A Diagnostic Loop-Mediated Isothermal Amplification Method to Distinguish Helicoverpa armigera (Lepidoptera: Noctuidae) From Other Related Species in the New World.

Helicoverpa armigera (Hübner) is a notorious agricultural pest native to the Old World. Recently, its invasion into South and Central America has become a serious problem in the New World. The rapid detection of invasive pests is essential to eradicate them and prevent establishment. However, an extremely similar species, H. zea (Boddie) distributed in the New World makes identification difficult. Helicoverpa armigera and H. zea have only minor differences in male genitalia to separate them morphologically. Both species are attracted to the same pheromone lure, and it takes considerable time and effort to identify them from bulk samples obtained during trap monitoring. Although several molecular approaches based on PCR have been reported, these methods require expensive equipment and are unsuitable for onsite diagnostics. Here, we developed a rapid and convenient diagnostic method based on the loop-mediated isothermal amplification to distinguish H. armigera from related species: H. zea, H. assulta (Guenée), H. punctigera (Wallengren), and Chloridea virescens (Fabricius). The diagnostic method makes it possible to detect H. armigera within 90 min only using simple equipment. The method also worked with mixed DNA templates containing excess DNA from H. zea at the ratio of 1:999 (H. armigera:H. zea). This method can be an effective tool for onsite diagnostics during monitoring surveys for invasive H. armigera.

Coil Embolization for Asymptomatic Low-grade Vertebral Artery Injury which Worsened after Cervical Decompression Surgery: A Case Report.

Introduction: Blunt cervical injuries rarely cause vertebral artery injuries (VAIs), such as vertebral artery (VA) dissection or occlusion. To prevent subsequent embolic infarction, embolization of the injured VA is needed before surgical fixation of the cervical spine. However, evidence on endovascular treatment for asymptomatic low-grade VAIs with blunt traumatic cervical injury is insufficient. Case Report: In the present case, a 79-year-old Japanese man presented tetraparesis after falling while walking. Digital subtraction angiography showed no intimal flap and only slight stenosis of the right VA. Embolization was not performed before spinal decompression surgery for this low-grade injury. However, on the 3rd day after surgery, diffuse-weighted imaging showed dot-like high signal intensity in the right thalamus and right posterior lobe, and magnetic resonance angiography (MRA) showed near occlusion of the right VA. 8 days after surgery, MRA showed recanalization of the right VA flow. We performed VA embolization to prevent emboli scattering to the distal region during recanalization of the intracranial blood flow. Conclusion: According to the relevant literature, prophylactic embolization may be indicated to prevent the embolic infarction not only in cases of VA occlusion requiring fixation of the cervical spine but also in cases of low-grade VAIs in which fixation is not required. Embolization of the VA before spinal surgery might be an aggressive treatment strategy that avoids serious embolic infarction disorder after VAIs.

[Trans-thoracic esophagectomy with 3-field lymph nodes dissection].

Although open-chest surgery is the mainstay treatment for esophageal cancer, the understanding of the context of the surgery differs in Japan and the rest of the world. Three-field lymph node dissection has been unique to Japan, although some reports on its benefits are emerging elsewhere. In addition to three-field lymph node dissection, various efforts are made during surgical procedures to reduce complications at high-volume Japanese healthcare institutions.

CD133 mRNA-transfected dendritic cells induce coordinated cytotoxic and helper T cell responses against breast cancer stem cells.

Breast cancer, a leading cause of death yearly, has been shown to be initiated and propagated by cancer stem cells. CD133, a cell surface antigen, has been shown to be present on cancer stem cells of many solid tumors, including breast cancer. A limitation to targeting CD133 is major histocompatibility complex (MHC)-restricted presentation of epitopes, leading to activation of only one arm of the immune system: either CD4+ helper T cells or CD8+ cytotoxic T cells. Thus, we hypothesized that by creating an MHC-independent vaccination, we would give rise to a sustained immune response against CD133 in triple-negative breast cancer (TNBCs). We transfected CD133 mRNA into dendritic cells and then tested this in animal models of TNBC. We showed in these models the activation of both CD8+ cytotoxic T cells and CD4+ helper T cells by dendritic cell vaccination with modified CD133 mRNA, with subsequent decrease in tumor growth. This study for the first time demonstrates in a syngeneic mouse model of TNBC that targeting CD133, in an MHC-independent manner, is an effective strategy against the cancer stem cell population, leading to tumor abrogation.

