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Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function., Author Summary The cytoplasmic dynein 1 motor complex is known to be essential for photoreceptor outer segment formation and function. NINL, an important interaction partner of three ciliopathy-associated proteins (lebercilin, USH2A and CC2D2A), was previously shown to associate with this motor complex. In this work, we scrutinize the role of NINL using a combination of affinity proteomics and zebrafish studies, in order to gain insight into the pathogenic mechanisms underlying these three associated hereditary disorders. We identify DZANK1 as an important interaction partner of NINL and show that loss of Ninl, Dzank1, or a combination of both synergistically results in impaired transport of trans Golgi-derived vesicles and, as a consequence, defective photoreceptor outer segment formation. Using affinity proteomics, we demonstrate that NINL and DZANK1 associate with complementary subunits of the cytoplasmic dynein 1 complex. Our results support a model in which the NINL-DZANK1 protein module is essential for the proper assembly and folding of the cytoplasmic dynein 1 motor complex, shedding light on the structure and stoichiometry of this important motor complex.

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment., Author Summary Ciliopathies are a group of disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in signal transduction. Mutations in CC2D2A cause two ciliopathies, Joubert and Meckel syndromes, and result in loss of ciliary protein localization. The mechanism by which CC2D2A, located at the ciliary transition zone, controls ciliary protein composition and its link to vesicular trafficking of incoming cargo remain largely unknown. Here, we identify a series of physical interactions linking CC2D2A to vesicular trafficking controlled by the small GTPase RAB8, suggesting a new model, whereby CC2D2A provides a specific docking point for ciliary-bound vesicles at the entrance to the ciliary compartment. We first identify NINL as a physical and genetic interaction partner of CC2D2A, show that both proteins co-localize at the entrance to the cilium and demonstrate that absence of Ninl or Cc2d2a result in similar retinal phenotypes in zebrafish, including mislocalization of Rab8. We further identify MICAL3, a protein known to bind RAB8, as another NINL interaction partner, thus linking CC2D2A to RAB8A-controlled trafficking. Finally, we describe an individual with Joubert syndrome, in whom combined CC2D2A and NINL mutations result in an enhanced phenotype, illustrating the impact of the detected interaction on the disease.

Contains fulltext : 51290.pdf (Publisher’s version ) (Closed access) Streptococcus pneumoniae is a major bacterial pathogen involved in the development of otitis media. The pathogenic mechanisms of this middle ear disease, including the bacterial adherence mechanisms to the mucosal epithelial cells of the host, are poorly understood. In this study, the role of glycosaminoglycans in the adhesion of pneumococci to mucosal epithelial cells is examined. Both nasopharyngeal epithelium from rats and an oral epithelial cell line were used for pneumococcal adherence experiments. Preincubation of pneumococci with heparin, heparan sulfate (HS) and to a lesser extent, chondroitin 4-sulfate (C-4S), was found to inhibit attachment of S. pneumoniae to oral epithelial cells, while dermatan sulfate and hyaluronate did not interfere with pneumococcal binding. Enzymatic removal of HS moieties by heparinase III from nasopharyngeal epithelial cells abolished the attachment of pneumococci to nasopharyngeal epithelium. This study demonstrates that heparin, HS and C-4S are involved in pneumococcal binding to mucosal epithelial cells. This knowledge may contribute to the development of a new prophylactic strategy for otitis media.

Previous studies have shown that Streptococcus pneumoniae exists in both middle ear effusions and the upper respiratory region from children with otitis media with effusion (OME), but it remains unclear whether these strains represent genetically identical clones. Therefore, it cannot be determined whether these bacteria originate from a common source. To determine the presence of pneumococci at different anatomical locations of OME patients, conventional culture and PCR techniques were used. To analyze the possible genetic relatedness between pneumococci from different anatomical sites, molecular typing by amplified fragment length polymorphism was utilized. The percentage of middle ear effusions of OME patients that are positive for pneumococci after PCR analysis (13%) was higher than after conventional culture (5%). Molecular fingerprints from pneumococci derived from two different anatomic sites within patients were very similar in 80% of OME patients and in 90% of acute otitis medium patients, indicating their genetic relatedness. Biofilm formation or pneumococcal L-forms probably play a role in OME, since culture-negative effusions prove to contain pneumococcal DNA. Bacteria involved in this process most likely originate from the nasopharynx since they show a close genetic relatedness with their nasopharyngeal counterparts.

