Increased permeability of the epithelium of middle ear cholesteatoma.

OBJECTIVE: We investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium to provide supporting evidence for the acid lysis theory of bone resorption in middle ear cholesteatoma. METHODS: Study subjects were patients with primary acquired middle ear cholesteatoma and those with non-cholesteatomatous chronic otitis media who underwent tympanomastoidectomy. The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy by loading alternating currents of 320 Hz and 30.7 kHz. The expressions of tricellulin (MARVELD2), claudin-1 (CLDN1) and claudin-3 (CLDN3) were examined by fluorescence immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. RESULTS: The electrical impedance of the cholesteatoma epithelium was significantly lower than that of the post-auricular skin and external auditory canal skin at both 320 Hz and 30.7 kHz. Immunoreactivity for MARVELD2, CLDN1 and CLDN3 was localised mainly in the granular layer, and to lesser degree, in the horny and spinous layers in both the cholesteatoma tissue and post-auricular skin. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. The expressions of MARVELD2, CLDN1 and CLDN3 mRNA were significantly lower in the cholesteatoma tissue than in the post-auricular skin. CONCLUSIONS: These results indicate the increased permeability of the cholesteatoma epithelium and suggest that this change is, at least partially, dependent on the decrease in the expressions of the tight junction molecules. This evidence supports the acid lysis hypothesis of bone resorption in cholesteatoma.

Expression of epidermal growth factors and a tight junction protein in the nasal mucosa of patients with chronic hypertrophic rhinitis.

BACKGROUND: ErbB family receptors and tight junction proteins participate in the pathologic process including tissue remodelling of inflammatory diseases in the upper and lower respiratory tracts. This study aimed at investigating the expressions of erbB1, 2, 3, 4, and a tight junction protein, claudin-1, in the nasal mucosa of patients with chronic hypertrophic rhinitis. METHODS: Inferior turbinates were collected from 10 turbinectomised patients with allergic and non-allergic chronic hypertrophic rhinitis. The expressions of erbB1, 2, 3, 4, and claudin-1 were examined by fluorescence immunohistochemistry and by quantitative real-time transcription-polymerase chain reaction (qRT-PCR). RESULTS: All erbB1-4 and claudin-1 were detected, and mainly localised in the epithelial cells and nasal gland cells. The immunoreactivity for claudin-1 was positively correlated with the expressions of erbB1, 2 and 4, but negatively correlated with that of erbB3. The mRNA expressions of erbB1, 2 and 4 were positively correlated with one another, whereas the expression of erbB3 showed negative correlation with the immunoreactivity for erbB2 and 4. CONCLUSIONS: These results suggest a possible participation of erbBs and claudin-1 in tissue remodelling in chronic hypertrophic rhinitis.

Origin of giant cells in osteoclast-like giant cell tumors of the pancreas.

To clarify the origin of giant cells in osteoclast-like giant cell tumors (OGCTs) of the pancreas, we performed microscopical, immunohistochemical, and K-ras gene mutation analyses with a microdissection approach in 3 cases, featuring 4 cellular components (osteoclast-like giant cells [OGCs], pleomorphic large cells [PLCs], mononuclear cells, and ductal carcinoma cells). Two cases had abundant OGCs, and 1 case contained large number of both OGCs and PLCs. In each, none of the microdissected OGCs contained any K-ras gene mutation while they were positive for a histiocytic marker (CD-68). In contrast, PLCs, when present, frequently harbored K-ras gene mutations and were negative for CD-68. In all cases, mononuclear cells, a mixture of histiocyte-like and atypical, from microscopic and immunohistochemical viewpoints, also frequently showed K-ras alteration. Histiocyte-like mononuclear cell was equipped with a regular and oval nucleus similar to those in OGCs and was positive for CD-68. Atypical mononuclear cell showed an irregular, pleomorphic, or sometimes bizarre nucleus similar to those in PLCs and was negative for CD-68. All of the K-ras gene mutations found in PLCs and mononuclear cells were the same as in the ductal carcinoma cells within the same tumor. Thus, OGCs differ in origin from ductal cells and are strongly suggested to be nonneoplastic and of mesenchymal origin, whereas PLCs, which harbor K-ras gene mutations, are neoplastic and presumably derived from ductal carcinoma cells. Moreover, mononuclear cells may be classified into 2 types, histiocyte-like and atypical.

