bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystallization of Lipopolysaccharide from a Salmonella typhimurium Semi‐Rough (SR) Mutant
Authors: Ito Hideo , Naito Setsuko , Arakawa Yoshichika , Sasaki Kyoyu , Kato Nobuo
Published: November 2013

Salmonella typhimurium SR‐form lipopolysaccharide (LPS), consisting of a single repeating unit of the O‐antigenic polysaccharide, linked to the R‐core consisting of oligosaccharide that is, in turn, linked to lipid A, formed crystals whose shapes were hexagonal plates, discoids, and solid columns when precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and kept in 70% ethanol containing 250 mM MgCl2 at 4 C for 10 days. Among these crystals, the basic form is considered to be the hexagonal plates. Analyses of hexagonal plate crystals showed that they consist of hexagonal lattices with a lattice constant (a axis) of 4.62 Å and longitudinal axis (c axis) of approximately 100 Å. In X‐ray diffraction patterns in the low‐angle region, crystals of S. typhimurium SR‐form LPS exhibited much less distinct reflections when compared with crystals of synthetic Escherichia coli‐type lipid A. In contrast to the previous finding that S. minnesota S‐form LPS possessing the O‐antigenic polysaccharide does not crystallize under the same experimental conditions as used in the present study, the presence of a single repeating unit of the O‐antigenic polysaccharide does not inhibit crystallization.
Cauliflower Mosaic Virus ORF III Product Forms a Tetramer In Planta: Its Implication in Viral DNA Folding during Encapsidation
Authors: Tsuge Seiji , Kobayashi Kappei , Nakayashiki Hitoshi , Mise Kazuyuki , Furusawa Iwao
Published: November 2013

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein; the function of which is as yet unknown. This protein has non‐sequence‐specific DNA binding activity and is associated with viral particles, suggesting that the ORF III product (P3) is involved in the folding of CaMV DNA during encapsidation. In this study, we demonstrated that P3 forms a tetramer in CaMV‐infected plants. A P3‐related protein with an apparent molecular weight of 60 kDa was detected by Western blotting analysis using anti‐P3 antiserum under non‐reducing conditions, while only 15 kDa P3 was detected under reducing conditions. Analysis of P3 using viable mutants with a 27‐bp insertion in either ORF III or IV revealed that the 60 kDa protein was a tetramer of P3. The P3 tetramer co‐sedimented with viral coat protein in multiple fractions on sucrose gradient centrifugation, suggesting that P3 tetramer binds to mature and immature virions. These results strongly suggested that CaMV P3 forms a tetramer in planta and that disulfide bonds are involved in its formation and/or stabilization. The finding of P3 tetramer in planta suggested that viral DNA would be folded compactly by the interaction with multiple P3 molecules, which would form tetramers, while being packaged into the capsid shell.
Prevalence of Maternal Cytomegalovirus (CMV) Antibody and Detection of CMV DNA in Amniotic Fluid
Authors: Nishimura Naoko , Kimura Hiroshi , Yabuta Yumi , Tanaka Naoko , Ito Yoshinori , Ishikawa Kaoru , Suzuki Chizuko , Morishima Tsuneo
Published: November 2013

The prevalence of cytomegalovirus (CMV) IgG antibody was determined in 573 pregnant women in the first trimester. The overall prevalence of CMV IgG antibody was 77.5%. The rate of seropositivity was 67.7% in women < 25 yr, and increased with age to 85.7% in women ≧40 yr. These results imply that young women in Japan are at increased risk for primary CMV infection during pregnancy and that congenital CMV infection rates might increase in the future. We conducted a prospective study of 75 pregnant women who underwent amniocentesis for various indications to determine if CMV DNA could be detected in the amniotic fluid. None had symptoms associated with CMV infection, CMV IgM antibody, or seroconversion to CMV IgG antibody during pregnancy. CMV DNA was not detected in the amniotic fluid using a polymerase chain reaction assay. The 65 fetuses, including 3 sets of twins, were followed through birth. CMV DNA was not detected in urine samples obtained within the first 2 weeks of life. In conclusion, CMV DNA was not detected in the amniotic fluid of women who did not have CMV infection. These results, however, suggest that the negative predictive value of prenatal amniotic fluid analysis is high and that the presence of CMV DNA in the amniotic fluid has clinical significance for the diagnosis of congenital CMV infection if detected in pregnant women.
Chemically Induced Infection of CD4‐Negative HeLa Cells with HIV‐1
Authors: Harada Shinji , Maeda Yosuke
Published: November 2013

