Expressions of isopeptide bonds and corneodesmosin in middle ear cholesteatoma.

OBJECTIVE: Isopeptide bonds form cross-links between constituent proteins in the horny layer of the epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes, which bind corneocytes together. Both play important roles in maintaining epidermal barrier functions. In the present study, we investigated the expressions of isopeptide bonds, CDSN, and related enzymes in middle ear cholesteatoma in comparison with the skin. DESIGN: Prospective case series of patients with middle ear cholesteatoma. SETTING: Tertiary medical institute. PARTICIPANTS: Cholesteatoma and normal postauricular skin were collected from patients with acquired middle ear cholesteatoma during tympanomastoidectomy. MAIN OUTCOME MEASURES: Expression of e-(g-glutamyl)lysine isopeptide bonds was examined by immunohistochemistry; Expressions of transglutaminase (TGase)1, TGase2, TGase3, and TGase5 by immunohistochemistry and quantitative RT-PCR (qRT-PCR); expression of CDSN by immunohistochemistry, qRT-PCR, and Western blot; and expressions of tissue kallikrein-related peptidase (KLK)5, KLK7, KLK14, and serine peptidase inhibitor Kazal type 5 (SPINK5) by qRT-PCR. RESULTS: TGase2 was higher (P=0.0046) and TGase5 was lower (P=0.0008) in cholesteatoma than in the postauricular skin. Immunoreactivity for isopeptide bonds was localized in the granular and horny layers, and was not different between the two tissues. Immunoreactivity for CDSN was localized in the granular layer, and was lower in cholesteatoma than in the skin (P=0.0090). Western blot and qRT-PCR confirmed that the expression of CDSN was lower in cholesteatoma than in the skin. Expressions of KLK5, KLK7, KLK14, or SPINK5 were not different between the two tissues. CONCLUSIONS: These results indicate that the production of CDSN is likely to be suppressed in cholesteatoma, which would account, at least in part, for the mechanical fragility and increased permeability of the cholesteatoma epithelium.

Fluorescent Visualisation of Oxytocin in the Hypothalamo-neurohypophysial/-spinal Pathways After Chronic Inflammation in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rats.

Oxytocin (OXT) is a well-known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT-mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT-mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up-regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN-spinal pathway may be involved in sensory modulation. OXT-mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in in vitro preparations.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

Electrophysiological effects of kainic acid on vasopressin-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 neurones isolated from the supraoptic nucleus in transgenic rats.

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 µm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1-containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.

A c-fos-monomeric red fluorescent protein 1 fusion transgene is differentially expressed in rat forebrain and brainstem after chronic dehydration and rehydration.

We have previously shown that an acute osmotic stimulation induces the expression of a c-fos and monomeric red fluorescent protein 1 (mRFP1) fusion transgene in osmosensitive rat brain areas, including the supraoptic (SON) and paraventricular nuclei (PVN). However, the effects of chronic stimuli, such as dehydration, have not been investigated. In the present study, the expression patterns of the c-fos-mRFP1 fusion gene in the forebrain and the brainstem of male and female transgenic rats were studied in seven experimental groups: ad lib. water (euhydration), water deprivation for 12, 24 or 48 h (dehydration) and water deprivation for 46 h + ad lib. water for 2, 6 or 12 h (rehydration). The number of cells that express nuclear mRFP1 fluorescence was quantified in the hypothalamus, the circumventricular organs and the brainstem. Compared to the euhydrated state, the number of transgene expressing cells significantly increased in all forebrain areas and in the rostral ventrolateral medulla after dehydration and 2 h of rehydration. In the nucleus of the solitary tract and area postrema, the number of mRFP1 fluorescent cells was markedly increased after 2 h of rehydration. Although the number of mRFP1 fluorescent cells in the organum vasculosum laminae terminalis, median preoptic nucleus and subfornical organ remained significantly increased after 6 h of rehydration, reaching control levels after 12 h of rehydration, the number of mRFP1 fluorescent cells in the SON and the PVN reached control levels after 6 h of rehydration. There were no significant differences between male and female rats. These results show that the expression of the c-fos-mRFP1 fusion gene changes in the forebrain and the brainstem not only after acute osmotic stimulation, but also after chronic osmotic stimulation. Interestingly, these studies reveal the differential activation of different neuronal groups over the time course of dehydration and rehydration.

Mitochondrial neurogastrointestinal encephalomyopathy associated with progressive hearing loss.

