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We investigated the localization of periostin in middle ear specimens from patients with eosinophilic otitis media (EOM) and from a newly constructed animal model for EOM. Periostin-positive immunostaining was observed in the middle ear sections obtained from the EOM patients. Immunoreactivity for periostin was also seen in the animal model. These results suggest that periostin plays an important role in subepithelial fibrosis in the middle ear in EOM.The purpose of the present study was to investigate the role of periostin in the middle ear of EOM patients and an animal model.Histological and immunohistochemical analyses for periostin were carried out in the middle ear specimens of six EOM patients with/without asthma. An animal model of EOM was constructed by intraperitoneal and topical sensitization with ovalbumin (OVA). Histological and immunocytochemical analyses for periostin were also performed in this model.Immunoreactivities for periostin were observed in the basement membrane and extracellular matrix of the middle ear sections obtained from all EOM patients with/without asthma. In the animal model, eosinophil infiltration and middle ear mucosa thickness were observed. Moreover, periostin-positive immunostaining was shown in the extracellular matrix of the middle ear mucosa on the side topically boosted by OVA.

Mutations in the connexin26 gene (GJB2) are the most common genetic cause of congenital bilateral non-syndromic sensorineural hearing loss. Transgenic mice were established carrying human Cx26 with the R75W mutation that was identified in a deaf family with autosomal dominant negative inheritance [Kudo T et al. (2003) Hum Mol Genet 12:995-1004]. A dominant-negative Gjb2 R75W transgenic mouse model shows incomplete development of the cochlear supporting cells, resulting in profound deafness from birth [Inoshita A et al. (2008) Neuroscience 156:1039-1047]. The Cx26 defect in the Gjb2 R75W transgenic mouse is restricted to the supporting cells; it is unclear why the auditory response is severely disturbed in spite of the presence of outer hair cells (OHCs). The present study was designed to evaluate developmental changes in the in vivo and in vitro function of the OHC, and the fine structure of the OHC and adjacent supporting cells in the R75W transgenic mouse. No detectable distortion product otoacoustic emissions were observed at any frequencies in R75W transgenic mice throughout development. A characteristic phenotype observed in these mice was the absence of the tunnel of Corti, Nuel's space, and spaces surrounding the OHC; the OHC were compressed and squeezed by the surrounding supporting cells. On the other hand, the OHC developed normally. Structural features of the lateral wall, such as the membrane-bound subsurface cisterna beneath the plasma membrane, were intact. Prestin, the voltage-dependent motor protein, was observed by immunohistochemistry in the OHC basolateral membranes of both transgenic and non-transgenic mice. No significant differences in electromotility of isolated OHCs during development was observed between transgenic and control mice. The present study indicates that normal development of the supporting cells is indispensable for proper cellular function of the OHC.

Patients with vertigo caused by psychological problems have increased in recent years. It has been reported that many outpatients with vertigo also suffer from depression. Selective serotonin reuptake inhibitors are widely used for the treatment of depression because of their efficacy and relatively favorable side-effects profile compared to those of tricyclic antidepressants. We used Paroxetine to treat patients with vertigo who were diagnosed with depression by self-rating questionnaires for depression (SRQ-D). Twenty-six patients experienced relief after taking Paroxetine. Fifteen of them were relieved of their vertigo symptoms, 8 were improved, one was not changed, and two were worse. SRQ-D scores were compared between the day of commencement and the day after 4 weeks. Fifteen patients were normalized. Side effects occurred in seven patients; nausea in five and sleepiness in two. The average duration of taking the medicine was 24.4 weeks. Since Paroxetine showed beneficial clinical effects for patients with vertigo, this medicine might be useful for vertiginous patients with depression.

A 12-year-old male experienced left-sided hearing impairment, tinnitus, and vertigo after being hit by a volleyball. Pure tone audiometry showed severe, mixed left hearing loss and nystagmus to the right side. We diagnosed perilymphatic fistula and treated it conservatively. After ten days, his vertigo had disappeared and hearing on his left side showed improvement except at 2,000 and 8,000 Hz. However, he experienced slight dizziness after discharge from our hospital. About eleven months later, because of severe vertigo, he again came to our hospital. We diagnosed benign paroxysmal positional vertigo (BPPV) based on his symptoms and the characteristic nystagmus. We performed the Epley maneuver, and his vertigo and nystagmus disappeared the next day. Exploratory tympanotomy was carried out, but no sign of a perilymphatic fistula was detected. He experienced BPPV again, and so we tried the Epley and Lempert maneuver. We believed his prolonged dizziness was due to a partial collapse of the utricular wall, a condition called floating labyrinth. This phenomenon may occur with migration of otoliths from the utricle, leading to BPPV.

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C (v)), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C (v) was about 70% of the mature level. C (v) saturated at P18 when hearing shows full maturation. On the other hand, C (lin), which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor's operating voltage midpoint, V (pkcm), tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V (pkcm) reached a steady-state level near -67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V (pkcm), may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.

