[A MULTICENTER CLINICAL SURVEY ABOUT THE PREVALENCE OF JAPANESE CYPRESS POLLINOSIS AND THE EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH JAPANESE CEDAR POLLEN EXTRACT DURING JAPANESE CYPRESS POLLEN DISPERSAL PERIOD].

BACKGROUND: Little is known whether sublingual immunotherapy using Japanese cedar pollen extract (cedar SLIT) is effective for not only Japanese cedar pollinosis but also Japanese cypress pollinosis. We investigated the prevalence rate of Japanese cypress pollinosis, efficacy of cedar SLIT on cypress pollinosis and patients' wish to receive cypress SLIT. METHODS: We investigated a multi-center (31 institutions), cross-sectional survey using a self-administrated questionnaire with four questions for patients received cedar SLIT aged from 5 to 69 years old. RESULTS: 2523 subjects were enrolled for analysis. 83.4% of them had pollinosis symptoms during cypress season before cedar SLIT. In such patients, 37.4% experienced lessened efficacy of cedar SLIT during cypress season. Both the prevalence of cypress pollinosis and the lessened efficacy of cedar SLIT on cypress pollinosis were significantly seen in western Japan as compared to eastern Japan. 76.1% of the subject having cypress pollinosis before SLIT wished to receive cypress SLIT if it is available. CONCLUSION: A lessened efficacy of cedar SLIT during cypress season was broadly seen in Japan, and further showed a regional difference. Together with the finding of high wish by patients, these results suggest a development of cypress SLIT is desirable.

Angioedema: 6 years experience with fourteen cases.

OBJECTIVE: To study the difference in the findings between the causes of angioedema and the characteristics of angioedema induced by angiotensin receptor II blockers (ARBs), and to investigate whether laboratory examinations for acute phase inflammatory markers can aid in diagnosis and predict airway risk. METHODS: We retrospectively reviewed fourteen cases of patients with angioedema that were treated from 2000 to 2006. Data were collected regarding age, sex, location of the edema, cause, time course of resolution and laboratory examinations (leukocyte counts, serum C-reactive protein (CRP) level, complement function and the activity of C1 esterase inhibitor). RESULTS: The causes of angioedema were ACEIs in six patients (42.9%), candesartan (ARB) in three (21.4%), HAE (types 1 and 2) in two, and unknown in three. Of these patients, 71.4% exhibited edema in the floor of the mouth, irrespective of the cause. Two patients with edema induced by candesartan exhibited both lingual and laryngeal edemas. The remaining one with candesartan-induced edema exhibited edema in the neck and mediastinum and pleural effusion. The average time to resolution was 4.1 days, ranging from one to twelve days. The edema in eleven patients resolved with conservative therapy, while three patients underwent tracheotomy. In two patients with candesartan-induced edema, although the edemas resolved completely after cessation of candesartan administration, the edemas reappeared in the same locations, two and thirty days after the cessation of candesartan for each patient. None of the patients with angioedema induced by ACEIs exhibited elevation of serum CRP levels. No significant differences were found for leukocyte counts and serum CRP levels between patients with angioedemas induced by ACEIs, ARB and those of unknown cause. No significant differences were observed in the above findings between the patients who underwent tracheotomy and those who did not. Two patients exhibited low C4 levels, and one of the two exhibited no activity of C1 esterase inhibitor. CONCLUSION: Consistent with previous reports, angioedema in the floor of the mouth extending to the tongue should be considered as a possible risk factor for airway compromise. Laboratory examinations for acute phase inflammatory markers are not useful for diagnosis and are not predictive for airway intubation and tracheotomy. Angioedema induced by candesartan can present in anomalous sites and reappear following drug cessation even if the edema has resolved completely.

Synergistic effect between proteasome and autophagosome in the clearance of polyubiquitinated TDP-43.

Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.

Axotomy alters alternative splicing of choline acetyltransferase in the rat dorsal motor nucleus of the vagus nerve.

Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6-9 of the common-type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT-positive neurons. Following nerve transection, we clearly detected pChAT-labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT-positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT-positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT-positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription-polymerase chain reaction (RT-PCR) by using laser capture microdissection. The number of pChAT-positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double-immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents.

