Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence

ユニバーサルグラビングにおける手指衛生遵守率

Background: The use of gloves for every patient contact (universal gloving) has been suggested as an infection prevention adjunct and alternative to contact precautions. However, gloves may carry organisms unless they are changed properly. In addition, hand hygiene is required before donning and after removing gloves, and there are scarce data regarding glove changing and hand hygiene in a universal gloving setting. Methods: This non-randomized observational before-after study evaluated the effect of education and feedback regarding hand hygiene. Compliance with hand hygiene and glove use was directly observed in a universal gloving setting at a 10-bed intensive care unit in a Japanese tertiary care teaching university hospital. Results: A total of 6,050 hand hygiene opportunities were identified. Overall hand hygiene compliance steadily increased from study period 1 (16.1%) to period 5 (56.8%), although there were indication-specific differences in the baseline compliance, the degree of improvement, and the reasons for non-compliance. There were decreases in the compliance for universal gloving and the incidence of methicillin-resistant Staphylococcus aureus. Conclusion: It is difficult to properly perform glove use and hand hygiene in a universal gloving setting, given its complexity. Direct observation with specific feedback and education may be effective in improving compliance.博士（医学）・甲第671号・平成29年6月28日Copyright © 2017 The Association for Professionals in Infection Control and Epidemiology, Inc. All rights reserved.Copyright © 2017 Elsevier Inc. All rights reserved

新生児マウス鼻腔への微生物抽出液の投与は気道中のオプソニン量及び、成熟CD1c+ 細胞を増加させる

Nasal exposure to the mixture of microbial extracts (MME) after ablactation enhanced airway resistance of newborn mice to Streptococcus pneumoniae (J Physiol Lung Cell Mol Physiol 298: L67, 2010). The present study was addressed to elucidate effective factors responsible for the enhanced innate resistance in the airway of MME-exposed newborn mice. MME exposure significantly increased the amount of pulmonary surfactants (SP-A and SP-D) in the airway. Bronchoalveolar lavage fluid of the exposed mice exhibited greater levels of opsonic activity, thereby enhancing the phagocytic and intracellular killing activities of alveolar macrophages (MØ) against S. pneumoniae. The exposure itself did not increase a complement component C3 and mannan-binding lectin-A (MBL-A) in the airway, whereas intratracheal infection with S. pneumoniae increased the quantity of SP-A, SP-D, C3, and MBL-A in the exposed mice to a significant extent compared with control mice. The exposure enhanced the expression of the class A scavenger MØ receptor with collagenous structure on alveolar MØ and also increased the frequency of major histocompatibility complex II+ CD11c+ cells in the lung; the cells were able to produce IL-10 and transforming growth factor-β in vitro. These results suggest that microbial exposure early in life increases the amounts of SP-A and SP-D and the number of scavenger MØ and also promotes maturation of CD11c+ cells in the airway of newborn mice, which may be involved in airway resistance to S. pneumoniae.博士（医学）・乙1323号・平成26年3月17日発行元の規定により、本文の登録不可。本文は以下のURLを参照 "http://dx.doi.org/10.1152/ajplung.00031.2012

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

BackgroundToca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model.MethodsFlow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC.ResultsTumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge.ConclusionsTreatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients

A CASE OF CORONAVIRUS DISEASE 2019 COMPLICATED BY VENTILATORASSOCIATED PNEUMONIA, LUNG ABSCESS, AND STAPHYLOCOCCUS AUREUS BACTEREMIA

Complications of healthcare-associated infections have been reported in patients with coronavirus disease 2019 (COVID-19). We encountered a case of ventilator-associated pneumonia and lung abscess, complicated with Staphylococcus aureus bacteremia and multiple abscesses, in a patient with COVID-19. Streptococci and anaerobes were cultured from the sputum, which was considered to be the causative organism of the lung abscess. In the management of severe COVID-19, care should be taken to prevent complications of healthcare-associated infections; when secondary respiratory tract infections are suspected, the presence of lung abscess and anaerobic culture should be considered

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

BackgroundProdrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects.MethodsHere we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts.ResultsIn both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells.ConclusionThese results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity

プラスミド性キノロン耐性遺伝子を保有するIMP-6産生腸内細菌科細菌でのイノカラムサイズ効果の比較

Almost all cases of carbapenemase-producing Enterobacteriaceae infections in Japan are caused by blaIMP-positive Enterobacteriaceae (especially blaIMP-6) and infections caused by other types of carbapenemase-producing Enterobacteriaceae are quite rare. We examined drug resistance genes co-harboring with blaIMP-6 and their inoculum size effects. We screened β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, and aminoglycoside-modifying enzyme genes by PCR and performed sequencing for 14 blaIMP-6-positive Enterobacteriaceae. Further, all PMQR-positive isolates were submitted to conjugation and inoculum effect evaluation. Our data showed that 13 of the 14 isolates harbored CTX-M-2 and one co-harbored CTX-M-2 and CTX-M-1 as extended-spectrum β-lactamases. All isolates carried one or more PMQRs; aac(6’)-Ib-cr was the most prevalent (92.8%), and was followed by oqxA (64.3%), qnrS (50%), oqxAB (21.4%), and qnrB (14.3%). However, Klebsiella pneumoniae contains chromosomal OqxAB. Inoculum size effects were significant in all strains for meropenem, 13 strains for imipenem, 7 for levofloxacin, and 3 for amikacin. We observed that 11 of the experimental strains (100%), 8 strains (72.7%), and 1 strain showed inoculum size effects for meropenem, imipenem, and amikacin, respectively. However, four strains harbored qnr genes and two strains harbored qnr genes and QRDR mutations concurrently; no inoculum size effect was seen for levofloxacin. The blaIMP-6-positive Enterobacteriaceae that we studied was found to harbor at least one plasmid-mediated drug resistance gene. The inoculum size effect for carbapenems was thought to be mainly due to IMP-6-type metallo-β-lactamase; however qnrB and qnrS also had a minimal impact on the inoculum size effect for levofloxacin.博士（医学）・乙第1463号・令和2年6月30日Copyright: © 2019 Ogawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited