Wholly vascularized millimeter-sized engineered tissues by cell-sized microscaffolds.

The in vitro fabrication of wholly vascularized millimeter-sized engineered tissues is still a key challenge in the tissue engineering field. Recently we reported a unique approach 'sedimentary culture' using a collagen microfiber (CMF) to fabricate large-scale engineered tissues. The millimeter-sized tissues with high extracellular matrix (ECM) density were easily obtained by centrifugation of cells and CMFs and subsequent cultivation because the CMFs acted as a micrometer-sized scaffold. However, cell distribution in the obtained tissues was not homogeneous because of the different sedimentation velocity of the cells and CMFs because of their size difference. Here we report the fabrication of wholly vascularized millimeter-sized engineered tissues using cell-sized CMFs. To avoid dissolving, vacuum drying was performed at 200 °C for 24 h for thermal crosslinking of primary amine groups of type I collagen. The 200- and 20-µm-sized CMFs (CMF-200 and CMF-20) were obtained by homogenization and subsequent sonication of the crosslinked collagen. Interestingly, the CMF-20 indicated a similar sedimentation velocity with cells because of their same size range, thus uniform millimeter-sized tissue with homogeneous cell distribution was fabricated by the sedimentary culture method. To form a whole blood capillary structure in the tissues, fibronectin (FN) was adsorbed on the surface of CMF-20 to stimulate endothelial cell migration. The distribution of the blood capillary network in 1.6-mm-sized tissues was markedly improved by FN-adsorbed CMF-20 (FN-CMF-20). Sedimentary culture using FN-CMF-20 will create new opportunities in tissue engineering for the in vitro fabrication of wholly vascularized millimeter-sized engineered tissues.

In-plane stress development in mesoporous thin films.

Ordered mesoporous thin films of TiO2 and CexZr1-xO2 (x = 0, 0.5, 1) were prepared via an evaporation-induced self-assembly (EISA) process and subsequently investigated in terms of the developing intrinsic and residual in-plane stress. These mechanical properties were determined by the curvature method, which is based on the determination of the deflection of light due to concave or convex bending of the films on a substrate. The films were investigated with regard to the intrinsic stress during heat treatment up to 500 °C and to the residual stress at room temperature for several annealing temperatures. Following this strategy, the influence of the decomposition of a block copolymer template on the intrinsic stress as well as the pore collapsing on the residual stress was analyzed. Nanoporous TiO2 thin films were prepared using two different block copolymers (PIB50-b-PEO45 and Pluronic® F127). A comparison between the templated and non-templated TiO2 films showed the lowest intrinsic and residual stress for the ordered mesoporous material prepared with PIB50-b-PEO45 indicating that the distributed polymer and the corresponding mesopores act as relaxing agents for the system. This was verified by mesoporous CexZr1-xO2 (x = 0, 0.5, 1) thin films showing a comparable behavior in terms of the experienced intrinsic stress. This work reveals an increase in the residual in-plane stress during pore collapse, which lays the foundation for further understanding of the stress-related mechanical properties of mesoporous thin films.

Mogamulizumab-induced photosensitivity in patients with mycosis fungoides and other T-cell neoplasms.

BACKGROUND: Mogamulizumab (Mog) is a defucosylated, therapeutic monoclonal antibody, targeting CCR4 and was first approved in Japan for the treatment of adult T-cell leukaemia/lymphoma (ATLL), followed by cutaneous T-cell lymphoma and peripheral T-cell lymphoma. OBJECTIVE: To retrospectively investigate development of photosensitivity in patients with mycosis fungoides and other T-cell neoplasms after treatment with Mog. METHODS: We treated seven cutaneous lymphoma patients with Mog. Upon combination treatment with narrow-band UVB, we noticed that four patients developed photosensitivity dermatitis following Mog therapy, including two cases of mycosis fungoides, one case of adult T-cell leukaemia/lymphoma and one case of EB virus-associated T-cell lymphoproliferative disorder. Phototest was performed with UVA and UVB, and immunohistochemical staining for CD4, CD8 and Foxp3 was conducted in both photosensitivity and lymphoma lesions. RESULTS: Phototest revealed that the action spectrum of the photosensitivity was UVB in three cases and both UVB and UVA in one case. Histopathologically, the photosensitive lesions were characterized by a lichenoid tissue reaction with a CD8+ T cell-dominant infiltrate, sharing the feature with chronic actinic dermatitis, an autoreactive photodermatosis with a cytotoxic T-cell response. Foxp3+ regulatory T cells (Tregs) were decreased in the photosensitivity lesions compared with the lymphoma lesions. CONCLUSION: Increased incidence of photosensitivity reaction was observed during Mog treatment. Decreased number of Tregs in the lesional skin suggests that this reaction is possibly induced by autoreactive cytotoxic T cells.

