bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Sequential Gene Regulation via FLP‐ and Cre‐Recombinase Using Adenovirus Vector in Mammalian Cells Including Mouse ES Cells
Authors: Kondo Saki , Takahashi Yuzuka , Shiozawa Seiji , Ichise Hirotake , Yoshida Nobuaki , Kanegae Yumi , Saito Izumu
Published: November 2013

Site‐specific recombinase is widely applied for the regulation of gene expression because its regulatory action is strict and efficient. However, each system can mediate regulation of only one gene at a time. Here, we demonstrate efficient “sequential” gene regulation using Cre‐ and FLP‐expressing recombinant adenovirus (rAd) in two different monitor cell lines, for regulation of one gene (OFF‐ON‐OFF) and for two genes (ON‐OFF and OFF‐ON, independently). Generally, serial use of Cre‐ and FLP‐expressing rAd tends to cause significant cytotoxicity, but we here described optimum dose of the rAds for serial regulation. We also established an efficient method of rAd infection to mouse ES cell lines after removing feeder cells, showing that this system is useful for removal of FRT‐flanked drug‐resistance gene cassette from recombinant ES cells prior to introduction of ES cells into blastocytes for chimeric mice production. Because our sequential gene‐regulation system offers efficient purpose‐gene regulation and strict OFF‐regulation, it is potentially valuable for elucidating not only novel gene functions using cDNA microarray analysis but also for “gene switching” in development and regeneration research.
Quantitative SNP‐Detection Method for Estimating HIV‐1 Replicative Fitness: Application to Protease Inhibitor‐Resistant Viruses
Authors: Ibe Shiro , Fujisaki Saeko , Fujisaki Seiichiro , Morishita Takayuki , Kaneda Tsuguhiro
Published: November 2013

We have improved the methods for the standard competitive growth assay of human immunodeficiency virus type 1 (HIV‐1). The cloning step for the mixed viral population and subsequent genotype analysis for arbitrary numbers of clones were excluded from procedures. Instead, a single nucleotide polymorphism (SNP)‐detection step was devised for the determination of viral populations. The quantitative SNP‐detection method can rapidly estimate the proportion of wild‐type and mutant populations with high reproducibility. Consequently, this method allows manipulation of many samples within a short period. Using this new competitive growth assay, replicative fitness of drug‐resistant HIV‐1 containing an M46I amino acid mutation in the protease was assessed in the presence or absence of indinavir. Without indinavir, replicative fitness of wild‐type HIV‐1 surpassed that of M46I‐mutated HIV‐1, and the fraction of mutated virus was reduced to about 10% at passage #9. In contrast, the fraction of M46I‐mutated virus increased to >90% at passage #5 in the presence of 26.4 nM indinavir. Almost identical results were obtained for L90M‐mutated HIV‐1 with or without saquinavir. HIV‐1 can survive under indinavir pressure by acquiring M46I mutation, as with acquisition of the L90M mutation under saquinavir pressure. However, these mutations damage viral replicative fitness under natural conditions without any drugs. Subtle differences between wild‐type and mutant viruses are thus easily detected using the improved method.
Proteolytic Activation of Vibrio mimicus (Vm) Major Outer Membrane Protein Haemagglutinin (HA) with Vm‐HA/Protease: Implication for Understanding Bacterial Adherence
Authors: Alam Munirul , Miyoshi Shin‐ichi , Ahmed Kabir Uddin , Hasan Nur A. , Tomochika Ken‐ichi , Shinoda Sumio
Published: November 2013

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm‐HA/protease) and a major outer membrane protein‐HA (Vm‐OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm‐HA/protease and Vm‐OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm‐OMPHA was confirmed by SDS‐PAGE. The proteolytic activation of the native cell‐associated Vm‐OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.
The ABCs (Antibody, B Cells, and Carbohydrate Epitopes) of Cholera Immunity: Considerations for an Improved Vaccine
Authors: Provenzano Daniele , Kováč Pavol , Wade William F.
Published: November 2013