Study of abnormal chromosome regions in esophageal squamous cell carcinoma by comparative genomic hybridization: relationship of lymph node metastasis and distant metastasis to selected abnormal regions.

Squamous cell carcinoma of the esophagus (ESCC) has a poor prognosis among digestive tract cancers. Lymph node metastasis and distant metastasis are the major factors determining its prognosis. We used comparative genomic hybridization (CGH) to evaluate primary tumor lymph nodes and metastatic areas from ESCC patients in order to determine the relationship between abnormal chromosome regions and outcome. Tumor tissues and lymph nodes were collected from 51 patients with ESCC, and abnormal chromosome regions were detected by CGH. We searched for regions that were significantly more common in patients with lymph nodes metastases (n>/= 6) or distant metastases, and correlated those chromosomal changes with survival. Regions showing amplification in more than 65% of esophageal squamous cell cancers were as follows: 17q12 (90.2%), 17q21 (86.3%), 3q29 (82.4%), 3q28 (78.4%), 8q24.2 (76.5%), 22q12 (76.5%), 3q27 (74.5%), 8q24.3 (74.5%), 1q22 (70.6%), 5p15.3 (70.6%), 22q13 (70.6%), 3q26.3, 8q23, 8q24.1, 9q34, 11q13, 17p12, 17q25, 20q12, 20q13.1 (68.6%), 1q32, 1q42, and 20q13.2 (66.7%). Regions showing deletion in more than 50% of the tumors were as follows: Yp11.3 (62.7%), 3p26 (56.9%), Yq12 (54.9%), 13q21 (52.9%), 4q32 (51.0%), and 13q22 (51.0%). When Fisher's test was used to assess associations of these regions with metastases to lymph nodes, amplification at 2q12-14 (P= 0.012), 3q24-26 (P= 0.005), and 7q21-31 (P= 0.026) were significant. Survival was worse for patients with amplification at all 3 regions. In patients with distant organ metastases, amplification at 7p13-21 was significant (P= 0.008), and survival was worse. Chromosomal amplifications in ESCC at 2q12-14, 3q24-26, and 7q21-31 were associated with lymph node metastasis, while amplification at 7p13-21 was related to distant metastasis. Amplification at these regions correlated with worse survival. Genes involved in the phenotype of ESCC may exist in these regions. Identification of these genes is a theme for future investigation.

CD133 mRNA-Loaded Dendritic Cell Vaccination Abrogates Glioma Stem Cell Propagation in Humanized Glioblastoma Mouse Model.

Cancer stem cells are initiating cells of cancer and propagate its growth through self-renewal and differentiation of its daughter cells. CD133 is a cell surface antigen that is present on glioma stem cells and has been used to prospectively isolate glioma stem cells. We hypothesized that a major histocompatibility complex (MHC)-independent and long-lasting immune response against CD133 could be generated by transfecting CD133 mRNA into dendritic cells and vaccinating animals with experimental gliomas. To test this hypothesis, we developed a novel humanized mouse model using CD34-positive hematopoietic stem cells. We confirmed the robust simultaneous activation of CD8- and CD4-positive T cells by dendritic cell vaccination with modified CD133 mRNA leading to a potent and long-lived immune response, with subsequent abrogation of CD133-positive glioma stem cell propagation and tumor growth. This study for the first time demonstrates in both a humanized mouse model and in a syngeneic mouse model of glioblastoma that targeting a glioma stem cell-associated antigen is an effective strategy to target and kill glioma stem cells. This novel and simple humanized mouse model for immunotherapy is a significant advance in our ability to test human-specific immunotherapies for glioblastoma.

