bluuebunny/crossref_metadata_2025 huggingface dataset:
Delayed Onset of Systemic Bacterial Dissemination and Subsequent Death in Mice Injected Intramuscularly with Streptococcus pyogenes Strains
Authors: Saito Mitsumasa , Kajiwara Hideko , Ishikawa Takahiko , Lida Ken‐ichiro , Endoh Miyoko , Hara Toshiro , Yoshida Shin‐ichi
Published: November 2013

Streptococcus pyogenes causes severe invasive diseases in humans, including necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome (STSS). We found that mice infected intramuscularly (i.m.) with S. pyogenes strains developed bacteremia and subsequent sudden death after at least 10 days of a convalescent period. Mostly, it occurred more than 21 days after muscle infection. We provisionally designate this phenomenon as “delayed death.” Just after muscle infection, all the mice lost weight and activity, but recovered completely within 3 days. They had kept good activity and a fine coat of fur till one or two days before their death. Some of the dead mice were found to have soft‐tissue necrosis. There was no correlation between the virulence leading to the delayed death and the severity of diseases from which strains were isolated. It was also found that the production of neither streptococcal pyrogenic exotoxin (SPE) A nor B correlated to the virulence leading to delayed death. The bacteria obtained from the organs of the mice with delayed death expressed capsule. We suggest that the mice with delayed onset of systemic bacterial dissemination and subsequent death after muscle infection with S. pyogenes are the animal models of STSS, because the pathophysiology is extremely similar to that of human STSS.
Ultrastructural Features of Borrelia garinii in Naturally Infected Voles, Clethrionomys rufocanus, with Special Reference to the Relationship with Peripheral Nerves
Authors: Yano Yasuhiro , Ishiguro Fubito , Nakata Keisuke , Takada Nobuhiro
Published: November 2013

The ultrastructural features of Borrelia garinii found in the ear tissues of the vole, Clethrionomys rufocanus, are described. The spirochetes were observed in the interstitium of connective tissue or in contact with fibroblasts and were occasionally situated in the endoneurium of the peripheral nerves. The spirochetes did not injure or enter into the fibroblasts or Schwann cells. The cytotoxicity and migration of the spirochetes are also discussed.
Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species
Authors: Kuwahara Tomomi , Norimatsu Izumi , Nakayama Haruyuki , Akimoto Shigeru , Kataoka Keiko , Arimochi Hideki , Ohnishi Yoshinari
Published: November 2013

The structural variation in 16S‐23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four‐nucleotide‐recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile‐tRNA and Ala‐tRNA in all strains tested. The nucleotide sequence, except for that in tRNA‐coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin‐labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.
Effect of Sealing and Tween 80 on the Antifungal Susceptibility Testing of Essential Oils
Authors: Inouye Shigeharu , Tsuruoka Tsutomu , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

Concentrations of essential oils showing high volatility decreased substantially in broth and agar media when incubated under open conditions. The decrease in the half life was from 0.7 to 38 hr in broth medium at 27 C. When evaporation was prevented by sealing, MIC values against Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes by broth or agar dilution assay were lowered two to eight‐fold, as compared with those obtained under open conditions. Addition of Tween 80 caused a rise of the MICs against A. fumigatus by two to four‐fold in broth dilution assay, but little affected the MICs in agar dilution assay.
Cluster Analysis of Human and Animal Pathogenic Microsporum Species and Their Teleomorphic States, Arthroderma Species, Based on the DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1
Authors: Makimura Koichi , Tamura Yoshiko , Murakami Arisa , Kano Rui , Nakamura Yuka , Hasegawa Atsuhiko , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otoe‐related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae‐related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1‐homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1‐homologous group of M. gypseum complex are more diverse. This ITS1‐based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time‐saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non‐culturable state.
Flow Cytometric Evaluation of Antiviral Agents against Human Herpesvirus 6
Authors: Amjad Muhammad , Gillespie Michael A. , Carlson Robert M. , Karim M. Reza‐ul
Published: November 2013