Cholera, a diarrheal disease, is known for explosive epidemics that can quickly kill thousands. Endemic cholera is a seasonal torment that also has a significant mortality. Not all nations with extensive rural communities can achieve the required infrastructure or behavioral changes to prevent epidemic or endemic cholera. For some communities, a single‐dose cholera vaccine that protects those at risk is the most efficacious means to reduce morbidity and mortality. It is clear that our understanding of what a protective cholera immune response is has not progressed at the rate our understanding of the pathogenesis and molecular biology of cholera infection has. This review addressesV. choleraelipopolysaccharide (LPS)‐based immunogens because LPS is the only immunogen proven to induce protective antibody in humans. We discuss the role of anti‐LPS antibodies in protection from cholera, the importance and the potential role of B cell subsets in protection that is based on their anatomical location and the intrinsic antigen‐receptor specificity of various subsets is introduced.
CrossLinking of the CD69 Molecule Enhances S100A9 Production in Activated Neutrophils
Authors: Shimada Sonoko , Nakamura Manabu , Tanaka Yasuhiko , Tsutsumi Kouichiro , Katano Masayoshi , Masuko Kayo , Yudoh Kazuo , Koizuka Izumi , Kato Tomohiro
Published: November 2013

Expression of CD69 on neutrophils and generation of anti‐CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti‐CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint‐composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte‐macrophage colony stimulating factor (GM‐CSF)‐activated neutrophils was crosslinked by anti‐CD69 monoclonal antibodies, and then intracellular proteins were detected using 2‐dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up‐regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS‐27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS‐27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL‐1β production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti‐CD69 autoantibodies possibly affect joint‐composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.
Direct Application of AvaII PCR Restriction Fragment Length Polymorphism Analysis (AvaII PRA) Targeting 644 bp Heat Shock Protein 65 (hsp65) Gene to Sputum Samples
Authors: Mun Ho‐Suk , Kim Hyun‐Ju , Oh Eun‐Ju , Kim Hong , Park Young‐Gil , Bai Gil‐Han , Do Junghwan , Cha Chang‐Yong , Kook Yoon‐Hoh , Kim Bum‐Joon
Published: November 2013

To evaluate the usefulness of the AvaII PRA method targeting 644‐bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS6110 PCR. Although this method showed a sensitivity slightly lower than IS6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.
Diversity in the Oligomeric Channel Structure of the Multidrug Efflux Pumps in Pseudomonas aeruginosa
Authors: Yoshihara Eisaku , Eda Shima
Published: November 2013

MexAB‐OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB‐OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel‐forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.
Mycobacterial Mammalian Cell Entry Protein 1A (Mce1A)‐Mediated Adherence Enhances the Chemokine Production by A549 Alveolar Epithelial Cells
Authors: Kohwiwattanagun Juthaporn , Kawamura Ikuo , Fujimura Takao , Mitsuyama Masao
Published: November 2013

Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non‐phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP‐1 and IL‐8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP‐1 and IL‐8 production. However, Mce1A‐coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells.
Genotyping and Quantitation of Noroviruses in Oysters from Two Distinct Sea Areas in Japan
Authors: Nishida Tomoko , Nishio Osamu , Kato Masahiko , Chuma Takehisa , Kato Hirotomo , Iwata Hiroyuki , Kimura Hirokazu
Published: November 2013

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real‐time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.
Infiltration of H‐2d‐Specific Cytotoxic Macrophage with Unique Morphology into Rejection Site of Allografted Meth A (H‐2d) Tumor Cells in C57BL/6 (H‐2b) Mice
Authors: Nomi Hayahito , Tashiro‐Yamaji Junko , Miura‐Takeda Sayako , Shimizu Tetsunosuke , Azuma Haruhito , Ueda Haruhiko , Katsuoka Yoji , Kubota Takahiro , Yoshida Ryotaro
Published: November 2013

It is assumed that CD8+ cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4+ helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL‐resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft‐induced macrophages (AIM) were cytotoxic against the allograft Bacterially‐elicited macrophages also exhibited cytotoxic activity (≈1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially‐elicited macrophages was completely inhibited by the addition of donor (H‐2d)‐type lymphoblasts, suggesting H‐2d‐specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three‐dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord‐like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H‐2d‐specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.
Molecular Characterization of Equine Herpesvirus 1 (EHV‐1) Isolated from Cattle Indicating No Specific Mutations Associated with the Interspecies Transmission
Authors: Pagamjav Ochir , Yamada Souichi , Ibrahim El‐Sayed M. , Crandell Robert A. , Matsumura Tomio , Yamaguchi Tsuyoshi , Fukushi Hideto
Published: November 2013

