bluuebunny/crossref_metadata_2025 huggingface dataset:
Time‐Kill Assay of Itraconazole against Malassezia Species
Authors: Sugita Takashi , Tsubuku Hisae , Tajima Mami , Tsuboi Ryoji , Nishikawa Akemi
Published: November 2013

The in vitro pharmacodynamic activity of itraconazole against Malassezia was determined by time‐kill methods. Itraconazole showed fungistatic activity at concentrations greater than 2× minimum inhibitory concentration (MIC) for each of the isolates of all nine Malassezia species associated with humans. A concentration exceeding 4× MIC decreased the growth to less than the inoculum amount (1×105 cells/ml).
Human Immunodeficiency Virus‐Type 1 Specific Cellular Immunity in Chronic Infected Patients on Prolonged Highly Active Antiretroviral Treatment and on Structured Treatment Interruption
Authors: Matsuda Reikei , Boström Ann‐Charlotte , Fredriksson Malin , Fredriksson Eva‐Lena , Bratt Göran , Hejdeman Bo , Sandström Eric , Okuda Kenji , Wahren Britta
Published: November 2013

We have followed 15 HIV‐1 chronically infected patients during prolonged highly active antiretroviral treatment (HAART) and subsequent long term structured treatment interruption (STI). We analyzed Nef, Tat, and p24 specific cellular immunity using IFN‐γ enzyme‐linked immunospot assays and T cell proliferation assays. Eight HAART patients showed IFN‐γ responses to at least one antigen, but no positive responses were seen during STI. We observed retained or increased p24 specific IFN‐γ responses in most patients during HAART with viral suppression. These results showed persisting HIV‐1 specific cellular immunity during HAART; however, in prolonged STI with viral rebound this immunity declined.
Genetic Diversity and Epizootiology ofChlamydophila psittaciPrevalent among the Captive and Feral Avian Species Based on VD2 Region ofompAGene
Authors: Chahota Rajesh , Ogawa Hirohito , Mitsuhashi Yoko , Ohya Kenji , Yamaguchi Tsuyoshi , Fukushi Hideto
Published: November 2013

To study genetic diversity and occurrence ofChlamydophila psittaci, a total of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and captive birds were examined usingompAgene based nested PCR. Three types of chlamydiae:C. psittaci(94.12%),C. abortus(4.41%) and unknownChlamydophilasp. (1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes‐59,Ciconiiformes‐8 andPasseriformes‐1). Based on nucleotide sequence variations in the VD2 region ofompAgene, all 64 detectedC. psittacistrains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and 7.35% samples respectively. A single strain of unknownChlamydophilasp. was found phylogenetically intermediate betweenChlamydophilaspecies infecting avian and mammalian hosts. AmongPsittaciformes, 28 out of 81 tested species including 10 species previously unreported were found to be chlamydiae positive. Chlamydiosis was detected among 8.97% sick and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was observed that though various genetically diverse chlamydiae may cause avian chlamydiosis, only a fewC. psittacistrains are highly prevalent and frequently associated with clinical/subclinical infections.
The Postantifungal Effect (PAFE) of Itraconazole, in Comparison with Those of Miconazole and Fluconazole, on Candida Species
Authors: Uchida Katsuhisa , Abe Shigeru , Yamaguchi Hideyo
Published: November 2013

To study the postantifungal effect (PAFE) of itraconazole (ITCZ) against Candida species, we quantitated it using a newly introduced parameter T/C (T=time required for the drug‐treated culture to reach 5‐fold increase in turbidity; C=time required for the drug‐free control culture to reach 5‐fold increase in turbidity) referred as PAFE index and compared the results with those obtained for two other azole antifungal drugs fluconazole (FLCZ) and miconazole (MCZ). The mean values of PAFE index for ITCZ against three C. albicans strains were 1.28, 1.45, and 1.60 when exposed to the drug for 1 hr at concentrations of 1×MIC, 2×MIC and 4×MIC, respectively. These values are similar to those for MCZ and appreciably higher than those for FLCZ. PAFE index values for ITCZ against each single strain of three non‐albicans Candida species exposed to 2×MIC of the drug for 1 hr were: 1.22 with C. glabrata, 1.63 with C. parapsilosis, and 3.90 with C. tropicalis. Again, comparable values were obtained for MCZ and lower values for FLCZ. The drug concentration for exposure and the duration of exposure only slightly or scarcely affected the PAFE of ITCZ, although the magnitude of the PAFE considerably varied among different strains or species tested. In general, ITCZ, as well as MCZ, produced greater extents of PAFE than those for FLCZ against C. albicans and several non‐albicans Candida species.
Mechanism of Enhanced Hematopoietic Response by Soluble β‐Glucan SCG in Cyclophosphamide‐Treated Mice
Authors: Harada Toshie , Kawaminami Hiromi , Miura Noriko N. , Adachi Yoshiyuki , Nakajima Mitsuhiro , Yadomae Toshiro , Ohno Naohito
Published: November 2013