Infection with human immunodeficiency virus type‐1 (HIV‐1) requires the presence of a CD4 molecule and chemokine receptors such as CXCR4 or CCR5 on the surface of target cells. However, it is still not clear how the virus enters the cells. Although CD4 was initially identified as the primary receptor for HIV‐1, the expression of CD4 or one of the chemokine receptors alone is not sufficient to render susceptibility to infection with the virus. To ascertain whether or not adsorption of the virus needs charge‐to‐charge interaction between viral envelope and host cell membrane protein(s) and if binding alone promotes penetration of the virus into the cells, we have developed a chemically induced infection system targeting a CD4‐negative and CXCR4‐positive HeLa cell clone (N7 HeLa) which is usually not susceptible to infection with the LAI strain of HIV‐1. Use of a poly‐L‐lysine (PLL)‐coated culture plate to enhance the attachment of the virus to the cells made N7 HeLa cells infectable with HIV‐1 at very low efficiency. PLL alone cannot fully substitute for the function of the CD4 molecule. However, trypsin‐treated viruses, which have largely lost infectivity to CD4‐positive MT‐4 cells that are highly susceptible to HIV‐1 infection, enhanced infectivity against N7 HeLa cells when the PLL‐coated plate was used. These results provide evidence that infection with HIV‐1 requires both high binding affinity between viruses and cells, and then needs a modification of the viral envelope such as cleavage of gp120/160 to enhance the infection, probably resulting in exposure of the hydrophobic fusion domain of gp41. HIV‐1 infection of N7 HeLa cells was also enhanced by treatment with low pH, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and some factor(s) from the MT‐4 cell culture supernatant. Not only tight viral adsorption with cleavage of the viral envelope but also some activated status of the cells may be required for sufficient HIV‐1 infection in this artificial condition.
Porphyromonas gingivalis Fimbriae Induce Adhesion of Monocytic Cell Line U937 to Endothelial Cells
Authors: Hirose Kimiharu , Isogai Emiko , Ueda Itsuo
Published: November 2013

This study used the human monocytic cell line U937 to examine whether or not Porphyromonas gingivalis fimbriae could induce the adhesion of monocytes to endothelial cells. An in vitro adhesion assay was used to investigate the effects of the fimbriae on U937 cell adhesion to human umbilical vein endothelial cells (HUVEC). The fimbriae enhanced U937 cell adhesion to HUVEC in a dose‐dependent manner. U937 cells adhered better to HUVEC pretreated with the fimbriae for a minimum of 2 hr than to untreated HUVEC. The enhanced adhesion was inhibited by a monoclonal antibody against P. gingivalis 381 fimbriae. Pretreatment of U937 cells with the fimbriae for 24 hr enhanced U937 cell adhesion to HUVEC approximately 4‐fold. This phenomenon was inhibited by an anti‐CD11b antibody, suggesting the involvement of CD11b. These results indicate that P. gingivalis fimbriae can induce monocyte adhesion to the endothelial cell surface. They also suggest that the fimbriae may be involved in the initial event for infiltration of monocytes into the periodontal tissues of individuals with adult periodontitis.
Effect of Antibiotics, Levofloxacin and Fosfomycin, on a Mouse Model with Escherichia coli O157 Infection
Authors: Isogai Emiko , Isogai Hiroshi , Hayashi Shunji , Kubota Toru , Kimura Koichi , Fujii Nobuhiro , Ohtani Tsuyoshi , Sato Kenichi
Published: November 2013

There have been some reservations about the treatment of enterohemorrhagic Escherichia coli (EHEC) infection with antibiotics to prevent the occurrence of hemolytic uremic syndrome (HUS). However, the administration of antimicrobial agents for EHEC infection is under discussion. Therefore, we used an experimental mouse model to assess the advantage/disadvantage of two major antibiotics, levofloxacin (LVFX) and fosfomycin (FOM). Germ‐free IQI mice were inoculated with EHEC O157 strain EDL931 or #7. Bacteria colonized feces at 109–1010 CFU/g, and Shiga toxins (STXs) were detected in the feces. From 1 day after infection, mice were assigned to LVFX (20 mg/kg) once daily or FOM (400 mg/kg) once daily. A significant decrease in overall mortality was observed after treatment of LVFX, with EHEC disappearing immediately from the feces of mice. FOM also reduced mortality for one strain, the STX level decreased gradually. LVFX exhibited higher therapeutic efficacy than FOM. Strain differences were observed in the model during the treatment.
Induction of High Levels of Epitope‐Specific Antibodies by Epitope/Peptide Candidate Vaccines against Human Immunodeficiency Virus Type‐1 (HIV‐1)
Authors: Yu Tianwei , Bai Yun , Dierich Manfred P. , Chen Ying‐Hua
Published: November 2013