Clinical Utility of Diagnostic Laboratory Tests in Dogs with Acute Pancreatitis: A Retrospective Investigation in a Primary Care Hospital.

BACKGROUND: Acute pancreatitis (AP) occurs frequently in dogs, but most previous studies examining the diagnosis of AP have used data from secondary care hospitals. HYPOTHESIS/OBJECTIVES: The aim of this study was to investigate the clinical utility of diagnostic laboratory tests in dogs with AP in a primary care hospital. ANIMALS: Sixty-four dogs with clinical signs suggestive of AP diagnosed with nonpancreatic disease (NP) or AP. METHODS: Medical records were retrospectively reviewed, including diagnostic laboratory tests considered potentially useful in the diagnosis of AP. The diagnostic accuracy of amylase and FUJI DRI-CHEM lipase (FDC lip) were investigated using receiver operating characteristics (ROC). In addition, we verified whether diagnostic laboratory tests were useful for evaluating duration of hospitalization and as biomarkers for monitoring recovery. RESULTS: Activities of amylase and FDC lip were significantly higher in the AP group than in the NP group (P = .001, P < .001, respectively). The sensitivity of FDP lip activity for diagnosing AP was 100% (95% confidence interval [CI], 87.7-100%); the specificity was 89.5% (95% CI, 66.9-98.7%). Area under the ROC curve for FDC lip activity was 0.98 (95% CI, 0.93-1). High alanine aminotransferase (ALT) activity was associated with extended duration of hospitalization (P = .04). A significant difference in C-reactive protein (CRP) concentration before and 5 days after treatment was found (P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of FDC lip activity appears useful for diagnosing AP. High ALT activity might be associated with prolonged duration of hospitalization, and CRP might be useful as a biomarker for monitoring recovery from AP.

Two-stage laparoscopic treatment for strangulated inguinal, femoral and obturator hernias: totally extraperitoneal repair followed by intestinal resection assisted by intraperitoneal laparoscopic exploration.

PURPOSE: Total extraperitoneal preperitoneal (TEP) repair is widely used for inguinal, femoral, or obturator hernia treatment. However, mesh repair is not often used for strangulated hernia treatment if intestinal resection is required because of the risk of postoperative mesh infection. Complete mesh repair is required for hernia treatment to prevent postoperative recurrence, particularly in patients with femoral or obturator hernia. CASES: We treated four patients with inguinocrural and obturator hernias (a 72-year-old male with a right indirect inguinal hernia; an 83-year-old female with a right obturator hernia; and 86- and 82-year-old females with femoral hernias) via a two-stage laparoscopic surgery. All patients were diagnosed with intestinal obstruction due to strangulated hernia. First, the incarcerated small intestine was released and then laparoscopically resected. Further, 8-24 days after the first surgery, bilateral TEP repairs were performed in all patients; the postoperative course was uneventful in all patients, and they were discharged 5-10 days after TEP repair. At present, no hernia recurrence has been reported in any patient. CONCLUSION: The two-stage laparoscopic treatment is safe for treatment of strangulated inguinal, femoral, and obturator hernias, and complete mesh repair via the TEP method can be performed in elderly patients to minimize the occurrence of mesh infection.

Standard of practice on laparoscopic gastric cancer surgery in Korea and Japan: experts' survey.