SCG is a major 6‐branched 1,3‐β‐D‐glucan in Sparassis crispa Fr. SCG shows antitumor activity and also enhances the hematopoietic response in cyclophosphamide (CY)‐treated mice. In the present study, the molecular mechanism of the enhancement of the hematopoietic response was investigated. The levels of interferon‐ (IFN‐) γ, tumor necrosis factor‐ (TNF‐) α, granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), interleukin‐ (IL‐) 6 and IL‐12p70 were significantly increased by SCG in CY‐treated mice. GM‐CSF production in the splenocytes from the CY‐treated mice was higher than that in normal mice regardless of SCG stimulation. Neutralizing GM‐CSF significantly inhibited the induction of IFN‐γ, TNF‐α and IL‐12p70 by SCG. The level of cytokine induction by SCG was regulated by the amount of endogenous GM‐CSF produced in response to CY treatment in a dose‐dependent manner. The expression of β‐glucan receptors, such as CR3 and dectin‐1, was up‐regulated by CY treatment. Blocking dectin‐1 significantly inhibited the induction of TNF‐α and IL‐12p70 production by SCG. Taken together, these results suggest that the key factors in the cytokine induction in CY‐treated mice were the enhanced levels of both endogenous GM‐CSF production and dectin‐1 expression.
Development of a Real‐Time PCR Assay for Detection of gyrA Mutations Associated with Reduced Susceptibility to Ciprofloxacin in Salmonella enterica Serovar Typhi and Paratyphi A
Authors: Morita Masatomo , Ito Kenitiro , Hirose Kenji , Takahashi Hideyuki , Shimuta Ken , Terajima Jun , Ohnishi Makoto , Harada Makoto , Matsuzaki Mitsuhiro , Watanabe Haruo , Izumiya Hidemasa
Published: November 2013

A real‐time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E‐test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.
Typing of bfpA Genes of Enteropathogenic Escherichia coli Isolated in Thailand and Japan by Heteroduplex Mobility Assay
Authors: Iida Mariko , Yamazaki Mitsugu , Yatsuyanagi Jun , Ratchtrachenchai Orn‐Anong , Subpasu Sarayoot , Okamura Noboru , Ito Kenitiro
Published: November 2013

We developed a rapid genetic approach for screening bfpA variants of enteropathogenic E. coli (EPEC) using a heteroduplex mobility assay (HMA). A total of 204 human EPEC strains were isolated in Thailand and Japan. Of 34 bfpA‐positive EPEC strains, bfpA variants were classified into 5 HMA‐types. Different HMA‐types were found in EPEC of the same serotypes. The results suggest that HMA is a simple and easy method to analyze polymorphism of bfpA gene, and can be used in laboratories without large apparatus such as sequencers.
CD8+ T Cells Play Disparate Roles in the Induction and the Effector Phases of Murine Experimental Allergic Conjunctivitis
Authors: Fukushima Atsuki , Yamaguchi Tomoko , Fukuda Ken , Sumi Tamaki , Kumagai Naoki , Nishida Teruo , Imai Shosuke , Ueno Hisayuki
Published: November 2013

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8α‐deficient (CD8KO) mice and wild‐type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW‐containing eye drops. Alternatively, EC was induced by transferring RW‐primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL‐5 and IL‐6 expression and greater upregulation of IL‐4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW‐primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.
Phylogenetic Analysis of Bacillus cereus Isolates from Severe Systemic Infections Using Multilocus Sequence Typing Scheme
Authors: Vassileva Maria , Torii Keizo , Oshimoto Megumi , Okamoto Akira , Agata Norio , Yamada Keiko , Hasegawa Tadao , Ohta Michio
Published: November 2013

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collection's strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.
Structural Requirements of Cholesterol for Binding to Vibrio cholerae Hemolysin
Authors: Ikigai Hajime , Otsuru Hiroshi , Yamamoto Koichiro , Shimamura Tadakatsu
Published: November 2013