Interspecies trasmission of equine herpesvirus 1 (EHV‐1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV‐1 isolates in cattle (BH1247, 3M20‐3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20–3 were the genome type EHV‐1 P, while G1753 was the genome type EHV‐1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission.
Gene Expression Profiling of Polymorphonuclear Leukocytes Treated with the Culture Filtrate of Aspergillus fumigatus and Gliotoxin
Authors: Higurashi Hiromi , Arai Makoto , Watanabe Akira , Igari Hidetoshi , Seki Naohiko , Kamei Katsuhiko , Kuriyama Takayuki
Published: November 2013

Pathogens of the Aspergillus species are frequently seen in deep‐seated mycoses. We previously demonstrated that the culture filtrate of Aspergillus fumigatus (CF) has immunosuppressive effects on polymorphonuclear leukocytes (PMNs), which act as the main phagocytes to hyphae of Aspergillus fumigatus (A. fumigatus). But little is known about the gene expression profiles involved in it. Therefore we investigated the changes in gene expression in human PMNs treated with CF or gliotoxin at two time points, using microarray analysis. CF and gliotoxin changed the expression of 548 and 381 genes, respectively. Only 51 genes showed the same expression patterns with the two stimulants, and CF‐induced changes in gene expression occurred comparatively earlier than those induced by gliotoxin. Among 31 genes encoding apoptosis, which were up‐ or down‐regulated in this assay, only 3 genes were similarly changed by both kinds of stimulation. Apoptosis was detected and quantified using two apoptosis assays. CF and gliotoxin changed the expessions of only 3 out of 19 regulated genes related to inflammatory mediators and receptors similarly. The up‐regulation of the gene encoding annexin 1 (ANXA1), which is known to be involved in extravasation and apoptosis of neutrophils, may play a role in the immunosuppressive effect of A. fumigatus. The difference in expression changes between CF and gliotoxin is presumed to be caused by the interaction among the components of CF and therefore the interaction is an area of interest for further investigation.
Simultaneous Detection and Genogroup‐Screening Test for Norovirus Genogroups I and II from Fecal Specimens in Single Tube by Reverse Transcription‐Loop‐Mediated Isothermal Amplification Assay
Authors: Fukuda Shinji , Sasaki Yukie , Kuwayama Masaru , Miyazaki Kazuo
Published: November 2013

We developed a reverse transcription‐loop‐mediated isothermal amplification (RT‐LAMP) assay for the rapid detection of noroviruses (NoVs) in a genogroup‐specific manner in a previous study. In this study, to detect NoVs more easily and simply, we have developed an RT‐LAMP assay for the simultaneous detection of NoV genogroup I (GI) and II (GII) genomes in a single tube. The genogrouping was achieved by using fluorescence‐labeled primers, and the green and red colors for GI and GII, respectively, sometimes with yellow color for GI and GII mixture, were indicated on the agarose gel under UV light at 312 nm.
A Murine Model of Esophageal Candidiasis with Local Characteristic Symptoms
Authors: Ishibashi Hiroko , Hisajima Tatsuya , Hu Weimin , Yamaguchi Hideyo , Nishiyama Yayoi , Abe Shigeru
Published: November 2013

A simple method to establish a murine esophageal candidiasis model that displayed characteristic symptoms of the condition was developed using the sedative agent, chlorpromazine. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. One day later, the mice received chlorpromazine to keep them in a sedated state for about 3 hr. Under the sedated condition, they were infected with 4× 107 viable cells of Candida albicans by intra‐esophageal injection with a round‐head needle on syringe. From day 3 to day 6 post inoculation, 105‐106 colony forming units of C. albicans were recovered from the esophageal tube of each mouse and whitish, curd‐like patches were observed on most of the inner surface of the tube. Histological examination showed that C. albicans in esophageal lesions grew mainly in mycelial form. In this experimental model, intragastric administration of an itraconazole oral solution (20 mg/kg/day) was clearly effective. This model would provide a useful tool to investigate the pathogenesis of C. albicans esophageal infection and the efficacy of various antifungal agents microbiologically and symptomatically.
Serotypes, Antimicrobial Susceptibility, and gyr A Gene Mutation of Campylobacter jejuni Isolates from Humans and Chickens in Thailand
Authors: Boonmar Sumalee , Morita Yukio , Fujita Masahiro , Sangsuk Leelaowadee , Suthivarakom Karun , Padungtod Pawin , Maruyama Soichi , Kabeya Hidenori , Kato Masahiko , Kozawa Kunihisa , Yamamoto Shigeki , Kimura Hirokazu
Published: November 2013