OBJECTIVE: We report a rare case of mitochondrial neurogastrointestinal encephalomyopathy with hearing loss. CASE REPORT: A 46-year-old woman presented with a three-year history of progressive, bilateral hearing loss and tinnitus. She had been suffering from unexplained abdominal pain and diarrhoea for 20 years. When first seen, her otoscopic findings were normal, and pure tone audiometry showed mild and moderate hearing loss in her right and left ears, respectively. She also had: bilateral ophthalmoparesis, neck and limb muscle weakness, and hypoactive deep tendon reflexes on neurological examination; diffuse leukoencephalopathy on magnetic resonance imaging of the brain; and markedly reduced leukocyte thymidine phosphorylase activity. On the basis of these findings, the patient was diagnosed with mitochondrial neurogastrointestinal encephalomyopathy. CONCLUSION: Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disease caused by mutation of the thymidine phosphorylase gene, and is characterised by ophthalmoparesis, peripheral neuropathy, leukoencephalopathy, gastrointestinal symptoms and abnormal mitochondria in muscle cells. Current advances in genetic research may reveal a higher prevalence of mitochondrial disorders than had previously been thought. Otolaryngologists should be aware of mitochondrial neurogastrointestinal encephalomyopathy and other rare genetic disorders when managing patients with progressive hearing loss.

Analysis of initial attachment of B cells to endothelial cells under flow conditions.

This study examined the adhesive interactions of peripheral blood B cells with TNF-alpha-activated endothelial monolayers, and analyzed the roles of E-selectin, P-selectin, or VCAM-1 molecules expressed on activated HUVEC. B cell interaction occurred on activated HUVEC, but not on resting HUVEC under flow conditions. The majority of peripheral blood B cells expressed P-selectin glycoprotein ligand-1 and alpha4 integrin. However, the expression of cutaneous lymphocyte Ag on B cells was low. Under flow conditions, B cells could bind to P-selectin-coated tubes and VCAM-1-transfected Chinese hamster ovary cells. In contrast, B cells could not bind to E-selectin-coated tubes. Adhesion activity of B cells to P-selectin-coated tubes was weaker than that of T cells. Furthermore, adhesion activity of B cells to VCAM-1-transfected Chinese hamster ovary cells was similar to that of T cells. Treatment of activated HUVEC with anti-VCAM-1 mAb reduced interaction of B cells under flow conditions. However, the treatment of activated HUVEC with anti-P-selectin mAb did not reduce interaction. These data indicated that the interaction of VCAM-1 with alpha4 integrin plays a major role in an initial attachment of B cells to endothelial monolayers under flow conditions.

Epidemiological study of severe cases of Meniére's disease in Japan.

In order to clarify the characteristics of severe cases of Meniére's disease (MD), we analyzed various epidemiological factors such as sex ratio, past history, complication, cause of onset of vertiginous attacks, etc., in a series of 958 patients with definite MD. Data were obtained from the three Japan-wide surveys of MD conducted by the Meniére's Disease Research Committee of Japan (1975-76) and the Vestibular Disorders Research Committee of Japan (1982-84 & 1990). Following the ideas proposed by the members of the Vestibular Disorder Research Committee of Japan, we divided severe cases into three categories according to the following criteria i) bilateral MD cases (BMD), ii) unilateral MD cases with prolonged disabled vertigo (UPDV), iii) unilateral MD cases with profound hearing loss (UPHL). About 40% of the subjects were classified as severe cases (UPDV: 23%; BMD: 9%; UPHL: 6%). The ratio of otitis media in past history was statistically different between severe cases and non-severe patients (p < 0.05), suggesting that otitis media in the past may contribute to the severity of Meniére's disease.

[Pneumatization of the petrous apex].

Pneumatization of the petrous apex was investigated in 226 subjects without middle ear disease by means of target imaging CT. The degree of pneumatization of all subjects was 32.7% (148/452 ears), but no difference in degree was revealed with distinction of bilateral ears or between sexes. In 148 ears with pneumatization of the petrous apex, a higher degree of pneumatization was found in larger mastoid cavities, suggesting that pneumatization of the petrous apex correlates with pneumatized air cells in other parts of the temporal bone. Pneumatization of all parts of the petrous apex was found in about 40% (58/148 ears), and of some parts in about 60% (90/148 ears). In the latter cases, pneumatized air cells were more often found in the lower portions of the CT slices than in the higher ones. These results indicate that pneumatization of the petrous apex must be taken into consideration in studies measuring the gas composition and volume of the middle ear.

Detection of the unilateral vestibular recruitment phenomenon using the rotation test.

The possibility of detecting unilateral vestibular recruitment using the rotation test was examined. According to the analogy model of cochlear recruitment, the directional difference of rotation nystagmus reaches its maximum at the threshold stimulus intensity of the affected ear when recruitment is present, and the predicted maximum directional difference remains within the normal variation range when the contribution from the spontaneous nystagmus is removed. Moreover, there was no difference in the positive ratio of recruitment in the rotation test between the two groups evaluated as having positive and negative recruitment on the caloric test. From these results, detection of the unilateral vestibular recruitment phenomenon, when similar to the cochlear recruitment, was concluded to be difficult using the rotation test.