Presence of hBD-1 and hBD-2 in human cerumen and external auditory canal skin.

CONCLUSION: Human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) antimicrobial peptides present in the cerumen, which is composed of exfoliated epithelial keratin and gland secretion, might provide the first line of defense against microbes in external auditory canal (EAC) skin. OBJECTIVES: Cerumen (earwax) plays a primary role in protecting the EAC skin and tympanic membrane. Even though the protection by antimicrobial peptides present in the skin secretion has been well established, little is known about the intrinsic role of the peptides in the EAC skin and cerumen. The aim of this study was to examine the presence of important antimicrobial peptides, hBD-1 and hBD-2, in the cerumen and EAC skin. MATERIALS AND METHODS: Cerumen was collected from 20 healthy adults, and the EAC skins were obtained from 12 patients who underwent middle ear surgery with canaloplasty. The presence of hBD-1 and hBD-2 was analyzed using immunohistochemistry and Western blotting. RESULTS: In the immunohistochemical study of the EAC skin, expression of hBD-1 and hBD-2 was observed in both the epithelium and the glands. The presence of hBD-1 and hBD-2 peptides in the cerumen was confirmed by Western blotting.

Human ceruminous gland: ultrastructure and histochemical analysis of antimicrobial and cytoskeletal components.

The ceruminous glands in the skin of the human external auditory canal are modified apocrine glands, which, together with sebaceous glands, produce the cerumen, the ear wax. Cerumen plays an important role in the protection of the ear canal against physical damage and microbial invasion. We studied the morphology of the glandular cells by light and electronmicroscopy. Antimicrobial and cytoskeletal components of the ceruminous glands were investigated by immunohistochemical methods. Numerous antimicrobial proteins and peptides are present in the ceruminous glandular cells: beta-defensin-1, beta-defensin-2, cathelicidin, lysozyme, lactoferrin, MUC1, secretory component of IgA. These data indicate a crucial role in the innate host defense against diverse pathogens. The apocrine secretion mechanism is a special mode of secretion by which the apical part of the cell cytoplasm surrounded by a membrane is pinched off. We could show that the presence of actin filaments, CK 19 and CK 7, seems to play a role in the pinching-off mechanism. Finally, we showed the secretion of lipid vesicles from the ceruminous gland. We could extend the number of detected antimicrobial peptides and proteins in human ceruminous glandular cells that protect the surface of the external auditory meatus. In addition, we detected proteins involved in the apocrine secretion mode of the ceruminous gland.

Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma.

Human toll-like receptors (TLR 1-10) are crucial in the induction and activation of innate immunity in the course of an infection. They are expressed mainly on the cells of the immune system, and also on some epithelia and endothelia. Their ligands so called pathogen associated molecular patterns are abundant on invading microbes. TLR-ligand binding results in cell signal transduction and subsequent production of various proinflammatory cytokines such as IL-1 and TNF-alpha. Acquired cholesteatoma is formed during chronic otitis media in the proportion of cases. It has adverse effects on ear structures, resulting in osteolysis and bone resorption. Its formation and pathogenesis are not fully understood. The current study attempted to search the possible role of TLRs in this somewhat awkward pathological condition. Surgical specimens of human acquired cholesteatoma (n=15) and normal external auditory canal skin (n=5, control tissues) were tested by immunohistochemistry for the presence of TLRs. Three TLRs were examined: TLR-2, TLR-3 and TLR-4. All TLRs tested were demonstrated in matrix (layer of keratinizing epithelium) and perimatrix (granulation tissue) of this inflammatory tumour. Expression of particular TLRs within the keratinizing epithelium was distinct and uneven. In the perimatrix, numerous T (CD3+) cells were seen and relatively few macrophages (CD11c+, HLA-DR+). There was a weak expression of all TLRs on normal (non-inflammatory) skin. Expression of TLR-3 both on the epithelium and some cells within the perimatrix and the presence of T cells may suggest that apart from innate immune responses, mechanisms of adaptive immunity also operate in cholesteatoma. Weak expression of these receptors on normal skin may also suggest the important role of TLRs in the etiopathogenesis of cholesteatoma.

Otitis externa maligna / Malignant external otitis

La otitis externa maligna es una infección del conducto auditivo externo que puede propagarse a hueso y tejidos blandos adyacentes. Poco frecuente, en general de etiología pseudomónica, compromete habitualmente a ancianos diabéticos. Presentamos el caso de una mujer adulta joven y sana, con compromiso de ambos conductos auditivos y pabellones auriculares. En la literatura dermatológica consultada no encontramos casos publicados por lo que nos parece de interés considerar a esta patología en los diagnósticos diferenciales de las afecciones del pabellón auricular (AU)

Air-exposed tissue culture of human middle ear epithelium and meatal epidermis: a method to study the advancing front of cholesteatoma.

The suitability of an air-exposed culture model consisting of a collagen matrix was investigated for constructing an advancing front (AF) of human middle ear epithelium (MEE) and meatal epidermis (ME). Three different culture settings were used: (i ) MEE; (ii) ME; and (iii) AF (MEE + ME). Small tissue biopsies were placed on a fibroblast-populated collagen matrix and grown at the air-liquid interface. After 3 weeks of culture, the MEE and ME outgrowth was differentiated. Light, scanning electron and transmission electron microscopy showed no visible differences compared to native MEE and ME. Cytokeratin 8 and cytokeratin 10 expressions were comparable to the expression seen in the native MEE and ME tissues. Proliferation, which was demonstrated by the expression of Ki-67, was present in the basal layers of cultured MEE and ME. A double layer of cells in which the ME covered the MEE formed the AF. In the AF, the MEE and ME showed the same morphological and immunohistochemical characteristics as in their native tissues. The results of the study show that this in vitro system is a well-defined model system offering the possibility to study the effects of external stimuli on the different epithelia of the AF involved in the pathogenesis of cholesteatoma.

