA antibody were significantly greater in cholesteatoma as compared with retroauricular skin. In cases of cholesteatoma, ErbB-2 protein was overexpressed and cell proliferation and apoptosis of keratinocytes were accelerated. ErbB-2 protein could modulate terminal differentiation and apoptosis in the keratinocytes of all layers in cholesteatoma matrix and cell proliferation in the keratinocytes of the basal and lower spinous layer in normal skin.

Expression of Rb in Human Middle Ear Cholesteatoma

Expression of Rb in Human Middle Ear Cholesteatoma

Detection of human papillomavirus in cholesteatomas.

The presence of human papillomavirus DNA in cholesteatoma may have some role in the development of middle ear cholesteatoma as well as in papilloma. In the present study, polymerase chain reaction and in situ hybridization with human papillomavirus (HPV)-6 and -11 DNA probes were used to detect the presence of HPV DNA in 32 human middle ear cholesteatomas. Only one specimen contained HPV-6 DNA. Although its occurrence may have been coincidental, it is also possible that the hyperproliferative epithelium of cholesteatomas might have some relationship with HPV infections.

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)