INTRODUCTION: As the laparoscopic approach has become a popular gastric cancer treatment in Korea and Japan, the need for sharing current practices of surgeons who are experienced in laparoscopic gastric cancer surgery has increased. METHODS: We sent a questionnaire on laparoscopic instruments, image documentation, preoperative evaluation, surgical indication, operative methods, and postoperative management to laparoscopic experts in Korea and Japan, and 24 (14 from Korea and 10 from Japan) responded. RESULTS: Endoscopic ultrasound and preoperative endoscopy-guided clipping are routinely employed, respectively, by 14 (58%) and 20 (83%) of the surgeons. Surgeons perform laparoscopy-assisted distal gastrectomy (LADG) based on varying indications. Five surgeons (21%) performed LADG only for cases of stage T1 cancer, 15 (63%) performed LADG on patients with less than T2 lesions, and 4 (17%) performed LADG on patients with less than T3 lesions. With regard to postoperative anastomosis, 18 surgeons (75%) preferred extracorporeal anastomosis and 6 (25%) preferred intracorporeal anastomosis. The mean postoperative hospital stay was 6.5 days in Korea and 10.1 days in Japan (P < 0.001). CONCLUSION: This survey can help to inform the current practice of laparoscopic gastric cancer surgery in Korea and Japan, where laparoscopic surgery is frequently performed.

The maneuver to release an incarcerated obturator hernia.

An obturator hernia occurs through the pelvic obturator canal, a rigid ring made up of the underside of the superior pubic ramus and the obturator fascia. Obturator hernias have been associated with a high mortality due to the difficulty in diagnosis and the population in which it occurs. We examined four patients diagnosed with incarcerated obturator hernia, and showed that the strangulated intestine was not necrotic. We flexed the diseased leg calmly and repeatedly with slight rotation toward the outside and slight adduction toward the inside at supine position. The pain vanished suddenly during this maneuver. After this maneuver, the patients were able to undergo elective surgery after a certain interval. We discuss the possible use of this maneuver to release an incarcerated obturator hernia.

Remote access laparoscopic (REAL) surgery: a novel approach for single-port surgery without a visible scar.

INTRODUCTION: Single-port laparoscopic surgery usually uses the transumbilical approach. However, the transumbilical incision may be associated with a high frequency of wound-related complications, including trocar site hernia and infection. Therefore, we developed remote access laparoscopic (REAL) surgery, a new access technique for single-port surgery within the pubic hairline. This study reports on a technique developed at our institution and describes our preliminary results. MATERIALS AND SURGICAL TECHNIQUE: Ten patients with cholecystolithiasis underwent REAL surgery. A single 2.5-cm transverse incision was made medially within the pubic hairline area, and a SILS Port was inserted. A 5-mm flexible endoscope and an articulating grasper or a long laparoscopic grasper were used. Dissection of the gallbladder was achieved with laparoscopic ultrasonic shears just as in the conventional laparoscopic cholecystectomy. All procedures were successfully performed without conversion to conventional laparoscopic technique. There were no postoperative complications, and outpatient evaluation showed a clean scar within the pubic hair area in all patients. DISCUSSION: Our early experience shows that REAL surgery is feasible and safe. This novel access technique can preserve the native umbilicus and provides an invisible scar that is concealed within the pubic hair.

Feasibility of fibrin glue versus endoclips to close the transgastric peritoneal access site in NOTES in a survival porcine study.