Antiviral activities of acyclovir (9‐[(2‐hydroxyethoxy) methyl] guanine, ACV), penciclovir (9‐[4‐hydroxy‐3‐(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9‐(1,3‐dihydroxy‐2‐propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV‐6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV‐6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV‐6 followed by PFA, PCV and ACV. For HHV‐6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 μm and 34.7 μm respectively, whereas the IC50 of ACV and PCV were found to be 53.7 μm and 37.9 μm respectively. For HHV‐6B, the IC50 of GCV and PFA were found to be 5.7 μm and 71.4 μm respectively, whereas the IC50 of ACV and PCV were found to be 119.0 μm and 77.8 μm respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV‐6.
Cross‐Linking of β2 Integrins Caused Diminished Responses of Neutrophils to Priming Agents Like Lipopolysaccharide or Tumor Necrosis Factor‐α: Possible Involvement of Tyrosine Kinase Syk
Authors: Kusumoto Keisuke , Aida Yoshitomi , Maeda Katsumasa , Pabst Michael J.
Published: November 2013

Neutrophils up‐regulate β2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up‐regulation of β2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2–). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2–, but instead they are primed for enhanced release of O2– after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP‐triggered O2– release by LPS‐primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), β2 integrins were cross‐linked by mouse monoclonal antibodies that had been immobilized by surface‐bound anti‐mouse antibody. When unprimed neutrophils were trapped on the surface by these cross‐linked monoclonal antibodies, O2– release was triggered, and priming by LPS for fMLP‐triggered O2– release was diminished, indicating that this cross‐linking of β2 integrins mimicked adherence. Alkaline phosphatase is up‐regulated by LPS or tumor necrosis factor‐α, and this response was also diminished by the cross‐linking antibodies. The diminished alkaline phosphatase up‐regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross‐linking of β2 integrins. These results suggested that adherence‐induced triggering and Syk kinase activation might be responsible for the diminished response of LPS‐primed neutrophils to fMLP when neutrophils were adherent.
Rapid Detection of Human Fecal Eubacterium Species and Related Genera by Nested PCR Method
Authors: Kageyama Akiko , Benno Yoshimi
Published: November 2013

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species‐specific primers, would allow rapid detection of Eubacterium species in human feces.
Much Higher Risk of Premalignant and Malignant Cervical Diseases in Younger Women Positive for HPV16 than in Older Women Positive for HPV16
Authors: Tanaka Hidenori , Karube Akihiko , Tanaka Toshinobu , Nakagomi Osamu
Published: November 2013

Human papillomavirus (HPV), particularly HPV16, is strongly associated with premalignant lesions of the uterine cervix and cervical cancer. However, HPV infection is a common sexually transmitted disease and only a few women develop cervical cancer. Although the presence of HPV and abnormal cytology are independent risk factors for cervical diseases, implementing both tests on every woman is argued not to be recommended mainly in terms of cost‐effectiveness. During a 20‐month period between October, 1994, and May, 1996, cervical swabs from 207 women who were referred for colposcopy because of cervical dyskaryosis (Papanicolaou class IIIa or higher) were examined by PCR for the presence of HPV16. When these women were divided into two groups; i.e., group A consisting of women who were 44 years old or younger (n = 111), and group B consisting of women who were 45 years old or older (n = 96), the risk of having premalignant and malignant cervical diseases upon infection with HPV16 was approximately 8 times higher in group A than in group B. Thus, we conclude that HPV‐testing should be implemented on every young woman with an abnormal Papanicolaou smear test.
Mechanism for Maintenance of Dominant T Helper 1 Immune Responses in Lewis Rats
Authors: Sakamoto Shuji , Fukushima Atsuki , Ozaki Akemi , Ueno Hisayuki , Kamakura Maki , Taniguchi Taketoshi
Published: November 2013

Lewis rats are prone to T helper (Th) 1 immune responses, whereas Brown Norway (BN) rats are susceptible to Th2 immune responses. Yet, the precise mechanism of induction of the different outcome between these two strains remained elusive. We investigated the expression levels of some cytokines, their receptors and accessory molecules responsible for the polarization of antigen‐specific immune response into a predominant Th1 or Th2 profile in Lewis and BN rats. Lymph node (LN) cells collected from rats immunized with short ragweed (RW) were used directly or after stimulation in vitro with RW for 3 days. Expression of cytokines, their receptors and accessory molecules in these LN cells were tested by reverse transcriptase‐PCR. Culture supernatant was used for ELISA to detect IL‐12 protein. We observed clear differences between these strains in the expression of IL‐12p40, which was high in LN cells of Lewis rats even before stimulation in vitro. In addition, a higher amount of IL‐12 was present in the culture supernatant in Lewis rats. Upregulation of the expression of IL‐12 receptor β1, β2, IFN‐γ receptor α and β genes were more prominent in Lewis rats rather than BN rats. Furthermore, attenuated expression of CD40 and CD40 ligand by stimulation in vitro was noted only in BN rats. Changes in expression of these molecules by stimulation as well as higher basal level of IL‐12p40 might have led to the activation of Th1 cells in Lewis rats.
Resistance to Antibiotics in Injured Coliforms Isolated from Drinking Water
Authors: Cordoba María Alejandra , Roccia Irene Laura , De Luca Maria Marta , Pezzani Betina Cecilia , Basualdo Juan Angel
Published: November 2013