Contains fulltext : 139980.pdf (Publisher’s version ) (Closed access) OBJECTIVES/HYPOTHESIS: The observation that during otitis media many different types of micro-organisms have been cultured from effusions indicate that, once present in the middle ear cavity, most types of micro-organisms are able to trigger an inflammatory reaction leading to otitis media. The present study was designed to determine the middle ear response after injection of different substances into the middle ear cavity. STUDY DESIGN: To determine whether and to what extent an inflammatory response of the middle ear depends on the entering agent, the response in the tympanic cavity was studied by otomicroscopy and histological examination after inoculation of various substances. METHODS: Lewis rats were inoculated in transtympanic fashion either with live or heat-killed bacteria (pathogenic and nonpathogenic), Keyhole limpet hemocyanin, active charcoal, or saline. The mucosal response of the challenged middle ears was studied histologically. RESULTS: Irrespective of the inoculated substance, no essential differences in the mucosal response were found. The intensity of the inflammatory response was greater when live bacteria were inoculated. CONCLUSIONS: The present study demonstrates that any substance reaching the middle ear cavity is likely to induce otitis media. These observations emphasize the role of the eustachian tube as "porte d'entree" in the pathogenesis of this disorder. Determination of specific aspects of the eustachian tube involved in protection or in facilitating bacterial translocation will be important for the understanding of the pathogenesis of otitis media and the subsequent development of new therapeutic strategies. In addition, elucidation of bacterial factors involved in the process of colonization and translocation will be of equal importance.

Hypothesis and Background: The Eustachian tube has three important functions with respect to the middle ear: ventilation, clearance, and protection. Surfactants are assumed to be important to maintain these functions. The administration of exogenous surfactant may therefore be effective to improve the function of the Eustachian tube. This randomized, doubleblind, placebo-controlled study was designed to investigate the effect of exogenous surfactant on the function of the Eustachian tube in rats. Materials and Methods: Exogenous surfactant was administered into the middle ear of 10 otologically healthy rats, and 10 other rats received placebo. The effect on the opening and closing pressure (passive ventilatory function) and the dye clearance time (clearance function) of the rat’s Eustachian tube was measured. Results: A significant decrease in the opening pressure was seen after the administration of surfactant. Both surfactant and placebo caused an increase in the closing pressure. A serious disturbance of the dye clearance time was induced in 13 rats, and the test failed in 1 rat. In the remaining 6 rats, no significant differences in the dye clearance time were observed between the two groups. Conclusions: Exogenous surfactant decreased the closing forces of the Eustachian tube even in otologically healthy rats. No significant effect on the mucociliary clearance was observed, but this may have resulted from the small number of rats. Additional randomized, double-blind, placebo-controlled trials should be conducted to determine the clinical relevance of these changes and to further assess the effect of surfactant on the function of the Eustachian tube. Key Words: Surfactant— Eustachian tube—Ventilatory function—Clearance function. Otol Neurotol 24:6–10, 2003.

It has recently been demonstrated that endolymphatic sac (ES) ribosome-rich (dark) cells respond to induced endolymph changes and are thus likely to be involved in endolymph homeostasis. Therefore, we studied the ultrastructural characteristics of rat ES ribosome-rich cells during development in order to determine the cellular distribution of organelles involved in protein metabolism, secretion and absorption, indicative for their contribution to endolymph homeostasis. During embryonal stages ribosome-rich cells contain a limited number and variety of organelles and are predominantly involved in the production of components for cell growth and differentiation. In the young adult stage (P60) three different states of ribosome-rich cells may be distinguished. State A resembles a cell with only limited metabolic activities whereas state B is characterized by numerous different intracellular organelles and is considered to be involved in production and secretion as well as absorption and degradation of complex proteins. A third cellular state, state C, is filled with phagolysosomes and contains very few other organelles. This is considered to be a final (pre)apoptotic state. Autoradiography data suggest that ES ribosome-rich cells are capable of synthesis and secretion of tyrosine-containing proteins and may thus be involved in regulation of the osmolarity of endolymph based on the capacity to bind cations as well as water molecules. In addition, ES ribosome-rich cells appear to synthesize and secrete fucosylated glycoproteins into the endolymph. In conclusion, the present data suggest that ES ribosome-rich cells are actively involved in endolymph homeostasis through secretion and absorption of complex proteins and it is hypothesized that they are able to adapt their function or activities in response to changes in endolymph composition.

Contains fulltext : 139981.pdf (Publisher’s version ) (Closed access) This study describes the development of a physiological rat model for otitis media. The model is based on the assumption that bacteria, intranasally introduced into the nasopharynx, will be transferred into the middle ear cavity during swallowing provided that the ambient air pressure is higher than the middle ear pressure. This model demonstrates that small pressure changes, generated in a pressure cabin under controlled conditions, can be used as driving force for the transfer of bacteria into the middle ear cavity resulting in bilateral otitis media. Because invasive techniques or biochemical agents are not applied, this model is suited to investigate immunological aspects of otitis media, including the effects of vaccination.

Item does not contain fulltext Otitis media with effusion is highly prevalent among young children. Adverse effects of this disorder are mainly restricted to the group of children with a history of recurrent or persistent otitis media with effusion. Early identification, assessment and intervention might prevent these adverse effects. Up to now it is not possible to distinguish these children from those with transient otitis media with effusion. This article presents a comprehensive model for the aetiology of otitis media with effusion. Eustachian tube functioning and the immunological response to environmental pathogens are the two core elements. This model can be used to formulate specific hypotheses about the interaction of several factors that may lead to the early identification of children who are likely to develop persistent or recurrent otitis media with effusion.

Item does not contain fulltext OBJECTIVE: To study the effect of various middle ear effusions on the structure of the lamina propria of the tympanic membrane. METHODS: Sterile and infective middle ear effusions were induced by obstruction of the eustachian tube in specific pathogen-free (SPF) rats and in rats with upper airway infections (URI), respectively. The condition of the tympanic membrane was monitored at regular intervals. After varying survival times, the animals were killed and the tympanic membranes processed for light and electron microscopy. RESULTS: Sterile effusions always resulted in tympanosclerotic lesions. These lesions did not develop in the presence of primary-infected effusions. These effusions had a severe destructive effect on the lamina propria, followed by fibrosis. Generally, secondary infection did not markedly affect preexisting tympanosclerotic lesions. Moreover, calcification disappeared when re-aeration of the middle ear occurred, but the abnormal collagen depositions persisted. CONCLUSIONS: Both sterile and infective effusions result in comprehensive irreversible changes in the lamina propria of the pars tensa. The development of tympanosclerosis is confined to sterile effusions. Mechanical injury and compromised vascularization of the lamina propria are likely to be important etiological factors in the development of tympanosclerosis.

In this developmental study, the distribution and features of melanocytes in the inner ear of pigmented and albino rats was investigated with the use of an antibody, which specifically reacts with a melanocyte differentiation antigen present in the membranes of (pre)melanosomes. Melanocyte precursors could be traced from 13 days post conception onwards and the course was followed to their targets in the inner ear. Melanocytes which settle in the modiolus appeared to reach their target along another pathway than strial and vestibular melanocytes. No difference was observed in the melanocyte distribution between pigmented and albino rats. The integration of melanocytes into the stria vascularis was associated with an increased rate of melanosome production in both strains, but in the albinos far fewer melanosomes were produced. After the stria had reached maturity, melanosome production was arrested and melanosomes were subject to lysosomal digestion. In the stria of the pigmented rats, cells with aggregations of disintegrating melanosomes appeared and persisted into adulthood. In the adult, the majority of the intermediate cells contained only a few scattered melanosomes, while melanosomes could only rarely be detected in the albinos. These observations indicate that there is a close relationship between melanosome production and the process of interdigitation of melanocytes with the marginal cells. It seems unlikely that melanosomes or melanin make any important contribution to the function of the adult stria vascularis. Outside the stria, the features of melanocytes in both strains were similar to skin melanocytes.