Identification of enzymes responsible for dantrolene metabolism in the human liver: A clue to uncover the cause of liver injury.

Dantrolene is used for malignant hyperthermia during anesthesia, and it sometimes causes severe liver injury as a side effect. Dantrolene is metabolized to acetylaminodantrolene, which is formed via the reduction of dantrolene to aminodantrolene and subsequent acetylation. Formation of hydroxylamine during the metabolic process may be associated with liver injury. We identified the enzymes responsible for dantrolene metabolism in humans to elucidate the mechanism of liver injury. Dantrolene reductase activity was not detected in human liver microsomes, but it was detected in cytosol. Formation was increased in the presence of N1-methylnicotineamide, which is an electron donor to aldehyde oxidase 1 (AOX1). Potent inhibitors of AOX1 and a correlation study with a marker of AOX1 activity, namely phthalazine oxidase activity, in a panel of 28 human liver cytosol samples supported the role of AOX1 in dantrolene reduction. Acetylaminodantrolene formation from aminodantrolene was highly detected in recombinant N-acetyltransferase (NAT) 2 rather than NAT1. A glutathione trapping assay revealed the formation of hydroxylamine via an AOX1-dependent reduction of dantrolene but not via hydroxylation of aminodantrolene. In conclusion, we found that AOX1 and NAT2 were responsible for dantrolene metabolism in humans and that AOX1-dependent metabolism determines dantrolene-induced liver injury.

Antitumor effects of vaccination with dendritic cells transfected with modified receptor for hyaluronan-mediated motility mRNA in a mouse glioma model.

OBJECT: The receptor for hyaluronan-mediated motility (RHAMM) is frequently overexpressed in brain tumors and was recently identified as an immunogenic antigen by using serological screening of cDNA expression libraries. In this study, which was conducted using a mouse glioma model, the authors tested the hypothesis that vaccination with dendritic cells transfected with RHAMM mRNA induces strong immunological antitumor effects. METHODS: The authors constructed a plasmid for transduction of the mRNAs transcribed in vitro into dendritic cells, which were then used to transport the intracellular protein RHAMM efficiently into major histocompatibility complex class II compartments by adding a late endosomal-lysosomal sorting signal to the RHAMM gene. The dendritic cells transfected with this RHAMM mRNA were injected intraperitoneally into the mouse glioma model 3 and 10 days after tumor cell implantation. The antitumor effects of the vaccine were estimated by the survival rate, histological analysis, and immunohistochemical findings for immune cells. Mice in the group treated by vaccination therapy with dendritic cells transfected with RHAMM mRNA survived significantly longer than those in the control groups. Immunohistochemical analysis revealed that greater numbers of T lymphocytes containing T cells activated by CD4+, CD8+, and CD25+ were found in the group vaccinated with dendritic cells transfected with RHAMM mRNA. CONCLUSIONS: These results demonstrate the therapeutic potential of vaccination with dendritic cells transfected with RHAMM mRNA for the treatment of malignant glioma.

[MALT-type lymphoma of lacrimal gland: case report].

A 72-year-old female presented with a lump in the left superior-lateral eyelid. The magnetic resonance imaging showed a well-delineated mass in the left lacrimal gland. The tumor was isointense on both the T1 and T2 weighted images, and it was homogenously enhanced with Gd-DTPA. Surgery via the trans-cranial approach revealed a pinkish and elastic-hard tumor. Total resection was successfully performed. The hematoxilyn-eosin staining of the surgical specimen showed a dense infiltrate of lymphocytes, which were composed predominantly of small lymphocytes, centrocyte-like cells, monocytoid cells, and occasionally transformed lymphocytes. The immunohistochemical findings for CD20, CD3, UCHL-1, CD23, CD5, cyclinD1, and bcl-2 were compatible with Mucosa Associated Lymphoid Tissue (MALT)-type lymphoma. The patient received local radiation therapy (30 Gy/15 fractions). She remained in complete clinical remission of the disease about one and a half years after treatment.

