bluuebunny/crossref_metadata_2025 huggingface dataset:
2‐Aminopurine Inhibits Lipopolysaccharide‐Induced Nitric Oxide Production by Preventing IFN‐β Production
Authors: Sugiyama Tsuyoshi , Fujita Megumi , Koide Naoki , Mori Isamu , Yoshida Tomoaki , Mori Hiroshi , Yokochi Takashi
Published: November 2013

2‐Aminopurine (2‐AP) is widely used as a specific inhibitor for double stranded‐RNA dependent protein kinase (PKR). Here we report that 2‐AP can inhibit lipopolysaccharide (LPS)‐stimulated nitric oxide (NO) production through the prevention of interferon (IFN)‐β production. 2‐AP significantly inhibited NO production in LPS‐stimulated RAW 264 murine macrophage cells. 2‐AP also reduced the expression of IFN‐β and IFN‐inducible genes, such as IFN‐γ‐inducible protein (IP)‐10 and immune‐responsive gene (IRG)‐1, and the inducible type of NO synthase (iNOS) mRNA in response to LPS. The addition of exogenous IFN‐β restored 2‐AP‐inhibited NO production in response to LPS. On the other hand, there was only partial inhibition by 2‐AP of nuclear factor (NF)‐κB activation, IL‐6 mRNA expression and tumor necrosis factor (TNF)‐α production. These results suggested that 2‐AP inhibited LPS‐induced IFN‐β production by preventing Toll/IL‐1 receptor domain‐containing adaptor‐inducing IFN‐β (TRIF)‐dependent signaling rather than myeloid differentiation factor (MyD) 88‐dependent signaling, resulting in the inhibition of NO production.
Recombinant Cholera Toxin B Subunit Activates Dendritic Cells and Enhances Antitumor Immunity
Authors: Isomura Iwao , Yasuda Yoko , Tsujimura Kunio , Takahashi Toshitada , Tochikubo Kunio , Morita Akimichi
Published: November 2013

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non‐toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7–2 on DC was upregulated and the secretion of IL‐12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB‐treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB‐treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB‐treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell‐depleted mice, although substantial antitumor effect was observed in CD4+ T cell‐depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.
Autoantibodies to Peroxiredoxin I and IV in Patients with Systemic Autoimmune Diseases
Authors: Karasawa Rie , Ozaki Shoichi , Nishioka Kusuki , Kato Tomohiro
Published: November 2013

Anti‐oxidative enzymes protect living bodies from various oxidative stresses. In the systemic autoimmune diseases, autoantibodies to oxidized molecules and to anti‐oxidative enzymes have been reported. To promote understanding of the relationships between autoimmunity and oxidative stress, we here investigate whether autoimmunity to the anti‐oxidative peroxiredoxin (Prxs) enzymes exists in patients with systemic autoimmune diseases. Specifically, we detected autoantibodies to recombinant Prx I and Prx IV respectively by ELISA and western blotting. Next, clinical parameters were compared between the anti‐Prx I or IV‐positive and ‐negative patients. We found that 33% of the 92 patients with autoimmune diseases tested possessed autoantibodies to Prx I (57% in systemic lupus erythematosus (SLE), 19% in rheumatoid arthritis (RA), 5% in Behçet disease, and 46% in primary vasculitis syndrome). In contrast, autoantibodies to Prx IV were detected in only 17% of the same patients. No significant correlation was found between occurrence of the two autoantibodies. Clinically, possession of anti‐Prx I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Taken together, our data demonstrate the existence of autoantibodies to Prxs for the first time. The autoantibodies to Prx I may be involved in the pathophysiology of systemic autoimmune diseases such as SLE and vasculitis.
A Novel Diagnostic Method for Human Immunodeficiency Virus Type‐1 in Plasma by Near‐Infrared Spectroscopy
Authors: Sakudo Akikazu , Tsenkova Roumiana , Onozuka Taisuke , Morita Kazuo , Li Shuming , Warachit Jiranan , Iwabu Yukie , Li Guimei , Onodera Takashi , Ikuta Kazuyoshi
Published: November 2013