Contains fulltext : 89602.pdf (Publisher’s version ) (Closed access) Detailed understanding of neurofilament protein distribution in the inner ear can shed light on regulatory mechanisms involved in neuronal development of this tissue. We assessed the spatio-temporal changes in the distribution of neurofilaments in the developing rat inner ear between embryonic day 12 and 30 days after birth, using antibodies against phosphorylated as well as non-phosphorylated light (NFL), medium (NFM) and heavy (NFH) neurofilament subunits. Our results show that during development, the onset of neurofilament expression in the rat inner ear is on embryonic day 12, earlier than previously shown. We demonstrate that neurofilament subunits of different molecular weight emerge in a developmental stage-dependent order. In addition, we determined that neurofilaments of the vestibular nerve mature earlier than neurofilaments of the cochlear nerve. Cochlear neurofilament maturation progresses in a gradient from base to apex, and from inner to outer hair cells. The sequential pattern of neurofilament expression we describe may help understand the consequences of certain mutations, and contribute to develop therapeutic strategies. 01 augustus 2010

In the present study the expression of vimentin-type intermediate filament proteins and cytokeratins was studied immunohistochemically in the rat inner ear from 12 days postconception up to 40 days after birth. With the use of a broad spectrum monoclonal antibody, cytokeratin expression was found to be present in the whole epithelial lining except for the sensory cells, throughout all the developmental stages examined. Vimentin was detected in the mesenchymal cells, the mesenchyme-derived tissues and the intermediate cells of the stria vascularis, confirming their origin from melanocyte precursor cells. In addition, the coexpression of vimentin and cytokeratins in the epithelial lining of the membranous inner ear was found to be developmentally regulated. During the final stages of differention, vimentin expression disappeared from the majority of the cell types. In the mature cochlea the coexpression of vimentin and cytokeratins was still found in the supporting cells of the organ of Corti, in the cells of Claudius and in external sulcus cells. As far as we could conclude from this study, the sensory cells showed only vimentin expression but not cytokeratin expression. A possible relationship between vimentin expression in adult epithelial cells of the inner ear and a specialised function of these cells is discussed.

Contains fulltext : 80244.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To assess whether free-living and/or biofilm bacteria are present in the putative sterile middle ear cavity before insertion of the electrode array during cochlear implantation. DESIGN: Prospective study. SETTING: Tertiary academic hospital. PATIENTS: The study included 45 healthy children (with or without a history of otitis media) undergoing cochlear implantation. INTERVENTIONS: Transmission electron microscopy or scanning electron microscopy was used to detect the presence of bacteria. MAIN OUTCOME MEASURE: Presence of both free-living bacteria and biofilm bacteria on the epithelial surface of biopsy specimens of middle ear mucosa. RESULTS: A majority of all mucosal specimens from clinically healthy tympanic cavities displayed inflammatory areas as well as dispersed, nonmatrix-enclosed bacteria. Also, rarely, fragments of biofilms were found. CONCLUSIONS: The presence of bacteria in the tympanic cavity, which is generally assumed to be sterile in healthy individuals, may provide an explanation for infectious complications after cochlear implantation. However, the possibility that the electrode array of a cochlear implant will actually become contaminated during insertion is unlikely because of the small amounts and dispersed presence of bacteria, which may account for the relatively low incidence of infectious complications after cochlear implantation.

The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes.

Contains fulltext : 50542.pdf (Publisher’s version ) (Closed access) In this review, a state of the art on otitis media research is provided with emphasis on the role of Streptococcus pneumoniae in the pathogenesis of this disease. Articles have been selected by MEDLINE search supplemented with a manual crosscheck of bibliographies. Pathogenic mechanisms in middle ear and eustachian tube are described. Furthermore, pneumococcal characteristics and pneumococcus-host interactions are highlighted as well as the possible role of biofilms in persistence or recurrence of otitis media. Because of the availability of new techniques, an increasing number of pneumococcal features contributing in the pathogenesis of otitis media are identified and in-depth knowledge of pneumococcus-host interactions has been gained. The present advances in research on otitis media open up new perspectives for therapeutic or preventive strategies.