Cholesterol is necessary for the conversion of Vibrio cholerae hemolysin (VCH) monomers into oligomers in liposome membranes. Using different sterols, we determined the stereochemical structures of the VCH‐binding active groups present in cholesterol. The VCH monomers are bound to cholesterol, diosgenin, campesterol, and ergosterol, which have a hydroxyl group at position C‐3 (3βOH) in the A ring and a C–C double bond between positions C‐5 and C‐6 (C–C Δ5) in the B ring. They are not bound to epicholesterol and dihydrocholesterol, which form a covalent link with a 3αOH group and a C–C single bond between positions C‐5 and C‐6, respectively. This result suggests that the 3βOH group and the C–C Δ5 bond in cholesterol are required for VCH monomer binding. We further examined VCH oligomer binding to cholesterol. However, this oligomer did not bind to cholesterol, suggesting that the disappearance of the cholesterol‐binding potential of the VCH oligomer might be a result of the conformational change caused by the conversion of the monomer into the oligomer. VCH oligomer formation was observed in liposomes containing sterols with the 3βOH group and the C–C Δ5 bond, and it correlated with the binding affinity of the monomer to each sterol. Therefore, it seems likely that monomer binding to membrane sterol leads to the assembly of the monomer. However, since oligomer formation was induced by liposomes containing either epicholesterol or dihydrocholesterol, the 3βOH group and the C–C Δ5 bond were not essential for conversion into the oligomer.
Genetic Variability of Human Cytomegalovirus UL132 Gene in Strains from Infected Infants
Authors: Sun Zheng Rong , Ji Yao Hua , Ruan Qiang , He Rong , Ma Yan Ping , Qi Ying , Mao Zhi Qin , Huang Yu Jing , Wang Yue Ping
Published: November 2013

Human cytomegalovirus (HCMV) displays genetic polymorphisms. HCMV infects a number of organs and cell types, leading to the hypothesis that HCMV disease and tissue tropism may be related to specific sequence variability. A gene in UL/b′ of HCMV, UL132 open reading frame (ORF), encodes glycoprotein (gpUL132) which is identified as a low‐abundance structural component of HCMV. In this study, the sequence variability of the UL132 gene was studied in 30 clinical strains. The results showed that a large number of nucleotide non‐synonymous substitutions occurred in the UL132 ORF, particularly in the 5′ half, in comparison to the UL132 of reference strain, Toledo. The UL132 variants of the clinical strains were clustered clearly into three major groups in the phylogenetic tree: G1(10/30), G2(9/30), and G3(11/30). The precise definition of UL132 genotypes and their putative functions would be helpful in a better understanding of the HCMV.
New Epitope Peptides Derived from Hepatitis C Virus (HCV) 2a Which Have the Capacity to Induce Cytotoxic T Lymphocytes in HLA‐A2+ HCV‐Infected Patients
Authors: Wang Yi , Takao Yukari , Harada Mamoru , Yutani Shigeru , Ide Tatsuya , Sata Michio , Itoh Kyogo , Yamada Akira
Published: November 2013

Because cytotoxic T lymphocytes (CTLs) play an important role in the specific immunotherapy of hepatitis C virus (HCV) infection, a series of CTL epitopes has been defined from HCV genotype 1a or 1b protein. Here, we attempted to identify HCV2a‐derived epitopes that are capable of inducing HLA‐A2‐restricted and peptide‐specific CTLs. Peripheral blood mononuclear cells (PBMCs) of HLA‐A2+ HCV2a‐infected patients or healthy donors were stimulated in vitro with each of the HCV2a‐derived peptides, which were prepared based on the HLA‐A2‐binding motif, and their peptide‐specific and HLA‐A2‐restricted cytotoxicities were examined. The HCV2a 432–441, HCV2a 716–724, and HCV2a 2251–2260 peptides were found to efficiently induce peptide‐specific CTLs from the PBMCs of HLA‐A2+ HCV2a‐infected patients. Cytotoxicity was mainly mediated by CD8+ T cells in a HLA class I‐restricted manner. These results indicate that the HCV2a 432–441, HCV2a 716–724, and HCV2a 2251–2260 peptides might be applicable for peptide‐based immunotherapy of HLA‐A2+ HCV2a‐infected patients.
Recombinant Vaccinia DIs Expressing Simian Immunodeficiency Virus Gag and Pol in Mammalian Cells Induces Efficient Cellular Immunity as a Safe Immunodeficiency Virus Vaccine Candidate
Authors: Okamura Tomotaka , Someya Kenji , Matsuo Kazuhiro , Hasegawa Atsuhiko , Yamamoto Naoki , Honda Mitsuo
Published: November 2013