Although dopaminergic neurons are thought to exist in the lateral olivocochlear efferent system and modulate the afferent nerve activity, the distribution of dopamine (DA) receptor subtypes is still obscure. In the present study, we investigated the localization of five subtypes of DA receptor (D1-5) by immunocytochemical analysis and the gene expression of D1-5 using RT-PCR procedure in the rat cochlea. Most, but not all, spiral ganglion neurons were immunolabeled with all the anti-DA receptor subunit antibodies and faint punctuate immunoreactivities were observed in inner hair cell regions. Gene expression for all receptors was detected. These results suggest that all DA receptor subtypes are present in spiral ganglion cells, and potentially regulate afferent neurotransmission.

When we treat patients with pollen allergy, we require correct information about pollen release. The total count of pollen and the date of first pollen release are strongly influenced by local weather conditions, so we wanted to predict local pollen release. Patients have degrees of sensitivity to pollen, with symptoms triggered in some cases by only small amounts. Therefore, we wanted to determine the previous dates of first pollen release (A), the first of two consecutive dates when one (count/cm2) or more grains were released (B), and the first of two consecutive dates when ten (count/cm2) or more grains were released (C). Aomori Prefecture has three different weather zones because it is surrounded by three oceans the Sea of Japan, the Pacific Ocean, and Mutsu Bay. We predicted pollen release in the major cities--Aomori, Hirosaki, and Hachinohe. Using nine years (1996-2004) of temperature data for each city from January 1 through February 1, we recorded the number of days in which the daily highest temperature was higher than each of five set temperatures (0 degree C, 1 degree C, 2 degrees C, 3 degrees C, 4 degrees C) up to the dates of A, B, and C. Multiple recordings were made for each year with the initial date of recording staggered at 10-day intervals. We then calculated the standard deviation and the efficient of variation of total days and totals of temperatures over set temperatures. For Aomori, results indicated that A was predicted as day 19 with temperatures over 4 degrees C after January 21. B was day 23 over 4 degrees C after January 21. C was the day 31 with over 4 degrees C after January 21. For Hirosaki, A was expected to be day 26 having temperatures over 0 degrees C after February 1. B was day 21 over 3 degrees C after February 1. C was day 30 over 3 degrees C after January 21. For Hachinohe, A was day 34 with temperatures over 0 degrees C after February 1. B was day 33 over 1 degree C after January 21. C was day 27 over 4 degrees C after January 21. We examined the day of pollen release in 2005.

Although apoptotic changes in auditory neurons induced by injury to peripheral processes (dendrites) have been intensively studied, apoptotic changes in auditory neurons induced by injury to central processes (axons of spiral ganglion cells, SGCs) have not been reported previously, probably due to lack of an experimental model. The present study reports for the first time the appearance, extent, and time course of SGC apoptosis following injury to the central processes. Apoptosis was studied in a rat model that consisted of compression of the auditory nerve in the cerebellopontine (CP) angle cistern with intraoperative recordings of auditory nerve compound action potentials (CAPs) to ensure highly reproducible results. Rats were killed between day 0 and day 14 after compression and apoptosis of SGCs was evaluated quantitatively as well as qualitatively by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, anti-activated caspase-3 immunostaining, Hoechst 33342 staining, and electron microscopy. The average number of TUNEL-positive apoptotic SGCs in each cochlear turn increased from day 1 to day 5 and then decreased gradually to an undetectable level on day 14 after compression. The average proportion of apoptotic SGCs identified in any cochlear turn on any day was always lower than 10%. The results of our present study should be useful in determining the therapeutic time window for rescuing auditory neurons undergoing apoptosis due to injury during surgery in the CP angle.

Reduced expression of the p16 gene product (protein), an inhibitor of cyclin-D-dependent protein kinase which regulates cell cycle at the G1/S boundary, is implicated in tumor progression in various neoplasms. Hypermethylation of the p16 promoter gene has recently been suggested to be one of the reasons for the reduced protein expression. To explore the role of p16 in the biological behavior of adenoid cystic carcinomas (ACC), we investigated the immunohistochemical expression of p16 protein in 38 ACC tumors (32 primary, 3 recurrent, and 3 metastatic tumors) and the methylation status of its promoter gene. We also examined their relationships to the histological grade of malignancy. Positive reaction of p16 protein was demonstrated in the nuclei of luminar cuboidal cells in areas with tubular patterns. The reactions were reduced in the areas with solid or large cribriform patterns. The levels of p16 expression correlated with the histological grade of malignancy. Recurrent or metastatic tumors did not differ with respect to histological grades from the original tumor except for 1 case, in which p16 expression was reduced compared to the primary tumor. Methylation-specific PCR demonstrated the hypermethylation status of the p16 promoter gene in 4 of 22 primary tumors (21%), all of which showed negative or low expression of the p16 protein. The study indicated that p16 expression was reduced in ACC cases of higher histological grade of malignancy and that hypermethylation of its promoter gene may be involved in its process in some cases.