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone‐resistant strains contained a mutation in the gyrA gene (T86→I86), suggesting that the strains were already widespread in Thailand.
Cryptococcus arboriformis Sp. Nov., a Novel Anamorphic Basidiomycetous Yeast Species Isolated from a Patient's Urine
Authors: Sugita Takashi , Takashima Masako , Sano Ayako , Nishimura Kazuko , Kinebuchi Takahiro , Yamaguchi Satoshi , Osanai Hiroaki
Published: November 2013

A basidiomycetous yeast strain isolated from the urine of a 73‐year‐old Japanese patient with chronic renal failure was revealed to be a novel species by sequencing the D1/D2 26S rDNA and ITS regions of the rRNA gene. The name Cryptococcus arboriformis sp. nov. is proposed for the isolate, with IFM 54862T (=CBS 10441T =JCM 14201T) as the type strain. A phylogenetic analysis positioned Cryptococcus arboriformis in the Trichosporonales lineage and showed that it is closely related to C. haglerorum.
Duration of Neutralizing Antibody Titer after Japanese Encephalitis Vaccination
Authors: Abe Motoharu , Okada Kenji , Hayashida Kenshi , Matsuo Fujio , Shiosaki Kouichi , Miyazaki Chiaki , Ueda Kohji , Kino Yoichiro
Published: November 2013

In paired serum samples collected from 17 children, we measured neutralizing antibody (NTAb) titers after the second series of routine Japanese encephalitis (JE) vaccination in Japan to estimate the duration of NTAb titer when children did not receive the third series of routine vaccination by applying a random coefficient model. We also measured NTAb titers in adult serum samples to confirm the duration of NTAb titer estimated in the analysis of pediatric serum samples. In the absence of the third series of routine vaccination, 18% (3/17), 47% (8/17), 82% (14/17) and 100% (17/17) of children were estimated to become NTAb negative at 5, 10, 15, and 20 years after the second series of routine vaccination, respectively. Of 38 adults, 39.5% (15/38) became NTAb negative; the percentage was somewhat lower than that of antibody‐negative children. The results suggested that JE vaccination schedule should be reevaluated in the future.
Real‐Time PCR Assay to Detect DNA in Sera for the Diagnosis of Deep‐Seated Trichosporonosis
Authors: Mekha Nanthawan , Sugita Takashi , Ikeda Reiko , Nishikawa Akemi , Poonwan Natteewan
Published: November 2013

Deep‐seated trichosporonosis due to Trichosporon asahii is life‐threatening and has high mortality. A real‐time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real‐time PCR assay may be used for diagnosing deep‐seated trichosporonosis due to T. asahii.
Characterization of Prion Susceptibility in Neuro2a Mouse Neuroblastoma Cell Subclones
Authors: Uryu Masahide , Karino Ayako , Kamihara Yukiko , Horiuchi Motohiro
Published: November 2013

In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrPSc) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrPC) at levels similar to the parental N2a cells. In contrast, there was a variation in PrPC expression in unsusceptible N2a subclones. For example, subclone N2a‐1 expressed PrPC at the same level as the parental N2a cells and prion‐susceptible subclones, whereas subclone N2a‐24 expressed much lower levels of PrP mRNA and PrPC than the parental N2a cells. There was no difference in the binding of PrPSc to prion‐susceptible and unsusceptible N2a subclones regardless of their PrPC expression level, suggesting that the binding of PrPSc to cells is not a major determinant for prion susceptibility. Stable expression of PrPC did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion‐unsusceptible N2a subclones that expressed PrPC at levels similar to prion‐susceptible subclones, indicated that a host factor(s) other than PrPC and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion‐susceptible and ‐unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.
Co‐Infection of Human Metapneumovirus with Adenovirus or Respiratory Syncytial Virus among Children in Japan
Authors: Kaida Atsushi , Kubo Hideyuki , Goto Kaoru , Shiomi Masashi , Kohdera Urara , Iritani Nobuhiro
Published: November 2013

Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients co‐infected with AdV were indistinct from those of patients mono‐infected with hMPV. The symptoms of the one patient co‐infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa.
Microbiological and Immunological Features of Oral Candidiasis
Authors: de Oliveira Marcos André M. , Carvalho Lucas P. , de S. Gomes Marcele , Bacellar Olívia , Barros Tania F. , Carvalho Edgar M.
Published: November 2013

Candida albicans (C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell‐mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2‐ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL‐5 and IL‐10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF‐α and IFN‐γ is observed in patients with oral candidiasis.
Identification and Characterization of a Dextranase Gene of Streptococcus criceti
Authors: Tamura Haruki , Yamada Arisa , Kato Hirohisa
Published: November 2013

The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene‐walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200‐amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell‐surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C‐terminus was partially purified and showed dextranase activity on blue‐dextran sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (BD‐SDS‐PAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD‐SDS‐PAGE, whereas no dextranase activity of the cells was observed on blue‐dextran brain heart infusion (BD‐BHI) agar plates. Furthermore, PCR‐based mutations of dextranase indicated that a deletion mutant of the C‐terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C‐terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp‐453 and Trp‐793 residues of S. criceti dextranase are critical to the enzyme's activity.
Differential Effect of Listeria monocytogenes Infection on Cytokine Production and Cytotoxicity of CD8 T Cells
Authors: Maruyama Saho , Shen Hua , Kanoh Makoto , Matsumoto Akira , Asano Yoshihiro
Published: November 2013

Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen‐dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen‐independent. Although Listeria is a TLR2 ligand, the immune system of the Lm‐infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen‐independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69‐positive CD8 T cells increase during Lm‐infection, cytotoxic activity of CD8 T cells was evaluated during Lm‐infection. Although CD8 T cells were activated to produce IFN‐γ, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR‐transgenic mouse was not augmented by Lm‐infection. Therefore, Lm‐infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.
Evaluation of a Cytolethal Distending Toxin (cdt) Gene‐Based Species‐Specific Multiplex PCR Assay for the Identification of Campylobacter Strains Isolated from Poultry in Thailand
Authors: Samosornsuk Worada , Asakura Masahiro , Yoshida Emi , Taguchi Takashi , Nishimura Kazuhiko , Eampokalap Boonchuay , Phongsisay Vongsavanh , Chaicumpa Wanpen , Yamasaki Shinji
Published: November 2013

We have recently developed a cytolethal distending toxin (cdt) gene‐based species‐specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter‐like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene‐based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene‐based multiplex PCR, respectively. Pulsed‐field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene‐based multiplex PCR, particularly cdtB gene‐based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.
Study of the Effect of Vaccination on Periodic Structures of Measles Epidemics in Japan
Authors: Sumi Ayako , Kamo Ken‐ichi , Ohtomo Norio , Kobayashi Nobumichi
Published: November 2013

The purpose of this paper is to investigate the effect of vaccination on periodic structures of measles epidemics in Japan. We carried out spectral analysis for time series data of measles notifications collected in Japan. It was confirmed that the interepidemic period, which corresponds to the interval between major epidemics of measles, increases as the vaccination ratio increases. This result was supported by a theory based on a mathematical model for epidemics of infectious diseases. It was suggested that the interepidemic period is useful to estimate the effect of vaccination on measles incidences quantitatively.
Pattern of Macrophage Activation in Yersinia‐Resistant and Yersinia‐Susceptible Strains of Mice
Authors: Tumitan Ana Rita Paladino , Monnazzi Luis Gustavo Silva , Ghiraldi Fabrício Rodrigues , Cilli Eduardo Maffud , de Medeiros Beatriz Maria Machado
Published: November 2013

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia‐susceptible) and C57BL/6 (Yersinia‐resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post‐infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFP‐1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN‐γ and IL‐4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF‐β1 and IL‐4 production by BALB/c mice and to an increase in the IFN‐γ levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.
Caspase‐Independent Cell Death by Fas Ligation in Human Thymus‐Derived T Cell Line, HPB‐ALL Cells
Authors: Nakayama Junichi , Ohtsuki Masahiko , Oda Tomiichiro
Published: November 2013

