Cholesteatoma epithelium is characterized by a keratinocyte dysregulation with aggressive growth subsequently destroying the middle ear mucosa. The monoclonal antibody Ki-67 recognizes a nuclear antigen expressed by cells in the G1, S, and G2/M phases being used to determine the growing cell fraction in tissue samples. Cryostat sections of skin and cholesteatoma biopsies were examined immunohistochemically for reactivity with Ki-67 using the alkaline phosphatase-anti-alkaline phosphatase method. Nuclear staining was seen in a small number of keratinocytes located in the basal cell layer of normal auditory meatal skin. In contrast, numerous cells of the basal and suprabasal layers in cholesteatoma were found to react with Ki-67. A cytoplasmic staining was also observed in both skin and cholesteatoma. In cholesteatoma, the cytoplasmic staining was stronger. These results clearly show that cholesteatoma epithelium proliferates at a higher rate than normal epidermis, confirming the hyperproliferative behavior of cholesteatoma.

Cholesteatoma in children is characterized by a more extensive and rapid growth in the middle ear and mastoid cavities. The growth characteristics of the cholesteatoma in 20 children were studied using the monoclonal antibody MIB 1, which recognizes a nuclear antigen expressed by cells in the G1, S, and G2/M phases. Specimens of normal adult auditory meatal skin (n = 15) and adult cholesteatoma (n = 15) served as controls. The tissue specimens were prepared for immunohistochemical examination using the alkaline phosphatase‐antialkaline phosphatase method and an automatic image analyzer. Specimens of normal skin revealed an average MIB 1 score of 9.2 ± 3.1%. Child and adult cholesteatomas showed higher values. The average MIB 1 score was higher in child cholesteatoma (42 ± 9.4%) than in adult cholesteatoma (28.2± 6%). This difference was statistically significant (P<.01). Our results confirm a significant increase of the proliferative rate of cholesteatoma keratinocytes in children, giving an explanation for the more aggressive clinical behavior observed in these patients.

We investigated the distribution of basement membrane zone (BMZ) components collagen type IV, collagen type VII, and fibronectin in human middle ear cholesteatoma, auditory meatal skin, and middle ear mucosa using both immunohistochemical and ultrastructural methods. Collagen type IV immunoreactivity of skin and middle ear mucosa is continuous in the BMZ, whereas cholesteatoma frequently showed absent immunoreactivity or focal discontinuities. Collagen type VII immunoreactivity is detected similarly within the BMZ of cholesteatoma and skin. Fibronectin immunoreactivity is observed within the dermoepithelial junction of skin and middle ear mucosa. In cholesteatoma, however, fibronectin immunoreactivity is markedly increased within the extrinsic BMZ and the subepithelial connective tissue. The ultrastructural arrangement of the BMZ of cholesteatoma is like that of skin; however, it exhibits distinct alterations of the lamina fibroreticularis and lamina densa. Our results outline cholesteatoma as a disease with disturbed cell matrix interactions analogous to those of wound reepithelialization.

Cholesteatoma epithelium is characterized by a keratinocyte dysregulation accompanied by destruction of the ossicles and temporal bone. Immunohistochemical methods using antibodies to cell-cycle-related antigens can be used as a means for assessing various aspects of proliferation in cholesteatoma tissue. They also have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Proliferating cell nuclear antigen (PCNA) is a 36 kDa DNA-delta-polymerase-associated protein that is directly involved in the mechanisms of DNA synthesis. in the present study the expression of PCNA was investigated in formalin-fixed, paraffin-embedded biopsy specimens of cholesteatomas and normal skin. Normal skin revealed nuclear staining in a small number of keratinocytes (PCNA grade, 1.5) located in the basal cell layer. In contrast, an increased number of PCNA-labeled basal and suprabasal epidermal cells (PCNA grade, 9.3) were found in cholesteatoma samples. Our findings indicate that PCNA represents a reliable marker for epithelial proliferation, showing that cholesteatoma epithelium proliferates at a higher rate than normal epidermis. These findings also support the concept of keratinocyte dysregulation in middle ear cholesteatoma.

Previous studies have shown an altered epithelial cell proliferation in middle ear cholesteatoma, reporting an aberrant expression of epidermal growth factor receptor (EGF-R) glycoprotein by immunohistochemistry. In this study, we quantified the presence of EGF-R using enzyme-linked immunosorbent assays (ELISAs) on tissue extracts, as well as the EGF-R gene expression by in situ hybridization on frozen sections. Human skin obtained from the external ear canal was used as control. The amounts of EGF-R glycoprotein in cholesteatoma were very similar to those in human skin. Human skin showed EGF-R messenger RNA (mRNA) only in the basal layer. A higher percentage of cells hybridized for the anti-sense probes EGF-R was found in cholesteatoma epithelium. Furthermore, we could find suprabasal cells with EGF-R mRNA. Our results confirm that the abnormal growth of cholesteatoma epithelium is reflected in an aberrant expression of EGF receptor.

