bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of Migratory and Aggregate Activities of Human Peripheral Blood Monocyte‐Derived Dendritic Cells by Stimulation with RANTES
Authors: Enomoto Makoto , Nagayama Hitomi , Takahashi Tsuneo A.
Published: November 2013

We examined the effects of various chemokines on the functional activation of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) plus interleukin‐4 (IL‐4)‐generated human peripheral blood monocyte‐derived immature dendritic cells (iDC). Stimulation of iDC with regulated on activation normal T cell expressed and secreted (RANTES) resulted in the promotion of their chemotactic migratory capacity in response to RANTES when compared with that of unstimulated cells. TNF‐α induced a homotypic aggregated cluster formation of iDC in a dose‐dependent manner, whereas the combination of TNF‐α and RANTES exhibited more potent induction. IDC stimulated with RANTES were more efficient than unstimulated iDC in the production of endogenous RANTES. Treatment of iDC with the combination of TNF‐α and RANTES was just little effective for the enhancement of allogeneic T‐cell stimulatory capacity as compared with that of TNF‐α treated iDC. These results suggest that endogenous secretions of RANTES from iDC stimulated with RANTES be potentially involved in RANTES‐induced changes of properties with respect to morphology and function.
A Mathematical Analysis of the Interactions between Immunogenic Tumor Cells and Cytotoxic T Lymphocytes
Authors: Takayanagi Toshiaki , Ohuchi Azuma
Published: November 2013

Recent developments of biotechnology have enabled us to use immunotherapy against certain kinds of tumors in patients. However, it is reasonable to doubt if the immunotherapy can completely aid the rejection of tumors that have escaped from the immune system. In this paper, we propose a new mathematical model of tumor immunity by tumor‐specific cytotoxic T lymphocytes (CTLs), since tumor‐specific CTLs play an important role in tumor immunity. Using this model, we have mathematically investigated the interactions between immunogenic tumor cells (TCs) and tumor‐specific CTLs and evaluated the availability of immunotherapies for tumors. The findings herein demonstrate that three kinds of dynamics of tumor immunity exist: i.e. (1) TCs continue to proliferate with CTLs; (2) TCs are rejected by CTLs; and (3) TCs equilibrate with CTLs, but with little possibility of the equilibrium. The findings also demonstrate that a sufficient increase in CTLs by immunotherapy can aid the rejection of TCs, but an insufficient increase in CTLs by immunotherapy causes only a transient regression of TCs. Clinically the findings mean that increasing tumor‐specific CTLs, e.g., by vaccination or adoptive transfer of tumor‐specific CTLs expanded ex vivo, can theoretically aid the rejection of TCs.
Protective Effect of Wogonin on Endotoxin‐Induced Lethal Shock in D‐Galactosamine‐Sensitized Mice
Authors: Van Dien Mai , Takahashi Kazuko , Mu Mya Mya , Koide Naoki , Sugiyama Tsuyoshi , Mori Isamu , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)‐induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D‐galactosamine (D‐GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D‐GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)‐α. The reduction was more marked in mice injected with D‐GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D‐GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF‐α and nitric oxide in LPS‐stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D‐GalN and LPS is discussed.
Effects of Mutation in Hepatitis C Virus Nonstructural Protein 5A on Interferon Resistance Mediated by Inhibition of PKR Kinase Activity in Mammalian Cells
Authors: Noguchi Tohru , Satoh Shinya , Noshi Takeshi , Hatada Eriko , Fukuda Ryuji , Kawai Akihiko , Ikeda Satoru , Hijikata Makoto , Shimotohno Kunitada
Published: November 2013

The IFN‐induced double‐stranded RNA (dsRNA)‐activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A‐expressing cell lines derived from human osteosarcoma (Saos‐2). The cells expressing NS5A derived from an IFN‐resistant clone (NS5A‐1b) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A‐2a) from an IFN‐sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A‐1b (NS5A‐ΔISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A‐2a nor the C‐terminal truncated mutant of NS5A‐1b (NS5A‐ΔC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C‐terminal region of NS5A‐1b is important for the suppression of PKR activity.
Characterization of Mumps Virus Isolated in Saitama Prefecture, Japan, by Sequence Analysis of the SH Gene
Authors: Uchida Kazue , Shinohara Michiyo , Shimada Shin‐ichi , Segawa Yukari , Hoshino Yoji
Published: November 2013