Sealing the thoracic duct with ultrasonic coagulating shears.

BACKGROUND/AIMS: Ultrasonic coagulating shears were developed as an endosurgical device that allows cutting of vessels without ligation. In this study, we obtained basic data on the feasibility of dividing and sealing the thoracic duct by using ultrasonic coagulating shears. METHODOLOGY: We obtained the thoracic duct and the left gastric artery from surgical specimens of 27 patients. After one end of each vessel was sealed using ultrasonic coagulating shears, we recorded the bursting pressure. The sealed ends of the vessels were also examined histopathologically. RESULTS: The mean bursting pressure of the thoracic duct was high enough to support the clinical use of this device, and was significantly higher than that of the left gastric artery (p<0.001). Microscopic examination of the sealed vessels showed that degenerated collagen fibers were more homogeneous and covered a significantly larger area in the thoracic duct than in the left gastric artery (p<0.001). CONCLUSIONS: The present study provides a basis for using ultrasonic coagulating shears to seal the thoracic duct and possibly lymph node dissection.

Assessing water-related risk factors for Buruli ulcer: a case-control study in Ghana.

To assess water-related risk factors of Buruli ulcer, a case-control study of 102 patients (51 cases and 51 controls) was undertaken by matching age group, sex, and bacille Calmette-Guerin (BCG) vaccination history in Ghana. The factors used here for matching have previously been implicated as factors of Buruli ulcer, an emerging infectious disease. This is the first study to delineate a set of previously suspected, water-related risk factors, in a case-control study matching for age group, sex, and BCG vaccination status. The results of both bivariate and multivariate analyses presented a significantly high odds ratio (OR) only for swimming in rivers on a habitual basis (OR = 18.00, P < 0.01) among the major water-related risk factors. Use of water from rivers and ponds for drinking, cooking, bathing, and washing purposes were not significant risk factors. Our data suggest that swimming, or activities on riverbanks associated with it, is a risk factor.

Synthesis and Characterization of Mononuclear and Dinuclear Ruthenium Complexes with Tris(2-pyridylmethyl)amine and Tris(5-methyl-2-pyridylmethyl)amine.

Novel Ru(II) and Ru(III) complexes having TPA (tris(2-pyridylmethyl)amine, L1) and 5-Me(3)-TPA (tris(5-methyl-2-pyridylmethyl)amine, L2) were prepared to establish their synthetic routes and to elucidate coordination geometry and interactions between tightly bound tripodal tetradentate ligands and Ru(II)/Ru(III) centers. They include mononuclear Ru(II) complexes [RuCl(DMSO)(L)]ClO(4) (1 (L1), 2 (L2)), dinuclear bis-&mgr;-chloro Ru(II) complexes [RuCl(L)](2)(ClO(4))(2) (3 (L1), 4 (L2)), and mononuclear Ru(III) complexes [RuCl(2)(L)]ClO(4) (5 (L1), 6 (L2)). They were characterized by X-ray crystallography (for 2, 3, and 5), (1)H NMR spectroscopy, and cyclic voltammetry. For compound 2, the crystal structure was determined to possess S-bound DMSO ligand which was trans to pyridine and Cl(-) trans to the tertiary amino group of L2, and this isomer was obtained exclusively. Complex 1 was also isolated as a single isomer. Complex 3 was revealed to be a dinuclear bis-&mgr;-chloro Ru(II) species with the center of symmetry midway between two Cl(-). (1)H NMR spectra of Ru(II) complexes 1-4, the molecular structure of 2, and comparison of the molecular structure of 3 with 5 suggest that the interaction between the Ru(II) center, pyridyl moieties, and the DMSO ligand is strengthened by pi-back-bonding from the Ru(II) center to the ligands in addition to sigma-bonding of the tertiary amino group. Electrochemical measurements on 1-6 in CH(3)CN revealed that the methyl groups on pyridyl rings exert electron-donating effects onto the Ru centers to lower each redox process and such effect strengthens the Ru-S bonding in 2 compared with that in 1, accommodating pi-back-bonding from Ru(II) center to other pi-acceptors such as DMSO in 2 enough to prevent isomerization of DMSO binding mode. The dinuclear complexes 3 and 4 showed relatively large comproportionation constants, which suggest mixed-valent Ru(II)Ru(III) states would be stabilized.

