Posology and Serum-/Xeno-Free Engineered Adipose Stromal Cells Cell Sheets.

Well-characterized adipose stem cells and chemically defined culture media are important factors that control the production of the cell sheet, used in translational medicine. In this study, we have developed and engineered multilayer adipose stem cell cell sheets (ASCCSs) using chemically defined/serum-free culture media: undifferentiated or differentiated into osteoblasts and chondrocytes. In addition, using the cell sheet transmittance, we estimated the number of cells per cell sheet. Undifferentiated ASCCSs were engineered in 10 days, using serum-free/xeno-free culture media. They were CD29+, CD73+, CD90+, CD105+, HLA-A+, and HLA-DR-. ASCCSs differentiated into chondrocytes and osteoblasts were also engineered using chemically defined and animal-free culture media, in only 14 days. The addition of an ROCK inhibitor improved the chondrocyte cell sheet engineering. The decrease in the cell sheet transmittance rate was higher for the osteoblast cell sheets due to the intracellular Ca2+ accumulation. The estimation of cell number per cell sheet was carried out with the transmittance, which will provide important information for cell sheet posology. In conclusion, three types of ASCCSs were engineered using serum-free, xeno-free culture media, expressing their specific markers. Their transmittance measurement allowed estimating the number of cells per cell sheet, with a non-invasive methodology.

[Primary central nervous system post-transplant lymphoproliferative disorder with few specific abnormal findings observed on MRI and in the cerebrospinal fluid].

A 60-year-old woman, who had a kidney transplant 16 years ago, was admitted to our hospital owing to cognitive decline and gait disturbances. She developed ataxia, consciousness disturbances, and myoclonus, and died two years after the onset of symptoms. No specific findings were observed on MRI or in the cerebrospinal fluid and blood analyses. The patient was diagnosed with post-transplant lymphoproliferative disorder (PTLD) based on the results of the autopsy. Pathological findings revealed proliferating monoclonal B cells in the perivascular space that was confined to the central nervous system. PTLD is a serious complication of transplantation. Furthermore, PTLD of the central nervous system usually presents as nodular lesions on MRI. When neurological symptoms appear after transplantation, it is necessary to consider PTLD as a differential diagnosis even if abnormal findings cannot be pointed out on MRI.

Measurement of the Adipose Stem Cells Cell Sheets Transmittance.

In the field of cell therapy, the interest in cell sheet technology is increasing. To determine the cell sheet harvesting time requires experience and practice, and different factors could change the harvesting time (variability among donors and culture media, between cell culture dishes, initial cell seeding density). We have developed a device that can measure the transmittance of the multilayer cell sheets, using a light emitting diode and a light detector, to estimate the harvesting time. The transmittance of the adipose stromal cells cell sheets (ASCCS) was measured every other day as soon as the cells were confluent, up to 12 days. The ASCCS, from three different initial seeding densities, were harvested at 8, 10, and 12 days after seeding. Real-time PCR and immunostaining confirmed the expression of specific cell markers (CD29, CD73, CD90, CD105, HLA-A, HLA-DR), but less than the isolated adipose stromal cells. The number of cells per cell sheets, the average thickness per cell sheet, and the corresponding transmittance showed no correlation. Decrease of the transmittance seems to be correlated with the cell sheet maturation. For the first time, we are reporting the success development of a device to estimate ASCCS harvesting time based on their transmittance.

[A case of central nervous system primary lymphomatoid granulomatosis with multiple vascular lesions in the spinal cord and brain].

A 71-year-old man was hospitalized because of low back pain and weakness in both lower limbs. He presented with fever and stiff neck, and his cerebrospinal fluid sample contained blood. MRI revealed intramedullary and epidural hemorrhages in the spinal cord. Microhemorrhages occurred frequently in the central nervous system over a short period. A brain biopsy was performed. The diagnosis was primary lymphomatoid granulomatosis (LYG) of the central nervous system (grade 2). As a result of lymphocytic infiltration to the vascular walls in LYG, hemorrhages occurred in multiple sites in the central nervous system. The biopsy of samples from the sites of microhemorrhages proved useful for diagnosis even in the absence of mass lesions.