Mumps virus (MuV) strains isolated from cerebrospinal fluid and throat swabs from patients in Saitama Prefecture and Tokyo, Japan, from 1997 to 2000 were examined by analyzing the SH gene nucleotide sequence (316‐nt). Eighteen of the 20 strains studied were divided into three genotypes, recognized as B, G, and H in previous reports. Two genotypes (G and H) are believed to be new in Japan. Two of the 20 strains belonged to none of the previously reported genotypes (A–I), but were closely related to two known strains, MP94‐H and Lougl/UK97. We propose that the two strains identified in this study together with the previously reported strains, MP94‐H and Lougl/UK97, form a new genotype, designated J, based on the divergence of the SH gene nucleotide sequences between these four strains and other strains reported (genotypes A–I). Our results also suggest that more than two genotypes circulated in Saitama Prefecture from 1997 to 1999, but only one, genotype G, was in evidence in 2000. Genotype B was earlier reported as the predominant strain in Japan, but it became undetectable by the year 2000. These results provide important epidemiological data on mumps in Japan.
Location of the Epitope for 7D5, a Monoclonal Antibody Raised against Human Flavocytochrome b558, to the Extracellular Peptide Portion of Primate gp91phox
Authors: Yamauchi Akira , Yu Lixin , Pötgens Andy J.G. , Kuribayashi Futoshi , Nunoi Hiroyuki , Kanegasaki Shiro , Roos Dirk , Malech Harry L. , Dinauer Mary C. , Nakamura Michio
Published: November 2013

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X‐linked chronic granulomatous disease by flow‐cytometry. Therefore, we re‐evaluated the location of the 7D5 epitope using gene‐engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow‐cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the ~58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin‐treated PLB‐985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558‐deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.
Characterization of an Extended‐Spectrum Class C β‐Lactamase of Citrobacter freundii
Authors: Haruta Shin , Nukaga Michiyoshi , Sawai Tetsuo
Published: November 2013

Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino β‐lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended‐spectrum class C β‐lactamase encoded by chromosomal gene(s). The GC3 β‐lactamase showed high amino acid sequence homology to a known C. freundii β‐lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 β‐lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15–22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended‐spectrum resistance in class C β‐lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165–169, 1998). However, Asp198 was eliminated as a cause of the extended‐spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino β‐lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active‐site. Some or all of the replacements are assumed to delicately modify the active‐site configuration. The GC3 β‐lactamase is the first example of an extended‐spectrum class C β‐lactamase in which mutations are independent of the omega loop.
Nucleotide Sequence and Molecular Characterization of a Dextranase Gene from Streptococcus downei
Authors: Igarashi Takeshi , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

DNA fragments encoding the Streptococcus downei dextranase were amplified by PCR and inverse PCR based on a comparison of the dextranase gene (dex) sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex was determined. An open reading frame (ORF) of dex was 3,891 bp long. It encoded a dextranase protein (Dex) consisting of 1,297 amino acids with a molecular mass of 139,743 Da and an isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex had homology to those of S. sobrinus, S. mutans and S. salivarius Dex in the conserved region (made of about 540 amino acid residues). DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus as well as that of S. downei, but did not to other species of mutans streptococci. The C terminus of the S. downei Dex had a membrane‐anchor region which has been reported as a common structure of C termini of both the S. mutans and S. sobrinus Dex. The recombinant plasmid which harbored the dex ORF of S. downei produced a recombinant Dex enzyme in Escherichia coli cells. The analysis of the recombinant enzyme on SDS‐PAGE containing blue dextran showed multiple active forms as well as dextranases of S. mutans, S. sobrinus and S. salivarius.
Analysis of Seawaters for the Recovery of Culturable Vibrio parahaemolyticus and Some Other Vibrios
Authors: Alam Muhammad Jahangir , Tomochika Ken‐ichi , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non‐selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V. parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile‐salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.
Protective Effect of OK‐432 on Mice against Endotoxemia and Infection with Pseudomonas aeruginosa and Salmonella enteritidis
Authors: Hashimoto Masahito , Kirikae Fumiko , Toyooka Kiminori , Kaneko Aki , Yamasu Hiromi , Iwai Hiroki , Nakano Masayasu , Kirikae Teruo
Published: November 2013

OK‐432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK‐432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pre‐treatment of OK‐432 reduced the lethality of lipopolysaccharide (LPS)‐induced endotoxic shock in d‐(+)‐galactosamine‐sensitized C3H/HeN mice. OK‐432 did not affect the TNFα production in blood, but it did decrease the susceptibility to TNFα. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK‐432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK‐432 against the endotoxemia and bacteremia may depend on an up‐regulation of clearance of LPS and bacteria and the augmented resistance to TNFα.
Suppressive Mechanisms of EPA on Human T Cell Proliferation
Authors: Terada Sachiyo , Takizawa Mari , Yamamoto Shigeru , Ezaki Osamu , Itakura Hiroshige , Akagawa Kiyoko S.
Published: November 2013