Japanese cedar is the most common pollinosis allergen in Japan. Predictions of total pollen counts and the first 2 or more days of the Japanese cedar pollen season are beneficial to patients. We developed predictive methods for those 2 factors in Hirosaki. We conducted an atmospheric Japanese cedar pollen survey from 1996 to 2002 in Hirosaki and compared yearly data with the male flowering index counted in 50 cedar trees in the preceding autumn. The male flowering index correlated highly positively with total cedar pollen counts in regression analysis, indicating its usefulness in predicting total pollen counts. Although the number of days from January 1 to the first day of continuous pollen emission showed no correlation with the cumulative maximum temperature during these days, the number of days from the day of first detection of pollen emission (minimum of 1 particle per sq. cm) to the first day of continuous pollen emission correlated highly with the cumulative maximum temperature and cumulative amount of snowfall on these days, suggesting that it is possible to predict the first day of continuous pollen emission by the cumulated daily maximum temperature after the day of first detection of pollen emission.

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.

The excessive glutamate released from the type I and type II hair cells and the supporting cells injure the bouton-type endings and the nerve chalices in 30 min ischemia, and neuronal damage of glutamate was slight in 10 min ischemia.In the present study, we investigated by means of post-embedding immunoelectron microscopic analysis whether neuronal damage in the vestibular end organs is associated with the change of cellular glutamate concentration during ischemia.Transient local anoxia (10 min, 30 min) of guinea pig inner ear was induced by pressing the left labyrinthine artery. The right sides were used as controls. The morphological changes of the vestibular end organs and the areal gold particle densities representing glutamate were compared in the ischemia side and the control side.The areal gold particle densities of the type I and type II hair cells and the supporting cells in the ischemic side were lower than those of the control side. There were no remarkable morphological changes compared to the control side in 10 min ischemia. In 30 min ischemia, the bouton-type endings were swollen and intercellular spaces between the type I hair cells and the nerve chalices were enlarged.

The localization of three glutathione S-transferase (GST) isoforms in the rat cochlea was examined using specific antibodies against each isoform. GST immunoreactivities were found in particular parts of the cochlea, including the intermediate cells and the basal cells of the stria vascularis and various types of fibrocytes in the spiral ligament. The different cell types showed varying combinations of GST isoforms. The GST immunopositive cells identified in the present study may play a central role in the metabolism and inactivation of endogenous and exogenous ototoxic compounds. The specific arrangements also indicated a possible contribution to the detoxification process in the form of a blood-labyrinth barrier.

Advances in molecular genetics have recently revealed that mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome in European and other populations. This is the first report confirming that an EYA1 gene mutation is also disease-causing in an Asian population. We have described one Japanese BOR syndrome family showing a novel mutation in exon 7 of the EYA1 gene. There was extensive variation of clinical phenotypes within this family. When the physician is confronted with a BOR family showing a wide variation in clinical expression, molecular genetic testing helps to achieve accurate diagnosis.

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1–4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.

Glial cells play an important role in the removal and metabolism of synaptically released glutamate in the central nervous system (CNS). It is not clear how glutamate is handled at peripheral glutamate synapses, which are not associated with glia. Glutamate is a likely transmitter in the synapse between the hair cells and afferent dendrites of the vestibular end organ. Immunocytochemistry was performed to investigate the distribution at this site of the high affinity glutamate transporter GLAST and glutamate metabolizing enzyme glutamine synthetase. Confocal microscopy revealed that GLAST and glutamine synthetase were co-localized in supporting cells apposed to the immunonegative hair cells. Postembedding immunoelectron microscopy revealed that GLAST was heterogeneously distributed along the plasma membranes of the supporting cells, with higher concentrations basally (at the level of the afferent synapses) than apically. Both immunoreactivities were also present in non-neuronal cells in the vestibular ganglion. The present findings suggest that glutamate released at the afferent synapse of vestibular hair cells may be taken up by adjacent supporting cells and converted into glutamine. Thus, at this peripheral synapse, the supporting cells may carry out functions similar to those of glial cells in the CNS.

The expression of glutathione S-transferase (GST)-pi and four oncogene products, c-Jun, c-Fos, c-H-Ras, and c-Myc, in human squamous cell carcinomas of the head and neck was investigated immunohistochemically before and after radiation therapy, to examine whether these oncogene products might be involved in GST-pi expression, and also to examine the relationship between their expression and therapeutic response. Clinical response to radiation was evaluated in terms of both tumor regression and relapse over two-year follow-up periods. The overall positive rates in 83 carcinoma specimens before therapy were 60.2% for GST-pi and 28.9-51.8% for the individual oncogene products, the positive rates for the oncogene products being higher in GST-pi-positive than in GST-pi-negative cancers. c-Jun was most highly correlated with GST-pi expression. Following radiation, the expression of GST-pi and the oncogene products was altered in about a half of 30 patients. Eleven of the 18 patients who exhibited prior positivity for GST-pi showed negative conversion, while 4 of the 12 patients with prior negativity demonstrated positive conversion. In most cases, changes in c-Jun staining coincided with those in GST-pi. Regarding clinical response to radiation therapy, the positive rates for GST-pi and c-Jun before radiation were higher in the residual cancer or relapse cases than in the group showing complete response without relapse. Examination of 26 patients with laryngeal cancer revealed that relapse occurred more frequently in cases exhibiting positive reactions for GST-pi, c-Jun, or c-H-Ras. These results suggest a direct link between c-Jun and GST-pi in head and neck cancers before and after radiation. Although GST-pi and the oncogene products can be influenced by radiation, GST-pi and c-H-Ras expression may be a risk factor for relapse of laryngeal cancer.