INTRODUCTION: Safe peritoneal access and gastric closure are the most important concerns in clinical applications of NOTES. Our past study demonstrated usefulness of the submucosal tunnel technique for safe peritoneal access and closure with endoclips. However, such closure is sometimes difficult and time-consuming. This study investigated the feasibility of fibrin glue for submucosal tunnel closure in a NOTES porcine model. METHODS: In 10 female pigs each weighing 40 kg, transgastric peritoneoscopy was performed through a 60 mm-long submucosal tunnel created using the endoscopic submucosal dissection technique. After transgastric peritoneoscopy for 30 min, the submucosal tunnel was closed with endoclips in five pigs and fibrin glue in five pigs. After a 7 d follow-up period, the pigs were euthanized for post-mortem examination. Outcome measures included (a) technical feasibility of closure with endoclips versus fibrin glue, (b) clinical monitoring for 7 d, (c) follow-up necropsy at 7 d, and (d) histopathologic examination of the peritoneal access site. RESULTS: Transgastric peritoneoscopy with submucosal tunnel technique was successful in all pigs. Mean time required to close the mucosal incision site with fibrin glue was 1.6 ± 0.5 versus 19 ± 18.7 min with endoclips. All pigs survived well without complications. Necropsy revealed no peritonitis. There were no differences in transgastric peritoneal access sites between endoclips and fibrin glue. Histopathologic examination of the submucosal tunnel demonstrated wound healing with transmural fibrosis. No adverse effects from fibrin glue were noted. CONCLUSION: Compared with endoclips, the application of fibrin glue is easy and simple in the closure of transgastric peritoneal access in NOTES.

Results of a national survey on laparoscopic bariatric surgery in Japan, 2000-2009.

INTRODUCTION: In Japan, laparoscopic bariatric surgery has not been popular until recently because morbidly obese patients were infrequently encountered previous and Japanese health insurance does not cover this type of surgery. In 2010, the Japan Research Society for Endoscopic and Laparoscopic Treatments of Obesity undertook the first nationwide survey on laparoscopic bariatric surgery to evaluate its current status and outcomes. METHODS: A mail survey was sent to the society's 64 member institutions, which included almost all institutions in Japan actively performing laparoscopic gastrointestinal surgery. RESULTS: From 2000 to 2009, 340 laparoscopic bariatric procedures, in total, were performed in nine of the 64 institutions (14%). The most popular procedure was laparoscopic Roux-en-Y gastric bypass (LRYGB, n=147), second was laparoscopic sleeve gastrectomy (LSG, n=102), and third was laparoscopic adjustable gastric banding (LAGB, n=55). However, the number of LRYGB procedures has decreased while the number of LSG procedures has rapidly increased. Total morbidity rates were 12.2% for LRYGB, 10.9% for LAGB, and 7.8% for LSG. Percent excess weight loss was 78% at 5 years after LRYGB, 52% at 4 years after LAGB, and 68% at 2 years after LSG. Although the bariatric procedures frequently resolved or improved obesity-related comorbidities, LRYGB appears to be superior to LAGB, as was previously reported. CONCLUSION: The first nationwide survey of laparoscopic bariatric surgery in Japan clearly showed the current status and outcomes of this group of procedures. The Japanese results appear to be comparable to similarly undertaken surveys in Europe and the USA.

Contralateral occurrence after laparoscopic total extraperitoneal hernia repair for unilateral inguinal hernia.

PURPOSE: Although laparoscopic total extraperitoneal repair (TEP) has been reported to have a low recurrence rate and relatively little postoperative pain, there have been few studies reported regarding contralateral occurrence after TEP. Although a high incidence of occult contralateral hernias has been reported in the literature, it is unknown whether occult hernias have any significance in clinical settings. The aim of this study was to evaluate the incidence of contralateral occurrence after TEP for unilateral inguinal hernia. METHODS: We retrospectively reviewed the medical charts of 157 TEPs between April 2003 and May 2009. No patients had undergone contralateral exploration during TEP for unilateral inguinal hernias. RESULTS: Five (3.2%) of 157 unilateral TEPs developed a hernia on the contralateral side. In three patients, the initial hernia was on the right side, and in two it was on the left side. In four patients the initial hernia was indirect, and in one it was direct. The mean duration to contralateral occurrence was 12.2 months. Three patients had contralateral occurrence within 6 months after the primary TEP, while in two over a year passed before contralateral occurrence. All five patients had undergone TEP for contralateral occurrence. The mean operation time was 87.2 min, and there was little intraoperative blood loss. There were no complications during and after the second TEP. CONCLUSIONS: The incidence of contralateral occurrence after TEP was found to be low. TEP is a valuable procedure with a low contralateral occurrence rate, and repeated TEP for contralateral occurrence can be performed easily.

Submucosal tunneling using endoscopic submucosal dissection for peritoneal access and closure in natural orifice transluminal endoscopic surgery: a porcine survival study.