In vivo and in vitro experiments show that polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) inhibit mitogen‐ or antigen‐stimulated proliferation of T cells in rodents and humans. However, the exact manner and mechanisms by which PUFA inhibits T cell proliferation is not clear. In the present study, we investigated the suppressive effects of EPA, an n‐3 PUFA, on PHA stimulated human peripheral blood T cells. Our results showed that EPA suppresses mitogen‐ or antigen‐stimulated human T cell proliferation by at least 2 steps; step 1) EPA suppresses T cell proliferation by inhibiting IL‐2Rα expression and IL‐2 production; step 2) EPA induces cell death of blast T cells without reducing the expression of IL‐2Rα. We also showed that EPA selectively stimulates the cell death of blast T cells but not resting T cells. The suppressive effect of EPA was mediated via the production of reactive oxygen products, because EPA‐stimulated H2O2 production and the suppressive effect of EPA was restored by addition of catalase or NAC. These results taken together suggest that such immunosuppressive effects of EPA may explain the apparent benefits of EPA‐enriched diets for patients with inflammatory disorders.
Role of V3 Independent Domains on a Dualtropic Human Immunodeficiency Virus Type 1 (HIV‐1) Envelope gp120 in CCR5 Coreceptor Utilization and Viral Infectivity
Authors: Foda Mohamed , Harada Shinji , Maeda Yosuke
Published: November 2013

The molecular mechanism of human immunodeficiency virus type 1 (HIV‐1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV‐1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV‐1KMT isolate were introduced either alone or in combination into the T‐tropic HIV‐1NL4–3 background. These chimeric constructs were employed in cell‐cell fusion and cell‐free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV‐1KMT but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell‐free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV‐1KMT efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3‐V5 domain from HIV‐1KMT to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3‐V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.
Molecular Ecology of Toxigenic Vibrio cholerae
Authors: Faruque Shah M. , Nair G. Balakrish
Published: November 2013

Toxigenic Vibrio cholerae is the etiological agent of cholera, an acute dehydrating diarrhea that occurs in epidemic form in many developing countries. Although V. cholerae is a human pathogen, aquatic ecosystems are major habitats of Vibrio species, which includes both pathogenic and nonpathogenic strains that vary in their virulence gene content. V. cholerae belonging to the O1 and O139 serogroups is commonly known to carry a set of virulence genes necessary for pathogenesis in humans. Recent studies have indicated that virulence genes or their homologues are also dispersed among environmental strains of V. cholerae belonging to diverse serogroups, which appear to constitute an environmental reservoir of virulence genes. Although the definitive roles of the virulence‐associated factors in the environment, and the environmental selection pressures for V. cholerae‐carrying virulence genes or their homologues is not clear, the potential for origination of new epidemic strains from environmental progenitors seems real. It is likely that the aquatic environment harbors different virulence‐associated genes scattered among environmental vibrios, which possess a lower virulence potential than the epidemic strains. The ecosystem comprising the aquatic environment, V. cholerae, genetic elements mediating gene transfer, and the mammalian host appears to support the clustering of critical virulence genes in a proper combination leading to the origination of new V. cholerae strains with epidemic potential.
Association of a Regulatory Gene, slyA with a Mouse Virulence of Salmonella Serovar Choleraesuis
Authors: Kaneko Aki , Mita Masaharu , Sekiya Kachiko , Matsui Hidenori , Kawahara Kazuyoshi , Danbara Hirofumi
Published: November 2013

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA‐defective mutant. The defective mutant was constructed by the insertion of a kanamycin‐resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 105 higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild‐type strain of S. serovar Choleraesuis was 40‐fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence‐associated regulator also in S. serovar Choleraesuis.
Chemokine Production by Rat Macrophages Stimulated with Streptolysin O from Streptococcus pyogenes
Authors: Mitsui Ken‐ichiro , Takano Katsuhiko , Nakatani Sumie , Nambu Hidemi , Shibata Futoshi , Nakagawa Hideo
Published: November 2013