Programmed cell death (apoptosis) in the inner ear of senescence-accelerated mouse was identified using specific labeling of fragmented DNA (the TUNEL method). In spite of some inter-individual differences, the apoptotic cells were predominantly found in the phylogenetically newer part of the inner ear, the cochlea and the saccules. In the saccules, sensory hair cells as well as supporting cells were positively labeled. In the cochlea, positive staining was detected in inner and outer hair cells, pillar cells, Deiters' cells, interdental cells, the stria vascularis (marginal cells, intermediate cells, basal cells), and cells in Reissner's membrane. The present results suggest that age-related cell death, which may cause hearing impairment and dysequilibrium, is due to apoptosis occurring in the inner ear.

The present study was conducted to elucidate species differences in the distribution of neuroactive substances, including substance P (SP), calcitonin gene-related peptide (CGRP) and gamma-aminobutyric acid (GABA), in the vestibular periphery of various animals (chicken, pigeon, rat, guinea pig and squirrel monkey). SP-like immunoreactivity was found in a subset of the primary vestibular afferents with no marked species differences in the staining pattern. In contrast, the localization of CGRP- and GABA-like immunoreactivities in the efferent nerve fibers varied according to species. This difference in the distribution pattern of neuroactive substances, found in the efferent system, may indicate that each species has a chemically (and probably functionally) distinct efferent system which is related to its specific environment and/or evolution.

The distribution of one of the calcium-binding proteins, calbindin-D28K (CB-D28K), was studied in the adult human and squirrel monkey inner ear by means of immunocytochemical methods. Inner and outer hair cells in the organ of Corti and vestibular hair cells showed CB-D28K immunoreactivity, though some vestibular hair cells were devoid of immunoreactivity. In the spiral and vestibular ganglion, immunoreactive cells were found in both the squirrel monkey and human. The present results indicate that CB-D28K is localized within afferent neuronal components in these sensory organs and may regulate Ca++ levels for optimal neurotransmission in the primate auditory and vestibular systems. This study also provides evidence of two nonneuronal localizations of CB-D28K in the squirrel monkey. Subpopulations of fibrocytes in the spiral ligament and vestibular end organs were enriched with CB-D28K, suggesting that these cells are possibly equipped with the function to regulate Ca++ concentration in the perilymphatic fluid. In the maculae, many CB-D28K-immunoreactive particles were found in the otoconial membrane, indicating that CB-D28K may participate in the formation of otoconia.

Three patients with traumatic positional vertigo were neurotologically examined. The first and second patients were middle-aged females who had suffered head injuries in traffic accidents. The first patient had left total deafness, and the second patient had no cochlear symptoms. The third patient was a male worker who was injured by falling rocks and had right sensorineural hearing loss. In all the three patients, we found brisk rotatory nystagmus accompanied with vertigo in the head hanging position. The theory of free-floating particles in the posterior semicircular canal seems to explain the rotatory nystagmus and the gradual decrease in its intensity. On the basis of this theory, a canalith repositioning procedure (CRP) was performed in all three patients. It was effective in the second and third patients, but in the first patient positional nystagmus and vertigo were unchanged.

Using infrared CCD camera and electronystagmography, we analyzed positional nystagmus in 33 patients with peripheral positional vertigo.1) Patients were classified into three categories: (a) lateral type: horizontal nystagmus occurs in lateral position; (b) sagittal type: rotatory nystagmus occurs in head-hanging position (so-called benign paroxysmal positional vertigo); and (c) mixed type.2) Vertigo and nystagmus began with a few seconds latency after the head was tilted, briefly increased to a maximum, and then gradually decreased.3) In all types, the number of female patients was greater than that of male patients.4) In the lateral type, the mean period from the onset to the disappearance of vertigo was about 3 weeks. However, in those with the sagittal type or mixed type it was much longer.5) The intensity of positional nystagmus was influenced by the direction of gravity in all types.The free-floating particles theory seems to be able to explain the latency of the nystagmus, the gradual decrease in its intensity, and recurrence of the vertigo. Lateral type lesions are thought to be in the lateral semicircular canal, and sagittal type lesions perhaps in the posterior semicircular canal; mixed type lesios are thought to be in both.