Presently, the diagnosis of virus infections is based mainly on serological assays. Although polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) have been increasingly used for the diagnosis of such viral infections, the risk of transfusion‐transmitted blood‐borne viruses remains. Furthermore, PCR and ELISA are expensive and time‐consuming, and sometimes cause false‐positive or false‐negative results. Therefore, a rapid, accurate and cost‐effective diagnostic procedure is needed. We subjected plasma from individuals infected with human immunodeficiency virus type‐1 (HIV‐1), the causative agent of acquired immune deficiency syndrome (AIDS), as well as plasma from uninfected individuals as a control to near‐infrared (NIR) spectroscopy, which may provide a rapid diagnostic method for HIV‐1 infection without using any reagent. NIR spectra in the 600–1,000 nm region for plasma from pre‐serologically HIV‐1‐infected individuals and healthy donors were subjected to partial least squares (PLS) regression analysis and leave‐out cross‐validation to develop a multivariate model to estimate the concentration of HIV‐1. Simultaneously, the same plasma samples were examined for HIV‐1 p24 by ELISA. The results obtained by the NIR spectroscopy model for HIV‐1 yielded a good correlation with those obtained by the reference method (HIV‐1 p24 ELISA). These results suggest that NIR spectroscopy using plasma could provide a rapid, accurate, cost‐effective tool for large‐scale diagnosis of HIV‐1 infection.
Rapid Species Identification and Partial Strain Differentiation of Clostridium butyricum by PCR Using 16S‐23S rDNA Intergenic Spacer Regions
Authors: Nakanishi Shusuke , Kuwahara Tomomi , Nakayama Haruyuki , Tanaka Mamoru , Ohnishi Yoshinari
Published: November 2013

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain‐specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S‐23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.
Microarray Analysis of Host Gene‐Expression during Intracellular Nests Formation of Trypanosoma cruzi Amastigotes
Authors: Imai Kazuhide , Mimori Tatsuyuki , Kawai Makoto , Koga Hisashi
Published: November 2013

The intracellular pathogens utilize numerous cellular components of host cells to advance the infection as well as to enter the host cell. Analyzing the host cellular response enables us to get a better understanding of the pathogenesis, and subsequently indicate possible therapeutic targets. We therefore analyzed gene‐expression profile of NIH3T3 fibroblast cells infected by Trypanosoma, a representative intracellular pathogen similar to Leishmania, using custom‐designed cDNA microarray consisting of 1,701 mKIAA cDNAs. Focusing on intracellular nest formation of Trypanosoma cruzi amastigotes, we profiled the host gene‐expression at 8 days post‐infection and found several degrees of change in 16 mKIAA genes. Among these genes, 10 were up‐regulated and 6 were down‐regulated. Assuming that these genes had important roles in the infection's progression, we performed semi‐quantitative RT‐PCR analysis and confirmed the gene expression change of 4 genes. Furthermore, 5 genes were mapped on cadherin signaling pathway using pathway analysis software. These results indicate significance of the host cellular pathway in the proliferative stage of Trypanosoma cruzi amastigotes.
Association of Cytomegalovirus with Infantile Hepatitis
Authors: Shibata Yukiko , Kitajima Naoko , Kawada Jun‐ichi , Sugaya Naomi , Nishikawa Kazuo , Morishima Tsuneo , Kimura Hiroshi
Published: November 2013

Infantile hepatitis is occasionally seen in apparently healthy children. In most cases, the etiology of the infection is uncertain. However, cytomegalovirus (CMV), Epstein‐Barr virus (EBV), human herpesvirus‐6 (HHV‐6), human herpesvirus‐7 (HHV‐7), human parvovirus B19, and TT virus (TTV) are considered to be associated with hepatitis in children. The objective of this study was to investigate the correlations between these viruses and infantile hepatitis. Twenty‐six children from 1 to 24 months old (median age, 7 months) who had liver dysfunction of unknown etiology were enrolled in this study. Plasma samples were examined by a real‐time PCR assay for CMV, EBV, HHV‐6, HHV‐7, parvovirus B19, and TTV DNA. The DNA of CMV was detected in the plasma of four patients (15.4%) and was detected significantly more often in the patient group than in the control group. The CMV‐infected patients were 1 to 3 months old, which was significantly younger than the remaining patients. The serological findings did not always correlate with the results of the real‐time PCR assay. The DNA of TTV was detected in four patients (15.4%), while human parvovirus B19 DNA was detected in three (11.5%). However, the detection frequencies of these viral DNAs were not significantly different from those in the control groups, and some of these patients had co‐infections. These results indicate that CMV might be one of the major pathogens responsible for infantile hepatitis; however, serological tests have limited utility for the diagnosis of CMV infection in young children.
Japanese Babesia microti Cytologically Detected in Salivary Glands of Naturally Infected Tick Ixodes ovatus
Authors: Yano Yasuhiro , Saito‐Ito Atsuko , Anchalee Dantrakool , Takada Nobuhiro
Published: November 2013

