bluuebunny/crossref_metadata_2025 huggingface dataset:
T Cell Response to Borrelia garinii, Borrelia afzelii, and Borrelia japonica in Various Congenic Mouse Strains
Authors: Ishii Norihisa , Isogai Emiko , Isogai Hiroshi , Kimura Koichi , Nishikawa Takeshi , Fujii Nobuhiro , Nakajima Hiroshi
Published: November 2013

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross‐reactivity between Borrelia antigens was observed, but there was no cross‐reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed‐type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4+8− and Ia− T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8+ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.
Isolation of K88 Antigen Variants (ab, ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes
Authors: González Enrique A. , Vázquez Froilán , Garabal José Ignacio , Blanco Jorge
Published: November 2013

Fimbriae isolation by means of thermal shock was applied to fifteen K88‐positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88‐positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris‐HCl for isolation of K88 antigen. The molecular weights by SDS‐PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.
Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication
Authors: Kodama Eiichi , Igarashi Akira , Mori Shuichi , Hashimoto Kou‐Ichi , Suzuki Toshimitsu , De Clercq Erik , Shigeta Shiro
Published: November 2013

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV‐induced cytopathogenicity in gastric adenocarcinoma MKN‐28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV‐1 and HSV‐2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC50) values of the antiherpes agents in MKN‐28 cells were compared with those in human embryo lung MRC‐5 cells, all compounds, except for BVDU, showed higher EC50 values in MKN‐28 cells. For BVDU the EC50 values in MKN‐28 cells were 0.8 (HSV‐1) and 0.036 (HSV‐2) times the EC50 values in MRC‐5 cells. Thus BVDU was 27.5 times more active against HSV‐2 in MKN‐28 cells than in MRC‐5 cells. The MKN‐28 gastric cancer cells may be useful for the rapid screening of anti‐HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.
Stability of Hemagglutinating Activity of Extracellular and Intracellular Forms of Japanese Encephalitis Virus Exposed to Acidic pH
Authors: Nawa Masaru
Published: November 2013

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH‐dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA‐inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100‐ to 1,000‐fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell‐grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.
In Vitro Effects of Immunosuppressive Agents on Cytokine Production by HTLV‐I‐Infected T Cell Clones Derived from the Ocular Fluid of Patients with HTLV‐I Uveitis
Authors: Sagawa Kimitaka , Mochizuki Manabu , Katagiri Kazuko , Tsuboi Izumi , Sugita Sunao , Mukaida Naofumi , Itoh Kyogo
Published: November 2013

The present study was designed to investigate the in vitro effects of potential therapeutic agents on cytokine production by five HTVL‐I‐infected T cell clones (TCC) established from the ocular fluid of patients with HTLV‐I uveitis. Each of the five HTLV‐I‐infected TCC was cultured at 1 × 106 cells/ml with or without an immunosuppressive agent (hydrocortisone, FK506, rapamycin, indomethacin, or prostaglandin E2) for 22 hr in humidified 5% CO2 in air at 37 C. The production of various cytokines in the culture supernatant from each TCC was measured by ELISA. The HTLV‐I‐infected TCC produced high amounts of IL‐1α, IL‐3, IL‐6, IL‐8, TNF‐α, IFN‐γ, and GM‐CSF, and low but significant levels of IL‐2 and IL‐10 without any stimuli. Hydrocortisone severely depressed the production by these TCC of all the cytokines except for IL‐2, which was slightly increased. Prostaglandin E2 depressed the production of IL‐1α, while it up‐regulated the production of IL‐6, TNF‐α, and IFN‐γ. Rapamycin depressed the production of IL‐6 and TNF‐α, and FK506 depressed the production of TNF‐α. Hydrocortisone also severely depressed the cytokine production by PHA‐stimulated peripheral blood mononuclear cells obtained from healthy volunteers. Of the immunosuppressive agents tested, hydrocortisone exhibited the strongest suppression of cytokine production by HTLV‐I‐infected TCC. This result was in agreement with the in vivo effects of hydrocortisone in patients with HTLV‐I uveitis. These TCC will be useful in investigating the effects of potential therapeutic agents for HTLV‐I uveitis in vitro.
Differential Regulation of HLA‐DR Expression and Antigen Presentation in Toxoplasma gondii‐Infected Melanoma Cells by Interleukin 6 and Interferon γ
Authors: Yang Tian‐Hui , Aosai Fumie , Norose Kazumi , Ueda Masakatsu , Yano Akihiko
Published: November 2013

