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The first outbreak in Japan of GES-5 carbapenemase-producing Pseudomonas aeruginosa occurred in a long-term care facility in 2014. To assess the spread of GES-5 producing P. aeruginosa clinical isolates in medical settings in Japan, 1,476 carbapenem-resistant P. aeruginosa isolates obtained from 2012 to 2016 were characterized. Of these 1,476 isolates, 104 (7.0%) harbored blaGES-5. Southern blotting revealed that the blaGES-5 was located on the chromosome. The isolation rates of these GES-5 producers increased significantly every year, from 2.0% (6 of 295) in 2012 to 2.8% (8 of 283) in 2013 to 5.3% (16 of 303) in 2014 to 9.7% (29 of 300) in 2015 to 15.3% (45 of 295) in 2016. Of the 104 GES-5 producers, 102 belonged to clonal complex (CC) 235, including 99 belonging to ST235 and three belonging to ST2233). Whole genome sequence analysis revealed that CC235 P. aeruginosa harboring blaGES-5 spread in a clonal manner. These results indicate that these GES-5 producing CC235 P. aeruginosa clinical isolates have spread in medical settings throughout Japan. [ABSTRACT FROM AUTHOR]

Purpose: Radio-hyperthermo-chemo (RHC) therapy, which combines radiotherapy, hyperthermia, and chemotherapy, for malignant soft tissue tumors has been introduced with the aim of decreasing the possibility of local recurrence after surgery. To avoid unnecessary neoadjuvant therapy and to plan the appropriate surgical treatment, surveillance of RHC therapeutic efficacy during treatment is necessary. In this study, we determined the optimal response criteria to evaluate the efficacy of RHC by comparing preoperative images before and after RHC with pathological evaluation of necrosis in the resected tumor. Patients and methods: From 2004 to 2014, 20 patients were enrolled into this study. Needle biopsy revealed 6 cases of myxoid liposarcoma, 6 cases of undifferentiated pleomorphic sarcoma, 4 cases of myxofibrosarcoma, and 4 cases of synovial sarcoma. Based on the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or modified RECIST, we calculated the responses to RHC therapy by comparing pre- and post-RHC therapy images. In addition, resected specimens underwent pathological analysis to evaluate response based on tumor necrosis. The correlation between assessment based on preoperative images and resected tumors were evaluated by the Spearman’s rank-order correlation coefficient. Result: From the surgical specimens, pathological assessment of necrosis in resected tumor were assessed as less than 50% (2 cases), 50–90% (9 cases), 90–99% (6 cases), and total necrosis (3 cases). Use of the RECIST 1.1 underestimated good responders as stable disease (SD) or progressive disease (PD) in 5 out of 15 cases; on the other hand, use of the modified RECIST did not underestimate the pathological assessment of necrosis. The correlations between responses based on preoperative images and those based on histological assessments were 0.23 (RECIST 1.1) and 0.76 (modified RECIST). Conclusion: Because pathological responses can be underestimated using the RECIST 1.1, the modified RECIST, which take into consideration tumor viability, as assessed by contrast MRI, should also be considered when evaluating the efficacy of RHC. [ABSTRACT FROM AUTHOR]

Background: Postoperative deep infection after bone tumor surgery remains a serious complication. Although there are numerous reports about risk factors for postoperative deep infection in general surgery, there is only a small number of reports about those for bone tumor surgery. This retrospective study aimed to identify risk factors for postoperative deep infection after bone tumor resection. Methods: We reviewed data of 681 patients (844 bone tumors) who underwent surgery. Associations between variables, including age, recurrent tumor, pathological fracture, surgical site (pelvis/other), chemotherapy, biological reconstruction, augmentation of artificial bone or bone cement, the use of an implant, intraoperative blood loss, operative time, additional surgery for complications, and postoperative deep infection were evaluated. Results: The rate of postoperative deep infection was 3.2% (27/844 tumors). A pelvic tumor (odds ratio [OR]: 3.4, 95% confidence interval [CI]: 1.0–11.3) and use of an implant (OR: 9.3, 95% CI: 1.9–45.5) were associated with an increased risk of deep infection. Conclusions: This retrospective study showed that pelvic tumor and use of an implant were independent risk factors for deep infection. This information will help surgeons prepare an adequate surgical plan for patients with bone tumors. [ABSTRACT FROM AUTHOR]

Background: A variety of surgical procedures are now available for tissue reconstruction after osteosarcoma excision, and an important prognostic factor is the evaluation of response to chemotherapy using histology. Although tumor-bearing autografts are useful tools for reconstruction, re-use of the primary tumor may make it difficult to assess the histological response to chemotherapy, since the entire tumor cannot be analyzed. Here, we analyzed the prognostic value of the histological response in the patients who received frozen tumor-bearing autografts for reconstruction. Method: Retrospective analysis of the medical records of 51 patients with high-grade osteosarcoma of the extremities was performed. All patients received reconstruction using frozen tumor-bearing autografts. Tumor necrosis was evaluated in extraskeletal masses and cancellous bone. Results: Five-year overall survival of patients with good and poor response to chemotherapy was 82.9% and 46.4%, respectively (P = 0.044), and 5-year event-free survival was 57.7% and 36.0%, respectively (P = 0.329). Multivariate analysis revealed that a poor histological response to chemotherapy was a significant prognostic factor for overall survival (P = 0.033). Conclusion: Histological response is an important and reliable prognostic factor in patients undergoing reconstruction using frozen tumor-bearing autografts. [ABSTRACT FROM AUTHOR]