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field‐collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporo‐blast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood‐sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector‐pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.
Close Correlation of Streptococcal DNase B (sdaB) Alleles with emm Genotypes in Streptococcus pyogenes
Authors: Matsumoto Masakado , Sakae Kenji , Hashikawa Shinnosuke , Torii Keizo , Hasegawa Tadao , Horii Toshinobu , Endo Miyoko , Okuno Rumi , Murayama Shoko , Hirasawa Kyoko , Suzuki Rieko , Isobe Junko , Tanaka Daisuke , Katsukawa Chihiro , Tamaru Aki , Tomita Masaaki , Ogata Kikuyo , Ikebe Tadayoshi , Watanabe Haruo ,   The Working Group for Group A Streptococci in Japan, Ohta Michio
Published: November 2013

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non‐synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype‐specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.
Detection and Quantification of Four Species of the Genus Clostridium in Infant Feces
Authors: Tonooka Toshiki , Sakata Shinji , Kitahara Maki , Hanai Michio , Ishizeki Shinobu , Takada Masaaki , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species‐specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle‐ and mix‐fed infants were relatively higher than in breast‐fed infants (P < 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to real‐time PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1 × 105 to 3 × 107 cells/g wet feces.
Analysis of the Distribution of Neuropathogenic Retroviral Antigens Following PVC211 or A8‐V Infection
Authors: Nakai Ryuhei , Takase‐Yoden Sayaka , Watanabe Rihito
Published: November 2013

A8‐V and PVC211 are neuropathogenic strains of the Friend murine leukemia virus (Fr‐MLV) that cause spongiosis in the rat brain after infection at birth. PVC211 exhibited stronger neuropathogenicity than A8‐V, and induced more severe neurological symptoms such as hind‐leg paralysis. These symptoms correlated with the neuropathological spread and intensity, which were more severe in the spinal cord of rats infected with PVC211 than in those infected with A8‐V, without exhibiting neuropathological differences in other areas of the CNS. Interestingly, virus titers recovered from infected spinal cords were similar in PVC211 and A8‐V infected animals. However, in the spinal cord infected with PVC211, glial cells attained higher immunohistochemical expression scores for the viral surface antigen, gp70 (Env) than in the A8‐V infected spinal cord, although expression levels of viral antigens in blood vessel walls were similar in A8‐V and PVC211 infections. Furthermore, many of those glial cells which carried viral antigens were found, by double immunostaining, to be microglia. The results suggested that the spread of viral antigen positive microglia plays an important role in forming the different neuro‐pathogenicity observed in A8‐V and PVC211 infections.
Comparison of Effects of Nitric Oxide Synthase (NOS) Inhibitors on Plasma Nitrite/Nitrate Levels and Tissue NOS Activity in Septic Organs
Authors: Hayashi Yuri , Abe Masayoshi , Murai Akira , Shimizu Naomi , Okamoto Iku , Katsuragi Takeshi , Tanaka Keiichi
Published: November 2013

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar‐King rats. The rats treated with the three iNOS inhibitors (N‐(3‐(aminomethyl)benzyl)acetamidine (1400W), (1S, 5S, 6R, 7R)‐2‐aza‐7‐chloro‐3‐imino‐5‐methylbicyclo [4.1.0] heptane hydrochloride (ONO‐1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose‐dependent decreases in plasma NOx levels and NOS activity in the lungs. The non‐selective NOS inhibitor (NG‐methyl‐L‐arginine (L‐NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.
Facilitated Production of Secretory IgA against Shiga Toxin B Subunits by Intranasal Application of Antigen‐Coated Polystyrene Microspheres
Authors: Kurohane Kohta , Kobayashi Chie , Imai Yasuyuki
Published: November 2013

We examined the effects of microspheres as antigen carriers in mucosal immunization. Shiga toxin B subunits (Stx1B) were adsorbed on 6 μm polystyrene microspheres, which were then intranasally administered to mice together with cholera toxin (CT). Stx1B‐specific serum IgG production and secretory IgA production at local mucosal sites were enhanced by the use of microspheres. When OVA was used as a model antigen, secretory IgA production but not serum IgG production was enhanced on the use of microspheres. These results indicated that microspheres provide a useful means of potentiating the immune response against Stx1B with weak immunogenicity.
Peroral Infectivity of Toxoplasma gondii in Bile and Feces of Interferon‐γ Knockout Mice
Authors: Piao Lian‐Xun , Aosai Fumie , Mun Hye‐Seong , Yano Akihiko
Published: November 2013