Item does not contain fulltext OBJECTIVE/HYPOTHESIS: The purpose of the study was to examine the specific involvement of endolymphatic sac mitochondria-rich cells in endolymph homeostasis. STUDY DESIGN: Transmission electron microscopy and immunohistochemistry were performed on the endolymphatic sac of young adult rats, and two important developmental stages were also investigated. METHODS: Ultrastructural characteristics of endolymphatic sac mitochondria-rich cells were studied more concisely and compared with renal mitochondria-rich cells (i.e., the intercalated cells). In addition, expression of cytokeratins 7 and 19 was determined. RESULTS: Until birth, only one type of mitochondria-rich cell is observed in the rat endolymphatic sac. In young adult animals, distinct differences in mitochondria-rich cell ultrastructure in the endolymphatic sac enables classification into subtypes or configurations. Comparison of endolymphatic sac mitochondria-rich cells with renal intercalated cells reveals striking similarities and provides additional information on their specific function in endolymph homeostasis. Furthermore, differences in cytokeratin expression are determined in endolymphatic sac mitochondria-rich cells. CONCLUSIONS: Differences in morphology of endolymphatic sac mitochondria-rich cells develop after birth and may reflect a distinct functional or physiological state of the cell. In analogy to renal intercalated cells, the distribution patterns of H+-adenosine triphosphatase and Cl-/HCO3- exchanger may differ between subtypes. We propose that subtype A mitochondria-rich cells, from which protruding A mitochondria-rich cells are the activated state, are involved in proton secretion (apical H+-adenosine triphosphatase) and thus are potential candidates for hearing loss accompanying renal tubular acidosis. Subtype B mitochondria-rich cells are the most likely candidates to be affected in Pendred syndrome because of the assumed function of pendrin as apical Cl-/HCO3- exchanger.

Item does not contain fulltext The purpose of this study was to determine specific characteristics of endolymphatic sac (ES) cells of the developing rat that are considered to be involved in endolymph homeostasis. Because intermediate filament proteins (IFPs) are regarded as markers of cell differentiation and basal lamina proteins (BLPs) are essential in cellmatrix interactions, we determined the presence of IFPs [cytokeratins (CKs) and vimentin] and BLPs [collagen IV, heparan sulphate proteoglycan (HSPG) and laminin] at different developmental stages before and after birth. In addition, we studied the expression of two enzymes of oxidative metabolism: cytochrome oxidase and succinate dehydrogenase. The presence of CKs 8, 18 and 19 in all epithelial cells of the ES during the embryonic stage is characteristic of simple (glandular) epithelial cells. Interestingly, a distinct population of these cells shows additional expression of CK 7, which is a feature of secretory cells. These CK 7-positive cells also contain a high concentration of oxidative enzymes and are rich in mitochondria, indicating that they are light cells. It is suggested that light cells possess specific energy-requiring transport capabilities. Loss of CK 19 expression in the distal part and in a large region of the intermediate part of the ES implies that these cells do not differentiate any further and acquire the capacity to proliferate. Furthermore, prominent co-expression of vimentin with the CKs in the distal part of the ES may confer viscoelastic properties on this epithelium. This may facilitate expansion and thus enable cushioning of pressure fluctuations. Finally, the early prominent occurrence of HSPG in the basal lamina of the ES enables transport of ions. In this light our recent observations of early functioning NaK-ATPases in certain ES cells are interesting.

The nature of the insertion of the tympanic membrane into the tympanic bone was studied in the rat during the developmental period ranging from 18 days post conception (dpc) to 40 days after birth (dab). Techniques applied were light microscopy, electron microscopy and immunohistochemistry with antibodies to cytoskeletal proteins: vimentin, desmin and alpha-smooth muscle actin (sma) as fibroblast differentiation markers. It was established that the cartilaginous annulus of the pars tensa was connected to the tympanic bone by an interface of specialised connective tissue. Both the fibrocartilage and the interface were derived from the embryonal mesenchyme between the tympanic ring and meatal plate. Electron microscopy showed that the interface was composed of two types of fibroblast. The majority of these cells were myofibroblasts, which were interconnected by junctions and had intimate contact with the collagenous fibres. A small number were identified as genuine fibroblasts. Cytoskeletal characterisation revealed the presence of three types of cell: V cells which expressed vimentin, VA cells which expressed vimentin and alpha-sma and VAD cells which expressed vimentin, alpha-sma and desmin. The myofibroblasts expressed antigens of both smooth muscle cells (alpha-sma, desmin) and connective tissue cells (vimentin). It is suggested that the pars tensa is connected to the tympanic bone by a network of contractile cells and fibres. Contraction will move the membrane in an outward direction and antagonise the inward retraction by the tensor tympani.