CD4+ cytotoxic T lymphocytes (CTL) clones, YT‐4 and YT‐9, specific for Toxoplasma gondii (T. gondii)‐infected melanoma SK‐MEL 28 (P36), were generated from the peripheral blood lymphocytes (PBL) of a patient with chronic toxoplasmosis. These CTL clones were shown to secrete significant amounts of interleukin 6 (IL‐6) and interferon γ (IFN‐γ) upon antigen (Ag)‐specific stimulation. Downregulation of human leukocyte antigen (HLA)‐DR surface expression and HLA‐DR mRNA levels in P36 cells were observed when P36 cells were infected with T. gondii. Such downregulated HLA‐DR expressions of 71 gondii‐infected P36 cells were upregulated by treatment with both recombinant IL‐6 (rIL‐6) and recombinant IFN‐γ (rIFN‐γ). The antigen‐presenting ability of T. gondii‐infected P36 cells to T. gondii‐infected cell‐specific CTL was enhanced by rIFN‐γ but not by rIL‐6. The present study reveals the existence of differential regulation of HLA‐DR expression and Ag presentation in T. gondii‐infected melanoma cells by IL‐6 and IFN‐γ.
Hepatitis B Virus with X Gene Mutation Is Associated with the Majority of Serologically “Silent” Non‐B, Non‐C Chronic Hepatitis
Authors: Fukuda Ryo , Ishimura Norihisa , Kushiyama Yoshinori , Moriyama Nobuyuki , Ishihara Shunji , Chowdhury Aktaruzzaman , Tokuda Alejandro , Sakai Shino , Akagi Shuji , Watanabe Makoto , Fukumoto Shiro
Published: November 2013

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non‐B, non‐C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8‐nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C‐terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.
Quantification of the CD55 and CD59, Membrane Inhibitors of Complement on HIV‐1 Particles as a Function of Complement‐Mediated Virolysis
Authors: Nakamura Mariko , Okada Hidechika , Sasaki Hiroyuki , Yoshida Kiyotsugu , Kamada Minori , Okada Noriko , Terada Masaki , Ohno Tsuneya
Published: November 2013

Previous studies have demonstrated that the murine monoclonal antibody (MoAb) NM‐01 activates the human complement classical pathway resulting in lysis of human immunodeficiency virus (HIV). The present study was performed to determine the availability of the V3‐loop of gp120 relative to the complement regulatory proteins, CD55 (DAF) and CD59 (HRF20) molecules on HIV. The results demonstrate that CD55 and CD59 exist on HIV virions, along with gp120 molecules. These findings suggest that activation of human complement on free viral particles is induced by MoAb NM‐01 and that this occurs regardless of the presence of CD55 and CD59 molecules. The destruction of viral particles was demonstrated by a decrease in infectivity. The involvement of human complement in this process was confirmed with an immunoelectron microscopy technique by the presence of a human C9 to prove membrane attack complex (MAC). The results indicate that NM‐01 can induce complement activation because of the ratios of CD55 and CD59 to gp120 molecules on HIV virions. The availability of the gp120 V3 domain on the virion is sufficient for binding of NM‐01 and thereby the formation of MAC that results in virolysis.
Does Coinfection ofBartonella henselaeand FIV Induce Clinical Disorders in Cats?
Authors: Ueno Hiroshi , Hohdatsu Tsutomu , Muramatsu Yasukazu , Koyama Hiroyuki , Morita Chiharu
Published: November 2013

It was found thatBartonella henselae (B. henselae)may induce clinical disorders in cats in natural conditions from a comparison of the serological status forB. henselaewith the serostatus for feline immunodeficiency virus (FIV) and several clinical characteristics in 170 domestic cats. Seropositivity forB. henselaewas not significantly different between FIV antibody‐positive and ‐negative cats (18.4% vs 16.0%). The incidence of clinical characteristics were compared among four cat groups distinguished by the reactivity of sera againstB. henselaeand FIV. The incidence of lymph node swelling was lower in only FIV antibody‐positive cats (3.0%), but higher inB. henselaeantibody‐positive cats (13.6%) and significantly higher in bothB. henselaeand FIV antibody‐positive cats (42.9%) compared with the incidence of lymph node swelling in cats which were negative for both antibodies (5.5%). The same relation was also observed for the incidence of gingivitis among the 4 cat groups, suggesting that coinfection ofB. henselaeand FIV may be associated with gingivitis and lymphadenopathy in cats.
Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O‐Antigenic Factors
Authors: Hisatsune Kazuhito , Kondo Seiichi , Iguchi Takehiro , Ito Teruyo , Hiramatsu Keiichi
Published: November 2013