BACKGROUND AND STUDY AIMS: Safe peritoneal access and gastric closure are the most important concerns in the clinical application of natural orifice transluminal endoscopic surgery (NOTES). We aimed to clarify the feasibility of a submucosal tunnel technique using endoscopic submucosal dissection (ESD) for transgastric peritoneal access and subsequent closure for NOTES. METHODS: Seven female pigs, each weighing about 40 kg were included in the study. The following procedures were performed: (i) after injection of normal saline into the submucosa, the mucosa was cut with a flex knife; (ii) the submucosal layer was dissected using an insulation-tipped electrosurgical knife to make a narrow longitudinal 50-mm submucosal tunnel; (iii) a small incision was made at the end of the tunnel and enlarged with a dilation balloon. After transgastric peritoneoscopy, the mucosal incision site was closed with clips. The following outcome measures were used: (a) evaluation of the technical feasibility of making a submucosal tunnel; (b) clinical monitoring for 7 days; (c) follow-up endoscopy and necropsy; and (d) peritoneal fluid culture. RESULTS: Natural orifice transluminal endoscopic peritoneoscopy with a submucosal tunnel was successfully carried out in all pigs. The pigs recovered well, without signs of peritonitis. Follow-up endoscopy showed healing of mucosal incision sites without open defects. Necropsy revealed no findings of peritonitis, confirming completeness of gastric closure; there was a thin scar in one pig and adhesion of the omentum in six pigs. Peritoneal fluid culture demonstrated no bacterial growth. CONCLUSIONS: The submucosal tunnel technique is feasible and effective for transgastric peritoneal access and closure.

Predictors and prognostic significance of operative complications in patients with hepatocellular carcinoma who underwent hepatic resection.

AIMS: The morbidity rate of hepatic resection for hepatocellular carcinoma (HCC) remains high. To clarify predictors and the prognostic significance of operative complications in patients with HCC, we conducted a comparative retrospective analysis of 291 patients with HCC who underwent hepatic resection. METHODS: Operative complications included hyperbilirubinemia, ascites, hemorrhage, respiratory and cardiovascular diseases, bile leakage and abscess formation, renal failure, wound infection, and pleural effusion. Predictors of operative complications and their prognostic value for long-term survival were studied by univariate and multivariate analyses. RESULTS: Mortality and morbidity rates were 7.2% and 42.6%. The main operative complications were ascites (n = 30), intraabdominal abscess (n = 25), hyperbilirubinemia (n = 19), wound infection (n = 16), pleural effusion (n = 10) and intraabdominal hemorrhage (n=9). By a multivariate logistic regression model, Child-Pugh class B and increased operative blood loss (> or = 1200ml) were independent predictors of postoperative complications. Among 243 patients without operative death, the 5-year overall survival rate was significantly lower in patients with operative complications (34.3%) than in those without these complications (48.7%). By the multivariate Cox proportional hazards model, the presence of operative complications was an independent predictor of poor overall survival as well as presence of portal invasion. CONCLUSIONS: Child-Pugh class B and operative blood loss > or = 1200ml were independent predictors of complications after hepatic resection for HCC. Long-term survival is poorer in patients with postoperative complications. Decreasing operative blood loss may result in fewer postoperative complications and better long-term survival of HCC patients.

Rapid alphabeta TCR-mediated responses in gammadelta T cells transduced with cancer-specific TCR genes.

Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.

Ecotoxicity of the degradation products of triphenylborane pyridine (TPBP) antifouling agent.