[Symmetrical lesions of the cerebral peduncles and internal capsules on MRI in a patient with extranodal NK/T cell lymphoma].

A 72-year-old man was admitted to our hospital because of subacute progressive consciousness disturbance. FLAIR images showed diffuse high intensity areas in the bilateral thalamus, basal ganglia, and white matter. Chest CT revealed a mass in the right lung. The patient was diagnosed with extranodal natural killer/T cell lymphoma (ENKL) according to the results of a biopsy of the right pulmonary mass. The FLAIR images showed diffuse white matter lesions, without a mass such as lymphomatosis cerebri. On diffusion-weighted images, symmetrical high-intensity lesions of the cerebral peduncle and the internal capsule suggested high-density lymphoma cells around neurons. ENKL of the central nervous system and lung is a very rare neoplasm, and this case showed characteristic images.

An Autopsy Case of Intimal Sarcoma of the Abdominal Aorta with Bone Metastasis and Lymph Node Metastasis: A Case Report and Review of the Japanese Literature.

A 73-year-old man complained of sternoclavicular joint pain; blood tests revealed elevated C-reactive protein. The patient developed delirium; magnetic resonance imaging showed metastatic bone tumors. He died two weeks after admission. Autopsy revealed abdominal aortic intimal sarcoma with metastasis to the peritracheal lymph nodes and sternum. Peripheral arterial embolism and bone metastasis are common symptoms of aortic intimal sarcoma, which implies a place for aortic intimal sarcoma in differential diagnoses of embolism or bone tumors of unknown origin.

[Reliability and validity of the Japanese version of the Thinking Style Inventory].

This study examined the internal and external validity of the Japanese version of the Thinking Styles Inventory (TSI: Hiruma, 2000), which was originally developed by Sternberg and Wagner (1991) based on the framework of Sternberg's (1988) theory of mental self-government. The term "thinking style" refers to the concept that individuals differ in how they organize, direct, and manage their own thinking activities. We administered the Japanese version of the TSI to Japanese participants (N = 655: Age range 20-84 years). The results of item analysis, reliability analysis, and factor analysis, were consistent with the general ideas of the theory. In addition, there were significant relationships between certain thinking styles and 3 participant characteristics: age, gender, and working arrangement. Furthermore, some thinking styles were positively correlated with social skill. Implications of these results for the nature of Japanese thinking styles are discussed.

[A case of dural arteriovenous fistula at the craniocervical junction, which spinal MRI findings reveals increased intensity signal in Th3-medullary cone].

A 60-year-old woman had transient weakness of the legs and urinary retention for six weeks. She presented with a gait disorder and was admitted to the hospital. She showed symptoms of paraplegia, tingling in the lower extremities, dysuria. She underwent an MRI, and T2-weighted images showed an enlargement of the thoracolumbar spinal cord and high intensity signal from Th3 to the medullary cone, and a contrast-enhanced T1-weighted image showed abnormal vessels anterior to the spine cord. Cervical and spinal angiography documented a dural arteriovenous fistula at the craniocervical junction that was fed by the right vertebral artery and the right ascending pharyngeal arteries and drained into the perimedullary veins. Surgical therapy improved her symptoms and MRI images. Craniocervical junction DAVF with thoracic-medullary cones lesion is rare.

[A case of myelitis with eosinophilia of the cerebrospinal fluid].

A 25-year-old woman developed numbness of the right flank two weeks after a one-month stay in Thailand and Laos, which are known as areas of angiostrongylosis cantonensis infections. The signs were numbness, pain sense disorder, and vibration sense disorder on the region of the 6th to 12th dermatome. On a MRI T(2) weighted image (T(2)WI), signal hyperintensity in a longitudinal spinal lesion was seen. In the cerebrospinal fluid (CSF), eosinophils were detected. The patient was diagnosed with eosinophilic meningitis and myelitis, and then treated with intravenous methylprednisolone. This improved her signs, CSF and MRI findings. She took no drugs, did not have any allergies, any vasculitis, or neuromyelitis optica spectrum disorders. Although anti-parasite antibodies were not identified, she was probably infected by angiostrongylus cantonensis from her history and examinations.

