Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

Short-chain fatty acids induce tissue plasminogen activator in airway epithelial cells via GPR41&43.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease generally divided based on the presence or absence of nasal polyps (NPs). One of the features of NPs is excessive fibrin deposition, which is associated with down-regulation of tissue plasminogen activator (t-PA) in NPs. As t-PA is expressed in epithelial cells, and epithelium is readily accessible to topical therapies, identifying compounds that can mediate the induction of t-PA would be a potential new strategy for the treatment of NPs. OBJECTIVE: The objective of this study was to determine whether short-chain fatty acids (SCFAs) can induce t-PA in airway epithelial cells via their known receptors GPR41 and GPR43. METHODS: We performed immunohistochemistry (IHC) to determine whether receptors for SCFAs, known as G protein-coupled receptor 41/free fatty acid receptor 3 (GPR41/FFAR3) and GPR43/FFAR2, are expressed in nasal tissue. Primary normal human bronchial epithelial (NHBE) cells were stimulated with different concentrations of SCFAs to test induction of t-PA, which was analysed by expression of mRNA and protein. Mediation of responses by SCFA receptors was evaluated by specific receptor gene silencing with siRNA. RESULTS: Immunohistochemistry study revealed that airway epithelial cells expressed GPR41 and GPR43. Acetic acid, propionic acid, butyric acid and valeric acid significantly induced t-PA expression from two- to tenfolds. The strongest inducer of t-PA from NHBE cells was propionic acid; cells stimulated with propionic acid released t-PA into the supernatant in its active form. Gene silencing of GPR41 and GPR43 revealed that induction of t-PA by SCFAs was dependent upon both GPR41 and GPR43. CONCLUSIONS AND CLINICAL RELEVANCE: Short-chain fatty acids were shown to induce airway epithelial cell expression of t-PA via GPR41 and GPR43. Topical delivery of potent compounds that activate these receptors may have value by reducing fibrin deposition and shrinking nasal polyp growth.

Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study.

BACKGROUND: Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS. METHODS: This was a retrospective study conducted by 15 institutions participating in the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC). We evaluated patients with CRS treated with endoscopic sinus surgery (ESS), and risk of recurrence was estimated using Cox proportional hazard models. Multiple logistic regression models and receiver operating characteristics curves were constructed to create the diagnostic criterion for ECRS. RESULTS: We analyzed 1716 patients treated with ESS. To diagnose ECRS, the JESREC scoring system assessed unilateral or bilateral disease, the presence of nasal polyps, blood eosinophilia, and dominant shadow of ethmoid sinuses in computed tomography (CT) scans. The cutoff value of the score was 11 points (sensitivity: 83%, specificity: 66%). Blood eosinophilia (>5%), ethmoid sinus disease detected by CT scan, bronchial asthma, aspirin, and nonsteroidal anti-inflammatory drugs intolerance were associated significantly with recurrence. CONCLUSION: We subdivided CRSwNP in non-ECRS, mild, moderate, and severe ECRS according to our algorithm. This classification was significantly correlated with prognosis. It is notable that this algorithm may give useful information to clinicians in the refractoriness of CRS before ESS or biopsy.

[Extended thymectomy and intraoperative-intrapleural perfusion hyperthermo-chemotherapy for stage IVa invasive thymoma with myasthenia gravis].

A 37-year-old woman diagnosed with ocular myasthenia gravis was referred to our department. Chest computed tomography (CT) showed anterior mediastinal tumor and right pleural dissemination. Extended thymectomy and right intraoperative-intrapleural perfusion hyperthermo-chemothrapy (IPHC) were performed. Pathological diagnosis was invasive thymoma type B2 and stage IVa based on Masaoka's classification. The post operative course was uneventful. The patient underwent 4 cycles of adjuvant chemotherapy with doxorubicin, cisplatin, vincristine, and cyclophosphamide (ADOC), and is free from recurrence at 12 months postoperatively.

Associations between decay-accelerating factor polymorphisms and allergic respiratory diseases.