Triphenylborane pyridine (TPBP) is an alternative to organotin antifouling compounds. This work aimed to identify the unknown Peak #1, and to evaluate the ecotoxicity of TPBP and its degradation products. Peak #1 was produced from TPBP dissolved in acetonitrile under UV-A photolysis using a high-pressure mercury lamp. The Peak #1 fraction was purified using two-step column chromatography from a TPBP-acetonitrile solution. The major compound of the fraction was identified as being biphenyl from the 1H NMR and 13C NMR spectra. The ecotoxicity of four degradation products (diphenylborane hydroxide, phenylborane dihydroxide, phenol, and biphenyl) and TPBP towards two marine planktons were assessed. The 48 h LC(50) values of the crustacean, Artemia salina, were 0.13 mg L(-1) for TPBP, 14 mg L(-1) for biphenyl, 17 mg L(-1) for phenol, and > 50 mg L(-1) for the other degradation products. The 72 h EC(50) values of the diatom, Skeletonema costatum, were 0.0022 mg L(-1) for TPBP, 1.2 mg L(-1) for biphenyl, and > 2 mg L(-1) for the other degradation products. Thus, the ecotoxicity of biphenyl and the other degradation products were not high compared to the parent compound, TPBP.

Carcinoid tumors of the pancreas: dynamic CT and MRI features with pathological correlation.

BACKGROUND: Carcinoid tumor of the pancreas is rare, and there are few reports that described its CT or magnetic resonance imaging (MRI) findings. We describe the characteristic CT and MRI findings in four cases of carcinoid tumor of the pancreas. METHODS: Radiologic and pathologic features were analyzed in four patients. All patients underwent triple-phase dynamic CT and MRI. RESULTS: The tumor size in the four cases ranged 15-20 mm and intratumoral calcification was detected in one case. On triple-phase dynamic CT, the peak enhancement of the tumors was seen at the arterial dominant phase in three cases; the remaining one was at the portal venous phase with prolonged contrast-enhancement effect. The tumors showed low to high signal intensity on T2-weighted images. Dilatation of the main pancreatic ducts (MPDs) distal to the tumors was seen in three cases, in which tumor invasion into the MPDs was pathologically confirmed. Furthermore, the tumors having mild to severe fibrosis pathologically invaded into the peripancreatic lymphatics or nerves. CONCLUSION: It would be characteristic of carcinoid tumor of the pancreas to be well enhanced at the arterial dominant phase on dynamic CT, and to highly invade into the MPDs and the peripancreatic lymphatics or nerves.

Prognosis of patients with intrahepatic recurrence after hepatic resection for hepatocellular carcinoma: a retrospective study.

AIMS: Hepatic resection is the most effective therapy for hepatocellular carcinoma (HCC); however, intrahepatic recurrence is common. Predictors of survival after intrahepatic recurrence have not been fully investigated. To clarify the prognosis and choice of treatment of intrahepatic recurrence after hepatic resection, we conducted a comparative retrospective analysis of 147 patients with HCC who underwent hepatic resection. METHODS: We retrospectively examined the relations between clinicopathologic factors, including the number of recurrent intrahepatic tumors and long-term prognosis after recurrence in 147 HCC patients who underwent resection. We also examined long-term survival after recurrence based on treatment types and recurrence pattern. RESULTS: Patients with multiple tumors (n=83) showed less tumor differentiation, more frequent portal invasion, a higher alpha-fetoprotein level, and larger tumors than did patients with solitary tumor (n=64). In the solitary tumor group, local ablation therapy and repeat hepatic resection were performed in 25 and 10, respectively. In the multiple tumor group, 59 were treated by transarterial chemoembolization. Multivariate analysis showed intraoperative blood transfusion and multiple tumors to be independent risk factors for poor cancer-related survival after recurrence. By subset analysis based on treatment types and recurrence pattern, survival after recurrence was significantly better in patients treated by local ablation therapy than those treated by other therapies in both solitary and multiple tumor groups. CONCLUSIONS: For patients with solitary recurrence, a good prognosis is predicted. Local ablation therapy is a best candidate for treatment of solitary and multiple intrahepatic recurrences after hepatic resection.

Evidence for interspecific hybridization between native white-spotted charr Salvelinus leucomaenis and non-native brown trout Salmo trutta on Hokkaido Island, Japan.

Hybrids between native white-spotted charr Salvelinus leucomaenis and non-native brown trout Salmo trutta were identified in streams of Hokkaido, Japan, using both appearance and genetic characters. The DNA analyses indicated that the specimens were hybrids between female S. leucomaenis and male S. trutta. Occurrence of such hybrids implies increased mating opportunities between these species in wild streams.

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.