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O‐polysaccharide chain composed of an α(1 → 2)‐linked N‐(3‐deoxy‐L‐glycero‐tetronyl)‐D‐perosamine (4‐amino‐4,6‐dideoxy‐D‐manno‐pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N‐(3‐deoxy‐L‐glycero‐tetronyl)‐2‐O‐methyl‐D‐perosamine at the non‐reducing terminus of the O‐polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co‐existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O‐antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O‐forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2‐O‐methyl group at the non‐reducing terminus of the α(1 → 2)‐linked N‐(3‐deoxy‐L‐glycero‐tetronyl)‐D‐perosamine homopolymer that forms the O‐polysaccharide chain of LPS of V. cholerae O1 (Ogawa).
Kinetics of Organ‐Associated Natural Killer Cells and Intermediate CD3 Cells during Pulmonary and Hepatic Granulomatous Inflammation Induced by Mycobacterial Cord Factor
Authors: Tabata Akihiro , Kaneda Kenji , Watanabe Hisami , Abo Toru , Yano Ikuya
Published: November 2013

We investigated here the kinetics of natural killer (NK) cells and extrathymic T cells, which include intermediate CD3 cells and γδ T cells, in the cord factor‐induced granulomatous inflammation of the lungs and liver. In Balb/c mice, pulmonary inflammation elevated the proportion of NK cells and that of extrathymic T cells to mononuclear cells in the lungs. C3H/He mice exhibited shorter‐term inflammation of the lungs than Balb/c mice and accordingly showed a smaller increase in the proportions of pulmonary NK cells and intermediate CD3 cells. In the liver of Balb/c mice, hepatic NK cells increased as well with the granulomatous changes, while intermediate CD3 cells exhibited a transient decrease before they increased. The present study has demonstrated that granulomatous inflammation is accompanied by the increase of lung‐associated NK cells and extrathymic T cells and that there exists a difference between these two mouse strains in the induction of these lymphocyte subsets by cord factor.
Purified Protein from Salmonella typhimurium Inhibits the Interleukin‐2 Response of Murine Splenic T‐Lymphocytes Activated with Anti‐CD3 Antibody
Authors: Matsui Katsuhiko
Published: November 2013

Previously, we demonstrated that the immunosuppression induced by a purified preparation of Salmonella typhimurium‐derived inhibitor of T‐cell proliferation (STI) can be observed in terms of suppression of the proliferation of murine spleen cells stimulated with a mitogenic lectin. In the present study, I observed that STI inhibited the interleukin‐2 (IL‐2) response of purified murine splenic T lymphocytes stimulated with anti‐CD3 antibody. The flow cytometric analysis of IL‐2 receptor (IL‐2R) expression on T cells showed that STI specifically suppressed the expression of IL‐2Rβ and IL‐2Rγ. Furthermore, when the IL‐2‐dependent T‐cell line CTLL‐2 was incubated with STI, the growth of CTLL‐2 cells was significantly inhibited. These results suggest that the target cells for STI are T cells themselves, and that the suppression of T‐cell proliferation induced by STI might involve a defect in the IL‐2 receptor (IL‐2R) function of T cells.
Adhesiveness for Extracellular Matrices and Lysosomal Enzyme Release from Normal andβ2Integrin‐Deficient Bovine Neutrophils
Authors: Nagahata Hajime , Higuchi Hidetoshi , Noda Hiroshi , Tamoto Koichi , Kuwabara Mikinori
Published: November 2013

The adhesiveness of control and CD18‐deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release ofN‐acetyl‐β‐D‐glucosaminidase (NAGase) from control and CD18‐deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18‐deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P< 0.05); however, similar adhesion to laminin was observed in CD18‐deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P< 0.05) with the presence of PMA. The mean activities for NAGaserelease from CD18‐deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg‐IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg‐IgG‐induced NAGaserelease from control and CD18‐deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions.
Coxsackievirus B3‐Induced Chronic Myocarditis in Mouse: Use of Whole Blood Culture to Study the Activation of TNFα‐Producing Cells
Authors: Hober Didier , Andreoletti Laurent , Shen Lu , Copin Marie‐Christine , Desmidt Angelique , Wattré Pierre
Published: November 2013