In HPB‐ALL cells, a human thymus‐derived T‐cell line, Fas (CD95)‐mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl‐Val‐Ala‐Asp‐(O‐methyl)‐CH2F (zVAD‐fmk) sufficient to block the caspase activity. Fas‐mediated caspase‐independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB‐ALL cells. This suggests that Fas‐mediated caspase‐independent cell death is a common feature in thymocytes. The signaling pathway of caspase‐independent cell death has not yet been fully elucidated. In HPB‐ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase‐independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB‐ALL cells have a caspase‐independent RIP kinase‐dependent pathway for Fas ligation.
Production of Cross‐Reactive Peptide Antibodies against Viral Capsid Proteins of Human Enterovirus B to Apply Diagnostic Reagent
Authors: Jeong Soo‐Young , Ahn Jeonghyun , Cho Young‐Joo , Kim Yeun‐Jung , Kim Dae‐Sun , Jee Youngmee , Lee Heuiran , Nam Jae‐Hwan
Published: November 2013

The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single‐stranded positive‐sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide‐derived antibodies directed against coxsackievirus VP1 (NO1‐NO5), VP2 (B3), and VP3 (GL3). We developed a type‐specific antibody (NO1) and broadly cross‐reactive antibodies (NO3 and NO5) to VP1. Anti‐VP2 and anti‐VP3 antibodies (B3 and GL3, respectively) are also cross‐reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot‐blot analysis also showed that NO5 against VP1 is able to detect less than 1 μg [2× 106 plaque‐forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.
Dectin‐1 Is Not Required for the Host Defense to Cryptococcus neoformans
Authors: Nakamura Kiwamu , Kinjo Takeshi , Saijo Shinobu , Miyazato Akiko , Adachi Yoshiyuki , Ohno Naohito , Fujita Jiro , Kaku Mitsuo , Iwakura Yoichiro , Kawakami Kazuyoshi
Published: November 2013

Dectin‐1 is known as a sole receptor for β‐glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin‐1 gene‐deficient and control mice. These results indicate that dectin‐1 is not likely essential for the development of host protective responses to C. neoformans.
A Potential Blood Test for Transmissible Spongiform Encephalopathies by Detecting Carbohydrate‐Dependent Aggregates of PrPres‐Like Proteins in Scrapie‐Infected Hamster Plasma
Authors: Tsukui Kazuo , Takata Masuhiro , Tadokoro Kenji
Published: November 2013

PrPres has rarely been detected in blood (except in leukocytes) even in diseased animal models that are known to contain a large amount of PrPres in infected tissues. It seems likely that PrPres detection in blood is difficult because of the low titer of infectious material within the blood. Here, we demonstrate the detection of proteinase K‐resistant 3F4‐reactive protein in the plasma of scrapie‐infected hamsters but not in the plasma of mock‐infected hamsters by partial purification using a novel method termed “acidic SDS precipitation,” in conjunction with a highly sensitive chemiluminescence detection system used to show the presence of PrP at a concentration equivalent to 1.4 ×10–9 g of brain homogenate or 1.5 × 10–12 g (6.5 ×10–17 mol) of rPrP by conventional Western blotting. The 3F4‐reactive proteins in scrapie‐infected hamster plasma often resulted in multiple Mw protein bands occurring at higher Mw positions than the position of the di‐glycosyl PrP molecule. Mixing scrapie‐infected hamster brain homogenate with mock‐infected hamster plasma resulted in the formation of similar Mw positions for multiple 3F4‐reactive proteins. Predigestion of carbohydrate side chains from the proteins in the plasma or brain homogenate before mixing resulted in failure to obtain these multiple 3F4‐reactive proteins. These observations indicate that PrPres aggregated with other proteins in the plasma through carbohydrate side chains and was successfully detected in the plasma of scrapie‐infected hamsters. Counterparts in these aggregates with PrPres‐like proteins in scHaPl are not known but any that exist should resist the PK digestion.
Reliable Enzyme‐Linked Immunosorbent Assay Systems for Pathogenic Factors of Pseudomonas aeruginosa Alkaline Proteinase, Elastase, and Exotoxin A: A Comparison of Methods for Labeling Detection Antibodies with Horseradish Peroxidase
Authors: Shigematsu Takashi , Suda Nao , Okuda Kenji , Fukushima Jun
Published: November 2013