Circadian gene expression in the murine larynx.

OBJECTIVE: Symptoms of airway diseases are often associated with specific times of the day. For example, midnight worsening of cough is a frequent complaint of patients with laryngitis and bronchitis. We speculate that these symptoms are under the control of the circadian clock, and the clock genes in the airway epithelium play some important roles. In the present study, we tried to prove the time specific expressions of clock oscillating genes in the murine larynx. MATERIALS AND METHODS: Adult wild-type C57/Bl6 mice and mCry1(-/-)mCry2(-/-) mutant mice were used for this study. We employed immunohistochemistry and/or Northern blotting for examining the circadian expression of mPer1, mPer2, C/EBPbeta, HNF3beta, and MUC5b. RESULTS: The expression of mPer2 mRNA showed a strong day-night expression difference, which was abolished after the lesion of the suprachiasmatic nucleus, and in mCry1(-/-)mCry2(-/-) mutant mice. mPER1 and mPER2 proteins both showed very similar expression profiles in the epithelium and submucosal glands with a peak in the evening and a trough in the early morning. Other nuclear proteins such as C/EBPbeta and HNF3beta did not show the rhythms. MUC5b protein showed circadian oscillation in the laryngeal submucosal gland. CONCLUSION: In this study, we confirmed the existence of a local laryngeal clock which is controlled by the central clock in the suprachiasmatic nucleus. MUC5b protein in the submucosal mucous gland also showed circadian rhythm. We consider that these rhythmic expressions may cause the time specific symptoms among laryngeal diseases.

Age-related expression of alpha-gustducin in the rat larynx.

OBJECTIVES: The age-related changes in distribution of alpha-gustducin-immunoreactive structures in the larynx of Sprague-Dawley rats were studied. METHODS: For this purpose, tissues obtained from 12 male rats ranging in age from 5 to 21 weeks were compared with respect to the distribution and morphology of laryngeal taste buds immunoreactive for alpha-gustducin, the alpha-subunit of a taste cell-specific G protein. RESULTS: Three different morphological types of alpha-gustducin-immunoreactive structures were seen: typical gemmal forms, clusters composed of 2 or 3 cells, and isolated immunoreactive cells not associated with taste buds. alpha-Gustducin-immunoreactive structures were most abundant in the epiglottis in all age groups. alpha-Gustducin-immunoreactive cells in rats seem to be located along the lateral food channels, in which they may come in contact with food. The total number of these alpha-gustducin-immunoreactive structures did not show any age-related changes, but the percentage of solitary immunoreactive cells in 5-week-old rats was significantly larger than the percentages in 8-, 14-, and 21-week-old animals. CONCLUSIONS: Solitary alpha-gustducin-immunoreactive cells, which are abundant in 5-week-old rats but are found in fewer numbers along the base of the epiglottis in mature rats, may be nociceptic in function, whereas the chemosensory clusters or buds that contain alpha-gustducin-positive cells and are distributed along the lateral food channels on the pharyngeal side of the larynx may have a role in gustatory reception.

Localization of fibroblast growth factor-1 in cholinergic neurons innervating the rat larynx.

Cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) are particularly vulnerable to laryngeal nerve damage, possibly because they lack fibroblast growth factor-1 (FGF1). To test this hypothesis, we investigated the localization of FGF1 in cholinergic neurons innervating the rat larynx by immunohistochemistry using central-type antibodies to choline acetyltransferase (cChAT) and peripheral type (pChAT) antibodies, as well as tracer experiments. In the DMNV, only 9% of cChAT-positive neurons contained FGF1, and 71% of FGF1-positive neurons colocalized with cChAT. In the nucleus ambiguus, 100% of cChAT-positive neurons were FGF1 positive. In the intralaryngeal ganglia, all ganglionic neurons contained both pChAT and FGF1. In the nodose ganglia, 66% of pChAT-positive neurons were also positive for FGF1, and 90% of FGF1-positive ganglionic cells displayed pChAT immunoreactivity. Neuronal tracing using cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV and nucleus ambiguus to the superior laryngeal nerve were FGF1 negative and FGF1 positive, respectively. In the nodose ganglia, some FGF1-positive cells were labeled with CTb. The results indicate that for innervation of the rat larynx, FGF1 is localized to motor neurons, postganglionic parasympathetic neurons, and sensory neurons, but expression is very low in preganglionic parasympathetic cholinergic neurons.