We studied the antibiotic sensitivity of injured coliforms isolated from drinking water of La Plata, Argentina. The antibiotic sensitivity test by the agar diffusion method were proved in: Klebsiella oxytoca (14 strains), Enterobacter aerogenes (4 strains) and Enterobacter cloacae genomic group 3 (14 strains). We found that while these impaired total coliforms were sensitive to piperacillin‐tazobactam (TAZ), netilmicin (NTL), ofloxacin (OFLX), and norfloxacin (NFLX) (100%), they had resistant to aminopenicillin‐sulbactam (AMS) and nitrofurantoin (NIT) (100%). The resistance to antibiotics demonstrated in these strains would point to the need to promote a rational and judicious use of antimicrobial agents while at the same time implementing a program of active vigilance aimed at ensuring the highest quality of drinking water throughout the system.
Negative Incidence of Lyme Disease‐Related Borrelia Spp. in Alishan, Taiwan
Authors: Masuzawa Toshiyuki , Pan Ming‐jeng , Yu Qin , Kadosaka Teruki , Imai Yasuyuki , Yanagihara Yasutake
Published: November 2013

To investigate the prevalence of Lyme disease‐related Borrelia species, wild rodents were captured around Yushan National Park and Alishan Forest Recreation Area Park in Taiwan 2,000 to 3,000 meters above sea level. Borrelia was not isolated from 67 small mammals of 7 species. Sera from rodents showed no positive reactivity against whole cell antigens of B. garinii, B. afzelii or B. valaisiana by ELISA. These results suggested that Lyme disease is not endemic to the Alishan area.
Molecular Heterogeneity of Heat‐Stable Toxin‐Producing and Non‐Producing Escherichia coli O25 Strains Isolated in Japan
Authors: Kaneko Aki , Fujino Tomoko , Sugawara Chizu , Kagami Yutaka , Miyadai Hidenori , Kirikae Teruo , Kawahara Kazuyoshi
Published: November 2013

Escherichia coli O25 strains that produce heat‐stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST‐producing and non‐producing strains of E. coli O25 was investigated. All eight ST‐producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder‐banding of LPS in SDS‐polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed‐field gel electrophoresis (PFGE). In contrast, ST‐non‐producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.
Helicobacter pylori DNA in Drinking Water in Japan
Authors: Horiuchi Takao , Ohkusa Toshifumi , Watanabe Mamoru , Kobayashi Daisuke , Miwa Hiroto , Eishi Yoshinobu
Published: November 2013

Helicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water‐borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05‐ or 0.22‐μm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real‐time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past H. pylori DNA was detected in well water and the users had been infected, so water‐borne transmission via well water may occur even in towns in Japan.
Morphological Variety of Intracellular Microcolonies of Legionella Species in Vero Cells
Authors: Ogawa Midori , Takade Akemi , Miyamoto Hiroshi , Taniguchi Hatsumi , Yoshida Shin‐ichi
Published: November 2013

Intracellular microcolonies of six Legionella species growing in Vero cells showed distinctly varied morphologies. The varieties were observed by light microscopy of Gimenez‐stained, Legionella‐infected Vero cells and by electron microscopy (EM). Legionella pneumophila Philadelphia‐1 formed needle‐shaped crystal‐like microcolonies. Legionella bozemanii WIGA formed microcolonies like wool balls containing filamentous cells. In EM, these organisms proliferated in endosomes, which were adjacent to swollen rough endoplasmic reticula. Legionella oakridgensis OR‐10 showed serpentine chains. Many mitochondria were observed around the microcolonies. Legionella jordanis BL‐540 formed spherical moss‐like microcolonies which were or were not surrounded by endoplasmic membranes. Legionella feeleii WO‐44C spread throughout the cytoplasm without making clusters. Legionella dumoffii Tex‐KL made big clusters that spread in the cytoplasm, a portion of which was outside the endosome membranes. These different morphologies imply diversity in modes of intracellular multiplication of Legionella spp.
Effect of Anticoagulants in Colorimetric Assay for Basic Carboxypeptidases
Authors: Komura Hidefumi , Obata Kyoko , Campbell William , Yumoto Miho , Shimomura Yasuyo , Katsuya Hirotada , Okada Noriko , Okada Hidechika
Published: November 2013

Carboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl‐l‐arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.
Engineering of a Sagiyama Alphavirus RNA‐Based Transient Expression Vector
Authors: Yamaguchi Yuka , Shirako Yukio
Published: November 2013

Sagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full‐length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N‐terminal 9 amino acids and C‐terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N‐terminally‐fused capsid‐protease domain. GFP was also expressed efficiently without the 5′‐terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly‐to‐Leu substitution at the − 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 μg from 1 × 106 BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high‐level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.
Inactivation of C3a and C5a Octapeptides by Carboxypeptidase R and Carboxypeptidase N
Authors: Campbell William D. , Lazoura Eliada , Okada Noriko , Okada Hidechika
Published: November 2013

Pro‐carboxypeptidase R (proCPR), also known as thrombin‐activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.
Two‐Dimensional Analysis of Exoproteins of Methicillin‐Resistant Staphylococcus aureus (MRSA) for Possible Epidemiological Applications
Authors: Nakano Miyo , Kawano Yasushi , Kawagishi Mika , Hasegawa Tadao , Iinuma Yoshitsugu , Ohta Michio
Published: November 2013

We applied two‐dimensional gel electrophoresis (2‐DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N‐terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell‐associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC3, toxic shock syndrome toxin‐1 (TSST‐1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha‐hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta‐hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid‐specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2‐DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2‐DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST‐1 than the strains belonging to other coagulase types. Taken together, 2‐DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.
Cross‐Reactivity and Sensitivity of Two Legionella Urinary Antigen Kits, Biotest EIA and Binax NOW, to Extracted Antigens from Various Serogroups of L. pneumophila and Other Legionella Species
Authors: Okada Chikako , Kura Fumiaki , Wada Akihito , Inagawa Hiroko , Lee Gang‐hong , Matsushita Hiroshi
Published: November 2013

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non‐serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in‐vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L. pneumophila SG 1 antigens, but reacted to different sets of non‐SG 1 L. pneumophila with different sensitivity. No cross‐reactivity was observed to Legionella species other than L. pneumophila.
The Morbillivirus Receptor SLAM (CD150)
Authors: Tatsuo Hironobu , Yanagi Yusuke
Published: November 2013

Morbilliviruses are highly contagious pathogens that cause some of the most devastating viral diseases of humans and animals, including measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RPV). They replicate mainly in lymphoid organs throughout the body and cause severe immunosuppression accompanied with lymphopenia. We have recently shown that human, canine, and bovine signaling lymphocyte activation molecules (SLAMs; also known as CD150) act as cellular receptors for MV, CDV, and RPV, respectively. In these three morbilliviruses, all strains examined were shown to use SLAMs of their respective host species, and laboratory strains passaged on SLAM‐negative cells were found to use, besides SLAM, alternative receptors, such as human CD46 for the Edmonston strain of MV. The use of SLAM as a receptor may be a property common to most, if not all, of the members of morbilliviruses. Human SLAM is a membrane glycoprotein selectively expressed on the cells of the immune system (immature thymocytes, activated lymphocytes, activated monocytes, and mature dendritic cells) and seems to mediate lymphocyte activation and to control interferon‐γ production. The destruction and/or impairment of infected SLAM‐positive cells may be a mechanism for the immunosuppression induced by morbilliviruses, but other mechanisms may be also involved.
Fine Mapping of the Subunit Binding Sites of Influenza Virus RNA Polymerase
Authors: Ohtsu Yasushi , Honda Yoshikazu , Sakata Yasutaka , Kato Hirohisa , Toyoda Tetsuya
Published: November 2013

Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of the subunit assembly. The subunit binding sites were mapped at about several hundred amino‐acid size. Fine mapping of the subunit binding sites was determined. The PB1‐PA binding regions were mapped within in the N‐terminal 25 amino acids of PB1 and 668–692 of PA. PB1 and PB2 interacted within wider regions, 600–757 of PB1 and 51–259 of PB2. In these amino‐acid spans, 206–259 of PB2 may be the most important region of PB1 binding and 718–732 of PB1 may be the most important region of PB2 binding because the binding activity was lost when the regions were lost in the subunits. The additional regions contributed to strong binding of these subunits.
Detection of Herpesvirus DNA in the Serum of Immunocompetent Children
Authors: Hara Shinya , Kimura Hiroshi , Hoshino Yo , Tanaka Naoko , Nishikawa Kazuo , Ihira Masaru , Yoshikawa Tetsushi , Morishima Tsuneo
Published: November 2013

The DNA of herpesviruses such as Epstein‐Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus‐6 (HHV6), and human herpesvirus‐7 (HHV7) has been detected in the serum of patients with primary infection or with immunosuppression. However, it is unknown how frequently herpesvirus DNA can be detected in the serum of immunocompetent children, or whether the detection of herpesvirus DNA indicates an active infection or virus‐related diseases. Using a real‐time polymerase chain reaction assay, attempts were made to detect herpesvirus DNA in the serum of 176 ambulatory children who visited a hospital for various reasons. EBV was detected in 4 (2.2%), HHV6 in 4 (2.2%), and HHV7 in 2 (1.1%) of 176 children, but CMV was not detected. Of the 10 positive patients, only 4 were considered, by virtue of clinical and serological characteristics, to have primary infections. The other 4 positive patients had other infections, such as mycoplasma and salmonella. Although herpesvirus DNA could be detected in the serum of immunocompetent children, there was not always a relationship between clinical manifestations and the detection of virus DNA. When herpesvirus DNA is detected in the serum, a careful interpretation is necessary to diagnose a primary infection or a virus‐associated disease.
IL‐10 Producing CD14low Monocytes Inhibit Lymphocyte‐Dependent Activation of Intestinal Epithelial Cells by Commensal Bacteria
Authors: Haller Dirk , Serrant Patrik , Peruisseau Genneviève , Bode Christiane , Hammes Walter P. , Schiffrin Eduardo , Blum Stephanie
Published: November 2013

Intestinal epithelial cell (IEC) activation by non‐pathogenic, commensal bacteria was demonstrated to require the presence of immunocompetent cells. In this study, HT‐29 and CaCO‐2 transwell cultures, reconstituted with CD4+ and CD8+ T cells, CD19+ B cells and CD14high monocytes, were challenged with nonpathogenic Gram negative Escherichia coli and Gram positive lactobacilli. Cytokine expression was analysed by reverse transcription‐polymerase chain reaction (RT‐PCR) and enzyme linked immunosorbent assays (ELISA). Expression of tumour necrosis factor alpha (TNF‐α) and interleukin (IL)‐8 mRNA in E. coli or L. sakei challenged IEC was promoted by lymphocyte populations predominantly CD4+ T cells, while monocytes failed to mediate an inflammatory cytokine response. The monocyte phenotype and function were further characterised by flow cytometry and mixed lymphocyte reaction (MLR). During the co‐culture with IEC and bacterial stimulated IEC, CD14high peripheral blood monocytes acquired a CD14low CD16low phenotype with reduced expression co‐stimulatory (CD80, CD86, CD58) cell surface molecules. Immunosuppressive functions of IEC conditioned CD14low monocytes were demonstrated by the predominant secretion of IL‐10 and IL‐1Ra and their reduced potential to trigger an allogeneic lymphocyte response. In conclusion, IEC contribute to the development of CD14low CD16low monocytes with immunosuppressive function and antagonised a lymphocyte‐mediated activation of the intestinal epithelium in response to intestinal and food derived bacteria. These results strengthen the hypothesis that the gut epithelium constitutes an important functional element in the regulation of mucosal immune homeostasis towards commensal bacteria.
Phylogenetic Analysis of Spotted Fever Group Rickettsiae Isolated from Ticks in Japan
Authors: Ishikura Mitsuhiro , Fujita Hiromi , Ando Shuji , Matsuura Kumiko , Watanabe Mamoru
Published: November 2013

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase‐encoding gene (gltA) and 190‐kDa antigen‐encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT‐1 and FLA‐1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO‐1, IO‐2, IO‐25, IM‐1 and IP‐2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT‐1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT‐1 and FLA‐1 were placed as R. japonica, and strain AT‐1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT‐1 and FLA‐1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT‐1 should be considered as a new species among SFG rickettsiae.
Mycoplasmal Lipoproteins Induce Toll‐Like Receptor 2‐ and Caspases‐Mediated Cell Death in Lymphocytes and Monocytes
Authors: Into Takeshi , Nodasaka Yosinobu , Hasebe Akira , Okuzawa Tsugumi , Nakamura Jun‐ichiro , Ohata Noboru , Shibata Ken‐ichiro
Published: November 2013