The pathogenesis of CVB3‐induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNFα secretion in vitro using whole blood culture in CVB3‐induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5 × 105 pfu of CVB3, and mice were mock‐infected. Thirty‐one days post‐infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT‐PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 μl) was cultured for 4 hr and 24 hr in sterile 96 well‐plate containing 225 μl RPMI in the presence or the absence of activators (LPS + PHA). The TNFα levels in the whole blood from mock‐infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNFα was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNFα was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNFα was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNFα was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post‐infection. The increased TNFα level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNFα level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus‐induced disease in a small animal model.
Molecular Epidemiology of Japanese Encephalitis Virus in Okinawa
Authors: Ma Shao‐Ping , Arakaki Sakae , Makino Yoshihiro , Fukunaga Toshihiko
Published: November 2013

In order to elucidate the molecular characteristics of Japanese encephalitis (JE) virus in Okinawa, 23 strains of JE virus isolated in a 25‐year span were sequenced for the 240 nucleotides of the C‐preM junction region and 111 nucleotides of the E gene region and compared with those of reference strains isolated in mainland Japan. The results of phylogenic analysis showed that although all the Okinawan isolates showed more than 96% homology in the nucleotide sequence in each region, they were chronologically divided into two groups: the old group (nine strains) and a new group (14 strains). On the other hand, in a comparison with reference strains in mainland Japan, the Okinawan isolates showed more than 94% nucleotide sequence homology in both regions, indicating that the Okinawan strains belong to the same genotype as that of JE strains in mainland Japan. The nucleotide homology of the old group was relatively higher than that of the new group. Among the 14 strains in the new group, 13 strains were isolated from mosquitoes collected from a pig farm from 1986 through 1992. These strains showed higher nucleotide divergence than the old group strains, isolated from mosquitoes and swine sera collected at several sites, in both regions. A nucleotide substitution at the position 1920 in the E gene was identified in three isolates. This substitution generated an asparagine‐proline‐threonine sequence capable of serving as an attachment site of carbohydrate.
The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil‐DNA Glycosylase
Authors: Chung Yang‐Tsung , Hsu Weili
Published: November 2013

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV‐1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil‐DNA glycosylase (UDGase) signature sequence. To determine whether the BHV‐1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET‐28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β‐D‐thiogalactopyranoside these cells produced a 23‐kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one‐step purification procedure using nickel‐chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV‐1 UL2 gene does encode a UDGase.
Proliferation and Teratogenicity of Aino Virus in Chick Embryos
Authors: Kitano Yoshio , Ohzono Hiroyuki , Shimizu Tsutomu
Published: November 2013

Aino virus (AIV; JaNAr 28 strain) 103 TCID50/0.2 ml was inoculated in the yolk sac of 8‐day‐old chick embryos. Recovery and titration of the virus from various organs including the central nervous system (CNS) and skeletal muscle were performed at 2, 4, 7, 10 and 13 days after inoculation (PI). AIV was systemically disseminated and proliferated even 2 days PI. The titers of the recovered virus from the CNS and from skeletal muscle was the highest at 4 days PI and declined with time, whereas hydranencephaly, arthrogryposis and cerebellar hypoplasia developed at 7 days PI and gradually progressed until 13 days PI.
Crystallization of Synthetic Escherichia coli‐Type Lipid A
Authors: Kato Nobuo , Naito Setsuko , Arakawa Yoshichika , Sugiyama Tsuyoshi , Ito Hideo , Ohta Michio , Sasaki Kyoyu
Published: November 2013

Synthetic Escherichia coli‐type lipid A formed hexagonal plate crystals when it was precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 C for 10 days. Analyses of crystals by electron diffraction and synchrotron X‐ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 Å and the longitudinal axis (perpendicular to the basal plane) of 49.3 ±1.3 Å. Results suggest that the previous finding that various kinds of R‐form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J. Bacteriol., 172: 1516‐1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.
Tumor‐Specific Transplantation Resistance in Mice after Treatment of Initial Tumors with Streptococcus thermophilus
Authors: Kaklij Gangadhar Supadu , Kelkar Shashikant Madan
Published: November 2013

Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated. The mice cured from fibrosarcoma by treatment with heat‐killed preparation of S. thermophilus, when challenged with fibrosarcoma failed to take up the tumor. However, these cured mice when challenged with sarcoma‐180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls. Similarly, mice cured from sarcoma‐180 were challenged with fibrosarcoma, sarcoma‐180 or Ehrlich ascites carcinoma. Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma‐180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls. However, there was no difference in the growth rate of fibrosarcoma. Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors. These findings thus suggest that the antitumor response was tumor‐specific and that tumor‐associated antigens may have a role in imparting this specificity. Bacterial treatment non‐specifically augmented this primary response.
Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China
Authors: Xu Jian‐Guo , Cheng Bo‐Qun , Wu Yan‐Ping , Huang Li‐Bao , Lai Xin‐He , Liu Bing‐Yang , Lo Xing‐Zu , Li Hun‐Fen
Published: November 2013

One hundred and seventy‐two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp‐2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp‐2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non‐O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy‐two strains (41.9%) hybridized with shiga‐like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe‐positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe‐positive strains might represent a new variety of verotoxin‐producing E. coli (VTEC).
Previous Enteric Adenovirus Infection Does Not Protect against Subsequent Symptomatic Infection: Longitudinal Follow‐up of Eight Infants
Authors: Unicomb Leanne E. , Jarecki‐Khan Kerstin , Hall Andrew , Podder Goutham
Published: November 2013

Eight infants followed longitudinally were found to have enteric adenovirus (EAdv) infections: in 5 infants with diarrhea and in 3 with no accompanying diarrhea. Sequential stool samples prior to EAdv infections were tested for adenovirus antigen, anti‐adenoviral IgA and neutralizing antibodies to serotypes 40, 41 and 2 in order to ascertain whether protection from symptoms was due to prior infection. No difference was found in the number of adenoviral infections among infants prior to their EAdv infections with or without accompanying diarrhea. However, in 3 of the 5 infants in whom EAdv infection was accompanied by diarrhea and 2 of 3 control infants, previous EAdv infections had occurred as detected by serotype‐specific antibody rises.
Murine Humoral Immune Response against Recombinant Structural Proteins of Hepatitis C Virus Distinct from Those of Patients
Authors: Ahmed Mamun , Shikata Toshio , Esumi Mariko
Published: November 2013

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti‐E1 and anti‐E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.
Airway Interleukin‐8 in Elderly Patients with Bacterial Lower Respiratory Tract Infections
Authors: Ponglertnapagorn Preecha , Oishi Kazunori , Iwagaki Akitaka , Sonoda Fuminari , Watanabe Kiwao , Nagatake Tsuyoshi , Matsushima Kouji , Matsumoto Keizo
Published: November 2013

We investigated the interleukin‐8 (IL‐8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL‐8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL‐8 at a range of 6.25‐25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype‐1 strain by human neutrophils in the presence of a serotype‐specific anti‐lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL‐8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL‐8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.
Detection and Measurement of Staphylococcus epidermidis Slime Using an ELISA Technique
Authors: Mempel Martin , Günther Bernd , Müller Edgar , Grüter Ludwig
Published: November 2013

A polyclonal rabbit anti‐serum against the strong slime‐producing Staphylococcus epidermidis strain RP62A was absorbed with the slime‐negative phase variant of this strain PV1 in order to remove not slime‐specific antibodies. Using this antiserum we established an ELISA which enables detection of slime production in S. epidermidis extracts. The ELISA showed high absorbance when extracts from slime‐positive strains (confirmed in the tissue culture tube test) were used as antigens. The high absorbance of slime‐positive strains was greatly reduced by periodate oxidation of the extracts and was resistant to proteinase digestion suggesting that the detected antigen is composed of polysaccharides. In contrast to other rapid and simple laboratory detection methods for S. epidermidis slime, the slime‐specific ELISA gave positive results in the presence of human serum.
Isolation of Coxiella burnetii from Children with Influenza‐Like Symptoms in Japan
Authors: Nagaoka Hiromi , Akiyama Masato , Sugieda Masaaki , Nishio Tomohiro , Akahane Sousuke , Hattori Hiroshi , Ho To , Fukushi Hideto , Hirai Katsuya
Published: November 2013