Participation of TRPV1 and TRPV2 in the rat laryngeal sensory innervation.

Laryngeal sensory innervation is essential to the laryngeal defense system. We investigated the participation of TRPV1 and its homologue TRPV2 in the rat laryngeal sensory innervation using immunohistochemistry and the neuronal tracer, fluoro-gold (FG). After injection of FG into the internal branch of the superior laryngeal nerve, FG-labeled neurons were seen in the rostral part of the nodose ganglion (NG). Neurons immunoreactive for TRPV1 or TRPV2 were distributed throughout the NG. TRPV1 immunoreactivity was seen in 49.0+/-4.5% of the FG-labeled neurons, while TRPV2 immunoreactivity was seen in 12.5+/-4.1% of the FG-labeled neurons. These findings suggest that both TRPV1 and TRPV2 participate in laryngeal nociception, but that TRPV1 may have a particularly important role.

Vocal fold paralysis as a sign of chest diseases: a 15-year retrospective study.

BACKGROUND: Vocal fold paralysis (VFP) is sometimes the only sign of chest diseases. However, some patients with VFP due to chest diseases are not diagnosed correctly at the first examination, which may leave the patients untreated for a long time. Depending on the situation, chest x-ray is not enough for detecting the primary lesion. The objective of this study was to discuss the diagnostic procedure for VFP based on the retrospective analysis of the cases. METHODS: A total of 42 patients (29 males and 13 females) with VFP due to chest disease examined at the Department of Otolaryngology of Kyoto Prefectural University of Medicine between 1988 and 2002 were reviewed retrospectively. RESULTS: Of the primary chest diseases, lung cancer (15 cases) was the most common, followed by thoracic aortic aneurysm (TAA) (9 cases), metastatic tumor from other regions (6 cases), pulmonary and mediastinal tuberculosis (TB) (5 cases), and esophageal cancer (4 cases). While the primary lesions were easily detected with chest x-ray in most of the cases, some lesions in the aortopulmonary window were difficult to detect. Contrast-enhanced computed tomography (CT) was useful to detect any mass in this region. CONCLUSIONS: In the diagnosis of VFP due to chest diseases, chest x-ray was useful but not always enough for detecting the primary lesion. Necessity of further examinations including contrast-enhanced chest CT must be kept in mind for the cases with negative chest radiographs.

A newborn case of congenital laryngeal cyst complicated with pneumothorax and pneumomediastinum.

Benign congenital laryngeal cysts are rare entities. They often cause chronic hoarseness and severe stridor. Case reports of congenital laryngeal cyst complicated with pneumothorax and pneumomediastinum are very rare. A 3,112 g full-term male newborn developed stridor which got worse during crying for 12 h after birth. Chest retractions were present with inspiration. Chest X-rays showed the presence of right pneumothorax and pneumomediastinum. Transnasal flexible laryngoscopic examination revealed a large cystic mass, which occupied almost the entire supraglottic airway. The operation was performed with the techniques of laryngomicrosurgery under general anesthesia. The cystic wall was punctured and serous liquid contents were aspirated. Excision of the entire cystic lesion was performed. The next day, extubation was performed without any troubles. The stridor had disappeared and the pneumothorax and pneumomediastinum were improved without further medical intervention. The histopathological examination revealed that the cystic wall consisted of normal squamous epithelial cells. It is reasonable to think that the high airway pressure due to congenital laryngeal cyst was responsible for pneumothorax and pneumomediastinum.

Comparison of FGF1 (aFGF) expression between the dorsal motor nucleus of vagus and the hypoglossal nucleus of rat.

