bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between O‐Serogroup and Presence of Pathogenic Factor Genes in Escherichia coli
Authors: Sunabe Tsuyoshi , Honma Yasuko
Published: November 2013

A total of 383 isolates of serogroup‐based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes. The serogroup‐based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers. The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%). The difference was not significant. The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA‐positive EPEC strains from patients but was not detected in the 9 strains from carriers. In serogroup‐based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers. The 4 ipaH‐positive strains possessed the invasive plasmid. These results suggested that the serogroup‐based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene.
New Monoclonal Antibodies against a Recombinant Second Envelope Protein of Hepatitis C Virus
Authors: Inudoh Michiharu , Kato Nobuyuki , Tanaka Yuetsu
Published: November 2013

To study the immunological features of the hepatitis C virus (HCV) envelope protein (E2 protein), new specific monoclonal antibodies (mAbs) were generated. WKA/H rats were immunized with syngeneic cells infected with a vaccinia virus expressing the E2 protein and with soluble E2 protein obtained from Chinese hamster ovary cells with a plasmid‐based expression system. By screening hybridoma cells obtained from spleen cells of the immunized rats, three specific mAbs were obtained. One mAb was reactive to a peptide corresponding to the hypervariable region 1 (HVR1) in E2 protein, while the others reacted to regions outside HVR1. The significance of these antibodies for the diagnosis of HCV infection as well as for analysis of the structure of the HCV E2 protein will be discussed.
Anti‐Cord Factor (Trehalose 6,6′‐Dimycolate) IgG Antibody in Tuberculosis Patients Recognizes Mycolic Acid Subclasses
Authors: Pan Jiongwei , Fujiwara Nagatoshi , Oka Shiro , Maekura Ryoji , Ogura Takeshi , Yano Ikuya
Published: November 2013

The detection of anti‐cord factor (trehalose 6,6′‐dimycolate) IgG antibody in active (smear‐and/or culture‐positive) and inactive (smear‐ and culture‐negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium‐infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human‐type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC‐infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti‐cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (α‐, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti‐cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized α‐ and keto mycolic acids weakly. Pre‐absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti‐cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.
Nucleotide Sequence of the 5′Nontranslated and Virion Polypeptides Regions of Coxsackievirus B6
Authors: Kato Sakura , Tsutsumi Reiko , Sato Shigehiro
Published: November 2013

The nucleotide sequence of coxsackievirus B6 (CVB6) has been determined, and the nucleotides encoding the 5′ nontranslated region (5′ NTR) and virion polypeptides (VP4, 2, 3 and 1) were compared with other serotype CVBs. An Unweighted Pair‐Group Method Analysis (UPGMA) of phylogenetic trees indicated that the 5′ NTR of CVB6 locates on an independent branch from the other CVBs. The tree based on the amino acid sequences showed that CVB6 has close correlation with CVB4 in the VP4 and VP2 regions, with CVB1 and CVB5 in the VP3 region, and with CVB5 in the VP1 region. Amino acid sequences of variable regions within the VP2, VP3, and VP1 of CVB6 were unique among CVBs. Thus, by comparison of the nucleotide and amino acid sequences of these variable regions, CVB6 can be easily distinguished from other serotypes. In addition, serine, instead of glycine, was found to locate at the amino‐terminus of the VP1 region of CVB6, indicating that CVB6 has a unique cleavage site (i.e., glutamine/serine instead of glutamine/glycine) for proteinase 3C of Picornaviridae.
Theiler's Murine Encephalomyelitis Virus (TMEV) Subgroup Strain‐Specific Infection in Neural and Non‐Neural Cell Lines
Authors: Obuchi Masatsugu , Yamamoto Jiro , Uddin Md. Nasir , Odagiri Takato , Iizuka Hideaki , Ohara Yoshiro
Published: November 2013

