Takuya Fujita

Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A.

We investigated the molecular mechanism involved in the adaptive regulation of the amino acid transport system A, a process in which amino acid starvation induces the transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, amino acid transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an amino acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by amino acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylamino)isobutyric acid.

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens

u2002Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 × 106 OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.

Enhanced Absorption of Insulin and (Asu1,7)Eel‐Calcitonin using Novel Azopolymer‐Coated Pellets for Colon‐Specific Drug Delivery

The objective of this study was to estimate colon-specific delivery of insulin and (Asu(1,7))eel-calcitonin using novel azopolymer-coated pellets. In vitro drug-release experiments from the azopolymer-coated pellets containing fluorescein isothiocyanate dextran (MW 4400; FD-4) were carried out by the Japanese Pharmacopoeia (J.P.) XIII rotating basket method with some slight modifications. Little release of FD-4 from the pellets was observed in phosphate buffered saline. However, the release of FD-4 was markedly increased in the presence of rat cecal contents. The intestinal absorption of insulin and (Asu(1,7))eel-calcitonin after oral administration of the azopolymer-coated pellets containing these peptides with camostat mesilate was evaluated by measuring the hypoglycemic and hypocalcemic effects, respectively. A slight decrease in plasma glucose levels was observed following the oral administration of these pellets containing 12.5 IU of insulin compared with the same dose of insulin solution. Camostat mesilate, a protease inhibitor that is incorporated with insulin in these pellets, further decreased the plasma glucose levels in a dose-dependent manner. Similar results were also obtained with the oral administration of pellets containing (Asu(1,7))eel-calcitonin. These findings suggest that azopolymer-coated pellets may be useful carriers for the colon-specific delivery of peptides including insulin and (Asu(1,7))eel-calcitonin.

Absorption of water-soluble compounds with different molecular weights and [Asu1.7]-eel calcitonin from various mucosal administration sites

The absorption of water soluble compounds with different molecular weights, such as phenol red (MW 354), trypan blue (MW 960), fluorescein isothiocyanate dextrans, (MW 4400 and 9100) was studied in the lung, nasal cavity, buccal cavity, small and large intestine of rats. For all the compounds, maximal absorption was observed when administered to the lung. The rank order of absorption of each compound from various administration sites was lung>small intestine> or =nasal cavity> or =large intestine> or =buccal cavity. In addition, the relationship between logarithm absorption % of the compounds from various administration sites and logarithm molecular weights of these compounds was examined. The absorption of compounds gradually decreased with increasing molecular weight for each site of administration. Moreover, the absorption of [Asu1.7]-eel calcitonin (ECT) from these sites and the effect of 10 mM sodium glycocholate, an absorption enhancer, on its absorption were also investigated in rats. When ECT alone was administered into these sites, the lung had the best absorption site of ECT, followed by the nasal cavity, the large intestine, the small intestine and the buccal cavity. Therefore, the absorption of ECT was also dependent on the administration site, although the rank order of absorption % of ECT was different from the other compounds. Sodium glycocholate (NaGC) remarkably increased ECT absorption from the small intestine, while we found marginal increase in its absorption from the lung even in the presence of NaGC. These findings provided useful fundamental information that might aid in the selection of administration routes for drugs of differing molecular weights including peptide drugs as far as the degree of drug absorption is concerned.

Inhibition of Transforming Growth Factor-β–Mediated Immunosuppression in Tumor-Draining Lymph Nodes Augments Antitumor Responses by Various Immunologic Cell Types