Mechanistic insights into the catalytic reaction of plant allene oxide synthase (pAOS) via QM and QM/MM calculations.

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the Fe(III) and Fe(IV) pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members.

Open-label study to evaluate the pharmacodynamics, clinical efficacy, and safety of meropenem for adult bacterial meningitis in Japan.

The aim of this study was to assess the efficacy, safety, and concentration of meropenem in cerebrospinal fluid when meropenem (2 g every 8 h) was administered to Japanese adult patients with bacterial meningitis. Five Japanese patients (mean age 60.6 years [range 35-71]) were enrolled. Infection with Streptococcus pneumoniae (three patients), Streptococcus salivarius (one patient), and Staphylococcus aureus (one patient) was confirmed by cerebrospinal fluid culture. Meropenem (2 g every 8 h) was administered to all five patients. Treatment duration ranged from 14 to 28 days (mean 22.6 days). All the patients were successfully treated. The concentration of meropenem in cerebrospinal fluid ranged from 0.27 to 6.40 µg/ml up to 8.47 h and was over 1 µg/ml 3 h after starting meropenem infusion. In each patient, the present study confirmed for the first time that the concentration of meropenem in cerebrospinal fluid exceeded the minimal inhibitory concentration for these pathogens. Eleven clinical and laboratory adverse events considered to be related to meropenem were observed in all patients, but no serious adverse event and no discontinuance of treatment due to adverse events occurred. Thus meropenem appeared to be a well-tolerated and effective agent for Japanese adult patients with bacterial meningitis. 2 g every 8 h of meropenem was delivered to CSF and its concentration was exceed in MICs for the detected pathogens.

[Case of dermatomyositis with anti-CADM-140 antibody and alveolar hemorrhage].

A-47-year-old woman was admitted to our hospital because of eruption, severe arthritis, myalgia, pharyngalgia and the elevation of serum creatine kinase. She was diagnosed with dermatomyositis based on a biopsied specimen and clinical symptoms. Serum anti CADM-140 antibody was detected by immunoprecipitation. Pulse therapy with methylprednisolone and cyclophophamide could transiently improve myalgia and so on, but she died of rapidly progressive interstitial lung disease. Autopsy findings demonstrated diffuse alveolar damage and alveolar hemorrhage. It is said that patients with anti CADM-140 antibody show poor muscle symptoms and alveolar hemorrhage has been described very rarely. This patient is the rare case of dermatomyositis with anti CADM-140 antibody developing severe muscle symptoms, pharyngalgia and aleveolar hemorrhage in autopsy findings.

Development of a novel fusion imaging technique in the diagnosis of hepatobiliary-pancreatic lesions.

INTRODUCTION: Multi-row detector computed tomography (MDCT) and magnetic resonance cholangiopancreatography (MRCP) play an important role in the imaging diagnosis of hepatobiliary-pancreatic lesions. Here we investigated whether unifying the MDCT and MRCP images onto the same screen using fusion imaging could overcome the limitations of each technique, while still maintaining their benefits. Moreover, because reports of fusion imaging using MDCT and MRCP are rare, we assessed the benefits and limitations of this method for its potential application in a clinical setting. METHODS: The patient group included 9 men and 11 women. Among the 20 patients, the final diagnoses were as follows: 10 intraductal papillary mucinous neoplasms, 5 biliary system carcinomas, 1 pancreatic adenocarcinoma and 5 non-neoplastic lesions. After transmitting the Digital Imaging and Communication in Medicine data of the MDCT and MRCP images to a workstation, we performed a 3-D organisation of both sets of images using volume rendering for the image fusion. RESULTS: Fusion imaging enabled clear identification of the spatial relationship between a hepatobiliary-pancreatic lesion and the solid viscera and/or vessels. Further, this method facilitated the determination of the relationship between the anatomical position of the lesion and its surroundings more easily than either MDCT or MRCP alone. CONCLUSION: Fusion imaging is an easy technique to perform and may be a useful tool for planning treatment strategies and for examining pathological changes in hepatobiliary-pancreatic lesions. Additionally, the ease of obtaining the 3-D images suggests the possibility of using these images to plan intervention strategies.