GDVII subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. In contrast, DA subgroup strains are less virulent and establish a persistent central nervous system infection which results in demyelinating disease. We previously reported a subgroup‐specific infection in a macrophage‐like cell line, J774‐1 cells; i.e., GDVII strain does not replicate in J774‐1 cells, whereas the DA strain actively replicates in these cells. In addition, this subgroup‐specific virus growth is shown to be related to the presence of L* protein, a 17 kDa protein translated out‐of‐frame of the viral polyprotein from an AUG located 13 nucleotides downstream from the polyprotein's AUG. The present paper demonstrated that this subgroup‐specific infection is observed in murine monocyte/macrophage lineage cell lines, but not in other murine cell lines including neural cells. An RNase protection assay also suggested that L* protein‐related virus growth is regulated at the step of viral RNA replication. As macrophages are reported to be the major cell harboring virus during the chronic demyelinating stage, the activity of L* protein with respect to virus growth in macrophages may be a key factor in clarifying the mechanism(s) of TMEV persistence, which is probably a trigger to spinal cord demyelination.
Prevalence of the Virulence Plasmids of Nontyphoid Salmonella in the Serovars Isolated from Humans and Their Association with Bacteremia
Authors: Chiu Cheng‐Hsun , Lin Tzou‐Yien , Ou Jonathan T.
Published: November 2013

To determine if the virulence plasmid is one of the elements contributing to Salmonella bacteremia in humans, 436 clinical Salmonella isolates of different serovars were examined by a specific multiplex polymerase chain reaction assay for the presence of a virulence plasmid. These serovars showed differences in their ability to produce particular disease syndrome in humans. In the serovars usually causing bacteremia without concomitant gastroenteritis (primary bacteremia), i.e., S. choleraesuis, S. dublin, and S. enteritidis in this study, the rate of virulence plasmid carriage was 100%, while among those occasionally generating bacteremia following an episode of gastroenteritis (secondary bacteremia), the majority were plasmidless. Only a portion of S. typhimurium strains harbored a virulence plasmid; however, the rates of virulence plasmid carriage in S. typhimurium were not statistically different between non‐fecal and fecal isolates (90% vs. 85%, 0.1 < P < 0.9). These results indicate that the virulence plasmids may be important for primary bacteremia, but not secondary bacteremia, to occur.
Alteration of Cell Wall Composition Leads to Amphotericin B Resistance in Aspergillus flavus
Authors: Seo Kuniko , Akiyoshi Hiroto , Ohnishi Yoshinari
Published: November 2013

An amphotericin B (AmB)‐resistant mutant was isolated from a wild‐type AmB‐susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 μtg ml−1. The acquired resistance of my celia was specific for polyene‐antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild‐type. Chemical analysis of the cell wall revealed that levels of alkali‐soluble and ‐insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild‐type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild‐type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3‐α‐glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.
Characterization of Mouse DAF on Transfectant Cells Using Monoclonal Antibodies Which Recognize Different Epitopes
Authors: Ohta Rieko , Imai Masaki , Fukuoka Yoshihiro , Miwa Takashi , Okada Noriko , Okada Hidechika
Published: November 2013

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay‐accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI‐DAF) and a transmembrane form (TM‐DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI‐DAF or TM‐DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI‐DAF transfectant cells and established seven unique mAbs (RIKO‐1–7). Western blotting analysis using RIKO‐3, which reacts with both GPI‐DAF and TM‐DAF, and RIKO‐4, which is an anti‐GPI‐DAF specific mAb, indicated that GPI‐DAF was expressed on erythrocytes, spleen and testis, and that TM‐DAF was expressed only in testis.
Increased Sensitivity of Cholera Toxin Detection by Enzyme‐Linked Immunosorbent Assay with Chemical Immobilization of Antibody onto Nylon Surface Providing Hydrophobicity
Authors: Koike Norio , Yabushita Yasunori , Yan Zhang Dong , Lida Tetsuya , Honda Takeshi
Published: November 2013

We developed an improved method to chemically immobilize antibodies on a nylon surface using a styrene maleic anhydride copolymer having an aryl group, which provides hydrophobicity to the nylon surface. We applied it to a modified enzyme‐linked immunosorbent assay (slip‐ELISA) for the detection of cholera toxin (CT). The sensitivity of slip‐ELISA for CT detection was 1,000 times higher than that of conventional methods of physical adsorption using polystyrene plates, and 10 times higher than that of the method of chemical immobilization using maleic anhydride methylvinyl ether copolymer.
Comparison of T‐Cell Receptor Jβ Gene Usage in Spleen Cells of Different Mouse Strains
Authors: Kato Tomohiro , Sasakawa Hiroko , Kurokawa Manae , Masuko‐Hongo Kayo , Hamada Chikuma , Yamamoto Kazuhiko , Nishioka Kusuki
Published: November 2013