A highly attenuated vaccinia virus substrain of Dairen‐I (DIs) shows promise as a candidate vector for eliciting positive immunity against immune deficiency virus. DIs was randomly obtained by serial 1‐day egg passages of a chorioarantoic membrane‐adapted Dairen strain (DIE), resulting in substantial genomic deletion, including various genes regulating the virus‐host‐range. To investigate the impact of that deletion and of the subsequent insertion of a foreign gene into that region of DIs on the ability of the DIs recombinant to induce antigen‐specific immunity, we generated a recombinant vaccinia DIs expressing full‐length gag and pol genes of simian immunodeficiency virus (SIV) (rDIsSIV gag/pol) and studied the biological and immunological characteristics of the recombinant natural mutant. The rDIsSIV gag/pol developed a tiny plaque on the chick embryo fibroblast (CEF). Viral particles of rDIsSIV gag/pol as well as SIV Gag‐like particles were electromicroscopically detected in the cytoplasm. Interestingly, the recombinant DIs strain grows well in CEF cells but not in mammalian cells. While rDIsSIV gag/pol produces SIV proteins in mammalian HeLa and CV‐1 cells, recombinant modified vaccinia Ankara strain (MVA) expressing SIV gag and pol genes (MVA/SIV239gag/pol) clearly replicates in HeLa and CV‐1 cell lines under synchronized growth conditions and produces the SIV protein in all cell lines. Moreover, intradermal administration of rDIsSIV gag/pol or of MVA/SIV239gag/pol elicited similar levels of IFN‐γ spot‐forming cells specific for SIV Gag. If the non‐productive infection characteristically induced by recombinant DIs is sufficient to trigger immune induction, as we believe it is, then a human immunodeficiency virus vaccine employing the DIs recombinant would have the twin advantages of being both effective and safe.
Analysis of Bacterial Community Structure in the Natural Circulation System Wastewater Bioreactor by Using a 16S rRNA Gene Clone Library
Authors: Niu Shi‐Quan , Fukushima Jun , Jiang Ying , Ishikawa Yuichi , Ueda Tohru , Matsumoto Satoshi
Published: November 2013

A variety of physical and chemical parameters are routinely monitored during operation of the Natural Circulation System, a wastewater purification bioreactor in which only natural materials and no synthetic chemicals are used. However, the microbial community structures existing in the Natural Circulation System have not been well characterized. Thus, bacterial community structure and composition in this system were studied using clone library analysis of 16S ribosomal RNA genes amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into the pGEM‐T vector. Each unique restriction fragment length polymorphism pattern, created by using two pairs of restriction endonucleases, was designated as an operational taxonomic unit (OTU). The Natural Circulation System comprises five tanks, the second and third of which play a major role in the bioreactor. Clone library profiles and principal component analysis revealed differences in the bacterial community structures of the second (anaerobic chamber) and the third (aerobic chamber) tanks. However, the β‐proteobacteria, Bacteroidetes/Chlorobi and γ‐proteobacteria groups were dominant in both tanks. Bacterial composition was more complex in the second tank (107 OTUs) than in the third tank (68 OTUs). Of a total of 154 OTUs in the clone libraries, only 21 were common to the two tanks. The results obtained in this study should provide important information for future research into and management of the Natural Circulation System wastewater bioreactor.
Screening of Upregulated Genes in Suckling Mouse Central Nervous System during the Disease Stage of Rabies Virus Infection
Authors: Ubol Sukathida , Kasisith Jitra , Mitmoonpitak Channarong , Pitidhamabhorn Dhanesh
Published: November 2013

The pathogenesis of hydrophobia remains unclear. The aim of this study was to identify the differentially upregulated genes that correlated with disease development in an experimental mouse model to provide better understanding of pathological mechanisms in rabies. The present work employed Clontech mouse array 1.2 II containing 1,176 gene transcripts. Suckling mice were intracerebrally infected with canine rabies virus. The gene expression profiles on day 2, 4 and 6 post inoculation were followed. The results show genes whose expression increased at least twofold above the control, mock‐infected brain. The numbers of genes showing altered expression level were 29, 109 and 98 genes on day 2, 4 and 6, respectively. The genes with altered expression were classified into eight major groups, namely immune response, metabolism, receptor and transporter, growth factors, death mediated factors, transcription and translation factors, proteases, and kinases. The numbers of upregulated genes during the disease stage was much higher than during the asymptomatic stage. This suggested that direct interaction between RABV and target cells induced massive destruction of a cellular homeostasis which may lead to functional termination of the CNS.
Prion Protein Gene‐Deficient Cell Lines: Powerful Tools for Prion Biology
Authors: Sakudo Akikazu , Onodera Takashi , Ikuta Kazuyoshi
Published: November 2013