An AT1-receptor antagonist and an angiotensin-converting enzyme inhibitor protect against hypoxia-induced apoptosis in human aortic endothelial cells through upregulation of endothelial cell nitric oxide synthase activity.

The protective effects and roles of AT1-receptor antagonists (AT1-RA) or angiotensin-converting enzyme inhibitors (ACEI) on vascular endothelial cell (EC) injury during hypoxia are not entirely known. Therefore, we investigated these effects and mechanisms in human aortic (HA) EC. DNA fragmentation, Lactate dehydrogenase (LDH) release, and caspase-3 activity were measured in cultured HAEC after exposure to hypoxia in the presence or absence of an AT1-RA (candesartan, CS) and/or an ACEI (temocaprilat, TC). Next, we investigated endothelial cell nitric oxide synthase (ecNOS) and inducible (i) NOS to determine the role of the bradykinin(BK)-NO pathway in the protective effect on ACEI and AT1-RA in the setting of hypoxia-induced apoptosis. Exposure to hypoxia increased DNA fragmentation in HAEC associated with the activation of caspase-3, but did not affect LDH release. In addition, hypoxia induced ecNOS mRNA but not mRNA iNOS. CS and/or TC reduced apoptosis induced by hypoxia in a dose-dependent manner, and significantly increased BK and ecNOS expression. This effect was attenuated by the kinin B2 receptor antagonist, HOE 140, and the NOS inhibitor, N-nitro-L-arginine methylester (L-NMMA). Hypoxia activates the pathway leading to apoptosis by enhancing caspase-3 activity. Both CS and TC can ameliorate hypoxia-induced apoptosis in HAEC through inhibiting caspase-3 activation by enhancing ecNOS activity, via the accumulation of BK.

A novel water-soluble vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol, protects against acute lung injury and mortality in endotoxemic rats.

Satisfactory therapy for acute lung injury related to endotoxemia remains to be established. However, in vivo antioxidant treatment with N-acetylcysteine reportedly suppresses acute lung injury and proinflammatory cytokine production induced by endotoxin (lipopolysaccharide, LPS). In addition, intrinsic vitamin E is protective against LPS-induced insults. We determined the effects of a novel water-soluble vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), on acute lung injury and mortality induced by LPS in rats. Intravenous injection of TMG (4 or 40 mg/kg) effectively decreased mortality and prevented the increased alveolar permeability and pulmonary edema that were caused by intravenous injection of LPS (20 mg/kg). Treatment with TMG decreased the enhanced lung expression of TNF-alpha caused by LPS. TMG also suppressed the sequestration of neutrophils in the lung induced by LPS. These results indicate that TMG is a possible therapeutic agent for acute lung injury and mortality, especially that caused by gram-negative bacteria. The therapeutic effects could be mediated at least partly through suppression of the increased expression of TNF-alpha and neutrophil sequestration in the lung that are caused by LPS.

Alpha-tocopherol prevents apoptosis of vascular endothelial cells via a mechanism exceeding that of mere antioxidation.

Alpha-tocopherol has been reported to exert an anti-atherogenesis effect. We attempted to clarify the effect of alpha-tocopherol-both as an antioxidant and as a nonantioxidant--on apoptosis induced by oxidized low-density lipoprotein (LDL) or oxysterols. Oxidized LDL and oxysterols induced necrosis and/or apoptosis of vascular endothelial cells. The induction of apoptosis was associated with increased caspase-3 activity and the generation of intracellular reactive oxygen species, both the effects of which were attenuated by alpha-tocopherol. Apoptosis was also decreased by beta-tocopherol or intracellular radical scavengers, but these suppressive effects were less than those of alpha-tocopherol. Neither beta-tocopherol nor the scavengers had pronounced effect on caspase-3 activity, but each of them decreased the generation of reactive oxygen species to the same extent as alpha-tocopherol. Our study suggests that alpha-Toc protects against apoptosis not only by scavenging reactive oxygen species, but also by inhibiting caspase activity, which means that its activity may exceed that of a mere antioxidant.