To elucidate whether T‐cell receptor Jβ gene usage was affected by major histocompatibility complex haplotypes and other genetic backgrounds, we investigated such usage with Jβ‐specific probes in four different mouse strains. As a result, (a) frequent usage of Jβ2.1 and Jβ2.6, (b) infrequent usage of Jβ1.3, Jβ1.5 and Jβ1.6, and (c) predominant usage of the Jβ2 cluster compared to the Jβ1 cluster were found. Importantly, these biases were common to almost all the tested Vβ families of the four strains. Thus, TCR Jβ usage would be independent of the major histocompatibility complex haplotypes and other genetic backgrounds.
Serum Antibody Level against GroEL Type Heat‐Shock Protein of Campylobacter jejuni in Patients with Guillain‐Barré Syndrome
Authors: Fujimoto Shuji , Fujita Masaki
Published: November 2013

Recently there has been an increase in the number of cases reported of Guillain‐Barré syndrome (GBS) developed after Campylobacter jejuni infection. To investigate the role of a C. jejuni GroEL‐type heat‐shock protein (CjHsp60) in the infection and induction of GBS, we examined the antibody level against CjHsp60 in 27 human sera, including GBS and non‐GBS patients, by an enzyme‐linked immunosorbent assay. Sera from patients with C. jejuni infection, despite the development of GBS, had a higher titer of anti‐CjHsp60 antibody than those of patients without the infection and healthy control subjects. The patients with C. jejuni infection followed by GBS had slightly higher levels of this antibody than did the patients with infection who did not develop GBS, but there was no statistical significance. In conclusion, CjHsp60 is found to be one of the major immunogenic antigens in actual C. jejuni infection, but no evidence that supports the direct relationship between this protein and C. jejuni‐associated GBS was found in this study.
Assessment of Mucosal Immune Response in Genitourinary Tract Using Urine
Authors: Kohno Takeyuki , Tanaka Hiroko , Watabe Kazuhito , Yamashita Shinji , Sezaki Hitoshi , Nadai Tanekazu , Sugie Yohko , Ogouchi Toshiyo
Published: November 2013

A novel method to assess mucosal immune response in the genitourinary mucosa after immunization with a mucosal vaccine has been developed. In this method, secretory IgA antibody is measured by a highly sensitive enzyme immunoassay (immune‐complex transfer enzyme immunoassay) using urine as a specimen. The urinary IgA antibody response could be detected by the immune‐complex transfer enzyme immunoassay. In contrast, a conventional enzyme immunoassay (enzyme‐linked immunosorbent assay (ELISA)) could not detect this response because of its low sensitivity. Because urine samples can be collected easily and nontraumatically, not only from experimental animals but also from humans, both males and females, the present method may be applicable for assessing the protective efficacy of candidates for mucosal vaccines against sexually transmitted microorganisms, such as human immunodeficiency virus. Furthermore, the usefulness of this method for novel mucosal vaccine formulae was shown for a model in which vaccine antigen and Bordetella pertussis adjuvant were adsorbed onto CaCO3 and enclosed in enteric coated capsules.
Induction of Interleukin 8 (IL‐8) Production by Pseudomonas Nitrite Reductase in Human Alveolar Macrophages and Epithelial Cells
Authors: Sar Borann , Oishi Kazunori , Matsushima Kouji , Nagatake Tsuyoshi
Published: November 2013

Interleukin‐8 (IL‐8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL‐8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648 2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial‐like cells (A549) to induce IL‐8 production and mRNA expression as well as the production of TNFα and IL‐1β. We demonstrated a time‐ and dose‐dependent IL‐8 protein synthesis and IL‐8 mRNA expression, but no TNFα or IL‐1β production, by A549 cells in response to PNR. New protein translation was not required for PNR‐mediated IL‐8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNFα or IL‐1β resulted in additive IL‐8 production in A549 cells. In adherent AM, PNR enhanced IL‐8 protein synthesis and IL‐8 mRNA expression in a time‐dependent fashion. PNR similarly induced a time‐dependent production of TNFα and IL‐1β by human adherent AM. Neutralization of TNFα or IL‐1β did not influence the levels of IL‐8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti‐human IL‐8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 μg/ml of PNR, the mean percent reduction of NCF activities were 49‐59% in A549 cells and 2434% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL‐8. PNR also mediates neutrophil migration, in part, through IL‐8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.
Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry
Authors: Heinzelmann Michael , Gardner Sarah Appel , Mercer‐Jones Mark , Roll Amy J. , Polk Hiram C.
Published: November 2013

