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We previously developed a fully automated spherical aberration compensation microscope system, Deep-C, to obtain spherical aberration-free images, but the contrast-based algorithm (Peak-C) may limit applications for low signal-to-noise ratio images. Herein we propose a new spatial frequency-based algorithm called Peak-F and compared its performance to Peak-C. Unlike Peak-C, Peak-F is robust to any noise level since it is independent of the dynamic range of the images, and it does not suffer from image saturation. Finally, Peak-F was implemented in a two-photon microscope to observe living aged and young mouse brains. Consequently, the average refractive index of brain tissue was higher in old mice than in young mice. The Peak-F algorithm determines high-resolution microscopic images stably and robustly.

The present study investigated the volatile constituents of fermented meat sauce (FMS) and identified some of the potent odour-active components. The FMS was prepared by fermenting a mixture of ground pork, koji, and salt for 12 months. After 12 months' fermentation, four potent odour components: 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolon), 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (ethyl furaneol), 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and 3-methylbutanoic acid, were found in FMS by gas chromatography-olfactometry analysis. The most characteristic components were sotolon and ethyl furaneol and contributed strongly to the FMS odour. These two components were detected in the FMS after 6 and 9 months' fermentation, respectively; especially sotolon concentration was 147.43 mg/l after 12 months' fermentation. These components were generated by fermentation and might play an important role in the FMS odour after 12 months' fermentation.

a Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Metabolic Medicine, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan c Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan d Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan e Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan

Treatment of refractory Takayasu arteritis (TA) remains an unresolved clinical issue. Patients usually respond to glucocorticoid (GC) therapy, but often relapse on tapering of the GC dose. The aim of the present study was to assess the safety and efficacy of the interleukin-6 (IL-6) receptor antibody tocilizumab (TCZ) in patients with TA refractory to conventional therapies including GC. Four patients with TA who had shown GC resistance received TCZ infusions (8 mg/kg) every 4 weeks a total of at least 24 times (range, 24 to 51). Clinical symptoms, the serum levels of acute phase proteins and IL-6, GC dosage necessary to maintain remission, and cross-sectional imaging by enhanced CT and MRI were assessed. All patients achieved good clinical response and rapid normalization of the acute phase proteins such as C-reactive protein and serum amyloid A during the therapy with TCZ. The mean dosage of prednisolone could be reduced from 21.3 mg/day to 1.5 mg/day. Although the serum IL-6 level was transiently elevated in all patients after several TCZ infusions, it gradually recovered to the initial level. Along with the decrease of serum IL-6, two patients exhibited significant reduction in thickened arterial lesions. No drug-related adverse effects were noted. In this small group of patients with refractory TA, TCZ therapy was effective and well-tolerated. Further larger studies should be conducted to confirm this finding.

SummaryBackgroundEndothelin (ET)-1 has been shown to play a significant pathogenic role in pulmonary arterial hypertension (PAH). However, the pathobiological significance of increased ET-1 concentration after administration of ET receptor antagonist in patients with PAH has not yet been fully examined.MethodsIn 16 PAH patients, plasma ET-1 concentration was measured at 0, 1, 3, 6, and 24h after a single 62.5mg dose of bosentan, a dual ET receptor antagonist, and the peak and 24-h change in ET-1 concentration from baseline were examined. The severity of PAH was evaluated by hemodynamic parameters, 6-min walk distance, New York Heart Association (NYHA) functional class, and brain natriuretic peptide (BNP).ResultsPlasma ET-1 concentration significantly increased from 1.93±0.12 to 3.36±0.18pg/ml after bosentan administration in PAH patients (p

Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5–10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500–10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.

o-Nitrophenyl sulfenyl-modified ACTH (NPS-ACTH) stimulated steroidogenesis acutely in bovine fasciculata-reticularis cells without increase in cellular cAMP synthesis. Application of NPS-ACTH to the cultured cells induced Ca2+ signals in individual cells as detected by video-enhanced microscopic fluorescence measurements. The percentage of Ca2+ signaling cells corresponded well with the increase of steroidogenesis induced by NPS-ACTH below 1 nM. Treatment of the cells with nicardipine, a Ca2+ channel blocker, suppressed the Ca2+ signals except for the transient increase just after the addition of NPS-ACTH and also blocked completely the stimulative effect on the steroidogenesis of NPS-ACTH below 1 nM. At a dosage of NPS-ACTH higher than 10 nM, the stimulative effect of steroidogenesis was partly suppressed by nicardipine and also by AA-861, a lipoxygenase inhibitor. The action of NPS-ACTH might be mediated by both Ca2+ and lipoxygenase metabolite(s) of arachidonic acid as dual second messengers. The effect of ACTH in pM range on the steroidogenesis was suppressed completely by the treatment with nicardipine and AA-861 at the same time, indicating that the action was mediated by both Ca2+ and the lipoxygenase metabolite(s) but not by cAMP. cAMP plays a significant role as a second messenger for ACTH action only at ACTH concentrations greater than 10 pM.