Prion diseases are zoonotic infectious diseases commonly transmissible among animals via prion infections with an accompanying deficiency of cellular prion protein (PrPC) and accumulation of an abnormal isoform of prion protein (PrPSc), which are observed in neurons in the event of injury and disease. To understand the role of PrPC in the neuron in health and diseases, we have established an immortalized neuronal cell line HpL3–4 from primary hippocampal cells of prion protein (PrP) gene‐deficient mice by using a retroviral vector encoding Simian Virus 40 Large T antigen (SV40 LTag). The HpL3–4 cells exhibit cell‐type‐specific proteins for the neuronal precursor lineage. Recently, this group and other groups have established PrP‐deficient cell lines from many kinds of cell types including glia, fibroblasts and neuronal cells, which will have a broad range of applications in prion biology. In this review, we focus on recently obtained information about PrP functions and possible studies on prion infections using the PrP‐deficient cell lines.
Sporicidal Activity of the KMT Reagent in Its Vapor Phase against Geobacillus stearothermophilus Spores
Authors: Kida Nori , Mochizuki Yasushi , Taguchi Fumiaki
Published: November 2013

In an investigation of the sporicidal activity of the KMT reagent, a vapor phase study was performed using five kinds of carriers contaminated with Geobacillus stearothermophilus spores. When 25 ml of the KMT reagent was vaporized in a chamber (capacity; approximately 95 liters), the 2‐step heating method (vaporization by a combination of low temperature and high temperature) showed the most effective sporicidal activity in comparison with the 1‐step heating method (rapid vaporization). The 2‐step heating method appeared to be related to the sporicidal activity of vaporized KMT reagent, i.e., ethanol and iodine, which vaporized mainly when heated at a low temperature such as 55 C, and acidic water, which vaporized mainly when heated at a high temperature such as 300 C. We proposed that the KMT reagent can be used as a new disinfectant not only in the liquid phase but also in the vapor phase in the same way as peracetic acid and hydrogen peroxide.
Suppression of the Bacterial Antigen‐Specific T Cell Response and the Dendritic Cell Migration to the Lymph Nodes by Osteopontin
Authors: Begum Mst. Dilara , Umemura Masayuki , Kon Shigeyuki , Yahagi Ayano , Hamada Satoru , Oshiro Kiyotetsu , Gotoh Kazuyo , Nishizono Akira , Uede Toshimitsu , Matsuzaki Goro
Published: November 2013

Osteopontin (OPN) has been reported to enhance the interferon (IFN)‐γ‐producing Th1‐type T cell response through the induction of interleukin (IL)‐12 and the suppression of IL‐10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co‐inoculation of OPN suppressed the induction of IFN‐γ‐producing CD4+ T cells and T cell proliferative response after the subcutaneous heat‐killed Listeria monocytogenes (HKLM) immunization. These results suggest that OPN down‐regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow‐derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN‐treated DC showed a normal antigen‐presenting function to an established Listeria‐specific Th1‐type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM‐induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN‐mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration.
Virological and Immunological Characteristics of Fatal Virus‐Associated Haemophagocytic Syndrome (VAHS)
Authors: Kashiwagi Yasuyo , Kawashima Hisashi , Sato Satoshi , Ioi Hiroaki , Amaha Masahiro , Takekuma Kouji , Hoshika Akinori , Oshiro Hisashi , Matsubayashi Jun , Mukai Kiyoshi
Published: November 2013