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60‐min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate‐labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright‐red fluorescent PMNs. Our experiments with two different bacteria, various PMN‐to‐bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.
Modulation of Barrier Function of Small Intestinal Epithelial Cells by Lamina Propria Fibroblasts in Response to Lipopolysaccharide: Possible Role of TNFα in Inducing Barrier Dysfunction
Authors: Chakravortty Dipshikha , Kumar K. S. Nanda
Published: November 2013

Recent evidence suggests an interaction between immune, enteric neural and fibroblasts in the regulation of intestinal function. Earlier, we have reported that lipopolysaccharide (LPS) induced cell proliferation, collagen synthesis and production of proinflammatory mediators in lamina propria fibroblasts. In this report, we investigated the change in transepithelial resistance (TER) as a marker of epithelial barrier function by lipopolysaccharide (LPS) and its modulation by human small intestinal lamina propria fibroblasts (HSILPF). Epithelial cells incubated with LPS alone did not show any change in the TER at any concentration or prolonged exposure. However, co‐cultivation of epithelial cells with lamina propria fibroblasts which had been exposed to LPS resulted in a rapid decrease in TER by 2 hr. The decrease in the TER was continued till 8 hr followed by returning to the basal level by 24 hr. The supernatant of LPS‐treated HSILPF was less effective in causing a fall in the TER than HSILPF itself. The fall in TER was accompanied by loosening of tight junctions as depicted by increased penetration of horse radish peroxidase (HRP) across the epithelial cells from the apical to the basal side. Increased incorporation of 3[H]thymidine (tritiated thymidine) in epithelial cells was observed at 48 hr in the presence of LPS‐treated HSILPF. The decrease in TER during the early time period in epithelial cells was abrogated to 70% by incubating the LPS‐treated HSILPF and the conditioned medium of LPS‐treated HSILPF with anti‐TNFα antibody, and not with antibody to other cytokines like IL1α, IL1β, IL6 and IL8. Overall, these results suggest that TNFα produced by HSILPF in response to LPS as a soluble form cause a decrease in the TER and loosening of tight junctions, and such early changes in the epithelial barrier may contribute to local inflammation in the gut.
Ribotyping of Vibrio parahaemolyticus Isolates Obtained from Food Poisoning Outbreaks in Taiwan
Authors: Wong Hin‐chung , Ho Chia‐yun , Kuo Li‐ping , Wang Tien‐kuei , Lee Chi‐lung , Shih Daniel Yang‐chih
Published: November 2013

Vibrio parahaemolyticus is a prevalent food‐borne pathogen in Taiwan, Japan and other Asian countries. This work presents a novel ribotyping method for the molecular epidemiological examination of this pathogen. Genomic DNA was fragmented by HindIII digestion and hybridized with cDNA probe for Escherichia coli 16S and 23S RNA genes. A total of 121 isolates obtained from outbreaks during 1992 and 1994 in Taiwan were characterized by this ribotyping method. Four to seventeen restricted fragments were visualized in these isolates. After hierarchical cluster analysis, these isolates were grouped into thirty different ribotypes. In addition, A3, A7, E3 and F1 were the major ribotypes, consisting of 22.3, 13.2, 9.1, and 8.3% of the isolates, respectively. A, E, F, G and B were the major groups, consisting of 46.2, 14.0, 9.1, 6.7, and 6.7% of the isolates, respectively. The discriminatory ability of this ribotyping method, as determined by Simpson's index of diversity, was 0.93, which closely resembled that of a previously reported pulsed‐field gel electrophoresis method.
The Expression of the Pathogenic Yeast Candida albicans Catalase Gene in Response to Hydrogen Peroxide
Authors: Nakagawa Yoshiyuki , Watanabe Koji Koide, Kiyomi , Morita Yasuhiro , Mizuguchi Ikuyo , Akashi Tomohiro
Published: November 2013

