Cholesteatoma Samples Research Articles

Anaerobic bacteria dominate the cholesteatoma tissue of chronic suppurative otitis media patients

The aim of this study was to investigate both the microbial composition and absolute abundance of clinically relevant bacteria in tissue specimens from patients with chronic suppurative otitis media with cholesteatoma (CSOM with cholesteatoma). Mastoid mucosa and cholesteatoma tissue from eleven subjects with CSOM with cholesteatoma, and mastoid mucosa from ten controls were examined using standard hospital culture swabs, Gram staining, bacterial 16S rRNA gene sequencing, Droplet Digital PCR (ddPCR), and multiplex PCR. Positive results from culture swabs were reported in half the CSOM with cholesteatoma samples and 1 control sample. In contrast, ddPCR detected bacterial genes copies in all 11 mucosa and cholesteatoma of CSOM subjects and 3 control samples. The average bacterial gene copies in tissue samples with CSOM with cholesteaotoma (1.6 ± 0.7 log10) was significantly higher compared to healthy controls (0.3 ± 1.6). These results were corroborated with Gram-staining that identified the large presence of Gram-positive cocci cells in the cholesteatoma tissue of CSOM subjects which were not seen in the mucosa of controls. The most abundant genus detected by sequencing in the mucosa and cholesteatoma of CSOM samples was Anaerococcus (93.5% of all reads), and genus Meiothermus (0.9%) in the control sample. The 3 samples with the highest sequencing reads (>300) were further analysed using multiplex PCR to identify the dominant Anaerococcus species. Anaerococcus hydrogenalis was the dominant species identified in these samples. In contract, commonly named ear pathogens, genera Staphylococcus and Pseudomonas, were detected in low numbers (<0.001% of all sequencing reads) and low prevalence (2/16 samples) in the tissue samples of this study.The results show that culture severely underestimated the bacterial diversity in CSOM samples and investigating tissue rather than standard culture swabs might be advantageous to understanding the disease process. The high abundance of bacteria and the large presence of Gram-positive cells detected in the cholesteatoma tissue of CSOM compared to mucosa of CSOM or controls could be members from the genus Anaerococcus. Anaerococcus may well be a pathogen in CSOM with cholesteatoma, but their role in this condition requires further investigation.

Next-generation Sequencing of MicroRNA in Acquired Middle Ear Cholesteatoma.

The pathophysiology of cholesteatoma is not precisely understood, and research on the associated microRNAs (miRNAs) is also deficient. We demonstrated the expression of miRNA in normal skin and middle ear cholesteatoma by next-generation sequencing (NGS) technology. The profiles of miRNA and relevant molecular interaction pathways were investigated. Case-control experimental study. Middle ear cholesteatoma and post-auricular skin tissue specimens were collected from 13 adult patients. Total RNA was extracted, and miRNA expression profiles were analyzed by NGS technology. Functional gene classification to predict target genes and relevant biological pathways was performed using DIANA-microT-CDS and the Kyoto Encyclopedia Gene and Genome database (KEGG) pathways. The expression of 2588 miRNAs from middle ear cholesteatoma and skin tissue samples was analyzed. The expression of 76 upregulated and 128 downregulated miRNAs was identified in the cholesteatoma samples compared to normal skin (FC ≥2 and p < 0.05). Ninety-nine differentially expressed miRNAs (FC ≥4 and p < 0.05) were used to explore the biological pathways involved in the etiopathogenesis of cholesteatoma. The most predicted pathway in cholesteatoma in the upregulated miRNA group was the ErbB signaling pathway and it was extracellular matrix (ECM)-receptor interaction in the downregulated miRNA group. This was the first study investigating small miRNAs in human acquired cholesteatoma using NGS technique. We were able to identify new miRNAs and pathways related to cholesteatoma. The results of this study are expected to be helpful in revealing new pathophysiologies of cholesteatoma. N/A Laryngoscope, 134:4374-4382, 2024.

Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing loss and brain abscess formation. However, the pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a single-cell RNA sequencing analysis on human cholesteatoma samples and identify a pathogenic fibroblast subset characterized by abundant expression of inhibin βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in these fibroblasts results in decreased osteoclast differentiation in a murine model of cholesteatoma. Moreover, follistatin, an antagonist of activin A, reduces osteoclastogenesis and resultant bone erosion in cholesteatoma. Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone destruction via the induction of local osteoclastogenesis, suggesting a potential therapeutic target.

Study of Angiogenic, Pro-Apoptotic, and Pro-Inflammatory Factors in Congenital and Acquired Cholesteatomas.

Despite recent advances in biomolecular research that have improved our knowledge of cholesteatoma pathogenesis, the reasons behind its highly variable clinical course are still not clarified. It has been proposed that biological signaling between peri-matrix and matrix cells could play a critical role in disease homeostasis. The aim of our study was to analyze the expression of inflammatory (IL-1β), hyper-proliferative (STAT-3, TGF-β), and angiogenic (VEGF-C, PDGFr) factors in congenital and acquired cholesteatomas (both in adults and children), which might correlate with the clinical features observed. We performed an experimental study on 37 patients (29 males and 8 females, ranging from 4 to 66 years of age) who were diagnosed with cholesteatoma between 2020 and 2021 in our institution. All patients underwent clinical, audiologic, and radiologic assessments. Bone erosion grading and staging of cholesteatoma growth were assessed through preoperative evaluation and intraoperative middle ear findings, according to the PTAM System proposed by the Japan Otological Society (2016). Retro-auricular skin specimens were intraoperatively collected in all patients. Skin and cholesteatoma samples were analyzed through histopathological, western blot, and immunohistochemical evaluations. The expression rate was measured to find out the differences between congenital and acquired cholesteatomas as well as between the adult and pediatric populations. Expression of angiogenic, inflammatory, and proliferative biomarkers is significantly increased in acquired cholesteatomas in children as compared to congenital and acquired forms in adults, in accordance with the higher stage of disease shown by imaging, surgical, and histological features. Our data suggest that pathways already supposed to be involved in the pathogenesis of cholesteatomas could be differently activated in more destructive forms, typically found in children. The identification of potential biomarkers of cholesteatoma aggressiveness could lead to more personalized management (timing of intervention, recurrence prevention) and the future identification of anti-growth/anti-proliferative agents as non-surgery therapeutic options.

Dynamic Microscopic Optical Coherence Tomography as a New Diagnostic Tool for Otitis Media

Hypothesis: Otitis media (OM) can be successfully visualized and diagnosed by dynamic microscopic optical coherence tomography (dmOCT). Background: OM is one of the most common infectious diseases and, according to the WHO, one of the leading health problems with high mortality in developing countries. Despite intensive research, the only definitive treatment of therapy-refractory OM for decades has been the surgical removal of inflamed tissue. Thereby, the intra-operative diagnosis is limited to the surgeon’s visual impression. Supportive imaging modalities have been little explored and have not found their way into clinical application. Finding imaging techniques capable of identifying inflamed tissue intraoperatively, therefore, is of significant clinical relevance. Methods: This work investigated a modified version of optical coherence tomography with a microscopic resolution (mOCT) regarding its ability to differentiate between healthy and inflamed tissue. Despite its high resolution, the differentiation of single cells with mOCT is often impossible. A new form of mOCT termed dynamic mOCT (dmOCT) achieves cellular contrast using micro-movements within cells based on their metabolism. It was used in this study to establish correlative measurements with histology. Results: Using dmOCT, images with microscopic resolution were acquired on ex vivo tissue samples of chronic otitis media and cholesteatoma. Imaging with dmOCT allowed the visualization of specific and characteristic cellular and subcellular structures in the cross-sectional images, which can be identified only to a limited extent in native mOCT. Conclusion: We demonstrated for the first time a new marker-free visualization in otitis media based on intracellular motion using dmOCT.

Expression and Correlation Research of MicroRNA10a-5p and PIK3CA in Middle Ear Cholesteatoma.