The immunocytochemical localization of substance P (SP) in vestibular endorgans of various mammals (rat, guinea pig, rabbit, squirrel monkey, and man) was investigated. SP-like immunoreactivity was found in all the vestibular endorgans studied. Most SP immunoreactive fibers were thick and distributed around or beneath sensory hair cells. A smaller group of SP immunoreactive varicose thin fibers was present in the stroma of vestibular endorgans, nerve trunks, and along blood vessels around the vestibular nerves. The former, probably corresponding to the afferent nerve endings of hair cells, was found predominantly in the peripheral region of each endorgan. There were no great species differences in staining patterns, except in rat vestibular endorgans, where numerous thin SP immunoreactive fibers were distributed beneath the sensory epithelium. SP immunoreactive fibers were also found in human vestibular endorgans, suggesting that SP may play an important role in human vestibular function.

The value of the radio allergosorbent test (RAST) in the detection of specific IgE antibody has been investigated by many researches.However, the clinical usefullness of the CAP-fluoro enzyme immuno assay (CAP-FEIA) method recently introduced for the same purpose as the RAST method is still not clear. The authors used both the RAST and the CAP-FEIA methods to test the same patients with nasal allergy and compared the results.It was found that the CAP-FEIA method was more sensitive than the RAST method in the detection of specific IgE antibodies to single or multiple antigens in patients with nasal allergy, except for house dust antigens. Further investigation showed that the lower sensitivity to house dust antigen was due to the impurities in the house dust extract.Reexamination with a new standard house dust extract showed as high sensitivity as to the other antigens. The CAP-FEIA method showed a better correlation with the results of skin tests than did the RAST method.It is concluded that CAP-FEIA is an excellent method for the diagnosis of nasal allergy.

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most common GJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations. Keywords: connexin 26; GJB2; Japanese; non-syndromic hearing loss

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell-extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of beta1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosis-inducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells.

Inner ear hair cells play a major role in the auditory pathway that converts sound stimulation into electrical signals, and then into a neural code. However this function is often lost by aminoglycoside ototoxicity. The injury of inner ear hair cells from aminoglycoside treatment is considered apoptosis, and caspase is an important participant in the apoptosis pathway in many organs. It has been reported that calpain, a calcium-dependent protease, is essential for mediation and promotion of cell death. The purpose of the present study was to investigate effects of caspase and calpain inhibitors on the inner ear hair cells after aminoglycoside treatment, and to explore the cell death pathway. Cochlea explant cultures were prepared from mice of postnatal 6 days, cultured with neomycin and/or protease inhibitors, and then stained with phalloidin-fluorescein isothiocyanate (phalloidin-FITC), which was used as a marker to identify surviving hair cells. We demonstrated that neomycin (0.1-1 mM) reduced the number of outer hair cells in a dose-dependent manner. Furthermore, we showed that leupeptin, a calpain inhibitor, significantly protects against the neomycin-induced loss of outer hair cells, whereas a caspase inhibitor was effective only against a lower concentration of neomycin (0.2 mM). Using the TdT-mediated dUTP-biotin nick and labeling method, we also found that a calpain inhibitor, but not a caspase inhibitor, prevents apoptotic DNA fragmentation after treatment with 1 mM neomycin. These results suggest that calpain, rather than caspase, may be responsible for apoptosis induced by aminoglycoside. Thus, leupeptin may prevent hearing loss from aminoglycoside ototoxity.

Adenoid cystic carcinoma, a relatively uncommon tumor of salivary glands, is characterized by a prolonged clinical course and a fatal outcome. The molecular events underlying their progression are unknown. In this study, we examined the methylation status of E-cadherin gene and its protein expression in 23 cases of adenoid cystic carcinoma and correlated the results with the clinicopathologic factors to determine its role in these tumors. We also analyzed the effect of 5-azacytidine on the re-expression in a methylated cell line of adenoid cystic carcinoma for this gene. In our study, E-cadherin immunoreactivity, although heterogeneous, showed a progressive reduction with high histological grade and in metastatic and recurrent lesions. Promoter methylation was detected in 16 of 23 cases (70%), but there was no correlation with the histological grade or patient prognosis. Microdissection of immuno-negative cells in heterogeneous tumors showed positive methlyation. In the cell line from salivary adenoid cystic carcinoma with methylated E-cadherin, 5-azacytidine restored the E-cadherin expression. Our results indicate that: (1) E-cadherin gene promoter is frequently methylated in adenoid cystic carcinoma, leading to reduced E-cadherin expression, (2) variable E-cadherin expression might result from the intratumoral heterogeneity, and (3) increased extent of methylated areas may be associated with progression and advancement of the disease.

The expression of NMDA R1 receptor mRNA in the rat and guinea-pig vestibular ganglion was examined using in situ hybridization histochemistry. Most of the ganglion cells of both species were strongly labelled, supporting the view that NMDA-type glutamate receptors may be involved in the vestibular afferent neurotransmission.

GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.