Toxoplasama gondii appeared in the bile and feces of interferon‐γ knockout (GKO) but not wild type mice on days 7–8 after peroral infection with T. gondii cysts of Fukaya strain. Both tachyzoite‐specific SAG1 and bradyzoite‐specific T.g.HSP30 mRNAs were detected in the bile and feces of GKO mice. Tachyzoites converted to bradyzoites by culturing in the bile. By feeding uninfected mice with the bile and washed feces of T. gondii‐infected GKO mice, T. gondii‐specific antibody formation in the serum and cyst formation in the brain were observed. The novel migration route of T. gondii from liver to bile and feces in GKO mice was confirmed.
Influence of Additional Acylation Site(s) of Influenza B Virus Hemagglutinin on Syncytium Formation
Authors: Ujike Makoto , Nakajima Katsuhisa , Nobusawa Eri
Published: November 2013

We studied the effects of an increase in the hydrophobicity of the transmembrane domain (TM) and cytoplasmic tail (CT) of influenza B virus hemagglutinin (BHA) on fusion activities. For this purpose, we created mutant HAs with novel acylation site(s) in the TM and/or CT. All mutants were able to induce hemifusion and to form fusion pores as well as could wild type (wt) BHA. However, the ability of these mutants to form syncytia was impaired, indicating that the increase in the hydrophobicity of these domains (especially the CT) affected fusion pore dilation.
Cooperation between Alteration of DNA Gyrase Genes and Over‐Expression of MexB and MexX Confers High‐Level Fluoroquinolone Resistance in Pseudomonas aeruginosa Strains Isolated from a Patient Who Received a Liver Transplant Followed by Treatment with Fluoroquinolones
Authors: Niga Toshiyuki , Ito Hideaki , Oyamada Yoshihiro , Yamagishi Jun‐ichi , Kadono Manami , Nishino Takeshi , Gotoh Naomasa , Inoue Matsuhisa
Published: November 2013

Clinical isolates of highly fluoroquinolone‐resistant Pseudomonas aeruginosa had a mutation in either A or B subunit of DNA gyrase and over‐expressed MexB and MexX, the efflux system proteins. Introduction of wild‐type gyrase genes of Escherichia coli into the isolates made them as fluoroquinolone‐susceptible as the moderately fluoroquinolone‐resistant strains that only over‐expressed efflux system proteins. These findings demonstrate that high fluoroquinolone‐resistance in P. aeruginosa is attributed to cooperation between alteration in DNA gyrase genes and over‐expression of efflux systems proteins.
Regulation of CD93 Cell Surface Expression by Protein Kinase C Isoenzymes
Authors: Ikewaki Nobunao , Kulski Jerzy K. , Inoko Hidetoshi
Published: November 2013

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)‐mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte‐like cell line (U937), a human NK‐like cell line (KHYG‐1), and a human umbilical vein endothelial cell line (HUV‐EC‐C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H‐89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde‐fixed cellular enzyme‐linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI‐11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down‐regulated CD93 expression on the U937 cells in a dose‐dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down‐regulated by Go6976, but not by Rottlerin, in the KHYG‐1 cells and by both Rottlerin and Go6976 in the HUV‐EC‐C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up‐regulated CD93 expression on the cell surface of all three cell‐lines and induced interleukin‐8 (IL‐8) production by the U937 cells and interferon‐γ (IFN‐γ) production by the KHYG‐1 cells. In addition, both Go6976 and Rottlerin inhibited the up‐regulation of CD93 expression induced by PMA and IL‐8 or IFN‐γ production in the respective cell‐lines. Whereas recombinant tumor necrosis factor‐α (rTNF‐α) slightly up‐regulated CD93 expression on the U937 cells, recombinant interleukin‐1β (rIL‐1β), recombinant interleukin‐2 (rIL‐2), recombinant interferon‐γ (rIFN‐γ) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.
Involvement of Mannose Receptor in Glycopeptidolipid‐Mediated Inhibition of Phagosome‐Lysosome Fusion
Authors: Shimada Ken‐ichi , Takimoto Hiroaki , Yano Ikuya , Kumazawa Yoshio
Published: November 2013