This study aimed to examine the roles of miR-10a-5p and phosphatidylinositol-4,5-bisphosphonate 3-kinase catalytic subunit α in the pathogenesis of middle ear cholesteatoma. We enrolled 27 patients with middle ear cholesteatoma and collected samples of intraoperative cholesteatoma and normal posterior ear skin tissues. The mRNA expression levels of miR-10a-5p and PIK3CA were detected using real-time quantitative polymerase chain reaction. PIK3CA protein expression was measured by immunohistochemistry and western blotting. Middle ear cholesteatoma tissues showed significantly lower miR-10a-5p expression levels and significantly higher PIK3CA expression levels than normal posterior ear skin tissues (both P < .05). Furthermore, the miR-10a-5p and PIK3CA expression levels were significantly negatively correlated in middle ear cholesteatoma tissues (r = -0.926, P < .001). Low miR-10a-5p expression levels in middle ear cholesteatoma tissues may inhibit the growth and proliferation of cholesteatoma, whereas high PIK3CA expression level may promote its growth and proliferation. In addition, miR-10a-5p may affect the proliferation and differentiation of cholesteatoma by negatively regulating its target gene, PIK3CA.

High-Throughput Sequencing Investigation of Bacterial Diversity in Chronic Suppurative Otitis Media and Middle Ear Cholesteatoma.

Background Chronic otitis media is a common middle ear disease in otolaryngology. Bacterial infection is considered as the cause of the disease, but relying on conventional bacterial cultures can be problematic for identifying specific pathogens. Current research suggests that bacteria in microbial communities can only be identified by rDNA sequencing of bacteria. Methods This cross-sectional study utilized broad-range PCR amplification of 16S rRNA genes with clone analysis to compare bacterial diversity in lesions from 6 patients with chronic suppurative otitis media (CSOM) and 10 patients with cholesteatoma of middle ear lesions. Bacteria were analyzed at the levels of phylum, order, family, genus, and species. Results The age and sex difference between the patients with chronic suppurative otitis media and the patients with middle ear cholesteatoma were comparable (P > 0.05). Bacterial species abundance and species diversity were greater in cholesteatoma of the middle ear lesions than in CSOM lesions. The total number of detected operational taxonomic units (OTU) was 838, comprising 788 OTU detected in cholesteatoma pathological tissues, 230 in CSOM pathological tissues, and 180 OTU common to both groups. Proteus is a major part of CSOM (99.46%, P = 0.000321). The phyla detected in the Cholesteatoma samples were Proteus (Proteobacteria) (35.77%), thikum (Firmicutes) (44.21%, P = 0.001071), and Actinomycetes (Actinobacteria) (16.66%, P = 0.032464). At all bacterial taxonomic levels, the epithelial tissue of middle ear cholesteatoma was complex in terms of bacterial diversity, covering many Gram-positive and Gram-negative bacteria, likely related to bacterial microbiome formation. In contrast, the bacteriology of the CSOM lesions was relatively simple at all taxonomic levels, with all sequences characterized as belonging to Gram-negative bacteria. Conclusion Our results suggest that persistent middle ear cholesteatoma infection may be a microbial flora disorder related to conditional pathogenic bacteria rather than a single bacterial infectious disease. The pathogen is relatively single in the diseased tissue of chronic suppurative otitis media, which is the main reason for its effective antiinfection treatment.

Immunohistochemical localization of d-β-aspartic acid in congenital and acquired middle ear cholesteatoma.