Objective: To investigate the relationship between the anatomical maturation of the middle ear and that of the eustachian tube and paratubal muscles in the rat. Design: Wistar rats ranging from gestational day 12 to postnatal day 40 were used. Methods: Tissue specimens were examined with routine light microscopy and electron microscopy. Epithelial differentiation was studied immunohistochemically with antibodies to different cytokeratins. Results: The epithelial lining of the tubotympanum showed differentiation-related cytokeratin expression throughout the whole developmental period. The mucociliary epithelium reached mature features around birth. A dorsal extension and its framing cartilage started forming around 5 days after birth. This extension became lined by stratified nonciliated epithelium and attained maturity around 10 days after birth concurrently with the attachment of the dilatory muscles. This process was immediately followed by aeration of the middle ear cavity. Conclusions: The continuous expression of cytokeratins demonstrates that the epithelial lining of the tubotympanum is only derived from the embryonal endoderm. Furthermore, this study demonstrates that the eustachian tube shows a two-stage postnatal development. First, the mucociliary system matures, providing protection/clearance when the animal starts respiration and swallowing. Subsequently, the dorsal part attains maturity. The features of the epithelial lining of the dorsal part of the eustachian tube and the coincidence of the maturation of this part with the attachment of the dilating muscle fibers and the aeration of the middle ear indicates that this part provides ventilation. These findings support the authors' hypothesis that different parts of the eustachian tube serve different purposes: clearance, protection and ventilation.

Vom histologischen Aufbau der Eustachi-Röhre können deren Grundfunktionen (Schutz, Reinigung und Belüftung) abgeleitet werden. Neuere systematische histologische und Mikroschnittuntersuchungen sowie embryologische Untersuchungen zur Entwicklung der Tuba auditiva führten zu Ergebnissen, die unsere Einsicht in die Funktion der Eustachi-Röhre vertieft haben. Diese Befunde sind: 1. Squamöses, nicht sekretorisches Epithel kommt nur im dorsalen Teil der Tube vor. 2. Die Öffnungsmuskeln der Tube setzen ausschliesslich am dorsalen Abschnitt an. 3. Zur Zeit der Geburt ist lediglich das mukoziliare Epithel ausgereift. Das dorsale, squamöse hingegen erst kurz vor der Belüftung des Mittelohres (10 Tage postpartal). Diese Befunde führten zum Konzept der räumlich-funktionellen Zweiteilung des Tubenepithels: Der dorsale Teil spielt eine Rolle bei der Belüftung des Mittelohres, der ventrale dient der Clearance.

Objective: To evaluate the type of differentiation of keratinocytes of acquired cholesteatoma and its significance for cholesteatoma invasiveness. Design: Forty acquired cholesteatomas and 10 tympanic membranes with persisting perforations were snap frozen and processed for immunohistochemical studies. Cytokeratin antibodies that represented all subgroups and antibodies that were directed against collagen components of the basal lamina were applied. Expression of these constituents was scored by using light microscopy. Results: The phenotype of the matrix was generally characterized by an extension of expression of basal cell cytokeratin 14 and hyperproliferation-associated cytokeratins 6, 16, and 17 into the suprabasal cell layers, while the expression of keratinization marker cytokeratin 10 was down-regulated. These features varied greatly at different sites of the matrix and were most marked at the advancing front of the cholesteatoma. A comparable expression pattern, but less pronounced, was observed at the epidermal front of the mucocutaneous junction of the tympanic membrane perforations. This phenomenon was invariably associated with a mononuclear cell infiltrate in the dermis at both junctions. The basal lamina was always intact. Conclusions: Acquired cholesteatomas show hyperproliferative features. There is a striking similarity between the pronounced expression of this phenotype and the associated inflammation at the mucocutaneous junctions of cholesteatomas and tympanic membrane perforations and those that are observed after epidermal injury. This indicates that epidermis and middle ear epithelium do not form stable junctions and the front can be considered to be a persisting epidermal defect. This involves the permanent presence of "activated keratinocytes" in the junction area that will lead to proliferation and migration, when additional triggers are present. Arch Otolaryngol Head Neck Surg. 1996;122:825-832