We previously reported that glycopeptidolipid (GPL) isolated from Mycobacterium avium serovar 4 inhibited phagosome‐lysosome (P‐L) fusion when macrophages phagocytosed heat‐killed Staphylococcus aureus (SA). In the present study we analyzed the underlying inhibitory mechanism of GPL coated on SA. Elimination of oligosaccharide from GPL abrogated its inhibitory activity. GPL did not inhibit P‐L fusion of opsonized SA phagocytosed via complement receptors. The inhibitory activity of GPL was competitively reduced by the presence of α‐methyl‐D‐mannoside and anti‐mannose receptor antibody, suggesting that inhibition of P‐L fusion by GPL is mediated through mannose receptor. Recruitment of early endosome antigen 1 and Ca2+/calmodulin kinase II in human macrophage‐like THP‐1 cells were significantly suppressed by GPL, indicating that GPL inhibits steps for leading to the P‐L fusion.
Predominant T Helper 2 Immune Responses againstBartonella henselaein Naturally Infected Cats
Authors: Kabeya Hidenori , Sase Makiko , Yamashita Masaya , Maruyama Soichi
Published: November 2013

This study was conducted to explicate the mechanism of long‐term bacteremia inBartonella henselae‐infected cats by the examining host immune responses. Blood samples were collected from three naturally infected cats and the IgG antibody titers and the cytokine responses in peripheral blood mononuclear cells (PBMC) were examined by quantitative reverse transcriptase‐polymerase chain reactions (RT‐PCR). Relapsing bacteremia was found in two of the three cats during the examination period. The quantitative RT‐PCR analyses demonstrated that increases of the mRNA expressions in interleukin‐4 (IL‐4) but not in gamma‐interferon (IFN‐γ) were observed in PBMC from these infected cats after the bacteremia had peaked, showing that the T helper 2 (Th2) responses were specifically induced in the cats. Furthermore, the specific antibody titer increased, resulting in a decrease in the number ofB. henselaeto undetectable levels in these cats. However, the number of bacteria increased again in two of these cats at 90 and 45 days after the previous bacteremia, respectively. These results suggest thatB. henselaepredominantly induced IL‐4 production from PBMC and resulted in stimulation of the humoral immune responses, including the secretion of specific antibodies in the cats. Furthermore, the specific antibody may play a role in eliminating the bacteria from cats partially but not completely, because relapsing bacteremia was found in these two cats.
Chloroquine Inhibits Paracoccidioides brasiliensis Survival within Human Monocytes by Limiting the Availability of Intracellular Iron
Authors: Dias‐Melicio Luciane Alarcão , Moreira Ana Paula , Calvi Sueli Aparecida , de Campos Soares Angela Maria Victoriano
Published: November 2013

The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18‐infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose‐dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast‐killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb‐infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment.
Occurrence of the Fimbria Gene hifA in Clinical Isolates of Nonencapsulated Haemophilus influenzae
Authors: Nakamura Masahiko , Asaka Toshiyuki , Kirita Akiko , Miyazaki Hatsumi , Senda Yasuko , Fujita Shin‐Ichi , Fukushima Ritsuko , Watanabe Kishichiro , Karasawa Tadahiro , Kawahara Ei , Shimura Satoshi , Yamagishi Takayoshi
Published: November 2013

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA‐positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.
Rapid Detection and Quantification of Japanese Encephalitis Virus by Real‐Time Reverse Transcription Loop‐Mediated Isothermal Amplification
Authors: Toriniwa Hiroko , Komiya Tomoyoshi
Published: November 2013

We established a rapid, quantitative real‐time reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RT‐LAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT‐LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription‐polymerase chain reaction (RT‐PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT‐LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT‐LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT‐LAMP was strongly correlated (r=0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT‐LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.
Presence of LuxS/AI‐2 Based Quorum‐Sensing System in Vibrio mimicus: LuxO Controls Protease Activity
Authors: Sultan Zafar , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

Presence of the quorum‐sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI‐2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi‐like quorum‐sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.
Prevalence of Low Positive Anti‐HCV Antibodies in Blood Donors: Schistosoma mansoni Co‐Infection and Possible Role of Autoantibodies
Authors: Agha Salah , El‐Mashad Noha , El‐Malky Mohamed , El‐Shony Huda , El‐Sherif Mohamed Zaki , El‐Hasan Mohamed Abo , Tanaka Yasuhito , Mizokami Masashi
Published: November 2013