Objective/HypothesisMiddle ear cholesteatoma is characterized by abnormal growth of the keratinizing squamous epithelium of the temporal bone. d‐β‐aspartic acid is the major isomer of d‐aspartic acid found in elderly tissue. We assessed the immunoreactivity to k‐β‐aspartic acid of congenital and acquired middle ear cholesteatomas.Study DesignCase–control studies.Material and MethodsTissue samples were collected from 21 patients comprising 21 ears with congenital middle ear cholesteatoma and 26 patients comprising 29 ears with acquired type. Their clinical and histopathological features were investigated. We divided the middle ear cholesteatoma samples into three layers: the perimatrix, matrix, and cystic contents. The patterns of immunoreactivity to d‐β‐aspartic acid expression were then assessed immunohistochemically.ResultsTwo patterns of immunoreactivity to d‐β‐aspartic acid were detected in middle ear cholesteatoma: infiltrative and diffuse. In congenital middle ear cholesteatoma, d‐β‐aspartic acid expression was observed throughout all the layers (perimatrix, matrix, and cystic contents), and immunoreactivity to d‐β‐aspartic acid was dramatically strong in all layers. The expression levels of d‐β‐aspartic acid to the cystic content and perimatrix were significantly higher in congenital middle ear cholesteatoma than in the acquired type.ConclusionsThis study showed the expression levels of d‐β‐aspartic acid in middle ear cholesteatoma to differ significantly between congenital and acquired middle ear cholesteatoma. Our results indicate that overexpression of d‐β‐aspartic acid is likely to be involved in the pathogenesis of cholesteatoma, and we speculate that d‐β‐aspartic acid could be a novel biomarker for, and a therapeutic target in, congenital and acquired middle ear cholesteatoma.Level of Evidence4

Complex Evaluation of Tissue Factors in Pediatric Cholesteatoma.

The aim of this study was to describe the appearance and distribution of tissue remodeling markers (MMP-2, MMP-9, TIMP-2, TIMP-4), Sonic hedgehog gene protein (Shh), pro- and anti-inflammatory cytokines (IL–1, IL–10), transcription factor (NF-κβ), proliferation marker (Ki–67), angiogenetic factor (VEGF), tissue defensins (HβD–2, HβD–4) of the pediatric cholesteatoma. Sixteen cholesteatoma samples were obtained from children, eleven skin controls from cadavers. Tissues were stained for MMP-2, MMP-9, TIMP-2, TIMP-4, Shh, IL–1, IL–10, NF-κβ, Ki–67, VEGF, HβD–2, HβD–4. Non-parametric statistic, Mann–Whitney, and Spearman’s coefficient was used. A statistically significant difference was seen between Shh and HβD–2 in perimatrix and control connective tissue, between NF-κβ in cholesteatoma and control skin, and between HβD–4 in matrix and skin epithelium. Complex intercorrelations between MMPs, NF-κβ and VEGF cause the intensification of angiogenesis in cholesteatoma. The persistent increase in Shh gene protein expression in cholesteatoma perimatrix suggests the stimulation of the cholesteatoma growth in children. Similar expression of IL-1 and IL-10 and their intercorrelation, proves there is a balance between pro- and anti-inflammatory cytokines. NF-κβ, and not Ki-67, seems to be the main inducer of cellular proliferation. The main antimicrobial protection is provided by HβD-2.

Expression of Cytokeratin 6 and 16 in Middle Ear Cholesteatoma

BACKGROUND: Cholesteatoma is hyperproliferative because of the response of direct biomechanical trauma, and inflammation processes then lead to temporal bone destruction with some clinical manifestations of complications. The hyperproliferation mechanism occurred because of the activation of intermediate filament protein type I and type II known as cytokeratin (CK). AIM: This study aimed to examine the expression CK 6 and CK 16 in cholesteatoma. METHODS: This is a cross-sectional comparative study. Cholesteatoma specimens obtained from 15 patients who underwent surgery were considered as the case, and 15 normal retro-auricular skins were considered as the control. All samples were examined for expression through immunohistochemistry and scored using the immunoreactivity score. Data were analyzed using SPSS via χ2 test, and the difference was significant (p &lt; 0.05). RESULTS: The expression of CK 6 was high in cholesteatoma (33.3%) and low in retro-auricular skin. The expression of CK 16 was high in all samples of cholesteatoma and mostly high in the retro-auricular skin; both expressions were statistically significant (p &lt; 0.05). CONCLUSION: The expression of CK 6 and CK 16 in cholesteatoma was higher than in normal retro-auricular skin.

Autophagy Is Deficient and May be Negatively Regulated by SERPINB3 in Middle Ear Cholesteatoma.

