Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.     

Evaluation of the efficacy of an Helicobacter pylori eradication treatment for idiopathic thrombocytopenic purpura patients]

The relationship between Helicobacter pylori (H. pylori) and gastric diseases (e.g. peptic ulcer, MALT lymphoma, and stomach cancer) has been widely accepted. Recent studies have also suggested an association between H. pylori infection and idiopathic thrombocytopenic purpura (ITP). In this study, an H. pylori eradication treatment was administered to 20 ITP patients and elucidated for its effectiveness. Among those 20 patients, H. pylori infection was confirmed in 17 (85%) through a C14 urea breath test, a rapid urease test, or a culture examination of a biopsied sample obtained by gastrointestinal endoscopy. Although the other 3 were negative to H. pylori, the H. pylori eradication treatment was also attempted because no other effective treatments had been established at the time of this study. In the H. pylori eradication treatment, lansoprazole (LPZ) 60 mg bid, amoxicillin (AMPC) 1500 mg bid, and clarithromycin (CAM) 400 mg bid were given to each patient for 7 days. For 4 cases, CAM was replaced with metronidazole (MNZ) 750 mg bid. The patients whose H. pylori infection was not eradicated after the first treatment received the re-eradication treatment with LPZ 60 mg bid, AMPC 1500 mg bid, and MNZ 750 mg bid for 7 days. After the treatments, the success of eradicating H. pylori was confirmed in all 17 H. pylori positive patients. In addition, platelet recovery was obtained in 11/20 patients (55%), which included 2 H. pylori negative patients and 2 patients whose H. pylori eradication was not successful after the first treatment. No relationship was found between the eradication effectiveness and the following clinical parameters: age, gender, previous therapies, disease duration, presence of anti-nucleus antibody, endoscopic atrophic change in the stomach, or kinds of antibiotics used for the treatment. These results support the efficacy of an H. pylori eradication treatment for ITP patients. A noteworthy result of this study was that an increase of platelet count was observed not only in H. pylori positive ITP patients, but also in 2 out of 3 H. pylori negative ITP patients after H. pylori eradication. Further studies are required to elucidate the efficacy of H. pylori eradication therapy in the patients negative for H. pylori.     

Structural determination of a new mutagenic heterocydic amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), present in beef extract

We previously found two new mutagens, compounds I and II, inbacteriological-grade beef extract by monitoring the mutagenicityto a new Salmonella strain, YG1024; compound I was identifiedas 2-amino-4-hydroxymethyl-3,8-dimethyli-midazo[4,5-f]quinoxaline(4-CH₂OH-8-MeIQX) In the present study, we isolated compoundII from the beef extract, which accounted for 2% of the totalmutagenicity of materials adsorbed on blue cotton. Further,we found that a large quantity of compound II was produced byheating a mixture of creatine, threonine and glucose (1:1:0.5)at 200^°C for 5 h, the level being 860-fold of that in thebeef extract. The structure of this compound was determinedto be 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx)by X-ray crystallography. The amount of 7,9-DiMeIgQx in bacteriological-gradebeef extract was estimated to be 53 ng/g. This compound induced13 800 and 670 revertants of S.typhimurium YG1024 and TA98 respectively,per µg in the presence of S9 mix.     

Study of active substances involved in skin dysfunction induced by crowding stress. I. Effect of crowding and isolation on some physiological variables,skin function and skin blood perfusion in hairless mice

The effects of five levels of population density on various organs, the neuroendocrine system, skin function, skin blood perfusion, and blood parameters were studied in the hairless mouse. Skin barrier recovery was evaluated by measuring transepidermal water loss after tape stripping. Blood perfusion was measured by means of a laser Doppler imaging technique. The effect of a parasympathetic nerve stimulator, carpronium chloride, on skin function in the crowded animal model was also examined. A 7 d crowding (10, 15, 20 mice/cage) significantly increased the levels of corticosterone, catecholamines (norepinephrine, epinephrine and dopamine), glucose and serum lactate dehydrogenase activity in circulating blood, induced atrophy of kidney, ovary and thymus and hypertrophy of adrenal glands, and decreased body weight gain in comparison with the control (5 mice/cage). Crowding also increased epidermal thickness and epidermal proliferative activity, and decreased corneocyte size, rate of barrier recovery and skin blood perfusion. Most of these changes became more marked with increasing population density and/or longer exposure to a crowded environment. Isolation (1 mouse/cage) increased the level of norepinephrine and rate of skin blood perfusion, and significantly delayed barrier recovery. Repeated topical applications of carpronium chloride for 7 d improved the changes in skin blood perfusion, barrier recovery, kidney and ovary, and epidermal morphology induced by crowding. The crowded animal model could be useful for quantifying objectively the influence of crowded environment-induced stress on cutaneous function and blood perfusion.     