Human beta-defensin-1 mRNA is transcribed in tympanic membrane and adjacent auditory canal epithelium.

The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human beta-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.

Allergy for the otologist. External canal to inner ear.

Allergy may affect the outer, the middle, or the inner ear. Although the otologic manifestations of allergy are not by themselves diagnostic, the history, including family history and associated symptoms in other target organs, will often help lead to the correct diagnosis and institution of therapy. Patients with significant and chronic symptoms, including those with labyrinthine symptoms of allergy, will respond well to specific immunotherapy and/or dietary elimination.

Local immune response in the skin of the external auditory meatus: an immunohistochemical study.

Cerumen plays an important role in the protection of the external auditory meatus against several kinds of damage. Its hydrophobic properties, due to the high concentration of lipids, shelter the canal from physical damages, while other components probably protect against certain microbial strains. Nevertheless there has been considerable dispute in the literature with regard to the antibacterial activity of cerumen. Because of the importance of the role of immunoglobulins (Ig) in local defense mechanisms, we attempted to study, by immunohistochemical methods, the presence and localization of the cells necessary to activate an Ig-mediated immune response and the epithelial expression of immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) in order to obtain information about a local immune response in those areas of the skin that take part in cerumen production. Our findings indicate that in the human skin of the external auditory canal the cells necessary to activate an antibody-mediated immune response were localized in the different layers of the epidermis and/or in the dermis surrounding the sebaceous and ceruminous glands and the piliary follicle, while an intense immunoreactivity for IgA and IgG was observed in the epithelial layers of the skin. The results suggest that the external auditory canal is protected from the insults of pathogens by an antibody-mediated local immune response, because all the effector components of an active local immune system are present.

Auditory epidermal cell migration. VII. Antigen expression of proliferating cell nuclear antigens, PCNA and Ki-67 in human tympanic membrane and external auditory canal.

A location of proliferating cells was investigated in eight human normal tympanic membranes (TMs) and external auditory canals (EACs) by an immunohistochemical method using two different types of antibodies for nuclear antigens in proliferating cells: anti-PCNA monoclonal antibody, and anti-Ki-67 polyclonal antibody. Four specimens prepared for cryostat sections were immunostained by both antibodies. Another four were fixed in 4% formaldehyde solution, embedded in paraffin wax and were reacted only with anti-PCNA antibodies. The expression pattern of Ki-67 was basically the same as of PCNA. In the pars tensa (PT), immunoreactivities were expressed in the nuclei of basal layer cells and cells just overlying the basal layer of epidermis both in the handle of the malleus (HM) and annular regions. In the intermediate region of the PT, no immunoreactivity was found basically, apart from a few labelled cells observed in the upper-third of the superior quadrant. In the pars flaccida (PF) and in both the osseous and cartilaginous regions of the EAC, positive cells were also situated in the basal layer and the deeper aspect of the suprabasal layers without any specific distributing pattern. It was certified that the generation centre of epidermal cells (keratinocytes) in the PT was located in both the HM and annular regions, and that stem cells in the PF and the EAC were uniformly scattered in the basal layer and the deeper aspect of the spinous layer. According to these findings, the migratory patterns of auditory epidermal cells in the human TM and EAC were discussed.

[The structure of the skin of the external auditory canal and the antiviral activity of the cerumen in carnivorous mammals]. / Stroenie kozhi naruzhnogo slukhovogo prokhoda i antivirusnaia aktivnost' tserumena u khishchnykh mlekopitaiushchikh.

The structure of the meatus acusticus externus skin was studied in the sable, mink, and European polecat with a parallel estimation of antiherpetic activity of the cerumen. Morphologically, these species differ in the degree of fat cellulose development, vascularization, and association with the lymphoid tissue. Lymphocytes are present both in the tissue and along the skin gland ducts and hair follicles up to the external surface only in the sable. The cerumen samples have antiviral activity also in the sable alone. The relationship between physiological properties of the mammalian cerumen and morphogenetic features is discussed.

Expression of a cell-cycle-associated nuclear antigen (MIB 1) in cholesteatoma and auditory meatal skin.

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 for 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 1 score that was 2.3 times higher than the score for keratinocytes of normal external auditory meatal skin.

Localization of cytokines in cholesteatoma tissue.

Acquired cholesteatoma is associated with an intense inflammatory reaction with resultant tissue and bone destruction. Cytokines are molecules released by inflammatory cells at the site of infection and are potent mediators of inflammation and the immune response. Five cytokines, tumor necrosis factor-alpha, transforming growth factor-beta 1 and 2, and interleukin-1 and 6, were immunolocalized in human cholesteatoma epithelium and subepithelial stroma, with greater intensity of staining compared with noninflamed external auditory canal skin. Increased interleukin-6 activity in cholesteatoma epithelium and stroma correlated significantly with the presence of ossicular and bony erosion and granulation tissue noted intraoperatively. Transforming growth factor-beta 2 activity in cholesteatoma epithelium correlated significantly with bony erosion at surgery. Additionally, transforming growth factor-beta 1 activity in cholesteatoma epithelium correlated significantly with increased length of disease. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 appear to be involved in the inflammation and resultant remodeling associated with cholesteatoma. We hypothesized a protective function of transforming growth factor-beta 1 and 2 in the presence of cholesteatoma. The antiinflammatory and osteoclast and keratinocyte inhibitory actions of the transforming growth factor-beta s could potentially slow the proliferation and resultant tissue destructiveness associated with cholesteatoma.