Whereas autophagy has been linked to various human diseases, whether it also plays a role in cholesteatoma is virtually unknown. This study aimed to investigate the activity and regulation of autophagy in cholesteatoma. The treatment of middle ear cholesteatoma has been challenging due to an insufficient understanding of the underlying disease mechanism. Expression of microtubule-associated protein 1A/1B-light chain 3 (LC3), the autophagy protein marker, and phosphorylated Akt (p-Akt), and mammalian target of rapamycin (p-mTOR), the known autophagy regulators, in fresh retroauricular skin and cholesteatoma tissue samples was analyzed by immunoblotting. The results were further confirmed by immunohistochemistry and statistical analyses. Cell proliferation of primary retroauricular skin- and cholesteatoma-derived fibroblasts was evaluated by methyl thiazol tetrazolium (MTT) assay. Ectopic expression of serine proteinase inhibitor, clade B, member 3 (SERPINB3) in the fibroblasts was achieved by electroporation and the expression was detected by immunoblotting. LC3 expression was significantly decreased in cholesteatoma in most of the 15 paired retroauricular skin/cholesteatoma tissue samples. However, p-Akt and p-mTOR expression in the cholesteatoma samples was not significantly different from that in the control subjects. Immunohistochemical studies further demonstrated an inverse correlation between LC3 expression and cholesteatoma. The cholesteatoma fibroblasts proliferated faster than the retroauricular skin fibroblasts, and had higher SERPINB3 but lower LC3 expression. Furthermore, overexpression of SERPINB3 in the retroauricular skin fibroblasts enhanced cell proliferation and downregulated LC3 expression. Autophagy is significantly suppressed in cholesteatoma tissues, which may not involve the Akt/mTOR signaling pathway. More importantly, SERPINB3 may promote cell proliferation and negatively regulate autophagy in cholesteatoma fibroblasts. Together, these findings warrant further investigation into the pathogenic mechanism of cholesteatoma.

Analysis of KRT1, KRT10, KRT19, TP53 and MMP9 expression in pediatric and adult cholesteatoma.

Cholesteatoma is an epidermal cyst with still unknown pathomechanism. The aim of the current study was to investigate molecular differences in the background of the hyperproliferative property and aggressive behavior typical of the cholesteatoma epithelium. The expression of three cytokeratin genes (KRT1, KRT10 and KRT19), the matrix metalloproteinase 9 gene (MMP9) and the tumor suppressor TP53 gene was measured by qRT-PCR in surgical samples of pediatric and adult cholesteatoma cases and their expression level was compared to that of normal skin samples from the retroauricular region of control individuals. Cholesteatoma samples were stratified according to the age of onset and recurrence for more detailed analysis. Our results showed identical expression pattern for KRT1 and KRT10, their expression was higher in pediatric cases than in adults, especially in pediatric recurrent samples. The expression level of KRT19 was inversely proportional to that of KRT1/KRT10, it was lower in the more invasive recurrent cases both in our pediatric and adult groups. As it was expected from the bone destructive behavior of cholesteatoma, a significantly elevated expression of MMP9 was measured in cholesteatoma samples, the highest level was found in adult recurrent cases. Low expression levels characterize the TP53 gene without significant differences in our samples. These findings demonstrate that cytokeratin expression distinguishes between pediatric/adult, nonrecurrent/recurrent cases, suggesting that distinct differentiation state and cell division potential characterize these cholesteatoma cases. KRT19 with a tumor suppressor potential might restrict the recurrence of cholesteatoma. The differences observed in gene expression profiles between cholesteatoma and control samples support the notion that cholesteatoma is a cystic lesion with tumor-like behavior because it is characterized by invasive, destructive growth and high tendency for recurrence.

