Murine autoimmune hearing loss mediated by CD4+ T cells specific for ß-tubulin.

Autoimmune inner ear disease is described as progressive, bilateral although asymmetric, sensorineural hearing loss and can be improved by immunosuppressive therapy. We showed that the inner ear autoantigen ß-tubulin is capable of inducing experimental autoimmune hearing loss (EAHL) in mice. Immunization of BALB/c mice with ß-tubulin resulted in hair cell loss and hearing loss, effects that were not seen in animals immunized with control peptide. Moreover, the EAHL model showed that ß-tubulin responsiveness involved CD4(+) T cells producing IFN-γ, and T cell mediation of EAHL was determined by significantly increased auditory brainstem response after adoptive transfer of ß-tubulin-activated CD4(+) T cells into naive BALB/c recipients. The potential mechanisms responsible for the observed pathology of EAHL can be attributed to decreased frequency and impaired suppressive function of regulatory T cells. Our study suggests that EAHL may be a T cell-mediated organ-specific autoimmune disorder of the inner ear.

Identification of the hair cell soma-1 antigen, HCS-1, as otoferlin.

Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells.

Localization of gamma-aminobutyric acid A receptor subunits in the rat spiral ganglion and organ of Corti.

Gamma-aminobutyric acid (GABA) is thought to be the major inhibitory neurotransmitter in the central nervous system. Although it is distributed in the olivo-cochlear bundles, which constitute the mammalian cochlear efferent system, its function in the cochlea is still obscure. In this study, we investigated the localization of GABAa receptor subunits (alpha1-6, beta1-3, gamma) in the rat cochlea in order to determine the role of GABA in the cochlea. Most spiral ganglion cells were intensely immunolabeled with all the anti-GABAa receptor subunit antibodies. In the organ of Corti, punctate immunoreactivities were observed in inner hair cell regions corresponding to the distribution of GABA. These data suggest that GABAa receptor was present in afferent nerve terminals in inner hair cell regions, and that GABA regulated afferent nerve transmission contacting efferent nerve endings by means of the axo-dendritic synapse function.

Neurotransmission in the vestibular endorgans--glutamatergic transmission in the afferent synapses of hair cells.

In the sensory pathways the first synapse is that between hair cells and primary afferent neurons and its most likely neurotransmitter candidate has long been thought to be glutamate. A number of pharmacological and electrophysiological studies have lent credence to this theory (reviewed by Bledsoe et al. 1988, Bobbin 1979, Ehrenberger and Felix 1991, Puel et al. 1991; Puel 1995) as has recent neurochemical and immunocytochemical work (reviewed by Ottersen et al. 1998; Usami et al. 2000). These recent studies reveal that the afferent hair cell synapse resembles the central glutamate synapses in many ways. Of the proteins confirmed to be involved in signal transduction and transmitter metabolism at most central synapses, many are also seen in the afferent hair cell synapse, and have an analogous compartmentation. On the other hand, there are also important differences, especially those related to the molecular mechanisms that underlie transmitter release.

The ankle-link antigen: an epitope sensitive to calcium chelation associated with the hair-cell surface and the calycal processes of photoreceptors.

A monoclonal antibody, mAb E40, that specifically recognizes hair cells and photoreceptors was derived from a mouse immunized with a membrane fraction prepared from the sensory maculae of the chick inner ear. In the mature chick inner ear, punctate labeling is observed along each stereocilium, but staining is mostly concentrated around the basal end of the sensory hair bundles, where it is closely associated with surface specializations known as ankle links. The epitope recognized by mAb E40 is therefore referred to as the ankle-link antigen (ALA). During early embryogenesis, the ALA is initially distributed evenly over the surface of the hair bundle. As development proceeds, it becomes more restricted to the base of the hair bundle, although a spot of the ALA remains associated with the bundle tip until just before hatching. In the eye, mAb E40 stains the calycal processes of photoreceptors. When maculae and retinae are treated with the calcium chelator BAPTA at room temperature, the ALA disappears. BAPTA-induced loss of the ALA from the hair-bundle surface is substantially reduced by lowering the temperature to 2 degrees C. The ALA and ankle links reappear on the hair-bundle surface when cells are cultured for 20 hr after BAPTA treatment. BAPTA sensitivity and recovery after BAPTA-induced loss are properties similar to those described for the tip link, a surface structure thought to gate the mechanotransducer channel. However, unlike the tip link, the ALA and ankle links are sensitive to subtilisin treatment. The results define a new component of the hair-bundle surface, with properties both common to and distinct from those of the tip link.

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

Immunologically defined component of the circumferential ring around the cuticular plate in mammalian hair cells.

A monoclonal antibody (CAR) was raised by in vitro immunisation to a component of the circumferential actin ring that is associated with the apical junctions encircling the cuticular plates of mammalian hair cells. On western blots it bound a protein band at about 42 kD, equivalent to the normal location of actin, but it did not label the paracrystalline bundle of actin filaments in the stereocilia, the complex actin filament gel that forms the cuticular plate or the filamentous actin in the cell cortex. When applied to whole mounts of the auditory sensory epithelium in the guinea pig it provided a clear, unambiguous map of the distribution of inner and outer hair cells. In this respect, it can provide an accurate guide to patterns of hair cell differentiation and repair. CAR cross-reacted with the membrane-associated cytoskeleton in selected cells from a wide range of other tissues.

Caracterizaçäo imuno-histoquimica de ceratinócitos humanos epidérmicos e foliculares in vivo e in vitro / Immuno histochemistry characterization of human epidermics and follicular Keratnocytes in vivo and in culture

FUNDAMENTO - As citoqueratinas säo excelentes marcadores da diferenciaçäo das células epiteliais. OBJETIVO. Caracterizar a expressäo de citoqueratinas e filagrina em ceratinócitos in vivo e em cultura. MATERIAL E MÉTODOS - Ceratinócitos epidérmicos e foliculares (da bainha radicular externa), cultivados sob as mesmas condiçöes, e criocortes de couro cabeludo normal foram corados mediante a técnica APAAP, com 13 anticorpos monoclonais anticitoqueratinas e um antifilagrina. RESULTADOS In vivo, os ceratinócitos epidérmicos expressam as citoqueratinas 1,2,5,10 e 14, e os ceratinócitos foliculares, as citoqueratinas 5,6,14,16,17 e 19. Por outro lado, in vitro, ambas as subpopulaçöes de ceratinócitos demonstraram as mesmas citoqueratinas 2,5,6,14,16,17,19, embora a expressäo de 2 tenha sido mais intensa nos ceratinócitos epidérmicos. Em cultura, ambos expressam citoqueratinas que näo estavam presentes in vivo. O anticorpo monoclonal antifilagrina demarcou ambas as subpopulaçöes in vitro, e, in vivo, estava presente somente nos ceratinócitos epidérmicos. CONCLUSäO - Ceratinócitos de diferentes origens, da epiderme e do folículo piloso, adotam, em cultura, estágio intermediário de diferenciaçäo

Ontogenesis of type II spiral ganglion neurons during development: peripherin immunohistochemistry.

In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.