Yoichi Yamashita

In vivo gene transfer into muscle via electro-sonoporation

Among the nonviral techniques for gene transfer in vivo, electroporation is simple, potent, inexpensive, and safe. To upregulate the expression levels of the transferred gene, we investigated the applicability of in vivo electro-sonoporation, which consists of a combination of electric pulse and ultrasound, for gene transfer using plasmid DNA encoding luciferase and mouse interleukin-12 (mIL-12). The quadriceps muscles of mice were injected with plasmid DNA, then sonoporated for 5 min, and electroporated by a pair of electrode plates at the middle of the duration of sonoporation. Three days later, mice that had undergone electro-sonoporation demonstrated twofold higher luciferase activity and low tissue damage in quadriceps muscle compared to mice having undergone electroporation alone. Serum mIL-12 levels in mice that had undergone electro-sonoporation (peaking at 25.5 ng/ml) were twofold higher after gene transfer than were those in mice having undergone electroporation alone (peaking at 14.3 ng/ml), and maintained high serum level of 13.9 ng/ml at 28 days after gene transfer. The efficacy of gene transfer via electro-sonoporation was superior when the plasmid DNA solution was 0.85% NaCl compared to albumin microbubble echo-contrast material. These results demonstrated that gene transfer into muscle via electro-sonoporation could provide a new potent nonviral technique for gene transfer in vivo.

Electroporation-Mediated Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/Apo2L Gene Therapy for Hepatocellular Carcinoma

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L) has been identified as important in promoting programmed cell death in breast and colon cancer xenografts. More importantly, normal liver tissue appears not to be susceptible to the cytotoxic effects of TRAIL/Apo2L, although activation of the related Fas ligand receptor system is known to promote massive liver apoptosis terminating in fulminant hepatitis. In the present study, we investigated the therapeutic potential of TRAIL/Apo2L gene therapy in hepatocellular carcinoma (HCC) and evaluated its side effects in an immune-competent mouse model. Intratumoral administration of the TRAIL/Apo2L vector by electroporation elevated serum TRAIL/Apo2L through at least day 28 after gene therapy and significantly inhibited the growth not only of the HCC directly administered TRAIL/Apo2L vector, but also of distant subcutaneous HCC. In addition, intratumoral administration of the TRAIL/Apo2L vector inhibited spontaneous lung metastasis. Serum alanine aminotransferase was mildly elevated by TRAIL/Apo2L gene therapy, but without showing such histological signs as TUNEL staining. These results demonstrate that TRAIL/Apo2L gene therapy for HCC by electroporation in vivo is efficient without significant side effects, and is thus promising for use in future clinical trials.

The triangulating stapling technique for cervical esophagogastric anastomosis after esophagectomy.

PurposeTo evaluate the safety and feasibility of the triangulating stapling technique (TST) for cervical esophagogastric anastomosis after esophagectomy (CEGA).MethodsThe subjects were 123 patients who underwent transthoracic esophagectomy with three-field lymph node dissection and reconstruction with a 3.5-cm wide gastric tube, for thoracic esophageal cancer. We performed the TST for CEGA in 33 patients operated on after December, 2006 (TST group) and hand-sewn anastomosis in 90 patients operated on between 2002 and 2006 (HSA group).ResultsIn the TST group, CEGA was performed in an end-to-end fashion using three linear staplers. The first anastomosis was applied to the posterior walls of the remnant esophagus and gastric tube in an inverted fashion. The second and the third anastomoses were done in an everted fashion to make the anterior wall. The end-to-end HSA was performed with interrupted sutures using 4-0 absorbable material. Anastomotic leakage occurred in only 1 (3.0%) of the 33 TST patients, but in 13 (14.4%) of the 90 HSA patients (P = 0.07). The frequency of anastomotic stenosis was 9.1% and 25.6% in the TST and HSA groups, respectively (P < 0.05).ConclusionsCervical esophagogastric anastomosis using TST may reduce the frequency of anastomotic leakage and stenosis. This technique is a safe and reliable alternative for CEGA after esophagectomy.

Galatea: Open-Source Software for Developing Anthropomorphic Spoken Dialog Agents

Galatea is a software toolkit to develop a human-like spoken dialog agent. In order to easily integrate the modules of different characteristics including speech recognizer, speech synthesizer, facial animation synthesizer, and dialog controller, each module is modeled as a virtual machine having a simple common interface and connected to each other through a broker (communication manager). Galatea employs model-based speech and facial animation synthesizers whose model parameters are adapted easily to those for an existing person if his or her training data is given. The software toolkit that runs on both UNIX/Linux and Windows operating systems will be publicly available in the middle of 2003 [7, 6].

Plasma D-dimer level as a mortality predictor in patients with advanced or recurrent colorectal cancer.

Objective: Plasma D-dimer levels are elevated in patients with a variety of solid tumors. Recently, it has been reported that the level before curative surgery is a prognostic factor for colorectal cancer (CRC). We investigated whether the plasma D-dimer level before systemic chemotherapies is a predictor for advanced or recurrent unresectable CRC. Methods: This study included 42 patients treated with systemic chemotherapies for advanced or recurrent unresectable CRC. Variables including clinicopathological factors, plasma D-dimer levels and the modified Glasgow Prognostic Factor Score (mGPS) were evaluated. Results: The plasma D-dimer level was closely related to the mGPS. Survival was shorter for patients with plasma D-dimer levels >5 µg/ml than for those with lower levels. Compared with an mGPS of 0 or 1, an mGPS of 2 was predictive of poor prognosis (p < 0.0001). Old age, advanced stage, plasma D-dimer level and mGPS were significantly associated with mortality, but plasma D-dimer level was the only independent risk factor in multivariate analysis, and was significant related to the clinical response to chemotherapy (p < 0.05). Conclusions: Survival was significantly shorter in patients with elevated plasma D-dimer levels having advanced or recurrent CRC. The plasma D-dimer level before systemic chemotherapies was an independent mortality predictor.