Recently, four patients with congenital choanal atresia were operated upon using a carbon dioxide laser, in our department. All patients were female, and patient age at the time of surgery ranged from 13-days to 4-months after birth. Three patients had bilateral choanal atresia and one had unilateral atresia and contralateral stenosis. All patients suffered from dyspnea at birth that required intubation or opening of an oral airway. The atresia plate was bony in 2 sides, membranous in 3 sides, and mixed in 2 sides. Three cases were accompanied by CHARGE association: C-coloboma, H-heart disease, A-atresia choanae, R-retarded growth and retarded development and/or CNS anomalies, G-genital hypoplasia, E-ear anomalies and/or deafness. Surgery was performed via a transnasal approach under a surgical microscope. In 2 cases, the newly created choanae closed again, and re-operation was necessary. Respiratory condition improved after surgery in all cases.We emphasize that using a carbon dioxide laser via a transnasal approach under a microscope is a valuable method for treatment of small infants requiring early treatment. The advantages of this method include excellent visualization and minimal potential for damage. To prevent re-obstruction of choanae, a tubular stent should be in place for at least 6 weeks postoperatively.

The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV., The secreted ligand Angiopoietin-1 is essential for embryonic blood vessel development and adult vascular homeostasis. Here the authors show, using conditional knockout mice, that myocardium-derived Angiopoietin-1 is required for the formation of coronary veins, but not arteries.

A 61-year old male complained of rotatory vertigo with hearing disturbance, nausea, and vomiting. Although the symptoms suggested peripheral vestibular dysfunction, a diagnosis of cerebellar infarction was made from the MRI findings. The patient first showed deviation toward the right while walking, secondary rotatory vertigo, nausea, vomiting, and spontaneous horizontal and rotatory nystagmus toward the left. The nystagmus changed to gaze nystagmus during the clinical course. Vertebral angiography showed occlusion of the right vertebral artery near the foramen magnum and collateral branches from the left vertebral artery. It is considered that circulatory insufficiency of the inner ear caused the vertigo attacks, but signs or symptoms of cerebellar dysfunction were not apparent because of the presence of collateral branches. We conclude that MRI and MRA are useful in the early diagnosis of cerebellar infarction.

Treating patients with head and neck tumor occasionally requires permanent occlusion of the internal carotid artery (ICA). To assess whether a patient will tolerate this occlusion, we studied stump pressure, EEG and angiogram under direct clipping of the common carotid artery during surgery for severely invasive hypopharyngeal carcinoma. In other cases, we tried balloon test occlusion and measured cerebral blood flow (CBF) with positron emission tomography (PET) using C15O2. This direct clipping test can be performed with the usual surgical instruments and provides significant information on carotid ligation. However, we recognized that the information was not sufficient for a clear assessment even if the stump pressure and EEG were within the normal range. Balloon occlusion test and PET were performed in two cases. The first case showed temporary hemiplegia after a slight decrease in stump pressure, while the second case did not show any change despite a severe decrease in stump pressure. The second case showed an increase of CBF on PET. Stump pressure does not necessarily reflect changes in CBF, and PET should be used for more reliable assessment.

Rationale: Grb2-associated binder (Gab) docking proteins, consisting of Gab1, Gab2, and Gab3, have crucial roles in growth factor–dependent signaling. Various proangiogenic growth factors regulate angiogenesis and endothelial function. However, the roles of Gab proteins in angiogenesis remain elusive. Objective: To elucidate the role of Gab proteins in postnatal angiogenesis. Methods and Results: Endothelium-specific Gab1 knockout (Gab1ECKO) mice were viable and showed no obvious defects in vascular development. Therefore, we analyzed a hindlimb ischemia (HLI) model of control, Gab1ECKO, or conventional Gab2 knockout (Gab2KO) mice. Intriguingly, impaired blood flow recovery and necrosis in the operated limb was observed in all of Gab1ECKO, but not in control or Gab2KO mice. Among several proangiogenic growth factors, hepatocyte growth factor (HGF) induced the most prominent tyrosine phosphorylation of Gab1 and subsequent complex formation of Gab1 with SHP2 (Src homology-2–containing protein tyrosine phosphatase 2) and phosphatidylinositol 3-kinase subunit p85 in human endothelial cells (ECs). Gab1-SHP2 complex was required for HGF-induced migration and proliferation of ECs via extracellular signal-regulated kinase (ERK)1/2 pathway and for HGF-induced stabilization of ECs via ERK5. In contrast, Gab1-p85 complex regulated activation of AKT and contributed partially to migration of ECs after HGF stimulation. Microarray analysis demonstrated that HGF upregulated angiogenesis-related genes such as KLF2 (Krüppel-like factor 2) and Egr1 (early growth response 1) via Gab1-SHP2 complex in human ECs. In Gab1ECKO mice, gene transfer of vascular endothelial growth factor, but not HGF, improved blood flow recovery and ameliorated limb necrosis after HLI. Conclusion: Gab1 is essential for postnatal angiogenesis after ischemia via HGF/c-Met signaling.