The catalase gene of the pathogenic yeast Candida albicans was cloned and its expression was examined. Activity of the catalase was detected when cells which were in the early logarithmic stage were treated with hydrogen peroxide. Additionally, activity was detected without any treatment to cells in the late logarithmic and stationary phases. When cells were cultured in galactose, glycerol, or ethanol, catalase activity was always observed without the hydrogen peroxide treatment, suggesting that glucose represses the induction of catalase expression. To elucidate the molecular mechanism of catalase expression, the putative gene for catalase and its 5′ untranscribed region were cloned. Sequences of the gene and its potential regulatory region revealed several motifs, including a GC box‐like element and stress‐responsive element (STRE), which could be involved in the transcriptional regulation. Northern analysis showed that hydrogen peroxide and sorbitol activated transcription of the catalase. On the other hand, treatment of glucose strictly repressed the expression of the catalase even when co‐treated with hydrogen peroxide. The expression of catalase against treatment with hydrogen peroxide took place very quickly and decreased slowly in the experimental condition adopted here. From these results, we assumed that the expression of the catalase in Candida albicans is regulated by various environmental conditions via motifs for transcriptional activation as in other yeast catalases.
Repetitive Sequence of Leptospira interrogans Serovar Icterohaemorrhagiae Strain Ictero No. 1: A Sensitive Probe for Demonstration of Leptospira interrogans Strains
Authors: Takahashi Yukie , Kishida Masami , Yamamoto Satoru , Fukunaga Masahito
Published: November 2013

A 4.8‐kilobase (kb) repetitive sequence element generated with KpnI digestion was cloned from the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1. The sequence, repeated in tandem, was located on the 280‐kb fragment between the FseI and AscI sites on the chromosome by hybridization using the 4.8‐kb fragment as a probe. We cloned the fragment containing the element for the Ictero No. 1 strain in a lambda EMBL3 bacteriophage DNA, and one out of 5 clones was sequenced. Within the sequenced 9‐kb segment that partially repeated, 9 putative open‐reading frames and 2 transfer RNA genes, for alanine and isoleucine, were identified. A similarity search for the products deduced from the sequenced data revealed that the repeated sequence includes both beta‐oxidation enzymes, acyl‐CoA dehydrogenase and enoyl‐CoA hydratase, and hydroxythiazole kinase protein homologues. Hybridization experiments against different leptospiral strains using the element as a probe showed a similar sequence in the strains of L. interrogans and L. kirschneri, but not in any strains of L. borgpetersenii, L. weillii, L. meyeri or L. biflexa. Results indicated that the highly repeated element in the Ictero No. 1 strain exists as a well conserved sequence, though at a moderate level of repetition, in certain strains of L. interrogans and L. kirschneri. PCR amplification targeting the repetitive element was successful and indicated that the procedure provides a sensitive and specific probe to detect leptospires.
Human IgM Monoclonal Antibody to Ganglioside GM2 and Complement Suppress Virus Propagation in Ex Vivo Cultures of Lymphocytes from HIV‐1 Infected Patients
Authors: Okada Noriko , Wu Xiaoshan , Mizokami Masashi , Irie Reiko F. , Okada Hidechika
Published: November 2013

HIV‐1 infection induces aberrant ganglioside GM2 expression on infected cell lines, and human IgM anti‐GM2 monoclonal antibody (L55 Ab) together with normal fresh human serum (FHS) as a source of complement causes complement mediated cytolysis of HIV‐1 infected cells as well as HIV‐1 particles. We report here that high expression of GM2 was also detected on HIV‐1 infected lymphocytes from HIV‐1 seropositive patients. L55 Ab effectively suppressed the generation of HIV in the presence of FHS in primarily cultured lymphocytes from HIV‐1 infected patients in ex vivo experiments, and the suppression was enhanced additively by AZT. These data suggest that L55 Ab may increase the therapeutic effect of chemotherapy.
Protective Immune Response to 16 kDa Immunoreactive Recombinant Protein Encoding the C‐Terminal VP1 Portion of Foot and Mouth Disease Virus Type Asia 1
Authors: Bayry Jagadeesh , Prabhudas Krishnamsetty , Gopalakrishna Susarla , Patil Prasanna K. , Ramakrishna Chitimalla , Misra Laxmi Dutt , Suryanarayana Veluvarti V. S.
Published: November 2013

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C‐terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C‐terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 μg/animal followed by a booster of the same dose was injected after a 21‐day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme‐linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post‐vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C‐terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.
Cytokine Secretion by Stimulated Monocytes Depends on the Growth Phase and Heat Treatment of Bacteria
Authors: Haller Dirk , Bode Christiane , Hammes Walter P.
Published: November 2013