The prevalence of Coxiella burnetii antibodies was investigated by indirect immunofluorescence (IF) test in 55 paired sera (acute and convalescent phases) of school children who had influenza‐like symptoms. Of the convalescent serum samples examined, 18 (32.7%) sera reacted positively to phase II antigen of C. burnetii. Coxiella‐like organism was isolated from the sera of 13 children after injection of the 18 acute phase sera into mice. The organism was identified as C. burnetii by Giemsa staining and the IF antigen test of mouse spleen smears, the polymerase chain reaction (PCR) method, electron microscopic observations of the mouse spleen cells, and the IF antibody test of mouse sera. This is the first report of isolation of C. burnetii from serum specimens of children having influenza‐like symptoms. The evidence that C. burnetii was isolated from people indigenous to Japan at a considerably high incidence suggested that C. burnetii may be widespread as a cause of influenza‐like symptoms in Japan.
A Study of Two Procedures of HIV‐1 Isolation from Whole Blood Cultures
Authors: Shen Lu , Hober Didier , Benyoucef Samira , Ajana Faïza , Gérard Yann , Lion Georges , Vermersch Annie , Bocket‐Mouton Laurence , Mouton Yves , Wattré Pierre
Published: November 2013

Culture techniques for isolation of HIV‐1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV‐1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)‐stimulated peripheral blood mononuclear cells (PHA‐PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 μl of WB with 1 × 106 PHA‐PBMC from donors; and procedure II based on the culture of 500 μl of WB with 4× 106 PHA‐PBMC from donors. The cocultures were placed in 24‐well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL‐2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA‐PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV‐1 p24 antigen in an enzyme immunoassay. The kinetics of HIV‐1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV + patients the isolation rate was higher with heparin‐ than with EDTA‐treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV‐1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV‐I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV‐1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n=4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV‐1 isolation from WB increased in advanced‐stage patients. Further studies are needed to define the clinical applications of WB culture.
Detection and Sequencing of Norwalk‐Like Viruses from Stool Samples in Japan Using Reverse Transcription‐Polymerase Chain Reaction Amplification
Authors: Nakayama Miyuki , Ueda Yuhichi , Kawamoto Hiroyoshi , Han‐jun Yue , Saito Kunihiko , Nishio Osamu , Ushijima Hiroshi
Published: November 2013

Norwalk‐like viruses were detected in Japan in 12% (26/209) of patients with nonbacteria and non‐rotavirus gastroenteritis in an outpatient clinic of a hospital from 1991 to 1994 by reverse transcription‐polymerase chain reaction. They were also present in 7% (26/378) of total samples including those from rotavirus‐positive gastroenteritis patients. In addition, the viruses were recovered in samples from 15 of 17 patients which were collected during outbreaks of gastroenteritis in various places in Japan by the same method. The DNA sequence of the polymerase region from patients at the hospital (sporadic cases) showed that two subgenogroups, similar to UK1‐6 in genogroup G1 and to UK1‐1 in genogroup G2 (Ando et al, J Clin Microbiol, 1995, 33: 64‐71) exist in Japan. The latter was more frequently found.
Relationship of HIV‐1 Envelope V2 and V3 Sequences of the Primary Isolates to the Viral Phenotype
Authors: Yoshimura Kazuhisa , Matsushita Shuzo , Hayashi Azusa , Takatsuki Kiyoshi
Published: November 2013

We examined the relationship between the amino acid sequences of the V2 and V3 regions of the envelope protein and the biological properties of ten human immunodeficiency virus type 1 (HIV‐1) primary isolates. The infectivity, cytopathic effect (CPE), and syncytium forming activity of these primary isolates were tested against three T cell lines (CEM, MT2, and MOLT4/CL.8 cells), CD8‐depleted peripheral blood mononuclear cells (PBMC), and primary monocyte‐derived macrophages (MDM) from seronegative donors. In addition to the viral groups which had the syncytium inducing/T‐cell line tropic (SI/TT) phenotype or non‐syncytium inducing/non‐T cell line tropic (NSI/NT) phenotype (including the NSI/macrophage tropic (NSI/MT) phenotype), there was a group of viruses that infected one or two T cell lines and PBMC but could not mediate syncytium formation. We therefore classified this group of viruses as a non‐syncytium inducing/partial T‐cell line tropic (NSI/pTT) virus. To investigate the relationship between these viral phenotypes and the sequence variability of the V2 and V3 regions of the envelope, we cloned the viral gene segment and sequenced the individual isolates. The sequence data suggested that the SI/TT type changes in the V3 sequence alone mediate a partial T cell line tropism and mild cytopathic effect and that an isolate became more virulent (SI/TT phenotype) if there were additional changes in the V2 or other regions. On the other hand, sequence changes in the V2 region alone could not mediate phenotypic changes but some additional changes in the other variable regions (for example, V3) might be required for the phenotypic changes in combination with changes in V2. These findings also suggested that amino acid changes in both the V2 and V3 region are required for the development of virulent variants of HIV‐1 that outgrow during advanced stages of the disease.
Distribution of Borrelia Species Associated with Lyme Disease in the Subalpine Forests of Nagano Prefecture, Japan
Authors: Nakao Minoru , Uchikawa Kimito , Dewa Hiroshi
Published: November 2013