To test the immunogenicity of GPGRAFY‐epitope‐based candidate vaccines, a peptide with four repetitive GPGRAFY epitopes, V3‐P1 [C‐(GPGRAFY)4], and a peptide (PND) of the principal neutralizing domain (V3 loop: amino acid 301–328: C‐TRPNNNTRKSIRIQRGPGRAFYTIGKI) on gp120 were synthesized and covalently coupled to a carrier protein BSA. Immunization of BALB/c mice and New Zealand White Rabbits with these conjugate vaccines engendered strong antibody responses against the PND (mouse serum titer by 1:12,800–25,600; rabbit serum titer by 1:6,400–12,800). Interestingly, the V3‐P1‐BSA conjugates and the PND‐BSA conjugates could induce high levels of GPGRAFY‐epitope‐specific antibodies in the mice and rabbits (mouse serum titer by 1:25,600; rabbit serum titer by 1:12,800–25,600), while a recombinant gp160 subunit vaccine induced a low level of GPGRAFY‐epitope‐specific antibodies (serum titer by 1:400–1,600 in mice and rabbits). To confirm the above results, GPGRAFY‐epitope‐specific antibodies were isolated from rabbit sera induced by V3‐P1‐BSA, PND‐BSA conjugates and rgp160 vaccine. In fact, 23–38 and 13–22 μg epitope‐specific antibodies per milliliter serum were isolated from rabbit sera induced by V3‐P1‐BSA and PND‐BSA conjugate, respectively, while 1.34 μg epitope‐specific antibodies per milliliter serum were identified in rabbit serum induced by rgp160 vaccine. In the control group, only 0.069 μg proteins per milliliter serum were found in pooled pre‐immune serum (normal serum). These results from mouse and rabbit experiments indicate that epitope and peptide vaccines both induce high levels of GPGRAFY‐epitope‐specific antibodies in comparison with rgp160 subunit vaccine, suggesting that epitope/peptide vaccines may be a new strategy to induce protective activity.
Exposure of Normal Monocyte‐Derived Dendritic Cells to Human Immunodeficiency Virus Type‐1 Particles Leads to the Induction of Apoptosis in Co‐Cultured CD4+ as Well as CD8+ T Cells
Authors: Suzuki Satoko , Tobiume Minoru , Kameoka Masanori , Sato Katsuaki , Takahashi Tsuneo A. , Mukai Tetsu , Ikuta Kazuyoshi
Published: November 2013

The depletion of immune T cells by human immunodeficiency virus type‐1 (HIV‐1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte‐derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte‐macrophage colony‐stimulating factor and interleukin‐4. The DCs exposed to HIV‐1 particles were co‐cultured with healthy donor‐derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV‐1 particles.
SDF‐1 Has Costimulatory Effects on Human T Cells
Authors: Yonezawa Akihito , Hori Toshiyuki , Sakaida Hitoshi , Uchiyama Takashi
Published: November 2013

Stromal cell‐derived factor‐1 (SDF‐1) is an efficacious chemoattractant for lymphocytes, monocytes and hematopoietic progenitor cells. In the present study, we examined whether SDF‐1 has growth promoting activity on human peripheral T cells and analyzed the possible underlying signal transduction pathways. SDF‐1 augmented the proliferation of anti‐CD3‐ or PHA‐stimulated normal human PBMC in a dose‐dependent manner but not that of resting PBMC. It was noted that SDF‐1 alone could induce a significant proliferation of PHA‐preactivated T cells. Anti‐SDF‐1 sera could inhibit the augmentation of T‐cell proliferation in each experiment. Furthermore, Western blot analysis revealed that mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase 2 (ERK2), but not c‐Jun N‐termimal kinase (JNK) was activated by SDF‐1. Considering that costimulatory signals have been reported to involve ERK2 activation, these results indicate that SDF‐1 has costimulatory effects on T cells that are possibly mediated by ERK2 activation and may play a role in not only migration but also the potentiation or maintenance of T cells.
In Vitro Susceptibility of 7.5‐kb Common Plasmid‐Free Chlamydia trachomatis Strains
Authors: Miyashita Naoyuki , Matsumoto Akira , Matsushima Toshiharu
Published: November 2013

Using a new plaque cloning technique, we obtained unique Chlamydia trachomatis strains which were confirmed to be free of the 7.5‐kb common plasmid and glycogen in inclusions. The in vitro susceptibility of these strains to various chemotherapeutic agents was tested by comparison with their parent strains and clinical isolates possessing the common plasmid. No difference was detected for any of the agents tested, indicating that the 7.5‐kb common plasmid is unrelated to the drug resistance of C. trachomatis.
Identification of a Two‐Component Signal Transduction System Involved in Fimbriation of Porphyromonas gingivalis
Authors: Hayashi Jun‐ichiro , Nishikawa Kiyoshi , Hirano Risa , Noguchi Toshihide , Yoshimura Fuminobu
Published: November 2013

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic Gram‐negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon‐induced, fimbria‐deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two‐component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two‐component regulatory system is involved in fimbriation of P. gingivalis.
Burkholderia uboniae Sp. Nov., l‐Arabinose‐Assimilating but Different from Burkholderia thailandensis and Burkholderia vietnamiensis
Authors: Yabuuchi Eiko , Kawamura Yoshiaki , Ezaki Takayuki , Ikedo Masanari , Dejsirilert Surang , Fujiwara Nagatoshi , Naka Takashi , Kobayashi Kazuo
Published: November 2013