The consumption of food containing lactic acid bacteria (LAB) has been shown to exert immunomodulatory effects in humans. The specific cellular interaction of these bacteria with immuno‐competent cells has not yet been fully understood. Since the TNF‐α secretion of stimulated monocytes is an important initial response to a bacterial challenge, we investigated the potential of LAB originating from the human intestine or fermented food in comparison to the effect of invasive pathogens. The challenge of monocytes with three LAB strains, Listeria monocytogenes or enterohaemorrhagic Escherichia coli (EHEC) elicited a strain specific, dose‐dependent biphasic TNF‐α secretion. The concentration (EDmax) of bacteria or bacterial cell wall components necessary to induce maximal TNF‐α secretion (TNFmax) by monocytes was mathematically approximated. It was shown for exponentially growing LAB strains that the maximal TNF‐α secretion (TNFmax) was stronger (57 to 78%) upon stimulation with living bacteria than with heat killed cells. In contrast to log‐phase bacteria, the maximal TNF‐α secretion of monocytes (TNFmax) was higher (15 to 55%) after the stimulation with heat killed, stationary‐phase bacteria when compared to that of live LAB. Thus, monocyte stimulation was clearly affected by the growth phase of bacteria. Purified cell walls of LAB straines revealed only a limited potential for monocyte stimulation. LPS exhibited a higher capacity to stimulate monocytes than purified Gram positive cell walls or muramyldipeptide. In comparison to pathogenic bacteria, the maximal secretory TNF‐α response (TNFmax) was up to 2 fold higher with LAB strains. In general, the amount of bacteria (EDmax) necessary to induce maximal TNF‐α secretion (TNFmax) was approximatly 1 to 3 log higher for heat killed bacteria when compared to live bacterial cells illustrating the significant lower potential of heat killed bacteria to activate monocytes.
Role of the Cytomegalovirus (CMV)‐Antigenemia Assay as a Predictive and Follow‐Up Detection Tool for CMV Disease in AIDS Patients
Authors: Hoshino Yoshihiko , Nagata Yoichi , Taguchi Hitomi , Masunaga Atsuko , Fujino Yujiro , Mochizuki Manabu , Nakamura Tetsuya , Iwamoto Aikichi
Published: November 2013

Forty‐two patients were evaluated to determine the value of the CMV antigenemia (CMV‐Ag) test as a follow‐up marker as well as a prediction marker of CMV disease. Twenty patients were positive for at least one positive CMV‐Ag assay and 9 of them developed CMV retinitis. With the threshold value (10 positive cells), sensitivity was 56% and specificity was 94%. The CMV‐Ag assay, with the threshold value, produced high specificity, positive predictive value and negative predictive value but relatively poor sensitivity. Eight patients experienced CMV disease relapse a total of 16 times. At relapse, 8 of the 16 times showed negative for CMV‐Ag assay; 7 underwent systemic maintenance while 1 underwent local maintenance. It is inferred that the CMV‐Ag test is a poor follow‐up marker to detect the relapse of CMV disease, particularly in patients undergoing systemic maintenance.
Characterization of the Cyclophilin of Trichophyton mentagrophytes
Authors: Kano Rui , Nakamura Yuka , Watanabe Shinichi , Tsujimoto Hajime , Hasegawa Atsuhiko
Published: November 2013

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C‐terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione 5‐transferase (GST)‐T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl‐prolyl cis‐trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.
Exacerbation of Vascular Endothelial Injury in the Generalized Shwartzman Reaction by the Administration of Anti‐E‐Selectin Antibody
Authors: Koide Naoki , Kato Yutaka , Sugiyama Tsuyoshi , Chakravortty Dipshika , Mu Mya Mya , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS‐sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti‐E‐selectin antibody in GSR‐induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti‐E‐selectin antibody. On the other hand, the administration of anti‐VCAM‐1 antibody did not induce such injury of vascular endothelial cells. The possible role of E‐selectin in the exacerbation of vascular endothelial injury in GSR is discussed.
Intranasal Sensitization of Japanese Cedar Pollen by the Co‐Administration of Low Doses of Cholera Toxin but Not Its Recombinant B Subunit to Mice
Authors: Hirai Terumasa , Hashiguchi Shuhei , Torigoe Naohiko , Toda Yoshihiro , Ito Yuji , Sugimura Kazuhisa
Published: November 2013