The contribution of streptolysin O (SLO) from Streptococcus pyogenes to neutrophil infiltration in inflammatory lesions was determined by production of cytokine‐induced neutrophil chemoattractant (CINC)−1, −2 and −3, and macrophage inflammatory protein (MIP)‐1α by rat macrophages stimulated with SLO in culture. Active SLO induced the production of CINCs and MIP‐1α in dose‐ and time‐dependent manners. These inductions were ascertained by chemokine mRNA expression in macrophages. Streptolysin S was without effect. The SLO‐cholesterol complex induced the chemokine production in proportion to the residual hemolytic activity of the complex. In addition, the effects of SLO on the chemokine production were confirmed by the injection of active SLO into the preformed air pouch on the back of rats. The infiltration of neutrophils into the pouch fluid (exudate) increased steadily with a lag phase of about 2 hr. The major chemokine found in exudates was MIP‐1α but not CINCs. In this study, it became clear that active SLO, but not the inactive one, contributed to the production of MIP‐1α and CINCs in the conditioned medium and in exudates.
Characterization of the Outermembrane Proteins of Vibrio cholerae Expressed in In Vivo Culture
Authors: Nakasone Noboru , Iwanaga Masaaki
Published: November 2013

Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated. The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron‐regulated proteins. Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein. The N‐terminal amino acid sequence of the 15 kDa protein was homologous to V. cholerae OmpT. Anti‐serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not the organisms in in vitro culture. The antiserum was bactericidal, but it did not inhibit the adhesion of the organisms to the intestine and HEp‐2 cells. These findings suggest the possibility that the 15 kDa protein could be involved in post‐infection immunity.
Signaling Pathways Induced by Lipoproteins Derived from Mycoplasma salivarium and a Synthetic Lipopeptide (FSL‐1) in Normal Human Gingival Fibroblasts
Authors: Nakamura Jun‐ichiro , Shibata Ken‐ichiro , Hasebe Akira , Into Takeshi , Watanabe Tsuguo , Ohata Noboru
Published: November 2013

Lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL‐1) activate human gingival fibroblasts to induce IL‐6 production and ICAM‐1 expression. Human gingival fibroblasts were treated with lipoproteins or FSL‐1 and then examined for the activation of mitogen‐activated protein kinases (MAPKs), ERK1/2, p38, and SAPK/JNK, and transcription factors, AP‐1 and NF‐κB. Western blotting indicated that p38 and SAPK/JNK were activated in response to the stimulators, but the activation of ERK1/2 could not be discriminated because ERK1/2 was activated in the absence of stimulators. The p38 inhibitor SB 203580 also suppressed their IL‐6 production‐inducing activities, whereas the ERK1/2‐activating MAPK kinase (MEK1) inhibitor PD 98059 did not suppress their activities. Moreover, they were capable of inducing the activation of AP‐1 and NF‐κB. NF‐κB activation was also confirmed by the phosphorylation of IκB‐α. On the basis of these results, it was concluded that lipoproteins of M. salivarium and FSL‐1 are capable of activating the MAPKs p38 and SAPK/JNK and the transcriptional factors AP‐1 and NF‐κB in human gingival fibroblasts.
Serological Investigation ofBartonella henselaeInfections in Clinically Cat‐Scratch Disease‐Suspected Patients, Patients with Cardiovascular Diseases, and Healthy Veterinary Students in Japan
Authors: Kikuchi Eisaku , Maruyama Soichi , Sakai Takeo , Tanaka Shigeo , Yamaguchi Fukumitsu , Hagiwara Toshikatsu , Katsube Yasuji , Mikami Takeshi
Published: November 2013

Seroprevalence ofBartonella henselaewas investigated in Japan in 48 individuals clinically suspected of having cat‐scratch disease (CSD), 159 patients with cardiovascular diseases, and 129 healthy veterinary students. Of 48 CSD‐suspected patients examined, 19 (39.6%) were positive forB. henselae‐IgGand 4 (8.3%) forB. henselae‐IgM. Of 159 patients with cardiovascular diseases, 5 (3.1%) were positive forB. henselae‐IgG. In healthy veterinary students, 14 of 129 (10.9%) were positive forB. henselae‐IgGand 1 (0.8%) forB. henselae‐IgM. The positive rates ofB. henselae‐IgGand ‐IgM in CSD‐suspected patients were significantly higher than in other sources. Most CSD‐suspected and healthy individuals who were positive forB. henselaeantibody had had some contacts with cats. In CSD‐suspected patients, theB. henselaepositive rate in females was significantly higher than in males, and high seropositive rates toB. henselaewere found in younger age groups.
Anti‐Tumor Mechanism of Z‐100, an Immunomodulatory Arabinomannan Extracted from Mycobacterium tuberculosis Strain Aoyama B, on Pulmonary Metastases of B16F10 Melanoma: Restoration of Helper T Cell Responses via Suppression of Glucocorticoid‐Genesis
Authors: Oka Hideki , Emori Yutaka , Sasaki Hidetaka , Shiraishi Yumiko , Yoshinaga Koji , Kurimoto Tadashi
Published: November 2013