Intratumoral Neoadjuvant Immunotherapy Using IL-12 and Dendritic Cells Is an Effective Strategy To Control Recurrence of Murine Hepatocellular Carcinoma in Immunosuppressed Mice

Liver transplantation is accepted as an effective therapy for hepatocellular carcinoma (HCC). However, recurrence is one of the most fatal complications. The aim of this study is to evaluate the efficacy of intratumoral immunotherapy using IL-12 gene therapy and dendritic cell injection for the purpose of effective treatment for HCC under conditions of immunosuppression. We found that the combined immunotherapy significantly induced sustained and high amounts of intratumoral IL-12 and IFN-γ proteins and that it induced high HCC-specific CTL activity under immunosuppression as compared with each monotherapy or control. The combined immunotherapy also exerted effective antitumor effects on the immunosuppressed host, resulting in significant suppression of growth of the s.c. established tumor and complete suppression of lung and liver metastasis, without rejection of a fully allogeneic skin graft. These antitumor effects were dependent on both T cells and NK cells. Noteworthily, the combined intratumoral immunotherapy and tumor resection (that is, neoadjuvant immunotherapy) resulted in achievement of tumor-free and long-term survival of the some immunosuppressed mice, even when the mice were challenged with i.v. injection of HCC at the time of tumor resection. In contrast, all of the mice treated with neoadjuvant immunotherapy using monotherapy or control therapy suffered from lung and liver metastasis. These results suggest that intratumoral neoadjuvant immunotherapy using IL-12 gene therapy and dendritic cell therapy is a potent effective strategy to control recurrence of HCC in patients after liver transplantation for HCC and may be applicable to general cancer treatment.

Robust Talker Direction Estimation Based on Weighted CSP Analysis and Maximum Likelihood Estimation

This paper describes a new talker direction estimation method for front-end processing to capture distant-talking speech by using a microphone array. The proposed method consists of two algorithms: One is a TDOA (Time Delay Of Arrival) estimation algorithm based on a weighted CSP (Cross-power Spectrum Phase) analysis with an average speech spectrum and CSP coefficient subtraction. The other is a talker direction estimation algorithm based on ML (Maximum Likelihood) estimation in a time sequence of the estimated TDOAs. To evaluate the effectiveness of the proposed method, talker direction estimation experiments were carried out in an actual office room. The results confirmed that the talker direction estimation performance of the proposed method is superior to that of the conventional methods in both diffused- and directional-noise environments.

Efficacy of a larger version of the hybrid artificial liver support system using a polyurethane foam/spheroid packed-bed module in a warm ischemic liver failure pig model for preclinical experiments.

We have reported the usefulness of a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver support system (PUF-HALSS). The aim of this study was to evaluate in detail the efficacy in serum parameters regarding the liver function of a larger version of the PUF-HALSS containing 2 × 1010 porcine hepatocytes for clinical use in warm ischemic liver failure pigs. Warm ischemic liver failure pigs weighing 25 kg were divided into two groups: (1) a control group (n = 3), in which each pig was attached to a PUF-HALSS without hepatocytes, and (2) a HALSS group (n = 3), in which each pig was attached to a PUF-HALSS. In the HALSS group, the increase of blood ammonia was completely suppressed and blood lactate levels were significantly suppressed. The Fishers ratio was better maintained, and the increase of total bile acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid was significantly suppressed in the HALSS group. Serum creatinine levels were significantly lower, and blood glucose levels were significantly higher in the HALSS group. Serum levels of tumor necrosis factor- a were not elevated in either group. In conclusion, the larger version of the PUF-HALSS demonstrated many advantages as a liver support system in warm ischemic liver failure pigs.

Construction of a Test Collection for Spoken Document Retrieval from Lecture Audio Data

The lecture is one of the most valuable genres of audiovisual data. Though spoken document processing is a promising technology for utilizing the lecture in various ways, it is difficult to evaluate because the evaluation require a subjective judgment and/or the verification of large quantities of evaluation data. In this paper, a test collection for the evaluation of spoken lecture retrieval is reported. The test collection consists of the target spoken documents of about 2, 700 lectures (604 hours) taken from the Corpus of Spontaneous Japanese (CSJ), 39 retrieval queries, the relevant passages in the target documents for each query, and the automatic transcription of the target speech data. This paper also reports the retrieval performance targeting the constructed test collection by applying a standard spoken document retrieval (SDR) method, which serves as a baseline for the forthcoming SDR studies using the test collection.

Estimation of speech recognition performance in noisy and reverberant environments using PESQ score and acoustic parameters

The automatic speech recognition (ASR) performance is degraded in noisy and reverberant environments. Although various techniques against degradation of the ASR performance have been proposed, it is difficult to properly apply them in evaluation environments with unknown noisy and reverberant conditions. It is possible to properly apply these techniques for improving the ASR performance if we can estimate the relationship between the ASR performance and degradation factors including both noise and reverberation. In this study, we here propose new noisy and reverberant criteria which are referred as “Noisy and Reverberant Speech Recognition with the PESQ and the Dn (NRSR-PDn)”. We first designed the “NRSR-PDn” using the relationships among the D value, the PESQ score, and the ASR performance. We then estimated the ASR performance with the designed criteria “NRSR-PDn” in evaluation experiments. Experimental evaluations demonstrated that our proposed criteria make the well suited for robustly estimating the ASR performance in noisy and reverberant environments.