We evaluated the effects of cholera toxin (CT) and the B subunit of cholera toxin (CTB) on the intranasal sensitization of Japanese cedar pollen (JCP) in mice. JCP suspended in phosphate‐buffered saline was administered into the nostrils of mice in combination with varying doses of CT or recombinant CTB(r‐CTB) once a week for 5 weeks. Antibody responses specific to sugi basic protein (SBP) were monitored by ELISA for seven weeks. The sensitization of JCP alone did not induce IgGl, IgG2b, IgG2a, IgE or IgA. In contrast, sensitization of JCP in combination with CT (JCP/CT) elicited the prominent production of SBP‐specific IgG1 and low levels of IgG2b and IgG2a on Day 49. IgE production was detected only in the serum of mice which were treated with JCP/CT, and not under any other protocol. Using spleen cells from these mice, cytokine production was examined by ELISA in culture supernatants after they had been stimulated in vitro with major cedar pollen allergens, Cry j 1, Cry j 2 or SBP. Notable responses were an increase of IFN‐γ as well as IL‐4 in JCP/CT‐sensitized cells stimulated with Cry j 2, but not in those stimulated with Cry j 1. No significant differences were detected in IL‐5 production among the experimental groups. Histopathological examination, however, showed that eosinophil infiltration was evident in the nasal mucosa of the JCP/CT‐sensitized mice following challenge with JCP/CT, but weak with BSA/CT or CT alone. Thus, the immunological and histological analyses indicated that the co‐administration of a low dose of CT in combination with JCP allows the induction of pollen‐allergic states in mice.
Autocrine Interferon‐β Stimulation Augments Nitric Oxide Production by Mouse Macrophage J774A.1 Cells Infected with Herpes Simplex Virus Type 1
Authors: Fujioka Noboru , Ohashi Kunihiro , Ikeda Masao , Kurimoto Masashi
Published: November 2013

The pathogenic roles of nitric oxide (NO) in mouse models have been reported for herpes simplex virus type 1 (HSV‐1)‐induced pneumonia as well as endotoxin shock. We compared the mechanism of NO production induced by HSV‐1 with that induced by lipopolysaccharide (LPS) using a mouse macrophage cell line, J774A.1. Both HSV‐1 and LPS induced NO production as well as antiviral activity, which were attenuated by anti‐interferon (IFN)‐β treatment. These results suggest that autocrine IFN‐β plays a role in NO release by J774A.1 cells stimulated with HSV‐1 or LPS.
Reduction of Thymocyte Numbers in Transgenic Mice Expressing Viral FLICE‐Inhibitory Protein in a Fas‐Independent Manner
Authors: Ohyama Takahiro , Tsukumo Shin‐ichi , Yajima Nobuyuki , Sakamaki Kazuhiro , Yonehara Shin
Published: November 2013

A viral FLIP (FLICE/caspase‐8‐Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor‐induced apoptosis by suppressing the activation of FLICE/caspase‐8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck‐proximal promoter. Although E8‐expressing thymocytes were resistant to Fas‐mediated apoptosis, the total number of thymocytes in 4‐8‐week‐old E8 transgenic mice was more than 3‐fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas–/– background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3‐mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex‐mediated stimulation. Thus, the Death receptor‐mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.
The Role of Osteopontin in the Development of Granulomatous Lesions in Lung
Authors: Chiba Satoru , Rashid Mohammod Mizanur , Okamoto Hiroshi , Shiraiwa Hirotake , Kon Shigeyuki , Maeda Masahiro , Murakami Masaaki , Inobe Manabu , Kitabatake Akira , Chambers Ann F. , Uede Toshimitsu
Published: November 2013

Osteopontin (OPN) has been shown to be expressed by cells in granulomas of various origins, but whether it plays a functional role in granuloma formation is not known. Here we used a cardiomyopathic hamster (T02) model, to test the hypothesis that OPN contributes functionally to granuloma development. We immunized cardiomyopathic and normal hamsters by subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, and assessed various tissues for both OPN RNA expression and granuloma formation. Cardiomyopathic hamsters expressed OPN, and formed granulomatous lesions, in heart tissue in both immunized and untreated animals. In addition, immunization induced expression of OPN in lung and lymph nodes of cardiomyopathic (but not normal) hamsters, and also induced granuloma formation in these organs. To test whether OPN expression could play a functional role in inducing granulomas, we produced an adenoviral vector containing the murine OPN gene, and introduced this vector intratracheally into the lungs of normal hamsters. The OPN‐containing vector, but not the control vector, induced pulmonary granuloma formation. These studies provided direct in vivo evidence that OPN can contribute functionally to the formation of granulomatous lesions, and suggest that OPN expression may be a common factor involved in formation of granulomas of various origin.
Detection of Hantaviral Antibodies among Patients with Hepatitis of Unknown Etiology in Japan
Authors: Kariwa Hiroaki , Yoshimatsu Kumiko , Araki Koichi , Chayama Kazuaki , Kumada Hiromitsu , Ogino Michiko , Ebihara Hideki , Murphy Michael E. , Mizutani Tetsuya , Takashima Ikuo , Arikawa Jiro
Published: November 2013

Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to AG hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.
Protection Induced in Mice Vaccinated with Recombinant Collagen‐Binding Protein (CnBP) and Alpha‐Toxoid against Intramammary Infection with Staphylococcus aureus
Authors: Mamo Wubshet , Fröman Gunnar , Müller Hans‐peter
Published: November 2013

Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen‐binding protein (CnBP) and alpha‐toxoid (α‐toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/α‐toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or α‐toxoid or controls (P ≤ 0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with α‐toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or α‐toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.
Sequence of the Gene Encoding an Alkaline Serine Proteinase of Bacillus pumilus TYO‐67
Authors: Aoyama Misako , Toma Claudia , Yasuda Masaaki , Iwanaga Masaaki
Published: November 2013

The complete nucleotide sequence of the gene encoding an alkaline serine proteinase (aprP) of Bacillus pumilus TYO‐67 was determined. The sequence analysis showed an open reading frame of 1,149 bp (383 amino acids) that encoded a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of subtilases. The sequence around these residues revealed that APRP was a new member of the true subtilisin subgroup of the subtilisin family. The highest homology was found in subtilisin NAT at 64.4% in the DNA sequence. The residue S189 of APRP was different from those of other subtilases.
The IgM Antibody Level against Ganglioside GM2 Correlates to the Disease Status of HIV‐1‐Infected Patients
Authors: Wu Xiaoshan , Okada Noriko , Goto Mieko , Iwamoto Aikichi , Okada Hidechika
Published: November 2013

HIV‐1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)‐mediated cytolysis of HIV‐1‐infected cells. Since GM2 is immunogenic in human, we proposed that an anti‐GM2 IgM Ab may be produced by some HIV‐1‐infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV‐1‐infected patients and 111 seronegative donors. As expected, the anti‐GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4+ cell count and the HIV‐RNA load in the HIV‐1‐infected patients. The results showed a positive correlation between the anti‐GM2 IgM Ab titer and CD4+ cell count but a negative correlation between the anti‐GM2 IgM Ab titer and HIV‐RNA load. These suggest that anti‐GM2 IgM Ab induced and/or enhanced by HIV‐1 infection causes C‐mediated cytolysis of HIV‐1‐infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV‐RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV‐1 infected patients.
Development of Hyperfimbriated Strains of Vibrio cholerne O1
Authors: Ehara Masahiko , Iwami Mamoru , Ichinose Yoshio , Hirayama Toshiya
Published: November 2013

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR™ vector. These clones were sequenced. The fimA sequences were found to be identical between V. cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX‐3X. The plasmid pVPP thus obtained was transferred into strains of wild‐type V. cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl‐beta‐d‐thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose‐linear gradient centrifugation (7.8 mg of fimbriae/L‐culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to d‐mannose and d‐glucose and immunogenicity) were identical to those of the wild‐type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.
Phylogenetic and Taxonomic Heterogeneity of Cryptococcus humicolus by Analysis of the Sequences of the Internal Transcribed Spacer Regions and 18S rDNA, and the Phylogenetic Relationships of C. humicolus, C. curvatus, and the Genus Trichosporon
Authors: Sugita Takashi , Takashima Masako , Ikeda Reiko , Nakase Takashi , Shinoda Takako
Published: November 2013

The phylogenetic and taxonomic heterogeneity of a rare opportunistic yeast pathogen, Cryptococcus humicolus, was revealed by analysis of the sequence of the internal transcribed spacer (ITS) region. Sixteen strains of C. humicolus showed a wide diversity in their ITS sequences. In addition, their 18S rDNA sequences were determined and used to analyze the phylogenetic relationships among C. humicolus and related yeasts. On trees constructed by the Neighbor‐Joining and Maximum Parsimony methods, C. humicolus strains were phylogenetically closely related to each other with the exception of one strain, and they clustered with C. curvatus and Trichosporon species with high bootstrap values. Three C. humicolus strains obtained from humans belonged to the group of Trichosporon serotype I species. The results suggest that C. humicolus is a genetically heterogeneous species which should be reclassified on the basis of DNA sequence data.