We report three infants and one teenager with fatal virus‐associated haemophagocytic syndrome (VAHS). Two infants were admitted to our hospital because of cardio‐pulmonary arrest (CPA). The third infant was admitted to our department because of fever and liver dysfunction, and he was diagnosed as combined immunodeficiency with predominant T cell defect. The teenager was diagnosed as systemic lupus erythema (SLE) when she was 10 years old and admitted to our department because of fever and thrombocytopenia. The histological findings for the four patients' organs revealed many haemophagocytic cells. The patients were infected by Parainfluenza virus type 2 (PIV2), Enterovirus (EV), Cytomegalovirus (CMV), and Epstein‐Barr virus (EBV), respectively. Their laboratory data revealed elevated levels of ferritin and IL‐6, which also suggested virus‐associated haemophagocytic syndrome (VAHS). Although aggressive therapies were performed in all cases, the outcome was fatal. Further investigation would be needed to clarify the mechanism of VAHS and an effective therapeutic regimen is needed.
Autonomous Proliferation of HTLV−CD4+ T Cell Clones Derived from Human T Cell Leukemia Virus Type I (HTLV‐I)‐Associated Myelopathy Patients
Authors: Miyano‐Kurosaki Naoko , Kira Jun‐ichi , Barnor Jacob Samson , Maeda Naoyoshi , Misawa Naoko , Kawano Yuji , Tanaka Yuetsu , Yamamoto Naoki , Koyanagi Yoshio
Published: November 2013

That HTLV‐I infects CD4+ T cells and enhances their cell growth has been shown as successful long‐term in vitro proliferation in the presence of IL‐2. It is known that T cells isolated from HAM patients possess strong ability for cell proliferation in vitro and mRNA of various cytokines are abundantly expressed in CNS tissues of HAM patients. Hence, the cytokine‐induced proliferation could have an important role in pathogenesis and immune responses of HAM. In this study, we examined the relationship between cell proliferation and ability of in vitro cytokine production of CD4+ T cell clones isolated from HAM patients. We started a culture from a single cell to isolate cell clones immediately after drawing blood from the patients using limiting dilution method, which could allow the cell to avoid in vitro HTLV‐I infection after initiation of culture. Many cell clones were obtained and the rate of proliferation efficiency from a single cell was as high as 80%, especially in the 4 weeks' culture cells from HAM patients. These cells were classified as mainly Th0 phenotype that produce both IFN‐γ and IL‐4 after CD3‐stimulation. However, the frequency of proviral DNA in these cloned cells was significantly low. Our results indicate that the ability of cell proliferation in HAM patients is not restricted in HTLV‐I‐infected T cells. HTLV‐I‐uninfected CD4+ T cells, mainly Th0 cells, also have a strong ability to respond to IL‐2‐stimulation, showing that unusual immune activation on T cells has been observed in HAM patients.
Bacterial DNA Containing Methylated CpG Motifs Retains Immunostimulatory Activity in Synergy with Modified Lipopolysaccharides
Authors: Cho Yang Je , Ahn Bo Young , Song Eun Sun , Park Shin Ae , Lee Dong Hyeon , Kim Doo‐Sik , Lee Na Gyong
Published: November 2013

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF‐κB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.
Colonization and Turnover of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in Otitis‐Prone Children
Authors: Yokota Shin‐ichi , Harimaya Atsushi , Sato Kiyoshi , Somekawa Yukihiro , Himi Tetsuo , Fujii Nobuhiro
Published: November 2013

Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitis‐prone and 2 non‐prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitis‐prone children even after administration of antibiotics but was not from the non‐prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis‐prone children.
Occurrence of RD9 Region and 500 bp Fragment among Clinical Isolates of Mycobacterium tuberculosis and Mycobacterium bovis
Authors: Das Samir , Das Suresh Chandra , Verma Rishendra
Published: November 2013

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no amplification product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.
Identification of DDX1 as a JC Virus Transcriptional Control Region‐Binding Protein
Authors: Sunden Yuji , Semba Shingo , Suzuki Tadaki , Okada Yuki , Orba Yasuko , Nagashima Kazuo , Umemura Takashi , Sawa Hirofumi
Published: November 2013

To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV‐TCR). Using nuclear extracts from the JCV‐susceptible neuroblastoma cell line IMR‐32, EMSA revealed a 670 kDa JCV‐TCR‐binding protein complex designated as #3‐bp. This complex could not be detected in nuclear extracts from non‐susceptible cell lines. Using column chromatographic purification and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3‐bp. However, as CstF is present in many cell types, we speculated that the IMR‐32‐specific component(s) of #3‐bp bind CstF. We performed a yeast two‐hybrid assay using CstF‐77 as the bait against a HeLa cDNA‐subtracted IMR‐32 cDNA library. This analysis detected binding between CstF‐77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV‐TCR. Our findings indicate that an association between DDX1 and the JCV‐TCR may play a significant role in JCV infection in IMR‐32 cells.
Cytolethal Distending Toxin (Cdt)‐Producing Escherichia coli Isolated from a Child with Bloody Diarrhea in Japan
Authors: Hinenoya Atsushi , Nagita Akira , Asakura Masahiro , Tsukamoto Teizo , Ramamurthy Thandavarayan , Nair Gopinath Balakrish , Takeda Yoshifumi , Yamasaki Shinji
Published: November 2013