Changes in biofilm in chronic cholesteatomatous otitis media in children following the application of sodium 2-mercaptoethanesulfonate (MESNA)

BackgroundPediatric cholesteatoma is a clinically challenging disease entity. Its biological behavior in the pediatric population differs from its behavior in adult population in terms of aggressiveness and recurrence. Several studies have shown the presence of biofilms associated with cholesteatoma that hinder the management and eradication of the infection. This led is to study the use of non-antimicrobial treatments impacting on the structure or composition of biofilms. ObjectiveTo evaluate the changes that occur in the biofilm of cholesteatoma in pediatric patients after the application of sodium 2-mercaptoethanesulfonate (MESNA). MethodsA pilot study of 10 pediatric patients, with a median age of 10 years and a diagnosis of cholesteatomatous chronic otitis media, who underwent surgery for primary or revision mastoidectomy in the Otorhinolaryngology Service of the Hospital Infantil de México Federico Gómez between January 2016 and May 2017. During the surgery, basal samples of cholesteatoma and tissue were taken after topical application of 4% MESNA for 10 min. The samples were then processed for confocal laser microscopy. ResultsIn all samples structures compatible with bacterial biofilms were identified. The most relevant finding was the changes in the structure of the biofilm after the application of MESNA, such as disintegration and separation of the underlying tissue. ConclusionsThis is the first study that showing changes associated with cholesteatoma in the structure of the bacterial biofilm after the application of MESNA. The observed disintegration of cholesteatoma biofilm ultrastructure could aid in the management of the chronic infection associated with cholesteatoma.

Comparison of changes in mitochondrial bioenergetics between keratinocytes in human external auditory canal skin and cholesteatomas from normoxia to hypoxia

Cholesteatoma has attracted many studies seeking to uncover its nature and the pathogenesis of related diseases. However, no researchers have explored the mitochondrial bioenergetics of cholesteatoma. The aim of this study was to investigate the energy demand and differential mitochondrial respiration profiles between keratinocytes in external auditory canal (EAC) skin and cholesteatoma samples cultured in normoxic (20% O2) and hypoxic (5% O2) conditions. Enhanced cellular proliferation of both types of keratinocytes was found in hypoxia compared to normoxia. In 20% O2 conditions, cholesteatoma keratinocytes exhibited less mitochondrial mass, lower ATP levels, and significantly lower basal oxygen consumption rate (OCR) and reserve capacity compared to normal skin keratinocytes. In contrast, in hypoxic conditions, cholesteatoma keratinocytes showed markedly higher levels in maximal OCR and reserve capacity, as well as lower proton leak OCRs, compared to normal skin keratinocytes. Hypoxia induced the reverse mitochondrial bioenergy profile from that in normoxia between these two types of keratinocytes, implying that an adaptive change of mitochondrial respiration to oxygen fluctuations may develop in cases of cholesteatoma. Such adaptation in response to hypoxic conditions may play a role in explaining the pathogenesis of acquired cholesteatoma.

The significance of the expression of cell proliferation and inflammation markers in the development of acquired middle ear cholesteatoma

Background/Aim. Permanent proliferation and periodical infection are the main clinical characteristics of acquired middle ear cholesteatoma. The aim of this study was to research immunohistochemical characteristics of the skin along with the cholesteatoma process in the nearby tissue. This research should influence further studying of etiology and development of acquired middle ear cholesteatoma. Methods. We investigated clinical, histological and immunohistochemical characteristics of cholesteatoma in 50 samples from operated patients with acquired middle ear cholesteatomas. We classified all samples according to their clinical characteristics of cholesteatoma such as bone destruction, presence of infection or cholesteatoma extension and histological characteristics of cholesteatoma such as keratinisation, inflammatory process and extracellular matrix proliferation. We used mouse monoclonal antibodies for proliferating cell nuclear antigen (MAbs for PCNA), Ki-67, COX-2, CD 4 and CD 8 lymphocytes to investigate the expression of those characteristics in the cholesteatoma and in the control skin tissue. Statistical analyses were performed using SPSS for Windows version 16.0 (SPSS, Chicago, IL, USA). We used the independent group t-test, Spearman?s correlation analysis and Mann-Whitney U test to analyze statistical analysis. Results. Expression of PCNA, Ki-67, COX-2 and CD 8 lymphocytes in more serious clinical picture of cholesteatoma was almost equal as in less serious clinical picture of cholesteatoma. There was statistically significantly higher concentration of inflammation marker CD 4 lymphocytes, both in the acquired cholesteatoma and in the skin of bony portion of the external auditory canal near fibrocartilaginous annulus in more serious clinical picture of cholesteatoma than in less serious clinical picture of cholesteatoma (p &lt; 0.01). There was statistically significant difference of expression of PCNA, Ki- 67, COX-2, CD 4 and CD 8 lymphocytes between all cholesteatoma samples and the skin of bony portion of the external auditory canal (p &lt; 0.05) and statistically significant difference of expression of those markers between the skin of bony portion of the external auditory canal and retroauricular skin (p &lt; 0.05). Conclusion. Inflammation of the skin of bony portion of the external auditory canal is a milestone in pathogenesis of acquired middle ear cholesteatoma. Expression of CD 4 lymphocytes can be the prognostic factor for acquired cholesteatoma clinical picture development. We found so much diversity in biological behavior through very different levels of cholesteatoma development. Expression of Ki-67 in acquired middle ear cholesteatoma is a reliable and stable marker of proliferation for acquired middle ear cholesteatoma.