Accelerated reoxygenation of a murine fibrosarcoma after carbon-ion radiation.

PURPOSE: Reoxygenation of a murine tumour after irradiation with carbon ions was investigated and compared to that after gamma-rays. MATERIALS AND METHODS: NFSa fibrosarcoma cells were transplanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with either 290 MeV/u carbon ions or 137-Cs gamma-rays was delivered to the tumours at 8 mm diameter. At given times after irradiation the leg tumours, either clamped or not, received test doses of photons. Differences in tumour growth delay between the clamped and non-clamped tumours were interpreted in terms of reoxygenation. A lung-colony assay was used to obtain cell-survival curves. RESULTS: The oxygen enhancement ratio in the NFSa tumour for 74 keV microm(-1) carbon ions was 1.6 while that for gamma-rays was 3.4. The NFSa tumours reoxygenated 4 days after 30 Gy of gamma-ray irradiation, but reoxygenated as early as 1 day after 16 Gy of carbon ions. Reoxygenation after gamma-rays shortened to 1 day when the tumours were initially clamped for the conditioning irradiation. CONCLUSIONS: The fraction of surviving oxic cells in the NFSa tumours is larger after irradiation with carbon ions than with gamma-rays, resulting in accelerated reoxygenation.

Mouse skin reactions following fractionated irradiation with carbon ions.

PURPOSE: Skin reactions in the mouse leg following various daily doses given with 290 MeV/u carbon ions were investigated. MATERIALS AND METHODS: Seven different LET (linear energy transfer) values ranging from 14 to 100keV/microm were selected. The fractionation schedules were 1-, 2-, 4- and 8-daily fractions. The isoeffect doses to produce moist desquamation on the dose-response curves were calculated with 95% confidence limits. RESULTS: The isoeffect doses for carbon ions of 14 and 20 keV/microm increased with an increase in the number of fractions up to 4 fractions, but became constant when the number of fractions further increased to 8 fractions. This leveling off in isoeffect dose was more prominent for 40 keV/microm. Recovered dose per fraction was largest for 2 fractions of the 14keV/microm carbon beam. The isoeffect doses for 50, 60, 80 and 100keV/microm consistently increased with an increase in the number of fractions and did not show saturation up to 8 fractions. RBE (relative biological effectiveness) increased linearly with LET for all fractionation schedules. CONCLUSIONS: These results suggest that daily fractionation with carbon ions could spare radiation damage in patients, and that changes the fractionation schedule affect clinical outcome.

Lobectomy: video-assisted thoracic surgery versus posterolateral thoracotomy.

BACKGROUND: Video-assisted lobectomy has been adopted by many thoracic surgeons, because it is a less invasive approach to small peripheral lung cancers. However, some authors disagree that video-assisted lobectomy is less invasive than traditional thoracotomy and lobectomy. The purpose of this study was to evaluate the advantages of video-assisted lobectomy over posterolateral thoracotomy and lobectomy in terms of pain-related morbidity. METHODS: A total of 70 patients with clinical T1N0M0 non-small-cell lung carcinomas underwent lobectomy with complete mediastinal lymphadenectomy. Of these 35 underwent posterolateral thoracotomy (between April 1994 and December 1995; open group), and 35 underwent video-assisted thoracic surgery (VATS) (between January and December 1996; VATS group). RESULTS: Although the operative time was significantly longer in the VATS group (p = 0.04), the intraoperative blood loss was significantly less (p = 0.03). No significant differences were found for the two groups with respect to the total number of mediastinal lymph nodes dissected or duration of chest tube drainage. Postoperative pain was less severe as determined by the number of doses of analgesics required between postoperative days 0 and 7 (p < 0.0001), and the length of postoperative hospitalization was shorter in the VATS group (p < 0.0001). CONCLUSION: Video-assisted lobectomy is associated with decreased postoperative pain and shortened length of postoperative hospitalization, when compared with posterolateral thoracotomy and lobectomy.