The differentiation of epidermis in the various parts of the human ear canal was documented on the basis of cytokeratin (Ck) expression patterns. Immunohistochemistry was performed on cryostat sections of normal meatal skin using a comprehensive panel of monospecific Ck antibodies representing the main lines of epithelial differentiation. The epidermis of the cartilaginous part showed a Ck profile characteristic of normal skin type differentiation. The deep meatal skin, including the tympanic membrane, showed a peculiar type of differentiation: in addition to epidermal Cks, hyperproliferation-associated Cks 6, 16, and 17 were expressed in the suprabasal cells, while the simple epithelia cell marker Ck 19 was found in the basal cells. The presence of hyperproliferative Cks in the deep meatal skin could only partly be related to areas of proliferative activity. Keratinocytes, which express markers of hyperproliferation, are migratory. Therefore, their presence in the meatal skin is likely to be related to the peculiar pattern of keratinocyte migration, the purpose of which is to keep the meatus free from desquamation products.

Objective: To determine the behavior of epidermal cells after transplantation in the middle ear. Design: In a rat model, full-thickness meatal skin grafts were transplanted into the middle ear and studied morphologically and immunohistochemically with the use of antibodies directed against different cytokeratin (Ck) polypeptides, which are markers of different types of epithelial cell differentiation. Results: The grafts had either transformed into epithelial cysts or had become integrated into the middle ear epithelium. The epithelium of the integrated grafts showed gradual transition into the epithelium of the middle ear. A clear distinction between epidermal cells and middle ear epithelium could be made only on the basis of their Ck profiles. The Ck profiles of the grafts revealed a decrease in the expression of epidermal Cks, while nonepidermal Cks became expressed. These changes can be ascribed to replacement of the dermal mesenchyma by mesenchyma from the middle ear. In two ears with superimposed infection, the graft epithelium showed expansive growth. Conclusions: Meatal epidermis is well tolerated in the middle ear, but superimposed infection can induce expansive growth. These findings favor the concept that the progressive growth of cholesteatoma is related to the presence of inflammatory processes. (Arch Otolaryngol Head Neck Surg. 1994;120:1102-1111)

Zur weiteren Charakterisierung der verschiedenen Zelltypen des Saccus endolymphaticus wurde die Expression intermediarer Filamentproteine (IFP) immunhistochemisch bei der jugendlichen und erwachsenen Ratte sowie beim erwachsenen Meerschweinchen untersucht. Monoklonale Antikorper gegen verschiedene Zytokeratine (CK) und Vimentin wurden verwendet. Beide Tierspezies zeigten eine homogene Expression der sogenannten einfachen epithelialen CK8 und 18. Beide einfachen epithelialen CK7 und 19 zeigten ein Expressionsmuster von CK4 und 5 beim Meerschweinchen beobachtet, was auf ein komplexeres Epithel hinweist. Bemerkenswerterweise wurde das nicht epitheliale IFP Vimentin heterogen in der Epithelschicht exprimiert. Vorlaufige Untersuchungen uber die funktionelle Bedeutung einer IFP-Expression demonstrierten eine Korrelation zwischen der CK7-Expression und einem hohen Gehalt an Enzymen des oxidativen Metabolismus.

The expression of cytokeratins in the epithelium of the middle ear and external auditory meatus of the rat was studied on cryosections of ethylenediaminetetraacetic acid–decalcified specimens by use of a panel of monoclonal antibodies. The normal middle ear epithelium revealed a complex cytokeratin profile, including regional differences. The induction of sterile middle ear effusions resulted in increased cytokeratin expression. Infective effusions were accompanied by both quantitative and qualitative changes in the cytokeratin expression patterns. The difference observed between the cytokeratin profiles of external meatal skin and those of middle ear epithelium may form a useful tool for research into cholesteatoma development.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.

This study deals with an animal model where tympanosclerosis could be evoked with high reproducibility during the course of a sterile otitis media, induced by Eustachian tube obstruction. The histopathological features of this induced lesion were very similar to those reported in human specimens. It was concluded that this process is most probably triggered by a mechanical deformation of the tympanic membrane.