Patients infected with schistosoma frequently show a high seroprevalence of anti‐hepatitis C virus (anti‐HCV) antibodies. The aim of this study was to find the underlying reason for this phenomenon, and to examine a possible involvement of autoantibodies. Out of 2,400 Egyptian blood donors, 192 (8%) were anti‐HCV positive by ELISA. They were 133 males and 59 females with age ranging from 27 to 48 years. According to optical density ratio (ODR) of anti‐HCV antibodies, 96 cases were low positive (LP) with ODR (1–2) designated as group I, and 96 were high positive (HP) with ODR (≥2) (group II). Both groups were examined for quantitative HCV core antigen (HCVcAg), liver function (Albumin, ALT, AST) and anti‐Schistosoma mansoni (anti‐Sm) IgG. Group I cases were HCVcAg negative with normal liver function tests, and 44 of them were anti‐Sm positive. Ninety cases (93.75%) of group II were HCVcAg positive with markedly affected liver function tests and 72 cases were anti‐Sm positive. All group I cases were examined for autoimmune markers (ANA, AMA, SMA and LKM). In group I, 33 (75%) of anti‐Sm positive cases were positive for one or more of the autoimmune markers examined, while none of anti‐Sm negative was positive for any marker with significant difference between the two groups (P<0.0001). Our results primarily on blood donors indicate that LP anti‐HCV frequently represents false‐positive reactivity with a possible role of Sm‐induced autoantibodies in this phenomenon.
Unnoticed Spread of Class 1 Integrons in Gram‐Positive Clinical Strains Isolated in Guangzhou, China
Authors: Shi Lei , Zheng Meiping , Xiao Zenghuang , Asakura Masahiro , Su Jianyu , Li Lin , Yamasaki Shinji
Published: November 2013

A total of 46 gram‐positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1‐specific primers, and the intI1‐positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII‐orfF‐aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram‐positive clinical strains isolated in Guangzhou, China.
Cell Elongation and Cell Death of Helicobacter pylori Is Modulated by the Disruption of cdrA (Cell Division‐Related Gene A)
Authors: Takeuchi Hiroaki , Nakazawa Teruko , Okamoto Takeshi , Shirai Mutsunori , Kimoto Mitsuo , Nishioka Mitsuaki , Con Sergio A. , Morimoto Norihito , Sugiura Tetsuro
Published: November 2013

The cell division‐related gene A (cdrA) of Helicobacter pylori is dispensable in vivo and unique in having a repressive role on cell division and long‐term survival. To clarify its role, comparisons of the wild‐type HPK5 and isogenic cdrA ‐disrupted mutant HPKT510 were examined by ultrastructural morphology, PBP profiles, and susceptibility to β‐lactam antibiotics during long‐term cultivation. Ultrastructural analyses revealed that the shorter rods of HPKT510 had a slightly wider periplasmic space between the inner and the outer membrane than those of HPK5. Cell division of HPKT510 cells was complete even under high‐salt conditions in which HPK5 cells became filamentous due to inhibition of division. The filamentous HPK5 cells constructed an inner membrane without a cell wall at the presumed division site. After 4 days of cultivation (the late stationary phase), most of the HPK5 cells turned into ghosts and aggregates, while some of the HPKT510 cells remained as curved rods, which coincided with the results of cell viability. HPKT510 cells became resistant to ampicillin killing compared to HPK5 cells, although their minimum inhibitory concentrations (MICs) and PBP profiles were not significantly different. These results suggest that the cdrA product represses cell division via inhibiting cell wall synthesis at division site. During infection in both mice and humans, inactivation of cdrA eventually gains biological aspects such as increased viability, long‐term survival and tolerance to antibiotics and high‐salt condition, which might enhance a persistent infection.
Role of the Septin Cdc10 in the Virulence of Candida albicans
Authors: González‐Novo Alberto , Labrador Leticia , Jiménez Antonio , Sánchez‐Pérez Miguel , Jiménez Javier
Published: November 2013

The relationship between the morphology and virulence of Candida albicans has aroused interest in the study of the proteins involved in its morphogenesis. We present virulence data for one important element in fungal morphogenesis—septins. We disrupted CaCDC10 and studied the virulence in a mouse infection model and the different steps followed by the fungus during the infection: adherence to epithelial cells, organ colonisation, macrophage phagocytosis, and host survival. We found the altered subcellular localisation of Int1—a C. albicans adhesin— in the septin null mutants. The Int1 mislocalisation and the defects in the cell wall of defective CaCdc10 strains permit us to propose a model for explaining the biological meaning of the absence of virulence presented by these septin mutants.
Isolation and Identification of Mycobacteria from Soils at an Illegal Dumping Site and Landfills in Japan
Authors: Wang Yan , Ogawa Midori , Fukuda Kazumasa , Miyamoto Hiroshi , Taniguchi Hatsumi
Published: November 2013