Neurons in the dorsal motor nucleus of the vagus (DMNV) are more severely affected by axonal injury than most other nerves, such as those of the hypoglossal nucleus. However, the mechanism underlying such a response remains unclear. In this study, we compared the expression of fibroblast growth factor 1 (FGF1), a neurotrophic factor, between the DMNV and the hypoglossal nucleus by RT-PCR and immunohistochemical analyses. RT-PCR showed that the level of FGF1 mRNA expression in the DMNV was lower than that in the hypoglossal nucleus (P<0.01). Immunohistochemistry revealed that FGF1 was localized to neurons. FGF1-positive neurons in large numbers were evenly distributed in the hypoglossal nucleus, whereas FGF1-positive neurons were located in the lateral part of the DMNV. Double immunostaining for FGF1 and choline acetyltransferase demonstrated that 22.7% and 78% of cholinergic neurons were positive for FGF1 in the DMNV and hypoglossal nucleus, respectively. A tracing study with cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV to the superior laryngeal nerve were FGF1-negative. The results suggest that the low expression of FGF1 in the DMNV is due to severe damage of neurons in the DMNV.

Distribution of vanilloid receptors in the rat laryngeal innervation.

OBJECTIVE: Capsaicin is known to selectively activate nociceptic sensory neurons through vanilloid receptors. In this study we investigated the distribution of vanilloid receptor subtype 1 (VR1) and vanilloid receptor-like protein 1 (VRL-1) in the rat larynx. MATERIAL AND METHODS: The distributions of VR1 and VRL-1 were determined immunohistochemically. The colocalization of vanilloid receptors with common choline acetyltransferase (cChAT), vasoactive intestinal polypeptide (VIP), substance P (SP) and neuronal nitric oxide synthase (nNOS) was also studied using an immunohistochemical double-labeling technique. RESULTS: VRL-1-positive fibers were detected in the laryngeal epithelium and lamina propria. VR1-positive nerve fibers were seen in the lamina propria but not in the mucosal epithelium. VR1- and VRL-1-positive cells were distributed in the intralaryngeal ganglia and colocalization of capsaicin receptors with VIP, nNOS and cChAT was seen. CONCLUSION: These findings suggest that these capsaicin receptors participate in the parasympathetic innervation as well as in nociception of the rat larynx.

Capsaicin receptor expression in rat laryngeal innervation.

Capsaicin elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. Vanilloid receptor subtype 1 (VRI) and the vanilloid receptor-like protein 1 (VRL-1) are activated, not only by capsaicin, but also by noxious heat and protons, and it has been suggested that they are polymodal nociceptors. We investigated the expression of VR1 and VRL-1 in the rat larynx and nodose ganglion using VR1 and VRL-1 immunohistochemical analysis with visualization by diaminobenzidine reaction. Fibers positive for VRL-1 were detected in the laryngeal epithelium and lamina propria. Cells positive for VRL-1 were distributed in the intralaryngeal ganglia. Half of the neurons in the nodose ganglion had VR-1 immunoreactivity, and almost 10% of the nodose ganglion neurons were positive for VRL-1. These findings suggest that these capsaicin receptors play an important role in the nociception of the laryngeal innervation.

Growth associated protein-43 mRNA expression in nucleus ambiguus motoneurons after recurrent laryngeal nerve injury in the rat.

OBJECTIVE: To investigate the time-dependent differences in growth associated protein-43 (GAP-43) mRNA expression in the nucleus ambiguus (NA) motoneurons after recurrent laryngeal nerve (RLN) transection in the rat. MATERIAL AND METHODS: Animals were sacrificed on Days 1, 7, 14 and 21 after axotomy. The sections were then processed for in situ hybridization of GAP-43 mRNA. RESULTS: GAP-43 mRNA transiently increased after axotomy, reaching a peak on postoperative Day 7, and then decreased gradually at 14 and 21 days after axotomy. The peak period of GAP-43 expression in the NA is different from those in the facial and hypoglossal nuclei after axotomy. CONCLUSION: We have already reported that neuronal nitric oxide synthase (nNOS) is induced in the NA motoneurons after axotomy and reaches a peak on postoperative Day 14. This study shows that the expressions of GAP-43 and nNOS are chronologically different.