In a retrospective analysis by PCR, the cdtI gene encoding the cytolethal distending toxin (Cdt) was detected in Escherichia coli O2:H12 strain isolated from the bloody diarrheal stool specimen of a child. To our knowledge, this is the first report showing the possible association of Cdt‐producing E. coli in Japan, particularly in a child with bloody diarrhea.
Prediction of Efficient Virological Response to Pegylated Interferon/Ribavirin Combination Therapy by NS5A Sequences of Hepatitis C Virus and Anti‐NS5A Antibodies in Pre‐Treatment Sera
Authors: El‐Shamy Ahmed , Sasayama Mikiko , Nagano‐Fujii Motoko , Sasase Noriko , Imoto Susumu , Kim Soo Ryang , Hotta Hak
Published: November 2013

A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV‐1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV‐1b‐infected patients were treated with pegylated interferon/ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti‐NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non‐EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N‐terminally flanking region, which we refer to as interferon/ribavirin resistance‐determining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was significantly larger for EVR(16w) compared with Non‐EVR(16w). Moreover, HCV‐1b isolates with ≥5 mutations in V3 or those with ≥6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti‐NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (≥5) or IRRDR (≥6) and the presence of detectable levels of anti‐NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (≤4) or IRRDR (≤5) together with the absence of detectable anti‐NS5A antibodies could be a predictive factor for Non‐EVR(16w).
Isolation and Characterization of Virulent Yellowtail Ascites Virus
Authors: Hirayama Takeshi , Nagano Ichiro , Shinmoto Hajime , Yagyu Ken‐ichi , Oshima Syun‐ichirou
Published: November 2013

Yellowtail ascites virus (YTAV) is the causative agent of ascites and deformity in fish and causes serious losses to the fish‐farming industry of yellowtail fry and fingerling Seriola quinqueradiata in Japan. In 2006, cultured yellowtail died from ascites in Kochi, Japan. We isolated and characterized a virus from the diseased fish. Based on the pathogenicity, culture characteristics, morphological features, RT‐PCR results targeting VP2/NS region, phylogeny based on the VP1 amino acid sequence, and immunochemical reactivity of structural proteins, the virus isolate was identified as YTAV (designated as YTAV‐06). YTAV‐06 was a more virulent isolate than YTAV Y‐6, isolated originally from yellowtail with ascites. To our knowledge, this is the first report describing that YTAV isolates may vary in their virulence.
Trafficking of QD‐Conjugated MPO‐ANCA in Murine Systemic Vasculitis and Glomerulonephritis Model Mice
Authors: Hoshino Akiyoshi , Nagao Tomokazu , Ito‐Ihara Toshiko , Ishida‐Okawara Akiko , Uno Kazuko , Muso Eri , Nagi‐Miura Noriko , Ohno Naohito , Tokunaka Kazuhiro , Naoe Shiro , Hashimoto Hiroshi , Yasuhara Masato , Yamamoto Kenji , Suzuki Kazuo
Published: November 2013

In systemic vasculitis, the serum level of myeloperoxidase (MPO)‐specific anti‐neutrophil cytoplasmic autoantibodies (MPO‐ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO‐ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO‐ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD‐conjugated MPO‐ANCA (ANCA‐QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO‐ANCA and surface‐translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.
Contribution of Multi‐Antimicrobial Resistance to the Population of Antimicrobial ResistantEscherichia coliIsolated from Apparently Healthy Pigs in Japan
Authors: Harada Kazuki , Asai Tetsuo , Kojima Akemi , Sameshima Toshiya , Takahashi Toshio
Published: November 2013