Esophageal foreign bodies can be removed relatively easily with a rigid or flexible endoscope, but external incision of the neck is sometimes necessary when endoscopic removal is difficult.Case 1 was a 69-year-old female who noted a sore throat after swallowing a fish bone. A diagnosis of an impacted foreign body was made by plain X-ray of the neck, and the foreign body in the hypopharyngeal constrictor muscle was removed by external incision. Case 2 was a 53-year-old female who felt dyspnea from the day after swallowing a fish bone. Plain X-ray of the neck showed a marked thickening of the shadow of the postpharyngeal soft tissue. A giant hematoma was detected in the visceral fascia and resected by external incision of the neck.

The submandibular region is masked by the mandible and submandibular gland, thus making it difficult to grasp its lesions. The region often gives rise to inflammatory lymphadenopathy and, the ultrasonic diagnosis can be difficult sometimes. In the present study, we performed ultrasonography on the submandibular region of cases with known pathological diagnoses. Studies regarding ultrasonic diagnosis of (1) disease of the submandibular gland, (2) submandibular lymphnodes and (3) sialolith were carried out. This report reviews the problems and possible changes. A satisfactory accuracy rate of 82% was achieved for the diagnosis of disease of the submandibular gland. Percentage of accurate diagnosis of submandibular lymph node metastasis was considerably lower compared to other regions of the neck. Detection of sialolith in the warthin's duct was also low owing to the structure of the submandibular region. When ultrasonography was performed by pressuring the floor of the oral cavity, it was possible to observe the changes in the size, form, and position of the lesions. Thus, ultrasonography can be considered as a very effective diagnostic technique in that region.

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal alpha-actin (alpha-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in alpha-SKA gene expression. Thus, alpha-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of alpha-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in alpha-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes alpha-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate alpha-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2(WT)), dominant-negative SHP2 (SHP2(C/S)), or beta-galactosidase (beta-gal), on alpha-SKA gene expression. LIF did not upregulate alpha-SKA mRNA in cardiomyocytes overexpressing either beta-gal or SHP2(WT). In cardiomyocytes overexpressing SHP2(C/S), LIF induced upregulation of alpha-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2(C/S), but not in myocytes overexpressing beta-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates alpha-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.

Castleman’s disease is a highly heterogeneous clinical-pathological entity that belongs to the lymphoproliferative disorders and is associated with pulmonary arterial hypertension (PAH) in some patients. It is linked to excessive immune stimulation by interleukin-6 (IL-6), which is also involved in the pathogenesis of PAH. A 31-year-old woman with Castleman’s disease demonstrated PAH characterized by severe right heart failure. Since she was resistant to various conventional therapies including steroids, prostacyclins, bosentan, and sildenafil, tocilizumab (anti-IL-6 receptor antibody) therapy was started. Her clinical course was followed for 6 months, with significant improvement without any adverse effect. This is the first reported case of use of tocilizumab in addition to steroids and conventional PAH therapy in a patient with PAH associated with Castleman’s disease.

Background and purpose: More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance. Methods and results: Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs. Conclusion: We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.

With the advent of tissue regeneration and gene therapy for heart disease, evaluation of coronary circulation and cardiac function in vivo, especially in a disease model, is extremely important. Conventional methods such as microcomputed tomography, high-resolution magnetic resonance angiography, and high-resolution ultrasound have become invaluable tools in cardiovascular research. However, the disadvantages and limitations of these approaches sometimes preclude researchers from conducting important and specific studies on coronary circulation and cardiac function. Therefore, we developed and applied a novel real-time, in vivo fluorescent optical imaging system for use in the mouse cardiovascular system. We report the use of this system for repeatedly assessing coronary circulation, cardiovascular structure, and cardiac function in live mice. First, we evaluated whether our new system can detect coronary vasodilator responses and changes in cardiac wall motion. Anesthesia was induced in C57BL/6 mice by 3.0% sevoflurane inhalation, and the heart was exposed through a left thoracotomy. One hundred microliters of an optical imaging probe (AngioSense-IVM 680, VisEn Medical Inc, Woburn, …

Treatment of refractory Takayasu arteritis (TA) remains a challenging clinical issue. Patients usually respond to glucocorticoid (GC) therapy, but often relapse on tapering of the GC dose. Additionally, GCs therapy is accompanied with a variety of adverse events. The aim of this study was to assess