BACKGROUND: Allergic diseases such as asthma and allergic rhinitis are major causes of morbidity in developed countries. The pathology underlying allergic respiratory diseases is considered to be IgE-mediated type I allergy characterized by mucosal inflammation that occurs in response to allergen exposure. They are common diseases involving a complex inheritance. Complement systems are known to play an important role in allergic diseases. Decay-accelerating factor (DAF) is important for the regulation of the complement system and is a good candidate for determining the susceptibility to allergic diseases. OBJECTIVE: The present study aimed to investigate whether polymorphisms in the DAF gene are associated with allergic respiratory diseases in the Japanese population. METHODS: We performed mutation screenings of DAF and conducted a tag single-nucleotide polymorphisms (SNP) association analysis for 684 unrelated adult individuals with seasonal allergic rhinitis (SAR) with Japanese ceder pollen, 188 mite-sensitive adults with asthma, and 346 unrelated non-allergic healthy controls. RESULTS: DAF is located in the tight linkage disequilibrium (LD) block spanning 62 kb. The tag SNP analysis revealed that rs10746463 was significantly associated with SAR (P=0.00033) and mite-sensitive adult asthma (P=0.044). The rs2564978 and rs3841376 haplotypes, which are located in the promoter region of DAF, were in complete LD with rs10746463 (r2=1). Luciferase reporter assays with constructs containing the 5' flanking regions of DAF showed that the plasmid with rs2564978 C/rs3841376 deletion (the risk haplotype) had a statistically significantly lower transcriptional activity than that containing the rs2564978 T/rs3841376 insertion. CONCLUSIONS: Our results suggest that DAF is one of the genes involved in conferring susceptibility to allergic respiratory diseases and show that decreased levels of DAF may be associated with the enhanced specific IgE responses occurring in allergic diseases in the Japanese population.

[Emergency operation due to complications of cardiac catheterization].

From January 2001 to July 2005, 4,741 cardiac catheterizations were performed on adult patients at our institute. Five of them underwent emergency operation due to complications of cardiac catheterization. The causes of operations were acute coronary occlusion due to coronary arterial dissection in 3 patients and bleeding due to perforation of the coronary artery in 2. Intra-aortic balloon pumping was used preoperatively in 4 patients and percutaneous cardio-pulmonary support in 2. Coronary artery bypass grafting was performed on all patients. Perforation sites of the coronary arteries were closed. One patient died 4 months after surgery, due to neurological damage, but the other 4 patients recovered and have been doing well. As coronary artery disruption is one of the main complications of cardiac catheterization, it is necessary to recognize the risk of hemodynamic derangement and to give prompt and appropriate treatment.

[Two-stage repair of the transposition of great arteries with interruption/coarctation of the aorta].

We report on 10 patients who underwent two-stage repair of transposition of the great arteries (TGA) with interruption (IAA) or coarctation (CoA) of the aorta. First, an operation for aortic arch reconstruction was performed: Blalock-Park with pulmonary artery banding (PAB) for IAA (5 patients), subclavian flap with PAB for CoA (4 patients) and end-to-end anastomosis without PAB (1 patient). All survived the first operation and had no significant pressure gradient with good growth of the ascending aorta, except for the 1 case without PAB. Half of the 8 patients who underwent PAB developed migration of the PAB. The arterial switch operation (ASO) was performed 0.7-12.6 (5.6+/-4.7) months after the first surgery. One patient with an abnormal coronary artery tract was lost after ASO. Five developed pulmonary artery stenosis and 1 developed supra-aortic stenosis late after ASO. Two patients need reoperation, 1 for supra-aortic stenosis, and the other for reCoA. Two-stage repair for TGA with IAA/CoA is still a useful method with a good operative result. However, strict follow-up is necessary because of the high frequency of late morbidity.

A novel diagnostic method for evaluation of vascular lesions in the digestive tract using infrared fluorescence endoscopy.

BACKGROUND AND STUDY AIMS: We have developed an infrared fluorescence endoscope to evaluate gastrointestinal vascular lesions. Infrared endoscopy (IRE) after intravenous administration of indocyanine green (ICG) is used at present to examine vascular lesions such as esophageal varices. However, no previous study has compared the sensitivity of infrared fluorescence endoscopy (IRFE) with that of IRE. In this study, we compared the usefulness of IRFE and IRE. PATIENTS AND METHODS: For IRFE we used an infrared endoscope equipped with excitation and barrier filters and an intensified charge-coupled device camera. In preliminary experiments, the observable tissue depth was assessed by wrapping increasing numbers of layers of commercially available pork around a syringe containing a uniform concentration of ICG or by changing the concentration of ICG in a syringe covered by a piece of pork of uniform thickness. In the clinical part of the study, ICG was administered intravenously at different concentrations to patients with esophageal varices and the resulting infrared fluorescent images were evaluated. RESULTS: The preliminary experiments revealed that the depth of tissue that could be visualized was significantly greater in IRFE than it was in IRE (11.2 mm in IRFE vs. approximately 3.2 mm in IRE). Clear infrared fluorescence was obtained by IRFE at lower concentrations of ICG than the concentrations required to obtain clear images using IRE. In the clinical part of the study, clear infrared fluorescence was observed in a region where esophageal varices had been detected by conventional endoscopy when ICG was administered in doses of 0.005 mg/kg to 0.01 mg/kg, which was lower than the doses used in IRE. CONCLUSIONS: Compared with conventional IRE, IRFE facilitated the observation of deeper layers, and esophageal varices were observed by IRFE following the intravenous administration of a markedly reduced dose of ICG. IRFE, in combining the characteristics of reflected infrared light and fluorescence, may be a useful novel procedure in the diagnosis of vascular lesions in the gastrointestinal tract.