[New tactile sensor for thoracoscopic detection of intrapulmonary nodules].

We have developed a new tactile sensor which can be used for thoracoscopic detection of invisible intrapulmonary nodules. We applied this new device for consecutive ten cases to excise twelve intrapulmonary nodules thoracoscopically from August 1994 to January 1995. In this report, one of ten cases was presented. The patient was a forty-three-year-old female and admitted with an indeterminate nodule on chest X-ray and computed tomography. When the sensor probe quantifying the hardness of objects by the changes in resonance frequency of the sensor (delta f) passed above the nodule, a sudden jump was evoked in delta f curve on the computer screen. The nodule was resected thoracoscopically and proved pathologically to be adenocarcinoma. Thoracoscopic procedure was then converted to open thoracotomy and lobectomy with lymph node dissection was performed.

[Intentional limited operation for minute peripheral adenocarcinoma of the lung].

The purpose of this report is to evaluate the limited operation for peripheral minute adenocarcinoma of the lung. Firstly, 44 cases (47 lesions) of surgically resected minute peripheral lung adenocarcinoma, 10 mm or less in diameter, were reviewed using Noguchi's classification, and the correlation between high resolution CT (HRCT) images and the clinicopathological features was examined retrospectively. All type A and B adenocarcinomas (n = 14) had no recurrence and all cases were the air containing type by HRCT. Lymph node metastasis and lymphatic/vascular involvement were detected with type C, D, E, F and 3 cases among them were died for recurrence. Based on those results, from April 2000, intentional limited operation was prospectively performed for 14 patients (16 lesions) with peripheral nodule showing ground-glass opacity on HRCT. The pathological findings were type A (n = 9), type B (n = 5), and atypical adenomatous hyperplasia was one case (If the findings were confirmed type C, D, E, F by permanent section diagnosis, VATS lobectomy will be performed). We recommend limited operation should be performed in only type A or B adenocarcinoma and permanent section diagnosis is necessary to determine whether or not.

[Intrathoracic neurofibroma originating in the left vagus nerve].

A 20-year-old man was admitted because of an abnormal mass shadow on chest X-ray film. Computed tomography (CT) and magnetic resonance imaging (MRI) disclosed a mass lesion in the superior portion of the left mediastinum. CT scans showed a well-defined mass with low density. Axial MRI rendered the mass lesion with intermediate signal intensity on T1-weighted images and high signal intensity on T2-weighted images. The preoperative diagnosis was bronchogenic cyst. Video-assisted thoracic surgery revealed that the tumor originated in the truncus of the left vagus nerve. The resected tumor was 90 x 24 x 18 mm in size. The postoperative course was uneventful and hoarseness did not develop. The pathologic diagnosis was benign mediastinal neurofibroma without von Recklinghausen's disease. Such cases are extremely rare in the Japanese literature.

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.

Electrophysiological identification of the functional presynaptic nerve terminals on an isolated single vasopressin neurone of the rat supraoptic nucleus.

Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate-dependent excitation and GABA-dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole-cell patch-clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole-cell patch clamp technique. Neurones expressed an AVP-enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP-eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP-eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones.

Ghrelin potentiates miniature excitatory postsynaptic currents in supraoptic magnocellular neurones.

Ghrelin is an orexigenic peptide discovered in the stomach as a ligand of the orphan G-protein coupled receptor, and participates in the regulation of growth hormone (GH) release. Previous studies have demonstrated that ghrelin suppressed water intake and stimulated the secretion of arginine vasopressin in rats. We examined the effect of ghrelin on the excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) using whole-cell patch-clamp recordings in in vitro rat and mouse brain slice preparations. The application of ghrelin (10(-7) approximately 10(-6) m) caused a significant increase in the frequency of the miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The increased frequency of the spontaneous EPSCs persisted in the presence of tetrodotoxin (1 microM). Des-n-octanoyl ghrelin (10(-6) m) did not have a significant effect on the mEPSCs. The ghrelin-induced potentiation of the mEPSCs was significantly suppressed by previous exposure to the transient receptor potential vanilloid (TRPV) blocker, ruthenium red (10 microM) and GH secretagougue type 1a receptor selective antagonist, BIM28163 (10 microM). The effects of ghrelin on the supraoptic MNCs in trpv1 knockout mice were significantly attenuated compared to those in wild-type mice counterparts. These results suggest that ghrelin participates in the regulation of synaptic inputs to the MNCs in the SON via interaction with the GH secretagogue type 1a receptor, and that the TRPV1 channel may be involved in ghrelin-induced potentiation of mEPSCs to the MNCs in the SON.