[A case of Rathke's cleft cyst in association with bilateral unruptured aneurysms of internal carotid artery].

Recent progress in diagnostic imaging techniques, such as MRI and CT, has shown that Rathke's cleft cyst is more common than once thought. However, few cases have been reported in which a cerebral arterial aneurysm was merged with a symptomatic Rathke's cleft cyst. We present a case of bilateral internal carotid aneurysms associated with a symptomatic giant Rathke's cleft cyst. A 66-year-old-woman with a visual disturbance was admitted to our hospital. CT and MRI showed a large mass in the frontal base to the suprasellar region. The mass showed isosignal intensity in T1-weighted images (WI), a ringed enhancement with Gd-DTPA, and hyperintensity in T2WI. An angiography showed bilateral A1 elevation but no tumor stain. In addition, unruptured aneurysms appeared in the inside back C2 portion of the bilateral internal carotid arteries. For these lesions, a bifrontal craniotomy was performed. In a single operative approach, the mass lesion was removed and the aneurysms were successfully clipped. The aneurysms adhered strongly to the wall of the mass. The histological diagnosis of the mass was Rathke's cleft cyst. A precise pre-operative understanding of the anatomical structure and a careful operative procedure are important for the treatment of these complex lesions.

[Controversies in esophageal cancer surgery].

In esophageal cancer treatment, the choice of treatment modality and the indications and extent of lymph node dissection surgery are controversial. In terms of the biological characteristics, esophageal cancer is more virulent than any other gastrointestinal malignancy. The distribution of lymph node metastases is very wide, extending from the neck to abdominal regions, and the sizes of lymph node metastases are very small. Almost two-thirds of all metastatic lymph nodes showed minute metastases less than 5 mm in diameter. In patients with superficial cancer with only submucosal invasion, lymph nodes metastases were found in both the upper mediastinal and paracardial areas in up to 27% cases. Furthermore, the accuracy of preoperative diagnosis of lymph node metastasis in esophageal cancer is still unsatisfactory. False negative rates in preoperative diagnosis of lymph node metastases are 14% in the neck area, 36% in the mediastinal area, and 34% in the abdominal area. Therefore, in order to cure esophageal cancer by surgery, wide, precise and complete removal of possible metastatic lymph nodes is essential. It is at this time that the quality assurance of surgery is indispensable in reducing morbidity and mortality, and in improving the patient survival. Because both surgery and chemoradiotherapy are local treatments, we must recognize the limitation of these therapeutic modalities. To improve overall survival of esophageal cancer patients, we have to make a more concentrated effort toward the systemic control of this disease.

[Surgery for benign submucosal tumors of the esophagus (leiomyoma and hemangioma)].

Among the submucosal tumors of the esophagus, leiomyoma is the most frequently found. Esophageal leiomyoma usually originates from the muscle layer of the esophageal wall and grows spirally around the esophageal axis. In the surgical treatment of leiomyoma, we enucleate the tumor through video-assisted thoracic surgery. When we enucleate leiomyoma, we must be very careful to aviod perforation of the esophageal mucosa. Esophageal hemangioma is a relatively rare disease. The location of this disease is mainly within the submucosal layer, without invading the muscle layer proper. After confirming the localization within the mucosa or submucosa with endoscopic ultrasonography, esophageal hemangioma can be resected safely using the endoscopic mucosal resection technique. In the treatment of benign esophageal submucosal tumors, "informed consent" is as essential as in esophageal cancer surgery. We have no absolute criteria concerning the indications for surgery for benign esophageal submucosal tumors. We must give reasons why the operation is necessary and indicated to the patients. Surgical treatment of esophageal submucosal tumors should be as minimally invasive as possible.