A polar multitrichous Gram‐negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA‐DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, biochemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA‐DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone‐8(Q8). G+C content of DNA is 69.71%.
Protective Effect of C‐Reactive Protein against the Lethality Induced by Vibrio vulnificus Lipopolysaccharide
Authors: Chae Mee‐ree , Park Byung‐hyun , Kim Jong‐suk , Rho Hye‐won , Park Jin‐woo , Kim Hyung‐rho
Published: November 2013

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C‐reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine‐sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor‐α (TNF‐α) induced by LPS administration was not affected by CRP pretreatment. However, the LPS‐ or TNF‐α‐induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF‐α. The possibility that impaired production of acute phase proteins in patients with pre‐existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.
Mutation of Aromatic Amino Acid Residues Located at the Amino‐ and Carboxy‐Termini of Escherichia coli Heat‐Stable Enterotoxin Ip Reduces the Efficiency of the Toxin to Cross the Outer Membrane
Authors: Yamanaka Hiroyasu , Okamoto Keinosuke
Published: November 2013

Heat‐stable enterotoxin Ip (STIp) of Escherichia coli is synthesized as a precursor form consisting of pre‐ (amino acid residues 1 to 19), pro‐ (amino acid residues 20 to 54) and mature (amino acid residues 55 to 72) regions. Mature STIp (bioactive STIp) is formed in the periplasmic space after the precursor is proteolytically processed and the mature STIp translocates across the outer membrane through the secretory system including TolC, an outer membrane protein of E. coli. However, it remains unknown how the mature STIp is recognized by this secretory system. In this study, we investigated the amino acid residues of STIp involved in its translocation across the outer membrane. We prepared mutant STIp genes by site‐directed mutagenesis and analyzed translocation of the mutant STIps across the outer membrane. Deletion of the Phe or Tyr residue at position 3 or 18, respectively, decreased the efficiency of translocation of STIp across the outer membrane. To confirm the involvement of these amino acid residues, we further mutated the codons for these amino acid residues to that for Gly. These mutations also decreased the efficiency of extracellular secretion of STIp. In contrast, substitution of Phe‐3 and Tyr‐18 with Tyr and Phe, respectively, did not affect the efficiency of translocation of the toxin. These results indicated that the aromatic amino acid residues at positions 3 and 18 in the mature region are important for the ability of STIp to cross the outer membrane.
The Relative Frequencies of G Serotypes of Rotaviruses Recovered from Hospitalized Children with Diarrhea: A 10‐Year Survey (1987–1996) in Japan with a Review of Globally Collected Data
Authors: Koshimura Yumi , Nakagomi Toyoko , Nakagomi Osamu
Published: November 2013

Since rotavirus vaccines aim to protect children from severe diarrhea, knowledge of the prevailing G serotypes among rotaviruses from hospitalized children is essential. Thus, we determined the G serotypes of rotaviruses collected from children with acute diarrhea in a local referral hospital in Akita, Japan, over the 10‐year period between January 1987 and December 1996. Based on the assumption that rotaviruses with an identical electropherotype possess the same G serotype, the G serotypes of 488 rotavirus‐positive specimens that were classified into 63 electropherotypes were determined by enzyme‐linked immunosorbent assay with a supplementary use of G typing by reverse transcription‐PCR. The relative frequencies over the 10‐year period were 77.0 (G1), 14.5 (G2), 2.7 (G3) and 5.3% (G4), leaving the possibility that only 0.4% had G serotypes uncommon to human rotaviruses. Of 24,050 rotaviruses extracted by reviewing 63 serotyping studies in literature, the relative frequencies of the four major G serotypes were 50.6 (G1), 9.3 (G2), 7.2 (G3) and 11.6% (G4). As to uncommon G serotypes, only 0.9% were described as serotypes other than G1–4, and our estimate for potential uncommon serotypes were at most 8.1%. Thus, both this long‐term study focusing on the rotaviruses only from severe cases in a single hospital in Japan and the global review of G serotypes published to date indicate that the primary target of any rotavirus vaccines should be rotaviruses possessing serotypes G1–4.
Sensitive Enzyme‐Linked Immunosorbent Assays for the Detection of Bacterial Superantigens and Antibodies against Them in Human Plasma
Authors: Miwa Keishi , Fukuyama Mayumi , Sakai Rumiko , Shimizu Shinji , Ida Nobuo , Endo Miyoko , Igarashi Hideo
Published: November 2013

Enzyme‐linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic‐shock syndrome toxin‐1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme‐linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.
Characterization of Monoclonal Antibodies against Hokkaido Strain Tick‐Borne Encephalitis Virus
Authors: Komoro Kimiyo , Hayasaka Daisuke , Mizutani Tetsuya , Kariwa Hiroaki , Takashima Ikuo
Published: November 2013