Acoustical experiment of yogurt fermentation process.

One of the important factors through food manufacturing is hygienic management. Thus, food manufactures prove their hygienic activities by taking certifications like a Hazard Analysis and Critical Control Point (HACCP). This concept also applies to food monitoring. Acoustical measurements have advantage for other measurement in food monitoring because they make it possible to measure with noncontact and nondestructive. We tried to monitor lactic fermentation of yogurt by a probing sensor using a pair of acoustic transducers. Temperature of the solution changes by the reaction heat of fermentation. Consequently the sound velocity propagated through the solution also changes depending on the temperature. At the same time, the solution change its phase from liquid to gel. The transducers usage in the solution indicates the change of the temperature as the change of the phase difference between two transducers. The acoustic method has advantages of nondestructive measurement that reduces contamination of food product by measuring instrument. The sensor was inserted into milk with lactic acid bacterial stain of 19 degrees C and monitored phase retardation of propagated acoustic wave and its temperature with thermocouples in the mild. The monitoring result of fermentation from milk to Caspian Sea yogurt by the acoustic transducers with the frequency of 3.7 MHz started to show gradient change in temperature caused by reaction heat of fermentation but stop the gradient change at the end although the temperature still change. The gradient change stopped its change because of phase change from liquid to gel. The present method will be able to measure indirectly by setting transducers outside of the measuring object. This noncontact sensing method will have great advantage of reduces risk of food contamination from measuring instrument because the measurement probes are set out of fermentation reactor or food containers. Our proposed method will contribute to the hygienization for the food manufacture industry.

Concurrent infiltration by CD8+ T cells and CD4+ T cells is a favourable prognostic factor in non-small-cell lung carcinoma.

The purpose of this study was to clarify the relationship between the number of tumour-infiltrating T lymphocytes and the clinicopathological features and clinical outcome in patients with non-small-cell lung cancer (NSCLC). Tissue specimens from 109 patients who underwent surgical resection for NSCLC were immunohistochemically analysed for CD4 and CD8 expression. Patients were classified into two groups according to whether their tumours exhibited a 'high' or 'low' level of CD8(+) or CD4(+) lymphocyte infiltration. Although the level of infiltration by CD8(+) T cells alone had no prognostic significance, the survival rate for patients with both 'high' CD8(+) and 'high' CD4(+) T-cell infiltration was significantly higher than that for the other groups (log-rank test, P=0.006). Multivariate analysis indicated that concomitant high CD8(+) and high CD4(+) T-cell infiltration was an independent favourable prognostic factor (P=0.0092). In conclusion, the presence of high levels of both CD8(+) T cells and CD4(+) T cells is a significant indicator of a better prognosis for patients with NSCLC, and cooperation between these cell populations may allow a significantly more potent antitumour response than either population alone.

The effect of thorough preoperative disinfection on the incidence of postoperative mediastinitis.