Experience with the Norwood procedure without circulatory arrest.

OBJECTIVE: We evaluated a new cardiopulmonary bypass technique that allowed complete avoidance of circulatory arrest and deep hypothermia in the Norwood procedure for hypoplastic left heart syndrome. METHODS: A total of 10 patients were included in this study. The arterial line of the cardiopulmonary bypass circuit was divided in two in a Y shape; one branch was used for cerebral perfusion through the innominate artery and the other for lower body perfusion through the cannula inserted into the descending thoracic aorta. Moderate hypothermia (29 degrees C-31 degrees C rectal temperature) and high pump flow (150-180 mL. kg(-1). min(-1)) were used. A valveless conduit between the right ventricle and the pulmonary artery was used in 6 patients as an alternative pulmonary blood source to a conventional Blalock-Taussig shunt (n = 4). RESULTS: Circulatory arrest was completely avoided throughout the operation in all cases, and no complications from the new cardiopulmonary bypass technique were seen. Early deaths occurred in 3 cases. Neurologic deficits were not seen among the survivors, and the postoperative course was stable and uneventful, including satisfactory renal function. CONCLUSIONS: The Norwood procedure for hypoplastic left heart syndrome was successfully accomplished with complete avoidance of circulatory arrest by means of cerebral perfusion through the innominate artery combined with cannulation of the descending aorta. A conduit between the right ventricle and the pulmonary artery seems an excellent alternative pulmonary blood source, although right ventricular function needs to be carefully monitored.

Early and late results of total correction of congenital cardiac anomalies in infancy.

OBJECTIVE: We evaluated long-term results of surgical correction of congenital cardiac anomalies in infancy. METHODS: We reviewed cases of 856 patients who underwent complete correction of major cardiac anomalies in the first year of life during last 24 years at our institution, and analyzed results. Surgery involved ventricular septal defect (n = 453), tetralogy of Fallot (n = 92), atrioventricular canal defect (n = 93), and complete transposition of the great arteries (n = 218). RESULTS: Operative mortality was 2.2% in ventricular septal defect, 0% in tetralogy of Fallot, 8.6% in atrioventricular canal defect, and 4.1% in complete transposition of the great arteries. Freedom from reoperation at 20 years was 96.5 +/- 2.0% and actuarial survival was 94.2 +/- 1.3% in ventricular septal defect. Freedom from reoperation at 15 years was 90.5 +/- 3.9% in tetralogy of Fallot and 86.6 +/- 4.4% in atrioventricular canal defect. Actuarial survival at 15 years was 97.8 +/- 1.6% in tetralogy of Fallot, 85.7 +/- 3.7% in atrioventricular canal defect, and 89.9 +/- 2.2% in complete transposition of the great arteries. Actuarial survival in complete transposition of the great arteries was significantly better in arterial than in atrial switch operations. CONCLUSIONS: Total correction of ventricular septal defect, tetralogy of Fallot, atrioventricular canal defect, and complete transposition of the great arteries in infancy was conducted safely, but the incidence of reoperation in late follow-up must be reduced.

Synthesis of oligosaccharide with controlled sequence.

A simple method for the synthesis of alternate heteropolysaccharide was investigated. Two types of modified glucose were combined into a cellobiose-type disaccharide monomer having only acetyl groups except a free hydroxyl group at C-3'. Polycondensation of this monomer, using stannic tetrachloride as catalyst, easily gave a novel copolysaccharide having regularly alternating 1,3-beta and 1,4-beta linkages.

[Clinical laboratory data and reference intervals standardized in Fukuoka].

In Fukuoka whose population is approximately five million inhabitants, surveys on the accuracy of laboratory data have been performed by the Fukuoka Prefecture Medical Association for the last 30 years. We have been attempting to evaluate the data for routine use since 1988, and it has become possible to share laboratory data between all institutions in Fukuoka prefectures. As a result, reference intervals for 23 clinical chemistry analytes were established in 1995, to which were added in 1996 five serum protein constituents that have been utilized for clinical examinations. Methods for documentations and monitorings the data obtained in the prefecture were also established, standardization of the above analytes extended to 97% of the institutions in the prefecture. Results for 14 of the 23 clinical chemistry analytes have become highly reliable and clinically useful as differences between institutions in terms of results have narrowed. Standardization of other analytes is now in progress.