A tick‐borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5–10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5–10 strain. These Mabs were flavivirus genus‐specific, TBE virus complex‐specific or TBE virus type‐specific. The Mabs are applicable for identification of TBE virus strains.
Comparison of Anti‐Tumor Responses against TL Positive Lymphoma Induced by Skin Grafting and Dendritic Cell Immunization
Authors: Iwase Shigeru , Tsujimura Kunio , Matsudaira Yasue , Ozeki Satoshi , Onozaki Kikuo , Obata Yuichi , Takahashi Toshitada
Published: November 2013

When the skin of Tg. Con.3–1 transgenic mice expressing the TL (thymus leukemia) antigen in most tissues is grafted on syngeneic C3H mice, it is rejected, and a cytotoxic T cell (CTL) response against the TL antigen is induced. In this study, we first demonstrated that growth of TL positive lymphoma is suppressed in mice immunized by skin grafting. Immunization with bone marrow derived dendritic cells (DCs) from Tg.Con.3–1, was also found to be associated with an anti‐tumor response, but less potent than skin grafting. Relative CTL precursor frequency with DC immunization was also approximately only one third that of skin grafting. The numbers of IFN‐γ producing cells in responder CD8 and CD4 T cell populations were higher with DC immunization than with skin grafting. However, DC immunization seems to induce non‐specific immune responses, as re‐stimulation with TL negative C3H spleen cells resulted in induction of almost half the number observed with TL positive cells. Thus, the actual number of IFN‐γ producing cells in specific responses to TL is not necessarily larger than with skin grafting immunization. The present results altogether suggest that DC immunization is capable of inducing an anti‐tumor reaction, but also possibly unwanted immune responses. In vitro monitoring of specific and non‐specific responses in the immune system, thus, is of particular importance for future development of cancer immunotherapy.
Rotavirus Infection and Intussusception: A View from Retrospect
Authors: Nakagomi Toyoko
Published: November 2013

A live orally‐administrable rhesus rotavirus (RRV) tetravalent (TV) vaccine, licensed in the U.S.A. and the European Union, was recalled from the market because it was suspected to increase the risk of intussusception during the week following immunization. In contrast, natural rotavirus infection is generally believed not to cause intussusception. Because my experience contributed to the first paper that linked intussusception with rotavirus infection, I have re‐examined our own data published 22 years ago and other studies on this issue. I also made a case study of adenovirus and intussusception as a paradigm to establish an etiological association of viral infection and intussusception. My hypothesis postulated in this review is that natural infection of susceptible (or predisposed) infants with some rotavirus strains, probably serotype G3 rotaviruses, will result in an appreciable fraction of idiopathic intussusception. Thus, the number of rotavirus‐induced intussusception cases may change reflecting the relative frequency of G3 strains, which I believe was much higher in the 70s than during the last two decades. The epidemiological data indicate that the RRV‐TV vaccine triggers intussusception at a rate significantly higher than the background incidence rate following the week of vaccination, particularly after the first dose. In contrast, the data do not suggest that the cumulative incidence among the vaccine recipients increases accordingly, implicating that the risk of intussusception attributable to the RRV‐TV vaccine may be minimal.
Blood Lysate Staining, a New Microscopic Method for Diagnosis of Fungemia Using Peripheral Blood
Authors: Makimura Koichi , Yamamoto Kyoko , Sugita Chise , Shibuya Kazutoshi , Yamaguchi Hideyo
Published: November 2013

We developed a microscopy method for the detection of fungal cells in peripheral blood, termed blood lysate staining, using an approximately 5 × 5 mm dotted blood lysate. This method was able to detect the emerging fungal pathogen Trichosporon asahii in murine models of systemic fungal infection and fungemia in patients quickly and at minimal cost. Pathogenic yeasts were successfully detected in 6 of 8 blood samples which were taken from feverish immunocompromised patients who were clinically suspected of having fungal infections. Fungal cells were observed as ovoid to elongated, 3 × 3 to 7 × 10 μm, and occurred singly, budding, and in short chains and clusters in a periodic acid‐Schiff‐stained blood smear. The yeast cells were easily distinguished from blood‐cell debris by their size, shape and smooth yet rigid outline.
The Rabies Virion‐Associated 100‐kDa Polypeptide (VAP100) Is a Host‐Derived Minor Component of the Viral Envelope
Authors: Xiao Sa , Komiya Kazuo , Tochikura Tadafumi S. , Sagara Junji , Tsukita Shoichiro , Kawai Akihiko
Published: November 2013