Sensitive sandwich enzyme‐linked immunosorbent assay (ELISA) systems for the quantification of 3 pathogenic factors of Pseudomonas aeruginosa—alkaline proteinase (aeruginolysin), elastase (pseudolysin), and exotoxin A—were developed. The maleimide‐pyridyl disulfide method was applied for the labeling of rabbit anti‐each antigen IgG with horseradish peroxidase (HRP) and the conjugates were used as secondary antibodies (detection antibodies) in the ELISA systems. The EDTA, a chelating agent, was added to the buffers for sample and detection antibody, which inhibited the degradation of IgG by elastase derived from P. aeruginosa for improving the assay precision. The ELISA systems using the HRP‐labeled detection antibodies produced by the maleimide‐pyridyl disulfide method exhibited higher sensitivity than previously reported methods. The detection limits for alkaline proteinase, elastase, and exotoxin A were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively. The intra‐assay coefficients of variation for alkaline proteinase, elastase, and exotoxin A were 3.4%–5.0%, 1.9%–3.5%, and 1.3%–5.4%, respectively. These ELISA systems exhibited good inter‐assay precision, non‐cross‐reactivity, dilution linearity, and recovery. Employing these ELISA systems, we revealed that pathogenic factor concentrations were different among the P. aeruginosa strains tested, which may relate to the different pathogenicity of each strain.
Analysis of Risk Epitopes of Anti‐Neutrophil Antibody MPO‐ANCA in Vasculitis in Japanese Population
Authors: Suzuki Kazuo , Kobayashi Shigeto , Yamazaki Kazushige , Gondo Masaaki , Tomizawa Kazuo , Arimura Yoshihiro , Nakabayashi Kimimasa , Ozaki Shoichi , Yoshida Masaharu , Yoshida Toshiharu , Tsusaka Norimasa , Muso Eri , Okazaki Tomio , Hashimoto Hiroshi
Published: November 2013

Autoantibodies to myeloperoxidase (MPO) are a subset of anti‐neutrophil cytoplasmic antibody (ANCA, MPO‐ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO‐ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO‐ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO‐ANCA in different vasculitic syndromes. We screened the sera of 148 MPO‐ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I‐CrGN), classic polyangiitis nodosa (cPAN), Churg‐Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I‐CrGN and MPA sera mainly reacted to the Ha epitope at the N‐termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C‐terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO‐ANCA recognizing specific regions of the N‐terminus of the MPO H‐chain confer an increased risk of vasculitis RPGN, I‐CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO‐ANCA differentiates vasculitic from non‐vasculitic syndromes associated with MPO‐ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.
Pain: A quality of care issue during patients' admission to hospital
Authors: Carr Eloise C.J. , Meredith Paul , Chumbley Gillian , Killen Roger , Prytherch David R. , Smith Gary B.
Published: November 2013

Aim

To determine the extent of clinically significant pain suffered by hospitalized patients during their stay and at discharge.

Background

The management of pain in hospitals continues to be problematic, despite long‐standing awareness of the problem and improvements, e.g. acute pain teams and patient‐controlled analgesia, epidural analgesia. Poorly managed pain, especially acute pain, often leads to adverse physical and psychological outcomes including persistent pain and disability. A systems approach may improve the management of pain in hospitals.

Design

A descriptive cross‐sectional exploratory design.

Method

A large electronic pain score database of vital signs and pain scores was interrogated between 1st January 2010 and 31st December 2010 to establish the proportion of hospital inpatient stays with clinically significant pain during the hospital stay and at discharge.

Findings

A total of 810,774 pain scores were analysed, representing 38,451 patient stays. Clinically significant pain was present in 38·4% of patient stays. Across surgical categories, 54·0% of emergency admissions experienced clinically significant pain, compared with 48·0% of elective admissions. Medical areas had a summary figure of 26·5%. For 30% patients, clinically significant pain was followed by a consecutive clinically significant pain score. Only 0·2% of pain assessments were made independently of vital signs.

Conclusion

Reducing the risk of long‐term persistent pain should be seen as integral to improving patient safety and can be achieved by harnessing organizational pain management processes with quality improvement initiatives. The assessment of pain alongside vital signs should be reviewed. Setting quality targets for pain are essential for improving the patient's experience.