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT‐4 and Raji, and in one monocytic cell line, THP‐1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL‐60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase‐3 in both MOLT‐4 and HL‐60 cells, which was assessed by the cleavage of the synthetic substrate DEVD‐pNA and the endogenous substrate poly(ADP‐ribose) polymerase. The cytotoxicity to MOLT‐4 and HL‐60 cells was inhibited by various caspase inhibitors, Ac‐DMQD‐CHO, Ac‐IETD‐CHO, and Z‐VAD‐FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll‐like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases‐dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2‐mediated signaling.
Persistent Parapoxvirus Infection in Cattle
Authors: Iketani Yuko , Inoshima Yasuo , Asano Akihiro , Murakami Kenji , Shimizu Shinya , Sentsui Hiroshi
Published: November 2013

The possibility of persistent parapoxvirus (PPV) infection was investigated by serologically and genetically using cattle infected with the virus experimentally and naturally. Three cattle were inoculated with the virus subcutaneously at several spots in the lips and abdominal regions. Small papules developed in the inoculated regions, and antibodies to the virus developed and continued persistently. One animal, from which one PPV had been previously isolated, was also subjected to serological and viral detection tests as a naturally infected case. Two of these four cattle were injected with dexamethasone (DM), and one was injected with interferon‐γ (IFN‐γ). The viral genome was rarely detected from the peripheral blood leukocytes in the ordinary condition, but frequently when the animals were injected with IFN‐γ. The viral genome was also detected from the lymph nodes as these PPV infected animals were euthanized. These results indicated that cattle were infected with PPV subclinically and persistently, and the virus was activated in stressed or immunosuppressed animals. The virus would be harbored in the lymphotic tissues of the animals when they show no clinical symptoms.
Protection of Mice from Lethal Endogenous Candida albicans Infection by Immunization with Candida Membrane Antigen
Authors: Tansho Shigeru , Abe Shigeru , Mizutani Shigetoshi , Ono Yasuo , Takesako Kazutoh , Yamaguchi Hideyo
Published: November 2013

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA‐immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.
A Novel Mechanism of Fluconazole Resistance Associated with Fluconazole Sequestration in Candida albicans Isolates from a Myelofibrosis Patient
Authors: Maebashi Kazunori , Kudoh Michinari , Nishiyama Yayoi , Makimura Koichi , Uchida Katsuhisa , Mori Takeshi , Yamaguchi Hideyo
Published: November 2013

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis‐complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell‐free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P‐450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [3H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 × g pellet (P‐120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative‐staining method suggests that most of the vesicular vacuoles were recovered in the P‐120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P‐450.
Physicochemical and Biological Properties of an Extracellular Serine Protease of Aeromonas sobria
Authors: Yokoyama Ritsuko , Fujii Yoshio , Noguchi Yoko , Nomura Tomohiko , Akita Masahiko , Setsu Kojun , Yamamoto Shigeo , Okamoto Keinosuke
Published: November 2013

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.
Computer Simulations of Slow Progression of Human Immunodeficiency Virus Infection and Relapse during Anti‐HIV Treatment with Reverse Transcriptase Inhibitors and Protease Inhibitors
Authors: Takayanagi Toshiaki , Ohuchi Azuma
Published: November 2013

Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) have been very serious problems since the 1980s. The progression of HIV infection into AIDS can be suppressed to some extent with reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs); however, there are some serious problems with treatments using the anti‐HIV drugs (e.g. very high expense, complicated administration, and drug resistance). Hence, more studies on HIV and the development of more effective anti‐HIV treatments are required. We consider it important to understand the complex dynamics involved in HIV infection, and we therefore propose new mathematical models of HIV infection. In the modeling, we have paid attention to the nonlinear relations between stimuli and responses (i.e., when responses are plotted against the logarithm of stimuli, a sigmoid curve is obtained), and to lymphoid organs which seem more important than the blood compartment (i.e., lymphoid organs are major reservoirs of HIV virions and contain most of the lymphocytes). Using the models, we have found that viral antigenic mutation plays an important role in the slow progression in the chronic phase of HIV infection. We have also found that viral antigenic mutation can cause relapse of HIV infection when the inhibition rate of anti‐HIV drugs is low and that viral antigenic mutation cannot cause relapse when the inhibition rate is high.
In Vivo Fungicidal Effect of KP‐103 in a Guinea Pig Model of Interdigital Tinea Pedis Determined by Using a New Method for Removing the Antimycotic Carryover Effect
Authors: Tatsumi Yoshiyuki , Yokoo Mamoru , Arika Tadashi , Yamaguchi Hideyo
Published: November 2013