We investigated a minor polypeptide component of 100‐kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS‐denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin‐fixed normal BHK‐21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X‐100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus‐infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane‐associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.
Monoclonal Antibody against Mycoplasma fermentans‐Specific Aminoglycoglycerolipid
Authors: Matsuda Kazuhiro , Li Jin‐liang , Ichinose Shizuko , Harasawa Ryo , Saito Masaki , Yamamoto Naoki
Published: November 2013

Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholine‐containing glycoglycerolipids: GGPL‐I and GGPL‐III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF‐III‐1) specific to GGPL‐III (phosphocholine‐containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin‐layer chromatography immunostaining. The MF‐III‐1 antibody specifically recognized M. fermentans attached to the surface of HTLV‐I‐infected human helper T‐cells, and it did not cross‐react with other lipids nor with human T‐cell antigens. Since MF‐III‐1 distinguishes GGPL‐III from GGPL‐I, the binding site may include a serinol (2‐amino‐1,3‐propanediol) residue of GGPL‐III. MF‐III‐1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases.
Molecular Ratio between Borna Disease Viral‐p40 and ‐p24 Proteins in Infected Cells Determined by Quantitative Antigen Capture ELISA
Authors: Watanabe Makiko , Zhong Qiu , Kobayashi Takeshi , Kamitani Wataru , Tomonaga Keizo , Ikuta Kazuyoshi
Published: November 2013

We developed the antigen capture enzyme‐linked immunosorbent assay (ELISA) systems for quantification of Borna disease virus (BDV) major antigens, p40 and p24. Using these ELISAs, we quantified the two proteins in various BDV‐infected materials, including the cell lysates and culture supernatants as well as the homogenates of experimental animal brains. The ELISAs were also applied to measure the infectious titer of BDV in persistently infected cell lines. Quantitative analysis with these ELISAs allowed us to measure the molecular ratio between the p40 and p24 in infected samples. Interestingly, the ratio of p24 to p40 in persistently infected cells was much higher than that observed in acutely infected cells although the ratios in the supernatants from both cell lines were quite similar. BDV‐inoculated gerbil brain cells showed a relatively high ratio of p24 to p40 as compared with acutely infected cells. These observations suggested that the molecular ratio between the proteins strongly depended on the infectious status of BDV in the host cells. The ELISA system developed here could be a convenient method for the quantification of BDV infection and may also be beneficial for understanding viral replication and infectious status in the host cells.
Seroprevalence of Antibodies to Chlamydia spp. in Human Immunodeficiency Virus‐Infected Subjects in Japan
Authors: Miyashita Naoyuki , Niki Yoshihito , Iwamoto Aikichi , Yasuoka Akira , Oka Shinichi , Kawata Kaoru , Ito Akira , Tomono Kazunori , Kohno Shigeru , Matsushima Toshiharu
Published: November 2013

To evaluate the seropositivity of Chlamydia spp. in human immunodeficiency virus (HlV)‐infected subjects in Japan, Chlamydia‐specific antibodies in sera collected from 106 HIV‐infected subjects were measured by the microimmunofluorescence test. The prevalence of C. pneumoniae‐specific IgA, C. trachomatis‐specific IgG and IgA and mean titers were significantly higher in the homosexual and heterosexual HIV‐infected subjects than in the hemophilic patients and HIV‐negative controls. These data indicate that the higher C. pneumoniae and C. trachomatis seroprevalence among HIV‐infected subjects is probably due to an HIV risk factor, such as promiscuous sexual behavior, rather than to HIV infection itself.
Single‐Tube Multiplex PCR for Rapid and Sensitive Diagnosis of Subgenus B and Other Subgenera Adenoviruses in Clinical Samples
Authors: Fujimoto Tsuguto , Chikahira Masatsugu , Kase Tetsuo , Morikawa Saeko , Okafuji Teruo , Yokota Yoko , Nishio Osamu
Published: November 2013

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non‐nested polymerase chain reaction (PCR). Sequences of the conserved hexon‐coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single‐tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non‐Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty‐seven out of 71 (94%) Sub B Ad culture‐positive samples, and 15 out of 19 (79%) Sub C or E‐positive samples amplified products of the expected size. Two of 20 (10%) culture‐negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non‐Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.
Incidence of Amantadine‐Resistant Influenza A Viruses in Sentinel Surveillance Sites and Nursing Homes in Niigata, Japan
Authors: Masuda Hiroki , Suzuki Hiroshi , Oshitani Hitoshi , Saito Reiko , Kawasaki Satoshi , Nishikawa Makoto , Satoh Hiroshi
Published: November 2013