In the present study, the anti‐tumor mechanism of Z‐100 was investigated with the use of pulmonary metastasis of B16F10 melanoma. In B16F10 mice, Th1 cytokine production (IL‐2, IFN‐γ) was suppressed in comparison with normal mice. On the other hand, Th2 cytokine production (IL‐4, IL‐10) was increased in the B16F10 mice. The administration of Z‐100 to B16F10 mice restored the balance of Th1/Th2 cell responses from the Th2 dominant state to the normal state. Z‐100 significantly suppressed the pulmonary metastasis of B16F10 melanoma in a dose‐dependent manner. These results suggest that Z‐100 restored the breakdown of Th1 cell responses, resulting in the suppression of pulmonary metastasis of B16F10 melanoma. Moreover, Z‐100 decreased the corticosterone levels, which is known to suppress the Th1 cell responses, in both serum specimens and splenic tissue, and the steroidogenic CYP11A1 mRNA expression in CD4+ T cells. These results suggest that a suppression of pulmonary metastasis and restoration of Th1/Th2 cell responses by Z‐100 may be due to the decrease in the corticosterone levels and the steroidogenic CYP11A1 mRNA expression of CD4+ T cells in B16F10 mice. Further, the role of Th1 cytokine, IFN‐γ, on these activities of Z‐100 was examined. The suppressive effects of Z‐100 on pulmonary metastasis and restoration of Th1/Th2 cell responses were eliminated by the administration of anti‐IFN‐γ mAb. Moreover, the suppressive effects of Z‐100 on glucocorticoid‐genesis were eliminated by the administration of anti‐IFN‐γ mAb. These results suggest that Z‐100 restores the balance of Th1/Th2 cell responses via the suppression of glucocorticoid‐genesis by Z‐100‐induced IFN‐γ. IFN‐γ acts as a key cytokine in anti‐tumor activities of Z‐100.
KP‐103, a Novel Triazole Derivative, Is Effective in Preventing Relapse and Successfully Treating Experimental Interdigital Tinea Pedis and Tinea Corporis in Guinea Pigs
Authors: Tatsumi Yoshiyuki , Yokoo Mamoru , Arika Tadashi , Yamaguchi Hideyo
Published: November 2013

The therapeutic efficacy of KP‐103, a triazole derivative, for 10 guinea pigs with interdigital tinea pedis or tinea corporis was investigated. Topical KP‐103 solution (0.25 to 1%) was dose‐dependently effective in treating both dermatophytoses. A 1% KP‐103‐treatment rendered all infected skins culture‐negative on day‐2 posttreatment. A high negative‐culture rate was obtained with 1% solutions of butenafine and lanoconazole but not with 1% neticonazole solution. The follow up study performed on day‐30 and day‐9 posttreatment demonstrated that the relapse rates for 1% KP‐103‐treated animals with tinea pedis and for those with tinea corporis were 20 and 30%, respectively, and that these values were the same as those for 1% butenafine‐treated animals, but lower than those for 1% lanoconazole‐treated animals (55 and 80%, respectively). When a single dose of 1% KP‐103 was applied to the back skin 48 hr before fungal inoculation, 9 of the 10 animals were protected from the dermatophytosis, suggesting that active KP‐103 is retained in skin tissue for at least 48 hr after dosing. Moreover, it was suggested that KP‐103 retains a high activity in the horny layer because of its lower keratin‐affinity. The effectiveness of KP‐103 against dermatophytoses may be due to the favorable pharmacokinetic properties in the skin tissues, together with its potent antifungal activity.
Comparative Profiles of Intramacrophage Behavior of Mycobacterium tuberculosis and Mycobacterium avium Complex with Different Levels of Virulence
Authors: Sano Keisuke , Sato Katsumasa , Sano Chiaki , Shimizu Toshiaki , Tomioka Haruaki
Published: November 2013

Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) strains with different levels of virulence in mice were examined for profiles of interaction with murine peritoneal macrophages (MΦs). Their growth rates in MΦs were in these orders: H37Ra strain (attenuated) > H37Rv strain (virulent) for MTB, and N‐260 strain (moderate virulence) > MAC N‐444 strain (low virulence) for MAC. MTB but not MAC caused the necrotic death of host MΦs in terms of increased release of lactate dehydrogenase from infected MΦs. The MTB H37Ra strain induced a greater production of reactive nitrogen intermediates (RNI) by MΦs than the MTB H37Rv strain did. However, this phenomenon was not observed with MAC, implying less important roles of RNI in the expression of MΦ antimicrobial activity against MAC organisms.
Correlation between Secondary Structure of an Amino‐Terminal Portion of the Nonstructural Protein 3 (NS3) of Hepatitis C Virus and Development of Hepatocellular Carcinoma
Authors: Ogata Satoshi , Ku Yonson , Yoon Seitetsu , Makino Shigeru , Nagano‐Fujii Motoko , Hotta Hak
Published: November 2013

Correlation between sequence variation of hepatitis C virus (HCV) and development of hepatocellular carcinoma (HCC) has not yet been demonstrated. In the present study, we analyzed sequence diversity of the NS3 protein of HCV and its possible correlation with HCC. On the basis of secondary structure of an amino‐terminal portion of NS3, HCV subtype 1b (HCV‐1b) isolates were classified into two groups, A and B. Group A isolates were found in 4 (11%) of 36 patients with HCC, and 22 (63%) of 35 patients without HCC. On the other hand, group B isolates were found in 32 (89%) of 36 patients with HCC, and 12 (34%) of 35 patients without HCC. The distribution patterns of those groups were significantly different between patients with and without HCC (P<0.001). HCV isolates of group B were found in both tumor and adjacent non‐tumor tissues obtained from patients with HCC, suggesting that the emergence of group B isolates was not a result of, but rather a possible causative factor for development of HCC. Taken together, our present results suggest that HCV‐1b strains of group B are highly associated with HCC and that the secondary structure analysis of NS3 would be useful to predict high risk for development of HCC in HCV‐1b‐infected patients.
Genogroup Characterization of Reemerging Serotype G9 Human Rotavirus Strain 95H115 in Comparison with Earlier G9 and Other Human Prototype Strains
Authors: Nakagomi Toyoko , Nakagomi Osamu
Published: November 2013

Serotype G9 human rotaviruses have emerged globally since the mid‐1990s. The 95H115 strain was derived from a stool specimen collected in Japan in the 1994–95 season, thus it is the earliest of the globally reemerging G9 human rotaviruses that were adapted to cell culture. Genogrouping by RNA‐RNA hybridization was performed to examine the genetic background of 95H115. The 95H115 strain belonged to the Wa genogroup, the most common human rotavirus genogroup, and it had a high degree of homology with AU32 and WI61, the prototype G9 isolates in the 1980s. However, the divergent genomic RNA constellation as indicated by the aberrant hybridization patterns between 95H115 and earlier G9 strains served as further evidence that 95H115 was not a direct descendant of the prototype strains in the ′80s.
Outbreak of Central Nervous System Disease Associated with Hand, Foot, and Mouth Disease in Japan during the Summer of 2000: Detection and Molecular Epidemiology of Enterovirus 71
Authors: Fujimoto Tsuguto , Chikahira Masatsugu , Yoshida Shigeru , Ebira Hitomi , Hasegawa Ayako , Totsuka Atsuko , Nishio Osamu
Published: November 2013

Few outbreaks of the serious enterovirus 71 (EV71) infections, which affect the central nervous system (CNS), had been reported in Japan before 2000. During June through August 2000, a patient died of pulmonary edema caused by brainstem encephalitis accompanied by EV71‐induced hand, foot, and mouth disease (HFMD), and many patients complicated by serious CNS disease, including paralysis, were hospitalized in a restricted area in Hyogo Prefecture, Japan (K‐area). During the same period, endemics of HFMD were reported in other areas in Hyogo Prefecture, where EV71 was isolated from HFMD patients, but few patients developed aseptic meningitis. The isolations of EV71 from K‐area patients were difficult with the use of Vero cells, so the strains were isolated by use of GL37 cells; Vero cells, however, could isolate EV71 strains from other areas in Hyogo Prefecture. We sequenced VP4 coding regions of these EV71 isolates and found that the isolates from K‐area had the same sequence, which, except for one isolate, was different from the sequences of EV71 strains isolated from other areas of Hyogo Prefecture. Although these results were not enough to state that EV71 from K‐area was a virulent strain, it seemed reasonable to conclude that serious CNS diseases in K‐area were caused by EV71 because it was the only infectious agent detected in the inpatients of K‐area.
Role of the C‐Domain in the Biological Activities of Clostridium perfringens Alpha‐Toxin
Authors: Nagahama Masahiro , Mukai Masakazu , Morimitsu Shinsuke , Ochi Sadayuki , Sakurai Jun
Published: November 2013