High-mobility group box protein 1 expression in inflammatory diseases of the middle ear.

High-mobility group box 1 (HMGB1) is a nuclear non-histone protein, playing a critical role as a mediator between innate and acquired immunity; when released extracellularly, it coordinates the cellular stress response (under necrosis, bacterial lipopolysaccharide stimulation) and acts as an inflammatory marker and cytokine. The aim of the study was to demonstrate whether HMGB1 is over-expressed in chronic middle-ear pathologies and whether the entity of expression and the localization are correlated with the degree of the inflammatory reaction, thus suggesting that HMGB1 may play a crucial role in chronic inflammatory disorders of the middle ear, as already demonstrated in other airway diseases.We analyzed 30 samples of middle-ear mucosa in patients affected by chronic suppurative otitis media with ear drum perforation with/without cholesteatoma and otosclerosis as control. The distribution of HMGB1 was evaluated as nuclear, cytoplasmic, and/or extracellular staining.The inflammatory cells observed in the biopsies were mostly lymphocytes and plasmacells. A statistically significant difference in inflammation score between otosclerosis and chronic otitis samples (P < 0.01; Anova test) and between otosclerosis and cholesteatoma samples (P < 0.05; Anova test) was observed; the HMGB1 positivity was in accordance with the density of the inflammatory infiltrate.HMGB1 is over-expressed in chronic middle-ear pathologies and may play a role in the progression of the inflammatory process from recurrent acute otitis media to chronic suppurative otitis media.

Manifestation of first branchial anomaly:56 cases reportrhinitis

Objective:To sum up and conclude manifestation of congenital first branchial anomaly(CFBCA).Method:The clinical data of 56 patients from 2005 to 2015 in our hospital were retrospective reviewed.Result:Manifestation:mass without pain(26.8%),repeated sore and discharge(71.4%),otological symptom(external auditory discharge､hearing loss,28.6%).Eleven cases bacterial sample showed positive result,and most of them show pseudomonas aeruginosa and staphylococcus aureus.Auricular endoscopy typically performed stricture of external auditory canal,cholesteatoma samples accumulated in ear canal,fistula at the conjunction of the bone and cartilage and tympanic membranous attachment.Typical performance of CT(MRI)was that there were cystic,lobulated or tubular abnormal shadow related with ear canal in Pochet's triangle area whose cyst wall or pipe wall could been enhanced in enhanced CT(MRI) scans,and part of that could be connected with skin.The statistical difference between type Oslen and Work and clinical characteristics(P<0.01),and the relationship between type Oslen and Work(P<0.01).Most of Work Ⅰ were cyst type,and these two type often had no infected symptom.Most of them were young patients.Most of Work Ⅱ were sinus and fistula type ,and these two type often had infected symptom.Most of them were teenagers.Part of patients of type Work Ⅱ showed tympanic membranous attachment.Conclusion:CFBCA was rare,and it is more common in young patients and often in left part.It always performed as mass without pain､repeated sore and discharge､external auditory discharge.Most of Work Ⅰ were cyst type,and these two type often had no infected symptom and most of them were young patients .Most of Work Ⅱ were sinus and fistula type,and these two type often had infected symptom and most of them were teenagers.Auricular endoscopy,CT,MRI could help make diagnose.Doctors clinical need to differentiate it with related diseases according to different manifestations.