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co‐culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto‐unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.
Immunological Effects of Recombinant Feline Interferon‐ω (KT‐80) Administration in the Dog
Authors: Kuwabara Masato , Nariai Youko , Horiuchi Yutaka , Nakajima Yuko , Yamaguchi Yu , Horioka Eriko , Kawanabe Mikiko , Kubo Takuya , Yukawa Masayoshi , Sakai Takeo
Published: November 2013

The immunological effects of recombinant feline interferon‐ω (rFeIFN‐ω; KT‐80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT‐80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post‐administration survived. These results suggest that rFeIFN‐ω (KT‐80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.
Preparation and Functional Analysis of Tumor‐Infiltrating Stroma Cells Using Bone Marrow Chimera Mice
Authors: Ishigaki Hirohito , Yamamoto Yoshiko , Ishida Hideaki , Kajino Kiichi , Itoh Yasushi , Fujiyama Yoshihide , Ogasawara Kazumasa
Published: November 2013

Tumor‐infiltrating stroma cells (TISC) as well as tumors themselves are thought to be involved in tumor‐related immunosuppression, which is one of the critical mechanisms of tumor escape from immune surveillance. However, preparation of TISC is difficult because of the small proportion of TISC in established tumors. Thus, the cells thought to be involved in tumor‐related immunosuppression are generally prepared from spleens or draining lymph nodes in tumor‐bearing mice. In this study, we developed a method for directly preparing TISC from established tumors in order to analyze their function. Using green fluorescent protein (GFP) transgenic (Tg) mice and C57BL/6 mice transplanted with bone marrow (BM) cells of GFPTg mice, we detected three subpopulations of TISC: one is compatible with immature myeloid cells (ImC) derived from BM and the two other subpopulations, CD11b+ cells and CD11b– cells, do not originate from BM. The TISC including these subpopulations but not each subpopulation independently after culturing with tumors in the presence of GM‐CSF could suppress T cell proliferation induced by anti‐CD3. In our system, tumors did not inhibit T cell responses directly, but unknown factors from tumors affected immunosuppression by TISC.
Efficacy of Intracerebral Immunization against Pseudorabies Virus in Mice
Authors: Shin Jae‐Ho , Sakoda Yoshihiro , Kim Jae Hoon , Tanaka Tomohisa , Kida Hiroshi , Kimura Takashi , Ochiai Kenji , Umemura Takashi
Published: November 2013

To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with formalin‐inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 106 plaque‐forming units of PRV were introduced into the hindleg of the immunized or non‐immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non‐immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.
Contribution of L‐Alanine Dehydrogenase to In Vivo Persistence and Protective Efficacy of the BCG Vaccine
Authors: Scandurra Gabriella M. , Ryan Anthony A. , Pinto Rachel , Britton Warwick J. , Triccas James A.
Published: November 2013

The tuberculosis (TB) vaccine strain Mycobacterium bovis BCG is unable to utilise alanine and this deficiency is thought to inhibit the growth of the vaccine in vivo and limit vaccine efficacy. In this report we demonstrate that L‐alanine catabolism can be conferred on BCG by introduction of the gene encoding L‐alanine dehydrogenase (Ald) of Mycobacterium tuberculosis. Restoration of Ald activity did not change the in vivo growth of BCG in macrophages or mice, and protection against aerosol M. tuberculosis infection was not altered by addition of ald to the BCG vaccine. These results demonstrate that the inability to utilise L‐alanine is not a contributing factor to the attenuated phenotype of BCG and does not influence the protective efficacy of the vaccine against TB.
Evidence for Pestivirus Infection in Free‐Living Japanese Serows,Capricornis crispus
Authors: Harasawa Ryô , Aoyama Fumio , Goryo Masanobu , Okada Kosuke , Nishimura Takashi , Matsubara Kazuei , Tsujimoto Tsunenori , Giangaspero Massimo
Published: November 2013