Docetaxel, cisplatin and 5-fluorouracil adjuvant chemotherapy following three-field lymph node dissection for stage II/III N1, 2 esophageal cancer.

To determine the efficacy of postoperative adjuvant chemotherapy with docetaxel + cisplatin + 5-fluorouracil (DCF) in lymph node metastasis-positive esophageal cancer, we retrospectively analyzed 139 patients with stage II/III (non-T4) esophageal cancer with lymph node metastasis (1-6 nodes), who did not receive preoperative treatment and underwent three-field lymph node dissection in the Juntendo University Hospital between December, 2004 and December, 2009. The tumors were histologically diagnossed as squamous cell carcinoma. The patients were divided into two groups, a surgery alone group (S group, 88 patients) and a group that received postoperative DCF therapy (DCF group, 51 patients). The disease-free and overall survival were compared between the groups and a multivariate analysis of prognostic factors was performed. The same analysis was performed for cases classified as N1 and N2, according to the TNM classification. There were no significant differences between the S and DCF groups regarding clinicopathological factors other than intramural metastasis and main tumor location. The presence of intramural metastasis, blood vessel invasion and the number of lymph nodes were identified as prognostic factors. The 5-year disease-free and overall survival were 55.8 and 57.3%, respectively, in the S group and 52.8 and 63.0%, respectively, in the DCF group. These differences were not considered to be statistically significant (P=0.789 and 0.479 for disease-free and overall survival, respectively). Although there were no significant differences in disease-free and overall survival between the S and DCF groups in N1 cases, both disease-free and overall survival were found to be better in the DCF group (54.2 and 61.4%, respectively) compared to the S group (29.6 and 28.8%, respectively) in N2 cases (P=0.029 and 0.020 for disease-free and overall survival, respectively). Therefore, postoperative adjuvant chemotherapy with DCF was shown to improve disease-free and overall survival in moderate lymph node metastasis-positive cases (N2), suggesting that the DCF regimen may be effective as postoperative adjuvant chemotherapy for patients with lymph node metastasis from esophageal cancer.

Reactive oxygen species responsive nanoprodrug to treat intracranial glioblastoma.

Chemotherapy for intracranial gliomas is hampered by limited delivery of therapeutic agents through the blood brain barrier (BBB). An optimal therapeutic agent for brain tumors would selectively cross the BBB, accumulates in the tumor tissue and be activated from an innocuous prodrug within the tumor. Here we show brain tumor-targeted delivery and therapeutic efficacy of a nanometer-sized prodrug (nanoprodrug) of camptothecin (CPT) to treat experimental glioblastoma multiforme (GBM). The CPT nanoprodrug was prepared using spontaneous nanoemulsification of a biodegradable, antioxidant CPT prodrug and α-tocopherol. The oxidized nanoprodrug was activated more efficiently than nonoxidized nanoprodrug, suggesting enhanced therapeutic efficacy in the oxidative tumor microenvironment. The in vitro imaging of U-87 MG glioma cells revealed an efficient intracellular uptake of the nanoprodrug via direct cell membrane penetration rather than via endocytosis. The in vivo study in mice demonstrated that the CPT nanoprodrug passed through the BBB and specifically accumulated in brain tumor tissue, but not in healthy brain tissue and other organs. The accumulation preferably occurred at the periphery of the tumor where cancer cells are most actively proliferating, suggesting optimal therapeutic efficacy of the nanoprodrug. The nanoprodrug was effective in treating subcutaneous and intracranial tumors. The nanoprodrug inhibited subcutaneous tumor growth more than 80% compared with control. The median survival time of mice implanted with an intracranial tumor increased from 40.5 days for control to 72.5 days for CPT nanoprodrug. This nanoprodrug approach is a versatile method for developing therapeutic nanoparticles enabling tumor-specific targeting and treatment. The nontoxic, tumor-specific targeting properties of the nanoprodrug system make it a safe, low cost, and versatile nanocarrier for pharmaceuticals, imaging agents, and diagnostic agents.