Middle ear cholesteatoma is characterized by the presence of a keratinizing squamous epithelium with hyperproliferative features. Such growth can only be supported by abundant blood vessels. The presence and distribution of blood vessels in cholesteatoma was studied to determine the mechanisms responsible for its origin and maintenance. Cholesteatoma (n = 30) and retroauricular skin samples (n = 30) were studied with indirect immunoperoxidase and alkaline phosphatase anti-alkaline phosphatase methods. Antibodies were used to recognize endothelial cells (von Willebrand Factor VII), vascular basal membrane components (type VI collagen), intercellular adhesion molecules (ICAM-1, 1CAM-2), human histocompatibility antigen (HLA-II) as a marker for cellular activation, angiogenetic growth factors and their receptors (TGF-alpha and VEGF), lymphocytes (CD3), and macrophages (KiM8). The cholesteatoma stroma had numerous vessels with intact basal membrane. The vessel concentration was higher in regions with abundant macrophage infiltration. Perivascular cell infiltrates were positive for antibodies against angiogenetic factors and HLA-II. Endothelial cells had increased expression of intercellular adhesion molecules and angiogenetic growth factor receptors. These results confirm the presence of increased vascularization in cholesteatoma, which may play an important role in sustaining continuous abnormal growth.

Middle ear cholesteatoma is often invasive with consequent bone destruction. Inflammatory stimulation of the underlying connective tissue, as well as an autocrine mechanism, may be responsible far the dysregulation and abnormal proliferative features of the keratinocytes in cholesteatoma. Comparative investigations were performed to assess the epithelial cell kinetics of cholesteatoma and normal auditory meatal skin. Monoclonal antibody MIB 1 immunostaining (which recognizes a nuclear antigen expressed by dividing cells) was applied using the alkaline phosphatase antialkaline phosphatase immunolabeling method.Specimens of normal auditory meatal skin (n = 7) revealed an average MIB 1 score (quotient of the MIB 1-positive cells and the total number of cells) of 7.6 +/- 2.2%. Cholesteatoma samples (n = 13) showed an average MIB 1 score of 17.4 +/- 8.9% and a heterogeneity of proliferating epithelial areas. Epithelial cones growing toward the underlying stroma exhibited high mitotic activity. Statistically, the results of this study confirm a highly significant increase in the proliferation rate of cholesteatoma keratinocytes, which had an MIB I score that was 2.3 times higher than the score for keratinocytes of normal external auditory meatal skin.

Cholesteatoma epithelium is characterized by a dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. Expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kDa c-myc protein product and the alkaline phosphatase-antialkaline phosphatase method. For quantitative analysis a computer-linked analyzing system was used. In contrast to normal skin, keratinocytes of basal and suprabasal layers showed nuclear staining in cholesteatoma epithelium. The extent of nuclear staining of epithelial cells in the cholesteatomas studied was significantly increased. Concurrent cytoplasmic staining was observed in both skin and cholesteatoma, but with a stronger reactivity in the latter. These findings suggest participation of the c-myc oncogene in cholesteatoma epithelium.

PURPOSE:Because abundant fibronectin deposition is a hallmark of healing cutaneous wounds and provides a matrix for hyperproliferative and migratory epidermal cells, the distribution of fibronectin in aural cholesteatoma was investigated immunohistochemically. MATERIALS AND METHODS:A monoclonal antibody against the major cell binding domain of human fibronectin was used to stain 4-micron cryosections of cholesteatoma tissue by the alkaline phospatase-antialkaline phosphatase method. Section of normal retroauricular skin served as control. RESULTS:When processed in parallel, fibronectin staining was much stronger in the stroma of cholesteatoma than in normal dermis. The squamous epithelium of both tissues did not show any staining for fibronectin. CONCLUSIONS:These observations lend support to the view that the growth of cholesteatoma epithelium reflects an aberrant regenerative process.

Cholesteatoma epithelium is characterised by a keratinocyte dysregulation with a hyperproliferative growth and altered differentiation. Keratinocytes with a reduced turnover time show an increase of calcium binding proteins like calgranulin A and B. These proteins are highly linked with the modulation of cell growth and differentiation. The expression of calgranulin A and B was studied immunohistochemically using the monoclonal antibody F12 on cryosections of cholesteatoma biopsies. Normal skin served as control. The staining in normal skin was confined to the highly proliferative activated follicular keratinocytes, whereas most cholesteatomas showed a staining of all cell layers of the epithelium. Our findings show that the keratinocyte dysregulation of the cholesteatoma epithelium is reflected by variations of the expression of calcium binding proteins.