We surveyed the incidence of amantadine‐resistant influenza A viruses both at sentinel surveillance sites and at nursing homes, and verified their types of change by partial nucleotide sequence analysis of the M2 protein. Fifty‐five influenza A viruses from 27 sentinel surveillance sites during six influenza seasons from 1993 to 1999, and 26 influenza A viruses from 5 nursing homes from 1996 to 1999 were examined for susceptibility to the drug by virus titration in the presence or absence of amantadine. While amantadine‐resistant viruses were not found in sentinel surveillance sites, a high frequency of resistance (8/26, 30.8%) in nursing homes was observed. Resistant viruses can occur quickly and be transmitted when used in an outbreak situation at nursing homes, where amantadine is used either for neurologic indications or for influenza treatment. Eight resistant viruses had a single amino acid change of the M2 protein at residue 30 or 31. In vitro, all 11 sensitive viruses turned resistant after 3 or 5 passages in the presence of 2 μg/ml amantadine, and they showed an amino acid change at residue 27, 30, or 31. The predominant amino acid substitution in the M2 protein of resistant viruses is Ser‐31‐Asp (a change at 31, serine to asparagine). The results indicate that a monitoring system for amantadine‐resistant influenza viruses should be established without delay in Japan.
Detection of IgA‐Binding Sites on Human Immunodeficiency Virus Type‐1 Envelope Glycoproteins, Gp120 and Gp41
Authors: Matsuda Shunji , Noda Masahiro
Published: November 2013

IgA has been supposed to play an important role in the prevention of HIV‐1 infection. In this study, IgA‐binding sites on gp120 and gp41 of HIV‐1 envelope glycoproteins were analyzed using ELISA and overlapping synthetic peptides covering all of the gp120 and gp41 sites. IgA antibodies in plasma and saliva mainly bound to six and five sites on gp120 and gp41, respectively. Some of the IgA‐binding sites differed from those of IgG‐binding sites and the amount of IgA antibodies that bound to each site varied among samples. IgA antibodies in some plasma samples neutralized HIV‐1 infection, and those IgA antibodies contained the antibodies which bound to the V3, C3 and ELDKWA sites. The results suggest that IgA antibodies which bind to certain sites on HIV‐1 envelope glycoproteins may neutralize HIV‐1 infection, presumably at mucosal sites where most IgA antibodies are produced. The induction of IgA antibodies that bind specific sites and neutralize HIV‐1 infection at mucosal sites may be important in the development of a vaccine against HIV‐1 infection.
Spectrum of Gut Immunologic Reactions: Selective Induction of Distinct Responses to Vibrio cholerae WO7 and Its Toxin
Authors: Walia Kamini , Vohra Harpreet , Singh Harminder , Ganguly Nirmal Kumar
Published: November 2013

Past studies with Vibrio cholerae have shown that cholera toxin (CT) is mainly responsible for inducing T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (sIgA) antibodies. In this study, V. cholerae WO7, which produces novel toxin unrelated to CT, was given orally to mice in order to determine whether the strain V. cholerae WO7 differs from V. cholerae 569B, which produces CT, in the nature of responses generated at the gut and splenic level. The analysis of immune responses evoked by V. cholerae WO7 in the gut of mice revealed striking differences as compared to those elicited by V. cholerae 569B infection. To assess the T helper cell type responses, lymphocytes from Peyer's patches and the spleen were stimulated in vitro for studying the cytokine patterns. PP and SP lymphoid cells from V. cholerae WO7 infected animals elaborated significant amounts of IL‐2, IFN‐γ and IL‐12 by 7 days p.i., suggesting a Th1 type of response. However by 15 days p.i., the PP and SP lymphoid cells secreted only IL‐6 and IL‐10 with traces of IFN‐γ. On the other hand, infection with V. cholerae 569B yielded mainly Th2 type responses at Peyer's patches as well as the splenic level. Infection with both V. cholerae WO7 and 569B induced toxin‐specific IgA secreting cells at the gut and splenic level along with IgG1 secreting cells, indicating that both V. cholerae WO7 and 569B evoke an antigen‐specific Th2 type of response in the gut as well as spleen. The persistence of IgA along with Th1‐type cytokines indicates an alternate induction mechanism since mucosal IgA responses are usually associated with Th2‐type responses. These observations are suggestive of a common mechanism employed by the host to clear different strains of V. cholerae infection (569B and WO7 in this case), while the nature of toxins elaborated failed to modulate the net outcome of the infection caused by V. cholerae.
The O‐Polysaccharide of Lipopolysaccharide Isolated from Vibrio fluvialis O19 Is Identical to That of Vibrio Bioserogroup 1875 Variant
Authors: Kondo Seiichi , Haishima Yuji , Ishida Keiichi , Isshiki Yasunori , Hisatsune Kazuhito
Published: November 2013