Clostridium perfringens alpha‐toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C‐domain (251–370 residues; CP251–370) in biological activities of the toxin. The N‐domain (1–250 residues; CP1–250) of the alpha‐toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC‐CP251–370) consisting of BcPLC and CP251–370 bound to the red cells and lysed them. Incubation of CP1–250 with CP251–370 completely complemented hemolytic and PLC activities. CP251–370 also conferred hemolytic activity on BcPLC. CP251–340 (251–340 residues) significantly stimulated PLC activity of CP1–250, but did not confer hemolytic activity on CP1–250. Kinetic analysis suggested that CP251–370 increased affinity toward the substrate of CP1–250. The results suggested that CP251–370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1–250. Acrylodan‐labeled CP251–370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazolyl)ethylene diamine (NBD)‐labeled CP251–370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251–370 to a hydrophobic environment. These observations suggest that interaction of CP251–370 of alpha‐toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1–250.
Detection of Specific Helicobacter pylori DNA and Antigens in Stool Samples in Dyspeptic Patients and Healthy Subjects
Authors: Wiśniewska Monika , Nilsson Hans Olof , Bąlk‐Romaniszyn Leokadia , Rechciński Tomasz , Bielański Władysław , Płaneta‐Małecka Izabela , Płonka Małgorzata , Konturek Stanisław , Wadström Torkel , Rudnicka Wiesława , Chmiela Magdalena
Published: November 2013

In this study stool samples from dyspeptic patients and healthy subjects were used for detection of specific Helicobacter pylori antigens and DNA by immunoenzymatic test (PPHpSA) and semi‐nested PCR (ureA‐PCR), respectively. The H. pylori status was estimated by invasive endoscopy‐based rapid urease test and histology or noninvasive urea breath test (UBT), and by serology (ELISA, Western blot). The coincidence of H. pylori‐negative invasive tests or UBT and negative antigen or DNA stool tests was very high (mean 95%). The PPHpSA results were found positive for 56% and ureA‐PCR for 26% of individuals with H. pylori infection confirmed by invasive tests or UBT. The detection of specific H. pylori antigens and especially DNA in feces is not sufficient as a one‐step diagnosis of H. pylori infection.
A Novel Caspase Dependent Pathway Is Involved in Apoptosis of Human Endothelial Cells by Shiga Toxins
Authors: Yoshida Tomoaki , Koide Naoki , Sugiyama Tsuyoshi , Mori Isamu , Yokochi Takashi
Published: November 2013

Shiga toxins have been shown to induce apoptosis on primary cultures, but not passaged ones, of human umbilical vein endothelial cells, independent of cytokine pre‐treatment. Here, a peculiar pattern of caspase activation was observed; caspase‐3 and ‐2, but not conventional upstream caspases, were activated at the initial phase of 6 hr, whereas a broad range inhibitor of caspases, VAD‐fmk, but not mono‐specific ones, suppressed DNA fragmentation and cell death. These results suggest additional analogous molecules, which have yet to be delineated, are involved. The requirement of retrograde uptake of toxins was also proved by the intervening effect of brefeldin A.
Characterization of Monoclonal Antibody Reacting Exclusively against Intracellular Orientia tsutsugamushi
Authors: Kim Mee‐kyung , Odgerel Zagaa , Chung Moon‐hyun , Lim Byung‐uk , Kang Jae‐seung
Published: November 2013

Intracellular bacteria often change the expression of their genes in order to adapt to new environmental conditions. Here we describe a monoclonal antibody (MAb) that reacts exclusively against intracellular Orientia tsutsugamushi Although MAb applied to the 56‐kDa protein, a major outer membrane protein, reacted against a large number of bacteria that had attached to host cells at the early stage of infection, M686–13 reacted against only a minor portion of the attached bacteria. In the later stage of the intracellular growth cycle, both antibodies showed identical staining patterns by double immunofluorescent staining. These results suggest that M686–13 reacted to an epitope or a protein that had probably been expressed during the intracellular growth cycle and rapidly diluted or degraded upon release into the extracellular environment. Although its molecular characteristics remain unknown, the reactive antigen may prove to be a novel developmental antigen and this MAb could be used as reagent for the staining of viable O. tsutsugamushi.
Use of Immunoglobulin Enriched Bovine Colostrum against Oral Challenge with Enterohaemorrhagic Escherichia coli O157:H7 in Mice
Authors: Funatogawa Kenji , Ide Takayuki , Kirikae Fumiko , Saruta Katsutoshi , Nakano Masayasu , Kirikae Teruo
Published: November 2013