The frequency of three mitochondrial point mutations, 1555A→G, 3243A→G, and 7445A→G, known to be associated with hearing impairment, was examined using restriction fragment length polymorphism (RFLP) analysis in two Japanese groups: (1) 319 unrelated SNHL outpatients (including 21 with aminoglycoside antibiotic injection history), and (2) 140 cochlear implantation patients (including 22 with aminoglycoside induced hearing loss). Approximately 3% of the outpatients and 10% of the cochlear implantation patients had the 1555A→G mutation. The frequency was higher in the patients with a history of aminoglycoside injection (outpatient group 33%, cochlear implantation group 59%). One outpatient (0.314%) had the 3243A→G mutation, but no outpatients had the 7445A→G mutation and neither were found in the cochlear implantation group. The significance of the 1555A→G mutation, the most prevalent mitochondrial mutation found in this study of a hearing impaired population in Japan, among subjects with specific backgrounds, such as aminoglycoside induced hearing loss, is evident. Keywords: mitochondria; point mutation; hearing impairment; frequencies

The 1555A--G point mutation is associated with a susceptibility to aminoglycoside antibiotics, and is of particular interest, as it may cause hearing loss even without aminoglycoside exposure. There may be a considerably large high-risk population in Japan, and to avoid possible side effects in this group, a rapid mass screening system and careful counseling are recommended. We are currently using the mutant allele specific amplification (MASA) method to detect the 1555A--G mitochondrial mutation and we distribute a warning card to subjects found to bear this mutation.

Glutamate is thought to be a major neurotransmitter between hair cells and afferent dendrites in the inner ear. However, excessive glutamate is known to be excitotoxic, and may be involved in ischemic neuronal damage in the central nervous system. The glutamate concentration in the perilymph has been reported to increase during ischemia, but the source of glutamate is still unclear. In the present study, we have used post-embedding immunogold cytochemistry to analyse changes in the cellular distribution of glutamate in the guinea pig organ of Corti during ischemia. The areal gold particle densities in the inner hair cells of the ischemic side were lower than those of the control side, indicating that glutamate may be released from the hair cells during ischemia. Adjacent supporting cells (border cells) also showed a decrease in particle density, suggesting that they constitute an additional source of glutamate.

Thirteen Japanese families (ten of which were from the northern part of Japan), with sensorineural hearing loss associated with the 1555 A to G (A1555G) mitochondrial mutation, a known cause of non-syndromic hearing loss, were phylogenetically analysed using data obtained by restriction fragment length polymorphism (RFLP) and D-loop sequencing of mitochondrial DNA (mtDNA). Various types of mtDNA polymorphism were detected by restriction enzymes and D-loop sequence. No common polymorphic pattern throughout the 13 families was found, though three families exhibited the same restriction patterns and the same sequence substitution in the D-loop. To find where each of the 13 families are situated in the phylogenetic tree, the 482-bp of D-loop sequence were compared with those of 62 normal Japanese subjects. Despite the three families mentioned above appearing to be clustered, the remaining 10 families were scattered along the phylogenetic tree. This indicates that there was no common ancestor for the 13 Japanese families bearing the A1555G mutation except three families, and that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in Japan. The present results showed that the common pathogenicity (hearing loss associated with the A1555G mutation) can occur sporadically in families which have different genetic backgrounds, even in the Japanese population.

Enlarged vestibular aqueduct (EVA), known as the most common form of inner ear abnormality, has recently been of particular genetic interest because this anomaly is inherited in a recessive manner. The locus for non-syndromic sensorineural hearing loss with EVA has been mapped to the same chromosomal region, 7q31, as the Pendred syndrome locus. In the present study, seven mutations in the PDS gene (PDS), the gene responsible for Pendred syndrome, have been found in families of non-syndromic sensorineural hearing loss with EVA. One family is homozygous, three families are compound heterozygotes, and two families are heterozygous but with no other mutation detected. The present results provide evidence that mutations in PDS cause both syndromic and non-syndromic hearing loss.

Five Japanese families showing aminoglycoside-induced hearing loss were genetically as well as clinically investigated. A mitochondrial mutation at nucleotide 1555 was found in 28 out of 32 subjects. One hundred American control subjects did not show any evidence of the mutation at nucleotide 1555, suggesting that the 1555 A--G (A1555G) mitochondrial mutation may be found more frequently among populations in the Asian continent. Many subjects who harbor this mitochondrial mutation exhibit a mild, high-frequency, progressive hearing loss even without aminoglycoside injection. The results presented here appear to support the hypothesis that the A1555G mutation may play a more general role in causing hearing loss.

Using high-resolution computed tomography, we measured the cross-sectional area of mastoid air cells and the shortest distance between the external auditory canal and the anterior edge of the sigmoid sinus (DIST), and then compared the right-left difference in 70 patients with unilateral chronic otitis media and 23 cases without middle ear disease. DIST was significantly short where there was poor mastoid pneumatization (P < 0.0001), regardless of whether it was the right or left ear. Furthermore, on the well-pneumatized temporal bone, the increase in size of the cross-sectional area was closely correlated with the increase in DIST (r = 0.495). We suggest that the relative position of the external auditory canal and the sigmoid sinus is affected by middle ear inflammations in childhood.