Sixteen serum samples collected from free‐living Japanese serows,Capricornis crispus, between 2001 and 2004 in Morioka and its vicinity were examined for the presence of pestivirus by reverse transcription‐nested PCR procedure. Three out of the 16 samples produced a visible band in electrophoresed agarose gels. The nucleotide sequences of the three PCR products were found to be identical. The pestivirus found in the serow was identified asBovine viral diarrhea virus 1(BVDV‐1) based on nucleotide sequence analyses by phylogeny as well as palindromic nucleotide substitutions at the 5′ untranslated regions. Our data first indicated that BVDV‐1 infection occurred continuously among the free‐living serow populations though the role of BVDV‐1 in wild ungulates is currently unknown.
Detection and Quantification of Specific IgE Antibodies against Eight Malassezia Species in Sera of Patients with Atopic Dermatitis by Using an Enzyme‐Linked Immunosorbent Assay
Authors: Kato Hiroshi , Sugita Takashi , Ishibashi Yoshio , Nishikawa Akemi
Published: November 2013

The lipophilic yeast Malassezia, a member of the cutaneous microflora, is an exacerbating factor in atopic dermatitis (AD). Of the 11 currently recognized species, M. globosa and M. restricta are found to frequently colonize the skin of AD patients. In this study, we attempted to quantify specific IgE antibodies against eight Malassezia species, namely, M. dermatitis, M. furfur, M. globosa, M. obtusa, M. pachydermatis, M. slooffiae, M. sympodialis, and M. restricta, in sera from AD patients by using an enzyme‐linked immunosorbent assay (ELISA). The specific IgE value against M. restricta was greater than those against other Malassezia species. Competitive ELISA inhibition tests revealed that M. restricta contained species‐specific as well as shared antigens. Therefore, M. restricta could be considered as a candidate diagnostic antigen for detecting anti‐Malassezia IgE in sera from AD patients.
Protective Immunity against Bordetella pertussis by a Recombinant DNA Vaccine and the Effect of Coinjection with a Granulocyte‐Macrophage Colony Stimulating Factor Gene
Authors: Li Qingtian , Zhu Yongzhang , Chu Jiayou , Wang Yaying , Xu Yinghua , Hou Qiming , Zhang Shumin , Guo Xiaokui
Published: November 2013

A recombinant pertussis DNA vaccine was described here with its immunogenicity and the ability to induce protection against B. pertussis infection in mice. Three immunodominant antigen gene fragments of pertussis, pertussis toxin subunit 1 (pts1), fragments of pertactin (prn) and filamentous hemagglutinin (fha), were recombined as fragment pts1‐prn‐fha named ppf, and it was cloned to plasmid pVAX1 as pVAX1/ppf. Compared to those injected with pVAX1, the mice injected with pVAX1/ppf significantly elicited more antigen specific antibody anti‐PTS1, anti‐PRN, anti‐FHA and cytokine IL‐10, IFN‐γ. When pGM‐CSF was coinjected with pVAX1/ppf, the mice showed significantly increases of the three antibodies and cytokine IL‐10, IL‐4, IFN‐γ and TNF‐α compared to those injected with pVAX1 only. The mice in group pVAX1/ppf & pGM‐CSF, in particular; induced much more anti‐PTS1, IL‐4 and TNF‐α than those in group pVAX1/ppf. In the intracerebral mouse protection test, the mice immunized with pVAX1/ppf or pVAX1/ppf & pGM‐CSF induced protection to a lethal dose of B. pertussis. The results indicate that recombinant DNA vaccine and pGM‐CSF coinjection can induce protective immunity against B. pertussis, demonstrating a valuable method to prevent pertussis.
Sulfatide and Its Synthetic Analogues Recognition byMoraxella catarrhalis
Authors: Ozcelik Pinar , Bezirci Fatma Betul , Suzuki Yasuo , Uzawa Hirotaka , Nishida Yoshihiro , Kobayashi Kazukiyo , Suzuki Takashi , Miyamoto Daisei , Nagatake Tsuyoshi , Ahmed Kamruddin
Published: November 2013

Moraxella catarrhalisis one of the major pathogens of respiratory and middle ear infections. Attachment of this bacterium to the surface of human pharyngeal epithelial cells is the first step in the pathogenesis of infections. This study revealed that sulfatide might act as a binding molecule for the attachment ofM. catarrhalisto human pharyngeal epithelial cells. Furthermore, six different synthetic sulfatides were found to inhibit the attachment ofM. catarrhalissignificantly at an optimum concentration of 10 μg/ml. Synthetic sulfatides may have the potential to be used as a therapy to preventM. catarrhalisinfections.