A structural analysis has been carried out on the O‐polysaccharide of lipopolysaccharide (LPS) isolated from Vibrio fluvialis 181–86 (Kobe) serotype O19 (019) which has the Inaba antigen factor C of O1 V. cholerae and factors D and E in common with Vibrio bioserogroup 1875. The O‐polysaccharide of O19 was characterized as an α (1→2)‐linked homopolymer of N‐3‐hydroxypropionyl‐d‐perosamine (4‐amino‐4,6‐dideoxy‐d‐mannopyranose), which was identical to that of Vibrio bioserogroup 1875 Variant. Passive hemolysis and passive hemolysis inhibition analysis performed using anti‐factor D, E and anti‐factor E antisera, demonstrated that the LPS from O19 harbored O‐antigenic factors identical to those of the LPS from Vibrio bioserogroup 1875 Variant.
Apparent Re‐Emergence of Serotype G9 in 1995 among Rotaviruses Recovered from Japanese Children Hospitalized with Acute Gastroenteritis
Authors: Oka Toyonori , Nakagomi Toyoko , Nakagomi Osamu
Published: November 2013

Serotype G9 rotaviruses have been detected in about 0.5% of the circulating strains worldwide. However, G9 strains emerged globally in the middle of the 90s and thereafter. A rotavirus, contained in stool specimen 95H115, possessing a G9 VP7 emerged in Japan in the 1994–1995 season for the first time after a 9‐year interval since prototype G9 strains AU32 and F45 were discovered in the 1985–1986 season. In comparison with other G9 VP7 genes thus far published, the sequencing of the VP7 genes of AU32 and 95H115 revealed that the 95H115 VP7 gene did not directly evolve from the AU32 VP7 gene but was much more closely related to the contemporary G9 VP7 genes found in the United States of America. Thus, recently emerging G9 VP7 genes were not direct descendants of the VP7 genes of the prototype strains in the 80s, rather they evolved independently into 4 phylogenetic clusters from a common ancestor.
NK Cells Eliminate Cryptococcus neoformans by Potentiating the Fungicidal Activity of Macrophages Rather than by Directly Killing Them upon Stimulation with IL‐12 and IL‐18
Authors: Kawakami Kazuyoshi , Koguchi Yoshinobu , Qureshi Mahboob Hossain , Yara Satomi , Kinjo Yuki , Uezu Koari , Saito Atsushi
Published: November 2013

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)‐12 and IL‐18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN‐γ and exerted marked cytotoxic activity against YAC‐1 cells. On the other hand, NK cells significantly potentiated the nitric oxide‐mediated cryptococcocidal activity of thioglycolate‐elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL‐12 and IL‐18. The culture supernatants of NK cells stimulated with IL‐12 and IL‐18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN‐γ because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage‐mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.
RAPD‐ and actA Gene‐Typing of Listeria monocytogenes Isolates of Human Listeriosis, the Intestinal Contents of Cows and Beef
Authors: Inoue Satoshi , Katagiri Kazuhiro , Terao Michinori , Maruyama Tsutomu
Published: November 2013

Seventy‐five L. monocytogenes isolates of human listeriosis, the intestinal contents of cows and beef were divided into 5 major clusters, 17 sub‐clusters and 28 minor clusters by typing using random amplification of polymorphic DNA (RAPD). According to their major RAPD category, L. monocytogenes isolates serotyped as 1/2b and 4b were distinguished from L. monocytogenes isolates of serovars 1/2a and 1/2c. Moreover serovar 4b was distinguished from serovar 1/2b by a difference in the RAPD sub‐cluster category. All L. monocytogenes were found to possess either actA gene Type I or n, and only one actA gene type was detected in each RAPD minor cluster. actA gene Type II was observed in 32.0%, 38.5 % and 18.9% of isolates from humans, cows and beef, respectively, and was detected more frequently in serovar 4b (46.9%) than in serovars 1/2a (22.2%), 1/2b (7.7%) and 1/2c (0.0%). Twenty (80%) of 25 human isolates fell within three minor RAPD types (II‐d (16%), V‐p‐1 (36%), V‐p‐2 (28%)). Two isolates from humans and beef were found to have the same RAPD type (Type IV‐k‐1), actA gene type (Type I) and serovar (1/2b). Our results suggest that only a few genotypes of L. monocytogenes are predominant in human listeriosis in Japan, although the human isolates were collected over a broad span of time and a wide geographical range. Our results also suggest that RAPD‐, actA gene‐ and sero‐typing can be useful for epidemiological analysis.
CD3− Bone Marrow Cells Augment the Generation of Cytotoxic T Lymphocytes Showing a Preference for the X‐Chromosome Linked Gene Product of Stimulator Cells
Authors: Oshiro Yoshinori , Tanabe Masao J.
Published: November 2013

Responder cells, composed of both a limited number of nylon wool‐passed lymph node (NW‐LN) cells and an excess number of CD3+ cell‐depleted bone marrow (CD3− BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3− BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW‐LN cells. C3H/He (H‐2k, C3H background) responder cells were stimulated with either B10.D2 (H‐2d, B10 background) or BALB/c (H‐2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 × BALB/c) F1 male target cells than (BALB/c × B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c × B10.D2) F1 male than (B10.D2 × BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H‐2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He × B10.BR) F1 male target cells than B10.BR or (B10.BR × C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X‐chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.