An immunoglobulin enriched bovine colostrum preparation, IMMULAC™ (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin‐resistant EHEC O157:H7 strain (O157‐SMR). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3‐week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim‐milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin‐pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim‐milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food‐borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.
Stimulation of the Secretion of Pro‐Inflammatory Cytokines by Bifidobacterium Strains
Authors: He Fang , Morita Hirotsugu , Ouwehand Arthur C. , Hosoda Masataka , Hiramatsu Masaru , Kurisaki Jun‐ichi , Isolauri Erika , Benno Yoshimi , Salminen Seppo
Published: November 2013

To characterize the ability of bifidobacteria to affect the production of macrophage‐derived cytokines, a murine macrophage‐like cell line, J774.1, was cultured in the presence of 27 strains of heat‐inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult‐type bifidobacteria, induced significantly more pro‐inflammatory cytokine secretion, IL‐12 and TNF‐α, by J774.1 cells, than did the infant‐type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti‐inflammatory IL‐10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult‐type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down‐regulate the inflammatory response.
Complete Sequence Analyses of Enterovirus 71 Strains from Fatal and Non‐Fatal Cases of the Hand, Foot and Mouth Disease Outbreak in Singapore (2000)
Authors: Singh Sunita , Poh Chit Laa , Chow Vincent Tak Kwong
Published: November 2013

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non‐structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.
Fecal Microbial Diversity in a Strict Vegetarian as Determined by Molecular Analysis and Cultivation
Authors: Hayashi Hidenori , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T‐RFLP) analysis and a culture‐based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria‐specific primers. Randomly selected clones were partially sequenced. T‐RFLP analysis was performed using amplified 16S rDNA. The lengths of T‐RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel “phylotypes” (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T‐RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).
Molecular Epidemiology of Salmonella enterica Serovar Enteritidis Isolated in Taiwan
Authors: Su Lin‐hui , Chiu Cheng‐hsun , Wu Tsu‐lan , Chu Chishih , Chia Ju‐hsin , Kuo An‐jing , Lee Chia‐ching , Sun Chien‐feng , Ou Jonathan T.
Published: November 2013

Incidence of Salmonella enterica serovar Enteritidis infection seems to be on the rise in Taiwan, and therefore, the characteristics of the isolate, including genotypes, were epidemiologically investigated. Of the 71 clinical strains isolated in 1997–1999, 61 (86%) remained susceptible to the eight antibiotics tested, while the remaining ten, eight of which were isolated in 1999, were resistant to one to three of the agents including three multiply resistant strains. The majority, 69 or 97% of the isolates, harbored a 60‐kb spvC gene‐carrying virulence plasmid and 12 of them harbored one or two additional various‐sized plasmids. Strains with more than one plasmid were isolated mostly in 1999. Pulse‐field gel electrophoresis (PFGE) revealed three major genotypes (Types A, B and C), in which Type A was the predominant type. Of the 68 Type A, which contained 8 subtypes, 59 (83%) belonged to only two subtypes. Similar results were obtained with a PCR‐based typing method, the infrequent‐restriction‐site (IRS) PCR. All four methods detected types that were rarely seen before and most of these were of recent isolates, indicating that these unusual types were new or strains of foreign origin. Though all four methods discriminated types well, PFGE and IRS‐PCR showed higher sensitivity for classification. Between the two, the latter, though less discriminatory than PFGE, seems the method of choice, since it is simpler, less time‐consuming and above all easy to perform.
Distribution of Rat C5a Anaphylatoxin Receptor
Authors: Akatsu Hiroyasu , Abe Masayoshi , Miwa Takashi , Tateyama Hisashi , Maeda Seiji , Okada Noriko , Kojima Kiyohide , Okada Hidechika
Published: November 2013

The anaphylatoxin, complement 5a (C5a), plays a key role in mediating various inflammatory reactions following complement activation. Several investigators have reported that C5a receptor (C5aR) is expressed in non‐myeloid cells under certain conditions or in different cell lines. In our study, the abundance of C5aR‐positive myeloid cells in rats depended on the organs examined. C5aR was usually expressed at the site of exposure to pathogens, such as in salivary gland or lung, and was up‐regulated in liver in the inflammatory state induced by lipopolysaccharide (LPS) administration. Furthermore, the increased expression of C5aR antigen was not accompanied by an increase in C5aR mRNA in Kupffer cells following LPS challenge.