Using receptor subunit-specific antibodies, the cellular localization of NMDA and AMPA type glutamate receptor subunits was studied within the rodent (rat, guinea pig) and non-human primate (monkey) inner ear. In the spiral and vestibular ganglion, almost all cells were immunoreactive for the NMDAR1 subunit and the AMPA type receptor subunit GluR2/3. This indicates that both NMDA and non-NMDA type glutamate receptors may be co-distributed in the primary afferent neuronal components, and are possibly involved in neurotransmission in the primary auditory and vestibular systems. This study also indicated the possible localizations of glutamate receptors in the nonneuronal cells in the inner ear, suggesting that some nonneuronal cells may also have the ability to mediate glutamate signalling.

Using infrared CCD camera and electronystagmography we analyzed positional nystagmus in 33 patients with peripheral positional vertigo. Patients were classified into three types; (a) lateral type: nystagmus occurs in lateral position, (b) sagittal type: nystagmus occurs in head-hanging position (so-called benign paroxysmal positional vertigo, BPPV), and (c) mixed type. Nystagmus in the lateral type was horizontal and that in the sagittal type was rotatory. In all types, the vertigo and nystagmus began with a few seconds' latency after head tilt and briefly increased to a maximum, and then gradually decreased. The duration of horizontal nystagmus in the lateral and mixed type was significantly longer than that of rotatory nystagmus in the sagittal type mixed type. The mean value of the former was 90.6 s and that of the latter 18.1 s. We suggest that the velocity storage mechanism of the horizontal vestibulo-ocular reflex (VOR) is considerably different from that in the vertical VOR.

In order to evaluate the involvement of neuroactive substances in the human vestibular periphery, the immunocytochemical distribution of substance P (SP), calcitonin gene-related peptide (CGRP), and choline acetyltransferase (ChAT) was examined. SP-like immunoreactivity (LI) was present around and beneath sensory hair cells, probably corresponding to their afferent nerve endings. SP-LI was found predominantly in subpopulations of the primary afferents distributed in the peripheral region of the end organs. ChAT-LI and CGRP-LI were found throughout as small puncta below the hair cell layer, probably corresponding to efferent endings. The present results indicate that these neuroactive substances, previously described in animals, are also distributed in the human vestibular periphery, and almost certainly contribute to human vestibular function.

Whether or not the shape and size of the sigmoid sinus are affected by middle ear inflammation is still controversial. Using high-resolution computed tomography (CT), we examined the shape and cross-sectional area of the sigmoid sinus in 80 patients with unilateral chronic otitis media. Forty patients had right otitis media, and the others left otitis media. Whether chronic inflammation involved the right or left ear, a protrusive type of sigmoid sinus was found more frequently on the right than the left, while the cross-sectional area was significantly greater on the right. These findings suggest that a side difference affecting the sigmoid sinus would thus appear not to be caused by postnatal otitis media, but to originate in fetal life. The shape of the sigmoid sinus greatly varies among individuals and according to whether it is on the right or the left. Findings also indicate that a temporal bone CT is desirable to optimize surgical safety.

The present study examined the immunocytochemical localization of various intermediate filaments (IFs), 68 kDa, 160 kDa and 200 kDa neurofilament protein (NFP), cytokeratin (CK) 1, 8, 10 and 19, vimentin, and glial fibrillary acidic protein (GFAP) in the vestibular end-organs and ganglia of normal and streptomycin-treated guinea pigs. In normal animals, 68 kDa, 160 kDa and 200 kDa NFP were found in afferent nerve fibers and nerve terminals (probably nerve chalices). Fine nerve fibers (probably efferent and/or sympathetic nerve fibers) were also immunoreactive to NFP. In the vestibular ganglia, 68 kDa and 160 kDa NFP were predominantly distributed in larger cells, whereas 200 kDa NFP was also found in some small ganglion cells. Cytokeratin 8 and 19 were located in supporting cells, transitional cells, dark cells of vestibular end-organs, and the epithelial cell lining of the membranous labyrinth. Vimentin was observed in the hair cells distributed in the central region of the end organs, supporting cells, most connective tissue cells, and Schwann cells of the vestibular ganglion. Although GFAP-like immunoreactivity was evident in glial cells of the proximal vestibular nerve, no immunoreactivity was detected in the distal portion of the vestibular nerve, vestibular ganglion, or vestibular end-organs. These highly distinct staining patterns of IFs indicated that they may play different roles in the different cell types, and that they may serve as a specific marker for each cell type. In streptomycin-treated guinea pigs, immunoreactivities for NFP and vimentin (found in the hair cells) decreased after treatment, whereas immunoreactivities for the other IFs were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Guinea pig cochlear supporting cells have microtubules which are composed of 15, instead of the ordinary 13, protofilaments. The microtubules form a large and stiff bundle involving abundant actin filaments to provide effective rigidity to the cell specialized for transduction of extremely high-frequent mechanical stimuli. We examined microtubules in guinea pig vestibular end organs with the transmission electron microscopy after treatment with tannic acid. Solitary microtubules occurred abundantly in the supranuclear region of type I hair cells, but sparsely in type II hair cells. Individual microtubules consisted of an ordinary set of 13 protofilaments in these hair cells as well as in the supporting cells. Supporting cells were characterized by cytoskeletal bundles of 2 to 30 microtubules which were found to be connected with actin filaments via cross-linking units. Results from the present investigation indicate that such less-organized microtubules of 13 protofilaments are sufficient for vestibular supporting cells which are specialized for detection of lower-frequency vibration together with fellow sensory cells.