Differential Protein Expression in Congenital and Acquired Cholesteatomas.

Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ from those of acquired cholesteatomas, which originate from retraction pocket epithelia.

Molecular signaling of the HMGB1/RAGE axis contributes to cholesteatoma pathogenesis.

Cholesteatoma represents progressive expansion of the keratinizing squamous epithelium in the middle ear with subsequent chronic inflammation in subepithelial connective tissues. The hypothesis was tested that receptor for advanced glycation endproduct (RAGE) and its ligand, high-mobility box 1 (HMGB1), are overexpressed in cholesteatoma, and the RAGE/HMGB1 axis might contribute to its pathogenesis. Cholesteatoma samples (n = 36) and 27 normal skin specimens were studied by immunohistochemistry (IHC) for HMGB1 and RAGE expression. Effects of HMGB1 signaling on proliferation, migration, cytokine production, and apoptosis of human immortalized keratinocytes (HaCaTs) and normal keratinocytes were studied by quantitative reverse transcription (qRT)-PCR, IHC, Western blots, and flow cytometry after cell co-incubation with HMGB1. While all studied tissues expressed HMGB1, its expression was higher in cholesteatoma than in normal skin (p < 0.0001). All cases of cholesteatoma also showed elevated RAGE expression levels, and only 7/27 (26 %) of normal skin specimens were weakly positive for RAGE. Proliferation and migration of HaCaT cells incubated with HMGB1 were up-regulated (p < 0.05). HMGB1 also prevented HaCaT cell apoptosis and induced activation of several molecular signaling pathways in keratinocytes. The data suggest that in cholesteatoma, HMGB1 released from stressed or necrotic epithelial cells and binding to RAGE overexpressed in keratinocytes initiates molecular signaling that culminates in pro-inflammatory cytokine release and chronic inflammation.Key message HMGB1 signaling engages multiple activation pathways in RAGE-positive keratinocytes.HMGB1 protects RAGE-positive keratinocytes from drug-induced apoptosis.Keratinocyte proliferation is controlled via RAGE and HMGB1 molecular signaling.Molecular signaling of the HMGB1/RAGE axis contributes to cholesteatoma pathogenesis. Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-014-1217-3) contains supplementary material, which is available to authorized users.

The c-MYC Protooncogene Expression in Cholesteatoma

Cholesteatoma is an epidermoid cyst, which is most frequently found in the middle ear. The matrix of cholesteatoma is histologically similar to the matrix of the epidermoid cyst of the skin (atheroma); their epithelium is characterized by hyperproliferation. The c-MYC protooncogene located on chromosome 8q24 encodes a transcription factor involved in the regulation of cell proliferation and differentiation. Previous studies have found aneuploidy of chromosome 8, copy number variation of c-MYC gene, and the presence of elevated level c-MYC protein in cholesteatoma. In this study we have compared the expression of c-MYC gene in samples taken from the matrix of 26 acquired cholesteatomas (15 children and 11 adults), 15 epidermoid cysts of the skin (atheromas; head and neck region) and 5 normal skin samples (retroauricular region) using RT-qPCR, providing the first precise measurement of the expression of c-MYC gene in cholesteatoma. We have found significantly elevated c-MYC gene expression in cholesteatoma compared to atheroma and to normal skin samples. There was no significant difference, however, in c-MYC gene expression between cholesteatoma samples of children and adults. The significant difference in c-MYC gene expression level in cholesteatoma compared to that of atheroma implies a more prominent hyperproliferative phenotype which may explain the clinical behavior typical of cholesteatoma.