This study was conducted to evaluate the effectiveness of scrubbing the skin of patients scheduled to undergo elective cardiac surgery with disinfectant solutions in an attempt to reduce the incidence of the potentially fatal postoperative complication of mediastinitis. Since April 1991, we have routinely disinfected the skin of all such patients three times preoperatively. A retrospective comparison of the incidence of mediastinitis before and after April 1991 was carried out. Microbiological examinations of the anterior chest were conducted in ten patients admitted for elective operations, and in four patients admitted for emergency surgery. Specimens were obtained before each scrubbing, before the operation, and just after skin closure. There was a significant decrease in the general incidence of mediastinitis after April 1991 (P < 0.01). Microbiologically, significant decreases in colony counts were also observed after the first (P < 0.05) and second scrubbing (P < 0.05) in the ten patients specifically studied before elective surgery. No colonies grew in the postoperative specimens from any of these patients, but colony formations were observed in two of the four patients who underwent emergency surgery (P = 0.066). Thus, we conclude that our thorough disinfection method effectively achieves adequate, prolonged suppression of the skin flora, which explains the marked decrease in the incidence of mediastinitis in our hospital since its institution.

Technical feasibility of video-assisted lobectomy with radical lymphadenectomy for primary lung cancer.

The morbidity and mortality for video-assisted curative resection of lung cancer was evaluated retrospectively. Forty-one consecutive patients with stage I and II lung cancer underwent video-assisted curative lobectomy with complete hilar and mediastinal lymphadenectomy. Conversion to an open procedure was necessary in two patients. The operating times for the second half of the series were shorter than for the first half. Compared with patients receiving a standard open procedure, the video-assisted patients experienced satisfactory results. We conclude that video-assisted curative lobectomy with complete lymphadenectomy for stage I and II lung cancer is technically feasible in the majority of patients, although follow-up is required to determine the long-term prognosis. Comparative series between video-assisted and open procedures should not be conducted until the surgeon has acquired the necessary video-assisted skills. A prospective randomized trial will determine the actual value of video-assisted procedure for lung cancer treatment.

[Adverse effect of internal thoracic artery steal on perioperative hemodynamic condition--a report of three cases].

In three patients who had suffered from perioperative hemodynamic deterioration after myocardial revascularization with an internal thoracic artery to the left anterior descending coronary artery (LAD), blood steal from the ITA into large side branches was strongly suspected. In two of them an intractable ventricular fibrillation occurred before closure of the chest. Additional insertion of saphenous vein graft to the LAD enabled them to get out of jeopardy. Postoperative angiographic studies demonstrated large pericardial branches which had several fistulous communications with the pulmonary circulation. The other suffered from anteroseptal myocardial infarction immediately after an uneventful operation. Postoperative angiography revealed an enlarged lateral costal branch which gave off four intercostal arteries. Careful and meticulous harvesting of the ITA is mandatory to avoid tragic consequences caused by its large side branches.

[Characterization of oncogenes (K, N-ras and N, c-myc) in subcloned cell lines (KCC-1a and 1b) derived from same endometrial carcinoma presenting well differentiated adenocarcinoma and clear cell carcinoma].

Human endometrial adenocarcinoma cell lines (KCC-1a and KCC-1b) were established from the same endometrial carcinoma. 1) Amplification of the N-ras gene was not able to be observed in Southern blot hybridization. K-ras gene amplification of KCC-1b was more than 10 fold that of KCC-1a and the control. N-myc gene amplification of KCC-1b was more than 3-5 fold that of KCC-1a. c-myc gene amplifications of KCC-1a and KCC-1b were more than 5-7 fold that of the control DNA. 2) p21 was observed in tubular adenocarcinoma but not in clear cell carcinoma. p21 was detected in KCC-1b cells but not in KCC-1a cells. 3) KCC-1a and KCC-1b cells had quite different characteristics in molecular biology.

Eosinophilic granuloma of lymph node. A case report.

Eosinophilic granuloma confined to the lymph node of a 25-year-old Japanese woman is reported. She presented with a low-grade fever and pain in the left cervical region. A finger-tip-sized cervical lymph node was excised for histological study. Microscopically, the normal architecture of the lymph node was distorted, and histiocytoid cells had proliferated mainly in the sinus. Numerous eosinophils and an appreciable number of large multinucleated cells were also observed. Ultrastructurally, Birbeck granules were found in the cytoplasm of the histiocytoid cells. Immunohistochemically, the proliferating cells and multinucleated cells were found to be positive for S-100 protein but negative for lysozyme in the cytoplasm. The patient has been well without recurrence for 10 months after the lymph node biopsy.