Background: Chemotherapy is essential to improve the prognosis of the patients with osteosarcoma, and the response to chemotherapy is an important prognostic factor. In this study, the impact of various radiological examinations on overall survival (OS) and event-free survival (EFS) was evaluated. Method: Eighty-two patients with high-grade osteosarcoma were included in this study, and we evaluated the following factors for prognostic significance: age (≥40 years), gender (male), tumor location (truncal site), metastatic disease, histological response to chemotherapy, radiological response to chemotherapy assessed using X-ray, angiography, CT, MRI, 201Tl scintigraphy, and 99mTc-MIBI scintigraphy (99mTc-MIBI), and combined radiological score (CRS). Results: Univariate analyses revealed that metastatic disease, histological response, 99mTc-MIBI, and CRS were significantly correlated with OS. Multivariate analyses showed that metastatic disease (OS: HR 35.9, P<0.001; EFS: HR 17.32, P<0.001) was an independent predictor of OS and EFS. Tumor location (HR 36.1, P = 0.003), histological response (HR 31.1, P = 0.036), and 99mTc-MIBI (HR 18.4, P = 0.038) were significant prognostic factors for OS. Moreover, CRS was a marginally significant predictor of OS and EFS. Conclusion: The chemotherapeutic effects evaluated by 99mTc-MIBI and CRS could be considered as prognostic factors in osteosarcoma. [ABSTRACT FROM AUTHOR]

Bone loss/resorption or osteoporosis is a disease that is accelerated with aging and age-associated chronic diseases such as cancer. Bone loss has been linked with human multiple myeloma, breast cancer, and prostate cancer and is usually treated with bisphosphonates, and recently approved denosumab, an antibody against receptor activator of NF-κB ligand (RANKL). Because of the numerous side effects of the currently available drugs, the search continues for safe and effective therapies for bone loss. RANKL, a member of the TNF superfamily, has emerged as a major mediator of bone loss via activation of osteoclastogenesis. We have identified cardamonin, a chalcone isolated from Alpinia katsumadai Hayata that can affect osteoclastogenesis through modulation of RANKL. We found that treatment of monocytes with cardamonin suppressed RANKL-induced NF-κB activation and this suppression correlated with inhibition of IκBα kinase and of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. Furthermore, cardamonin also downregulated RANKL-induced phosphorylation of MAPK including ERK and p38 MAPK. Cardamonin suppressed the RANKL-induced differentiation of monocytes to osteoclasts in a dose-dependent and time-dependent manner. We also found that an inhibitor of NF-κB essential modulator (NEMO) blocked RANKL-induced osteoclastogenesis, indicating a direct link with NF-κB. Finally, osteoclastogenesis induced by human breast cancer cells or human multiple myeloma cells were completely suppressed by cardamonin. Collectively, our results indicate that cardamonin suppresses osteoclastogenesis induced by RANKL and tumor cells by suppressing activation of the NF-κB and MAPK pathway. [ABSTRACT FROM AUTHOR]

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines. [ABSTRACT FROM AUTHOR]

Background: Dendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli. Methods: Thirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-c and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy. Results: Approximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL- 12 showed significant elevation at one and three months after DC-based immunotherapy. Conclusions: Although TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors. [ABSTRACT FROM AUTHOR]

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3' untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3' untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production. [ABSTRACT FROM AUTHOR]

Mammalian cochlear sensory epithelial cells are believed to possess minimal regenerative potential because they halt proliferation during late stage of embryogenesis and never regenerate after birth. This means that sensorineural hearing loss caused by the death of cochlear sensory epithelial cells is a permanent condition. However, stem cells were recently identified in neonatal mice following dissociation of their inner ear organs. This suggests that regenerative therapy for sensorineural hearing loss may be possible. Unfortunately, dissociation distorts the microanatomy of the inner ear, making it difficult to determine the precise location of stem cells in unaltered specimens. To develop new therapeutic approaches based on sensory epithelial cell regeneration, the location of these stem cells must be elucidated. Stem cells normally proliferate at a slow rate in adult organs. In fact, so-called label-retaining cells, or slow-cycling cells, of the brain and skin are recognized as stem cells. In this study, using the exogenous proliferation marker, 5'-bromo-2'-deoxyuridine (BrdU) in combination with the endogenous proliferation marker Ki-67, we identified tympanic border cells. These cells, which are located beneath the basilar membrane in vivo, represent slow-cycling cells of the murine cochlea. Immunohistochemically, these cells stained positive for the immature cell marker Nestin. But it will be difficult to achieve regeneration of the cochlear function because these slow-cycling cells disappear in the mature murine cochlea. [ABSTRACT FROM AUTHOR]