Tumor-draining lymph nodes (DLN) are the most important priming sites for generation of antitumor immune responses. They are also the location where an immunosuppressive cytokine, transforming growth factor-beta (TGF-beta), plays a critical role in suppressing these antitumor immune responses. We focused on TGF-beta-mediated immunosuppression in DLNs and examined whether local inhibition of TGF-beta augmented antitumor immune responses systemically in tumor-bearing mice models. For inhibition of TGF-beta-mediated immunosuppression in DLNs, C57BL/6 mice subcutaneously bearing E.G7 tumors were administered plasmid DNA encoding the extracellular domain of TGF-beta type II receptor fused to the human IgG heavy chain (TGFR DNA) i.m. near the established tumor. In DLNs, inhibition of TGF-beta suppressed the proliferation of regulatory T cells and increased the number of tumor antigen-specific CD4(+) or CD8(+) cells producing IFN-gamma. Enhancement of antitumor immune responses in DLNs were associated with augmented tumor antigen-specific cytotoxic and natural killer activity in spleen as well as elevated levels of tumor-specific antibody in sera. The growth of the established metastatic as well as primary tumors was effectively suppressed via augmented antitumor immune responses. Inhibition of TGF-beta-mediated immunosuppression in DLNs is significantly associated with augmented antitumor responses by various immunocompetent cell types. This animal model provides a novel rationale for molecular cancer therapeutics targeting TGF-beta.

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation.

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.

Infected Bronchogenic Cyst Treated With Drainage Followed by Resection

Bronchogenic cysts originate from anomalous development of the ventral foregut. Although treatment of asymptomatic bronchogenic cysts remains controversial, symptomatic bronchogenic cysts should be surgically removed. We report a case of a 62-year-old man with an infected bronchogenic cyst. We drained the cyst using transesophageal endoscopic ultrasonography to control the inflammation and decrease the size of the cyst; we subsequently resected the cyst. Five months after resection, the patient was well, and computed tomography showed no evidence of cyst recurrence.

Thoracolithiasis diagnosed by thoracoscopy under local anesthesia.

A 68‐year‐old woman was referred to our hospital for a lung nodule identified on chest radiography. Computed tomography (CT) showed a 10‐mm calcified nodule in the left thoracic cavity. On follow‐up CT, the nodule was found in a different location within the left thoracic cavity. Thoracoscopy was performed under local anesthesia, removing a pearl‐like pleural stone. Thoracolithiasis was therefore diagnosed without any complications. To the best of our knowledge, this is the first report on thoracoscopy under local anesthesia for the diagnosis of thoracolithiasis. When thoracolithiasis is suspected, thoracoscopy under local anesthesia is minimally invasive and useful, and could be considered as an option for definite diagnosis.

Catamenial Pneumothorax With Bullae

The physiologic mechanisms and diagnostic approach of catamenial pneumothorax remain controversial. We report 3 patients with catamenial pneumothorax with bullae. Endometrial cells in these patients were located around the bullae, suggesting a possible new mechanism for catamenial pneumothorax in which cyclic endometrial shedding in the lung causes destruction of the lining of alveolar epithelial cells and forms bullae. Because intrathoracic endometriosis is considered an underrecognized cause of secondary spontaneous pneumothorax, we performed careful histologic examination for definitive diagnosis of thoracic endometriosis.

Single-trocar thoracoscopy under local anesthesia for pleural space infection

ObjectiveThe role of single-trocar thoracoscopy for complicated parapneumonic effusion (CPE) and pleural empyema is not established as yet. The aim of this study was to report our experience and analyze the efficacy and safety of debridement by single-trocar thoracoscopy for the patients with CPE and multiloculated empyema.MethodsWe performed a retrospective study reviewing the medical records of the patients treated parapneumonic effusion and multiloculated empyema by single-trocar thoracoscopy under local anesthesia at our department from January 2000 to December 2012.ResultsA total 29 patients with CPE and multiloculated empyema were treated by single-trocar thoracoscopy. As the staging of pleural infection, class 5 and class 7 by Light classification were 21 and 8 patients, respectively. The onset of the symptom was on average 13.9xa0±xa011.7xa0days before the procedure. This procedure was successful in 23 of 29 patients (79.3xa0%) without further operation under general anesthesia. Complication occurred in 1 case of 29 patients (3.4xa0%). Six patients required subsequently the operation under general anesthesia, and one of the 6 patients died to multiple organ failure caused by sepsis. A microbiological diagnosis could be made in fifteen patients (51.7xa0%).ConclusionsDebridement by single-trocar thoracoscopy can be an acceptable approach as the first-line procedure in patients with CPE and empyema. This procedure can provide not only appropriate and expeditious treatment but also information of pleural cavity to decide indication for thoracotomy under general anesthesia.