Mapping the HLA-A24-restricted T-cell epitope peptide from a tumour-associated antigen HER2 / neu: possible immunotherapy for colorectal carcinomas

HER2 / neu is a potential antigen candidate for immunotherapy because of its correlation to a poor prognosis and high expressions in many kinds of epithelial tumours. Especially in the colorectal carcinomas, the higher expression of HER2 / neu is recognized in metastatic regions as well as in primary sites. Several CTL epitopes restricted by HLA-A2.1 and -A3 were identified so far, however epitopes restricted by HLA-A24, that is one of the most common allele in Japanese and Caucasians, have not been identified. In this paper, we showed identification of a CTL epitope peptide of HER2 / neu restricted by HLA-A24. HLA-A24 binding peptides selected by an analysis based on HLA-A24 binding motifs were determined for their binding affinities to HLA-A24 molecules. The peptide with a sequence of RWGLLLALL (position 8-16) named HE1 showed the highest affinity. We induced CTLs from CD8(+)cells of HLA-A24 healthy donors by stimulation with HE1-pulsed autologous dendritic cells. The CTLs showed cytotoxic activity against not only the peptide-pulsed target cells but also HLA-A24 colorectal tumour cell lines that endogenously overexpressed HER2 / neu. The antigen-specificity was confirmed by cold target inhibition assay using HE1-pulsed target cells. In summary, HER2 / neu peptide, RWGLLLALL, may contribute to the induction of antitumour immunity with the peptide-based immunotherapy for the colorectal carcinomas.     

Self-assembled micellar formulation of chafuroside A with improved anti-inflammatory effects in experimental asthma/COPD-model rats

Chafuroside A (CFA), a poorly water-soluble flavone C-glycoside, was firstly isolated from oolong tea, and it acts as a potent anti-inflammatory agent. The present study was undertaken to develop a water-soluble formulation of CFA using a self-assembled micellar (SAM) system, with the aim of improved dissolution behavior and potent anti-inflammatory effects. The SAM formulation of CFA (CFA/SAM) was characterized in terms of its morphology, particle size distribution, crystallinity, and dissolution behavior. In dissolution testing, the CFA/SAM exhibited marked improvement in dissolution behavior when compared with crystalline CFA, and then, nano-micellar particles were constituted with a mean diameter of 84 nm. The therapeutic potential of the crystalline CFA and CFA/SAM was assessed using an experimental asthma/chronic obstructive pulmonary disease (COPD)-like model. Orally-administered CFA at 0.5mg/kg or higher could attenuate inflammatory symptoms in a dose-dependent manner, as evidenced by decreases of infiltrated granulocytes, including macrophages and neutrophils, and myeloperoxidase, a specific biomarker for neutrophilia. Biomarker profiling demonstrated that the CFA/SAM at 0.1mg CFA/kg was equipotent to CFA at 1.0mg/kg in ameliorating antigen-induced airway inflammation, suggesting the better pharmacological effect of CFA/SAM due to improved dissolution behavior. From these observations, the SAM formulation might be an efficacious approach for enhancing the therapeutic potential of CFA for treatment of inflammatory diseases.     

Long-term phenotypic, functional and genetic stability of cancer-specific T-cell receptor (TCR) alphabeta genes transduced to CD8+ T cells

In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.     

Intra-corporeal robot-assisted versus open radical cystectomy: a propensity score-matched analysis comparing perioperative and long-term survival outcomes and recurrence patterns

International Journal of Clinical Oncology - To compare perioperative and long-term oncological outcomes and recurrence patterns between robot-assisted radical cystectomy with intra-corporeal...     

Beer congener stimulates gastrointestinal motility via the muscarinic acetylcholine receptors

BACKGROUND: Ethanol and alcoholic beverages are known to affect upper gastrointestinal motility in humans. Beer has been reported to accelerate gastric emptying compared with other beverages that contain the same ethanol concentrations. In this study, we investigated the mechanism that underlies the effects of beer congener on gastrointestinal motility. METHODS: Gastric emptying activity was measured by means of movement of a semisolid test meal (0.05% phenol red/1.5% methylcellulose) in mice. To elucidate the mechanism for the effect of beer congener on gastrointestinal motility, we conducted receptor binding assays and contraction study by using longitudinal muscle from guinea pig ileum. RESULTS: Beer congener (1 g/kg orally) enhanced gastric emptying of a semisolid meal in mice. The receptor binding assay revealed that beer congener bound to dopamine D2 receptor and 5-hydroxytryptamine (5-HT)3 receptor. These IC50 values were more than 5 mg/ml. However, beer congener bound to 5-HT2 receptor, 5-HT4 receptor, and muscarinic M3 receptor with IC50 values of 2, 0.9, and 2 mg/ml, respectively. Beer congener (0.05-2 mg/ml) induced the contraction of longitudinal muscle from guinea pig ileum in a dose-dependent manner. This effect was not affected by either tetrodotoxin (10(-6)M) or ketanserin (10(-7)-10(-5)M), an antagonist for the 5-HT2 receptor. On the other hand, 4-DAMP (10(-8)-10(-5)M), an antagonist for the muscarinic M3 receptor, inhibited the contraction of the longitudinal muscle induced by beer congener (2 mg/ml) dose dependently. CONCLUSIONS: Beer congener stimulates gastrointestinal motility via the muscarinic M3 receptor.