We surveyed the natural distribution of Borrelia species associated with Lyme disease in the subalpine forests of Nagano prefecture, Japan, during 1993‐94. Tick‐derived isolates (n = 112) from Ixodes persulcatus and rodent‐derived isolates (n = 55) from Apodemus argenteus, Apodemus speciosus, Eothenomys andersoni, Eothenomys smithii, and Microtus montebelli were classified by rRNA gene restriction fragment length polymorphism analysis (RFLP ribotyping). Ribotype group IV (an intraspecific variant of Borrelia garinii) was predominant among the tick isolates. It was also isolated repeatedly from the rodents. Ribotype group III (Borrelia afzelii) was detected in low frequencies among the tick and rodent isolates. The data suggest that humans are likely to be exposed to the group IV when they are bitten by I. persulcatus ticks.
Monoclonal Antibody #5‐2‐26 Recognizes the Phosphatase‐Sensitive Epitope of Rabies Virus Nucleoprotein
Authors: Kawai Akihiko , Anzai Jun , Honda Yoshikazu , Morimoto Kinjiro , Takeuchi Kenji , Kohno Takashi , Wakisaka Koji , Goto Hideo , Minamoto Nobuyuki
Published: November 2013

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5‐2‐26) was shown to lack reactivity with the phosphatase‐treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)‐denatured N protein. The MAb did not recognize the N‐protein analogues produced in Escherichia coli (E. coli), indicating that the N‐gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N‐gene products produced in COS‐7 cells, but not with those produced in the presence of K‐252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross‐reactivity with the Triton‐insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5‐2‐26 preferentially recognizes a phosphatase‐sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N‐protein phosphorylation and its role(s) in virus replication.
A Monoclonal Antibody Modulates Neutrophil Adherence While Enhancing Cell Motility
Authors: Ohtake Kazuhisa , Takei Hiroshi , Watanabe Tadashi , Sato Yukiko , Yamashita Takao , Sudo Kazuhiko , Kuroki Motomu , Chihara Junichi , Sendo Fujiro
Published: November 2013

A monoclonal antibody (MoAb) to human neutrophils, designated 3H9, was established by screening for the inhibition of neutrophil adherence to plastic plates containing a medium supplemented with fetal calf serum (FCS medium). The antigen recognized by 3H9 was shown to be present on human leukocytes and found at the highest levels on granulocytes. On Western blotting, 3H9 reacted with a molecule having a molecular weight of 80 kDa. When this MoAb was added at the same time as a neutrophil stimulant (fMLP), the inhibition of neutrophil adherence to plastic plates in the presence of FCS medium was observed after 60 min incubation. Furthermore, this MoAb enhanced not only fMLP‐induced chemotaxis but random migration of neutrophils as well. The mechanisms of these phenomena are discussed.
Nitrogen‐Gas Bubbling during the Cultivation of Clostridium tetani Produces a Higher Yield of Tetanus Toxin for the Preparation of Its Toxoid
Authors: De Luca María Marta , Abeiro Héctor Dario , Bernagozzi Jorge Alberto , Basualdo Juan Angel
Published: November 2013

We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N2 was bubbled through nine 10‐liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.
In Vitro Amphotericin B Effects on Growth, Viability and Dimorphism of Paracoccidioides brasiliensis: Reversal of the Treatment
Authors: Jesuino Rosália Santos Amorim , de Bastos Ascenço Soares Renata , Salem‐Izacc Silvia Maria , Pereira Maristela , Felipe Maria Sueli Soares , de Almeida Soares Célia Maria
Published: November 2013

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 μg/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two‐dimensional gels of 35S‐labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.