The aim of this study was to clarify the involvement of tetracycline usage in resistance rates against other antimicrobials. Antimicrobial susceptibility testing was carried out on 545 porcineEscherichia coliisolates throughout Japan. As the result of analyzing by regions, resistance rates against kanamycin, oxytetracycline and trimethoprim in the Kanto/Koshinetu district were higher than those in some other districts. High resistance rates against kanamycin or trimethoprim in oxytetracycline‐resistant isolates were also observed in the Kanto/Koshinetu district The prevalence of multi‐antimicrobial resistance through co‐selection of resistances against kanamycin or trimethoprim by tetracycline usage could be the cause of regional differences in these resistances in porcineE. coli. By a communicative surveillance, kanamycin‐ and trimethoprim‐resistance rates were likely to be elevated with tetracycline usage. Thus, usage of specific antimicrobial(s) is a remarkable viewpoint to control antimicrobial resistant bacteria.
WT1 (Wilms' Tumor 1) Peptide Immunotherapy for Renal Cell Carcinoma
Authors: Iiyama Tatsuo , Udaka Keiko , Takeda Sachihiko , Takeuchi Tamotsu , Adachi Yoshihiro C. , Ohtsuki Yuji , Tsuboi Akihiro , Nakatsuka Shin‐ichi , Elisseeva Olga A. , Oji Yusuke , Kawakami Manabu , Nakajima Hiroko , Nishida Sumiyuki , Shirakata Toshiaki , Oka Yoshihiro , Shuin Taro , Sugiyama Haruo
Published: November 2013

Tumor‐specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA‐A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide‐specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.
Serological Evidence of Leptospiral Infection in Pig Populations in Different Districts in Japan
Authors: Naito Michiko , Sakoda Yoshihiro , Kamikawa Takayuki , Nitta Yoshiki , Hirose Kazuhiko , Sakashita Mitsuaki , Kurokawa Satoru , Kida Hiroshi
Published: November 2013

Serum samples were collected from 938 pigs of 24 farms in Hokkaido, Kagoshima, and Okinawa prefectures in Japan in 2001–2005. Enzyme‐linked immunosorbent assay (ELISA) was used for the detection of antibodies to LipL32 antigen which is common to Leptospira interrogans. Samples positive in ELISA were then investigated by microscopic agglutination test for the identification of causal leptospires. Antibodies specific to leptospires of serovars Copenhageni, Bratislava, Australis and Javanica were detected in serum samples of pigs from each of the three districts. In addition, antibodies to leptospires of serovars Autumnalis and Tarassovi were predominantly detected in those from Kagoshima. The present study, thus, revealed that leptospires belonging to different serovars prevail in the pig population in Japan. In addition, it is the first detection of antibodies to leptospires belonging to serovars Javanica and Tarassovi in pigs in Japan.
Characteristics of a Pink‐Pigmented Bacterium Isolated from Biofilm in a Cooling Tower in Tokyo, Japan
Authors: Furuhata Katsunori , Goto Keiichi , Kato Yuko , Saitou Keiko , Sugiyama Jun‐ichi , Hara Motonobu , Yoshida Shin‐ichi , Fukuyama Masafumi
Published: November 2013

Strain K‐20, a Gram‐negative, non‐motile, non‐spore‐forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3–94.6 sequence similarities. Growth occurred at 25–40 C and pH 5.0–10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of ≧2% NaCl. The API 20NE identification system gave a positive result for urease, L‐arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K‐20 were C18:1Δ11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q‐10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K‐20 (=JCM 14634) is a novel species of the genus Roseomonas.
Adjuvant Effect of CpG‐Oligodeoxynucleotide in Anti‐Fungal Chemotherapy against Fatal Infection with Cryptococcus neoformans in Mice
Authors: Kinjo Takeshi , Miyagi Kazuya , Nakamura Kiwamu , Higa Futoshi , Gang Xiao , Miyazato Akiko , Kaku Mitsuo , Fujita Jiro , Kawakami Kazuyoshi
Published: November 2013

Cryptococcal meningoencephalitis is a life‐threatening infectious disease in immunocompromised patients. Unmethylated CpG‐oligodeoxynucleotides (CpG‐ODN) protects hosts in a mouse model. In the present study, we tested the adjuvant effect of CpG‐ODN in anti‐fungal chemotherapy. Administration of either fluconazole (FLCZ) or CpG‐ODN was effective in extending survival, accelerating clearance of fungi and preventing disseminated infection. Combination of both agents provided more beneficial effect than either agent alone. Cytokine balance in the infected lungs was biased to Th1‐type response by CpG‐ODN, while FLCZ did not further promote. These results suggest that CpG‐ODN is a promising adjuvant in chemotherapy against this infection.
Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria
Authors: Soejima Takashi , Iida Ken‐ichiro , Qin Tian , Taniai Hiroaki , Seki Masanori , Takade Akemi , Yoshida Shin‐ichi
Published: November 2013

Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double‐stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single‐stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.