Cholesteatoma epithelium is characterised by a keratinocyte dysregulation with a hyperproliferative growth and altered differentiation. Keratinocytes with a reduced turnover time show an increase of calcium binding proteins like calgranulin A and B. These proteins are highly linked with the modulation of cell growth and differentiation. The expression of calgranulin A and B was studied immunohistochemically using the monoclonal antibody F12 on cryosections of cholesteatoma biopsies. Normal skin served as control. The staining in normal skin was confined to the highly proliferative activated follicular keratinocytes, whereas most cholesteatomas showed a staining of all cell layers of the epithelium. Our findings show that the keratinocyte dysregulation of the cholesteatoma epithelium is reflected by variations of the expression of calcium binding proteins.

Transforming growth factor alpha (TGF-α) wirkt auf den epidermal growth factor receptor (EGF-R) ein und ist ein wichtiger Regulator für die Proliferation von Keratinozyten. TGF-α wird in primären Zellkulturen humaner Keratinozyten produziert. Des weiteren ist TGF-α in der Lage, seine eigene Synthese in dieser Zellart zu stimulieren. In der vorliegenden immunhistochemischen Studie untersuchten wir die Verteilung und die Expressionsstärke von TGF-α und seinem Rezeptor in Gefrierschnitten von Cholesteatomgewebe, um die Faktoren herauszufinden, die in die Deregulation des Wachstumsverhaltens eingeschaltet sind.

Cholesteatoma epithelium is characterized by a keratinocyte dysregulation with an aggressive growth that leads to the destruction of normal middle ear mucosa. The abnormal behavior of cholesteatoma epithelium seems to be induced by the presence of a heavy immune cell infiltrate releasing different cytokines and growth factors in high amounts. Middle ear mucosa rests are often observed within the cholesteatoma stroma or adjacent to the advancing front of cholesteatoma epithelium. This study investigated the presence of interleukin-1 (IL-1), transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and epidermal growth factor-receptor (EGF-R) in the mucosa rests as well as the expression of an activation marker, 4F2. The findings were correlated with the features of a surrounding stroma with an enhanced immune cell infiltrate. Cholesteatoma epithelium showed a high staining intensity of IL-1, TGF-alpha, and EGF-R. In contrast to this, middle ear mucosa did not show any positive reactions for the mentioned factors. Epidermal growth factor immunoreactivity was found in neither cholesteatoma epithelium nor in middle ear mucosa residues. The authors found a high concentration of lymphocytes and macrophages in the surrounding stroma. Most of these cells expressed TGF-alpha, IL-1, and 4F2, suggesting an activated form. Results indicate that keratinocytes present in the middle ear mucosa do not appear to react to the stimuli released by the inflamed stroma, reflecting important differences in the cell biological features of the keratinocytes that form parts of both types of epithelium.

The stratum corneum (SC) antibodies are present in high titers in the sera of patients who have diseases in which cells containing keratin intermediate filaments have been damaged. Aural cholesteatoma is a skin-related disease of the temporal bone with an extensive production and accumulation of keratinizing epithelium in the middle ear. The aim of the present investigation was to study the humoral immune response to SC in these patients. Sera were obtained from patients with aural cholesteatoma (n=10) and from normal donors (n=8) of about the same sex and age distribution. All sera were analyzed for the existence of autoantibodies (IgG and IgM) against SC by both direct and indirect immunofluorescence, using skin and cholesteatoma frozen tissue sections as antigen substrate. The direct immunofluorescence showed a low level of staining intensity for nearly all of the cholesteatoma patients, indicating the in vivo absence of deposits of anti-SC autoantibodies. The indirect immunofluorescence demonstrated positive staining of the SC in both cholesteatoma and skin tissue sections. Furthermore, some patients showed for IgG anti-SC-autoantibodies a positive staining of the highest suprabasal layers of the epithelium of cholesteatoma. IgM anti-SC autoantibodies were always weaker in titer than IgG autoantibodies. Results show that cholesteatoma patients do not have particularly high levels of anti-SC autoantibodies. A humoral response against SC seems not to be a significant event in the pathogenesis of cholesteatoma disease.