Standardization of laboratory data and establishment of reference intervals in the Fukuoka Prefecture: a Japanese perspective.

Standardization of 22 clinical chemistry analytes and five serum protein constituents has been performed in the Fukuoka Prefecture, which has a population of approximately five million. The standardization project was established to determine reference intervals for these analytes by educating physicians, medical technologists and staff of medical institutions, and by daily or monthly monitoring the use of common control samples through e-mail. Standardization extended to 97% of the institutions in the prefecture. Results for 14 of the 22 clinical chemistry analytes have become highly reliable and differences between institutions decreased. Standardization of other analytes is now in progress. Regional collaboration based on international guidelines led to a significant improvement in interlaboratory comparability. Areas where further improvements are needed have been identified.

Successful staged-Fontan operation in a patient with ectopia cordis.

Ectopia cordis is a very rare and often fatal disease. We report a successful surgery case of thoracoabdominal ectopia cordis with univentricular heart. This patient underwent a three-stage Fontan procedure, a right-modified Blalock-Taussig shunt at the age of 1 month, bidirectional Glenn shunt and pulmonary arterioplasty at 2 years 8 months, and finally a total cavopulmonary connection at 4 years. This patient was discharged from the hospital in good condition and has been doing well since. Thus, ectopia cordis is not a contraindication for a Fontan operation.

[Mitral valve replacement 41 years after right pneumonectomy].

Mitral valve replacement was performed successfully on a 68-year-old patient who had undergone right pneumonectomy and thoracoplasty 41 years earlier. Preoperative pulmonary function tests revealed poor results; the forced vital capacity was 950 ml (28.0% of the predicted value) and the forced expiratory volume in 1 second was 750 ml (28.9% of the predicted value). Despite such poor pulmonary function, the patient tolerated the operation well and led uneventful course. Careful perioperative management to prevent pulmonary edema and aggressive postoperative pulmonary toilet to facilitate recovery of the pulmonary function seem important.

Mitral valve replacement using bileaflet mechanical prosthetic valve in the first year of life.

OBJECTIVE: The operative management and long term outcome of mitral valve replacement in infancy remain a therapeutic challenge. The selection of a prosthetic valve for this particular age group might affect the clinical outcome. Here we present our experience of mitral valve replacement in 6 infants using small bileaflet mechanical prosthetic valves. METHODS: Between January 1994 and August 1997, 6 infants (their age ranged from 3 months to 11 months, and their body weight from 2978 g to 7403 g) underwent mitral valve replacement using a mechanical valve prosthesis (16 mm CarboMedics prosthetic valve in 5, and 17 mm St. Jude Medical prosthetic valve Hemodynamic Plus in 1). The preoperative morphological features of the mitral valve were stenosis in 1, regurgitation in 3, and a combination of these in 2. The prosthesis was fixed at the annulus in 3, and at the supra-annular position in 3. Anticoagulation was performed using warfarin. RESULTS: There was no operative mortality. Postoperative catheterization revealed an acceptable wedge pressure in the pulmonary arteries, ranging from 10 to 12 mmHg. During the mean follow-up period of 36 months, late death due to residual pulmonary hypertension occurred in 1 patient at 10 months after surgery. Excluding this patient, all remaining patients are doing well with no need for repeated operation with no thromboembolic complication. The actuarial survival rate and the reoperation free rate at 70 months are 83 +/- 15% and 100%, respectively. CONCLUSION: Mitral valve replacement using a small size bileaflet mechanical prosthetic valves in infancy can be performed with low operative mortality and with satisfactory mid-term results.

[Patching of the raw surface of the posterior right ventricular wall in Ross procedure to prevent bleeding and to confirm anastomosis of the conduit].

An 8-year-old boy underwent Ross procedure to repair aortic regurgitation and subaortic stenosis. In reconstruction of the right ventricular outflow tract, the raw surface of the right ventricular posterior wall was covered with autologous pericardial patch to prevent bleeding and to confirm the anastomosis of the pulmonary tract conduit made of expanded polytetrafluoroethylene. Bleeding from the posterior wall was absent and he led uneventful postoperative course. Same technique was used in 4 other patients, and satisfactory results were obtained. This technique, first reported from India, seems very useful in Ross procedure.