We developed a new technique for culture study that successfully recovers fungi from drug‐treated skin tissues, in which tissue specimens were homogenized, dialyzed against water, digested with trypsin, and then washed with PBS, to eliminate the drug that remaining in the skin tissue specimens. With this modified culture method, we reevaluated the efficacy of KP‐103, neticonazole, and lanoconazole in a guinea pig interdigital tinea pedis model. Guinea pigs with tinea pedis were topically treated with a 1% solution of KP‐103 or a reference drug once a day for 10 consecutive days. Five days after the last treatment, left and right feet were subjected to culture study by the conventional and modified recovery culture methods, respectively. One hundred percent (20/20) of lanoconazole‐treated feet were judged as culture‐negative by the conventional culture method, but 85% (17/20) of the feet were shown to be culture‐positive when the modified recovery culture method was used. On the other hand, KP‐103 achieved high rates of culture‐negative rates, 95% (19/20) and 85% (17/20), in both conventional and modified culture methods, respectively. Furthermore, on day‐30 posttreatment, KP‐103 sterilized 14 of the 20 infected feet, whereas neticonazole and lanoconazole were not effective even in reducing fungal burden. KP‐103 proved to be highly effective in achieving mycological cure and preventing relapse against tinea pedis presumably because of its good bioavailability in the skin based on its low keratin‐affinity, along with its potent antifungal activity.
Intraperitoneal Immunization Led to T Cell Hyporesponsiveness to Helicobacter pylori Infection in Mice
Authors: Watanabe Koichiro , Murakami Kazunari , Maeda Kosaku , Fujioka Toshio , Nasu Masaru , Nishizono Akira
Published: November 2013

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori‐challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori‐challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized‐challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL‐2, IFN‐γ, and IL‐4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL‐2 and IFN‐γ. CTLA‐4 has been documented to be a negative regulator of IL‐2 production and lymphoproliferation that induces peripheral tolerance and functions 24–72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized‐challenged mice showed higher levels of CTLA‐4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori‐associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA‐4 following challenge.
Studies on the Rabies Virus RNA Polymerase: 3. Two‐Dimensional Electrophoretic Analysis of the Multiplicity of Non‐Catalytic Subunit (P Protein)
Authors: Eriguchi Yoshiro , Toriumi Harufusa , Kawai Akihiko
Published: November 2013

We described previously (Takamatsu et al., 1998. Microbiol. Immunol. 42: 761–771) the rabies virus P protein as being composed of several components of different sizes, among which the full‐sized major components were termed as p40 and p37 according to their electrophoretic mobilities, and radiolabeling studies with [32P]phosphate implied that p40 was a hyperphosphorylated form. We further examined here these proteins by two‐dimensional (2‐D) gel electrophoresis and immunoblotting, showing that a major component, p37, was composed of multiply modified subcomponents of different pIs (termed p37–1, p37–2, p37–3, etc., based on their acidity) in the virion and infected cells, but the unmodified precursor (termed p37–0) was little in amount. The viral nucleocapsid (NC)‐bound P proteins were composed of multiple forms of p37 (the major one was p37–1) and also a minor component, p40–1. P proteins which were bound to newly synthesized free N proteins were mostly composed of p37–1, indicating that hyperphosphorylation of P proteins occurred after their being used for the encapsidation. Treatment of the infected cells with okadaic acid induced accumulation of the more acidic forms of P proteins, suggesting that heterogeneity in the full‐sized P proteins is a reflection of their dynamic aspects of multiple cycles of phosphorylations and dephosphorylations in the cell. Two‐D gel analyses demonstrated also that p40 was not so acidic as we expected, and implied that our previous data of apparent hyperphosphorylation of p40 was due to very frequently recycled utilization of the protein, and preformed non‐labeled P proteins were also 32P‐phosphorylated in a radiolabeling period and were converted to the p40.