INTRODUCTION:Cell adhesion molecules are cell surface proteins that allow specific cell-cell interactions among leukocytes, as well as between leukocytes and other cells. Because middle ear cholesteatoma is characterized by the presence of leukocyte infiltrates, the presence of the two molecule types of intercellular adhesion molecules (ICAM-1 and ICAM-2) was investigated on cholesteatoma using monoclonal antibodies.METHODS:Tissue sections from 10 patients with cholesteatoma, and normal skin from 5 patients were prepared for alkaline-phosphatase--anti-alkaline-phosphatase (APAAP) staining.RESULTS:ICAM-1 and ICAM-2 were present in normal skin in microvascular endothelial cells and in intersticial cells of the dermis. Cholesteatoma showed a very important increase of the ICAM-1 expression with comparison to human skin. All infiltrating immune cells showed positive reactions for the antibody. Furthermore, the intensity of the staining of vessels cells was higher than in normal skin. Keratinocytes were only positive if a very heavy infiltrate was present subepidermally. ICAM-2 was present in endothelial and intersticial cells in normal skin and in cholesteatoma. Most of the infiltrating cells in the cholesteatoma stroma showed positive reactions for the anti-ICAM-2 antibody.CONCLUSION:Our results suggest that both ICAM-1 and ICAM-2 play a central role in the regulation of the inflammatory disorders observed in cholesteatoma.

Transforming growth factor alpha (TGF alpha) and interleukin 1 alpha (IL-1 alpha) are known to be produced by normal human keratinocytes stimulating their proliferation. The distribution and expression of TGF alpha and IL-1 alpha were examined in specimens of middle ear cholesteatoma by means of immunohistochemical methods using a monoclonal antibody against TGF alpha and a polyclonal one against IL-1 alpha. Normal retroauricular skin was stained for comparison. Staining for TGF alpha was consistently stronger in cholesteatoma epithelium than in normal epidermis, and encompassed all epithelial cell layers. Immune cells occurring in the stroma of cholesteatoma also reacted positively for TGF alpha. The intensity of staining for IL-1 alpha was markedly stronger in cholesteatoma tissue than in normal epidermis. All cellular layers of the squamous epithelium of cholesteatoma stained strongly and uniformly for IL-1 alpha, whereas the keratin layer was negative for IL-1 alpha. In the connective tissue beneath the cholesteatoma epithelium intensely positive cells were scattered between negative stromal cells. These data are consistent with autocrine stimulation of the squamous epithelium of cholesteatoma by TGF alpha and IL-1 alpha as well as with a paracrine stimulation by immune cells. Both factors contribute to the unrestrained growth of cholesteatoma in the middle ear cavity.

Introduction: Transforming growth factor alpha (TGF-α) is known to be produced by normal human keratinocytes and to stimulate their proliferation. The squamous epithelium of middle ear cholesteatoma is believed to exhibit hyperproliferative characteristics. This study was undertaken to determine if growth factors can be identified in cholesteatoma.

Cholesteatoma of the middle ear consists of keratinising squamous epithelium in the middle ear cavity. Many points of the pathogenesis of cholesteatoma seem to be related to immune cell infiltrates in stroma and to epithelial cell migration and proliferation. In our study we found that the vast majority of cells infiltrating the stroma consisted of T-cells and macrophages, showing an immunologically activated state. Furthermore, the cholesteatoma epithelium showed an enhanced simultaneous expression of keratine-16 and Ki-67 positive cells. This expression was accompanied by the over-expression of transforming growth factor (TGF)-alpha, and its receptor, epidermal growth factor receptor (EGF-R) and interleukin-1, suggesting that the proliferation of keratinocytes could be stimulated in an autocrine manner. Finally, we conclude that the presence of immunologically activated immune cells in the stroma may be responsible for keratinocyte dysregulation in cholesteatoma epithelium.

Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium is believed to have hyperproliferative properties. Keratin 16 is accepted as a molecular marker for hyperproliferative epithelia. Two monoclonal antibodies K8.12 (directed against keratin 13) and KS. 1A3 (directed against keratin 13 and 16) were used in an alkaline phosphatase anti-alkaline-phosphatase (APAAP)-technique to compare the expression of both keratin 13 and keratin 16 in normal human skin and aural cholesteatoma. Furthermore, the cytokeratin expression was compared to that of normal skin and palatine tonsil using one-dimensional gel electrophoresis. For both monoclonal antibodies, normal ear skin was stained only in the basal layer. In contrast, in the cholesteatoma samples the immunostaining of the antibody KS-1A3 was done not only in the basal cell layer but also in the suprabasal cells of the stratum spinosum and stratum granulosum. Using gel-electrophoresis, the presence of cytokeratin 16 was demonstrated in the cholesteatoma samples only. These results support the hyperproliferative character of cholesteatoma epithelium.