By tannic acid staining, the 13-protofilament composition of cochlear hair cell microtubules, an impressive contrast against the 15-protofilament microtubules in cochlear pillar cells, was verified. The 15-protofilament microtubules formed a large and stiff cytoskeletal bundle in pillar cell bodies involving abundant actin filaments. The bundles were always situated vertically, i.e., longitudinally to the cell body axis, and were most numerous in the outer as well as the inner pillar cells in the basal turn, decreasing gradually toward the apex. Such gradient architecture of the pillar cell cytoskeleton can be correlated with the tuning mechanism for traveling waves of sound containing variable frequencies.

Delayed endolymphatic hydrops (DEH) was proposed by Nadol (1975) and Schuknecht (1976) as a pathological diagnosis. Because of severe hearing loss, the presence of endolymphatic hydrops (EH) cannot be demonstrated by the glycerol test or electro-cochleography. This paper discusses a clinical method of examining EH in this disease. The authors' earlier study, reported in Acta Otolaryngol (Stockh) suppl. 435 (1987), showed that EH in Meniere's disease could be detected not only by audiometry but also by the trapezoid rotation test. The demonstration of ipsilateral labyrinthine preponderance (LPi) by this rotation test is a useful indicator of the development of EH. The authors performed this rotation test in seven patients who satisfied Schuknecht's criteria of DEH by careful history taking and audiometry, and LPi was detected in five of them, especially before the onset of vertigo. It was concluded that the detection of LPi by the trapezoid rotation test should be added to the tests used for the exact diagnosis of DEH.

BackgroundThymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer and high expression of TS has been associated with poor prognosis or refractory disease in several cancers including colorectal and head and neck cancer. Although TS is known to regulate cell cycles and transcription factors, its potency as a therapeutic target has not been fully explored in adenoid cystic carcinoma (ACC).MethodsAn ACC cell line (ACC3) was transfected with siRNA targeting the TS gene and inhibition of cell growth and induction of apoptosis-associated molecules were evaluatedin vitro. In addition, thein vivoeffect of TS siRNA on tumor progression was assessed using a xenograft model.ResultsOur results demonstrated that ACC3 cells showed significantly higher TS expression than non-cancer cell lines and the induction of TS siRNA led to inhibition of cell proliferation. The effect was associated with an increase in p53, p21, and active caspase-3 and S-phase accumulation. We also found up-regulation of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme. Furthermore, treatment with TS siRNA delivered by atelocollagen showed a significant cytostatic effect through the induction of apoptosis in a xenograft model.ConclusionTS may be an important therapeutic target and siRNA targeting TS may be of potential therapeutic value in ACC.

Background Salivary gland carcinomas are relatively uncommon heterogeneous malignancies characterized by locoregional invasion and distant metastasis. Topoisomerase IIα (topoIIα), located at chromosome 17q21-22, is considered a major mediator of cell proliferation and DNA replication. The purpose of this study was to evaluate the expression of topoIIα in various types of salivary gland tumors and its biological significance. Methods The protein expression of topoIIα was evaluated immunohistochemically in formalin-fixed, paraffin-embedded tissue from 54 salivary gland carcinomas and 20 benign tumors (10 pleomorphic adenomas and 10 Warthin's tumors). The primary salivary gland carcinoma specimens consisted of 17 adenoid cystic carcinomas, 7 adenocarcinomas not otherwise specified, 7 mucoepidermoid carcinomas, 6 salivary duct carcinomas, 3 acinic cell carcinomas, 3 carcinomas ex pleomorphic adenomas, 3 epithelial-myoepithelial carcinomas, 2 carcinosarcomas, 2 lymphoepithelial carcinomas, 2 myoepithelial carcinomas, 1 oncocytic carcinoma, and 1 squamous cell carcinoma. The associations between clinicopathological factors and outcome were analyzed. Results Of the 54 primary salivary gland carcinomas, 38 (70%) showed positive expression (≥10%) of topoIIα protein, and 16 carcinomas (30%) and all benign tumors were negative (p < 0.001). Expression of topoIIα was more frequently observed in salivary duct carcinoma, carcinoma ex pleomorphic adenoma, adenocarcinoma, and adenoid cystic carcinoma, solid type, and it was associated with advanced stage and shortened survival. Conclusion The results of the present study suggest that topoIIα expression is associated with histologically aggressive subtypes and shortened survival. Furthermore, it may provide useful prognostic information and suggests the potential efficacy of topoIIα-targeting therapy in patients with salivary gland carcinoma.