bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in T‐Cells Bearing CD25 in Bronchoalveolar Lavage Fluid from HAM/TSP Patients and HTLV‐I Carriers
Authors: Mukae Hiroshi , Kohno Shigeru , Morikawa Nobuo , Kadota Jun‐ichi , Matsukura Shigeru , Hara Kohei
Published: November 2013

To determine the immunologic characteristics of T‐cells in local pulmonary lesions of human T‐cell lymphotropic virus type I (HTLV‐I) carriers, we investigated lymphocyte surface markers in peripheral blood and bronchoalveolar lavage fluid (BALF) of 38 HTLV‐I carriers, 8 HTLV‐I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 44 HTLV‐I seronegative patients with pulmonary diseases and 7 healthy volunteers using two‐color flow cytometric analysis. In peripheral blood, activated T‐cells, CD4+HLA‐DR +, CD8 + HLA‐DR + and CD3 + CD25 +, and CD4+CD29+ cells increased significantly in carriers and HAM/TSP patients compared with healthy volunteers and seronegative patients. In BALF, T‐cells, especially CD25+ cells, increased significantly in carriers and HAM/TSP patients, compared with healthy volunteers and seronegative patients. These findings indicated that T‐cells in the lungs, as well as in peripheral blood, are activated in carriers and HAM/TSP patients. Interestingly, there was dissociation between expression of CD3 + CD25+ cells in BALF and peripheral blood from these patients. These results suggest that T‐cells activated probably by HTLV‐I accumulate in the lungs in some carriers and HAM/TSP patients, and HTLV‐I may be involved in the immunologic dysfunction in the lungs of these patients. However, we did not find any correlation between the degree of clinical features and the elevation of CD3 + CD25+ cells in BALF, or its characteristic features on chest roentgenograms.
Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa, Subtropical Area in Japan. I. Investigations on Antibody Levels to Japanese Encephalitis Virus in Swine Sera and Vector Mosquito in Okinawa, Miyako and Ishigaki Islands
Authors: Tadano Masayuki , Kanemura Kazumi , Hasegawa Hideo , Makino Yoshihiro , Fukunaga Toshihiko
Published: November 2013

From 1985 to 1989, serum specimens of swine raised in the northern, central and southern areas in Okinawa island were examined for Japanese encephalitis (JE) virus antibody by ELISA and hemagglutination‐inhibition test. The antibody positive rate was found to be higher in the north and central than in the south. The 2‐mercaptoethanol sensitive antibody to JE was detected mostly in June and July, and occasionally in other months except February and March. There was no month when all specimens from three areas turned antibody‐negative simultaneously, indicating that JE virus transmission to swine lasted longer in Okinawa island than in other temperate areas in Japan. From 1986 to 1991, the vector mosquitoes (Culex tritaeniorhynchus) were collected in a pig farm in the south of Okinawa island. A total of 153 strains of JE virus was isolated from the vector mosquitoes mainly in June. In Miyako and Ishigaki islands, the antibody positive rate in swine sera was found to be extremely low, compared with that in Okinawa island. In Miyako island, where no paddy rice field is cultivated, a few adults as well as larvae of the vector mosquito were collected, while in Ishigaki island, where there are many watered rice fields, a lot of adults as well as larvae were collected. Although the environmental situation is quite different between the two islands, JE virus transmission appeared to be very low in both islands.
Induction of Yersinia enterocolitica Stress Proteins by Phagocytosis with Macrophage
Authors: Yamamoto Tomoko , Hanawa Tomoko , Ogata Sachio
Published: November 2013

Yersinia enterocolitica is a facultative intracellular pathogen which invades to epithelial cells and survives in phagocytes. Since the internal environment of phagocytes should be stressful conditions for the phagocytosed Yersinia, the bacteria should respond to protect themselves from otherwise lethal results. We analyzed the stress‐induced proteins which possibly contribute to survival of Yersinia within the phagocytes. Y. enterocolitica was radiolabeled during the growth in macrophage‐like J774‐1 cells, and the bacterial proteins were analyzed by two‐dimensional gel electrophoresis. At least 16 proteins were selectively induced in response to phagocytosis, and several out of 16 proteins were also induced by heat shock at 42 C or oxidative stresses in vitro. Of those, two major stress proteins were identified to be homologues of DnaK and CRPA by immunoblotting analysis. These results have indicated that Y. enterocolitica exhibits a global stress response to the hostile environment in the phagocytosed macrophage.
Modulation by Polymyxin B of the Effects of Interferon on Human Myelogenous Leukemia Cells
Authors: Tamura Minori , Nagano Yasuiti
Published: November 2013

Natural and recombinant human interferon‐α (IFN‐α) and ‐γ (IFN‐γ) exert differentiation‐inducing and cytocidal effects in vitro on cells of the human myeloblastic leukemia cell line ML‐2. These activities of IFNs are modulated by polymyxin B (PMB), a cyclic polycationic peptide antibiotic effective on Gram‐negative bacilli. The modulating effect of PMB varies according to the species of IFN, namely, PMB enhances the activities of either natural IFN‐γ or recombinant IFN‐γ, while it inhibits the effects of either natural IFN‐α or recombinant IFN‐α. The cause of this variety in PMB effect on IFNs remains to be clarified.
Relationship between Hemolytic Activity and Adsorption Capacity of Aluminum Hydroxide and Calcium Phosphate as Immunological Adjuvants for Biologicals
Authors: Kato Hiroshi , Shibano Michiko , Saito Takuya , Yamaguchi Junji , Yoshihara Shinobu , Goto Norihisa
Published: November 2013

Aluminum hydroxide (Al) and calcium phosphate (Ca) gels have been used as vaccine adjuvants for many years. We investigated mechanism of the hemolytic activities of both adjuvant materials. The hemolytic activity of each gel depended on the gel dose. The adsorption capacities and the hemolytic activities of both adjuvants decreased as the concentration of phosphate increased in a gel‐washing solution. A positive correlation between the hemolytic activity and the adsorption capacity was found in Al‐gel. A disruptive effect of Ca‐gel on membrane of erythrocytes was shown by electron microscopy. Ca‐gel required more than 10 times as much pre‐adsorbed ovalbumin as did Al‐gel to inhibit the hemolysis. These results suggest that the hemolytic activity of both adjuvant materials depended mainly on the adsorption ability, and it may be useful to control the adsorption ability of adjuvants to reduce their hemolytic activity.
A Sandwich Cup Method for the Penetration Assay of Antimicrobial Agents through Pseudomonas Exopolysaccharides
Authors: Kumon Hiromi , Tomochika Ken‐ichi , Matunaga Tosiyuki , Ogawa Masatoshi , Ohmori Hiroyuki
Published: November 2013

We developed new sandwich cup method to assay the penetration of various antimicrobial agents through Pseudomonas exopolysaccharides. Using alginate extracted from mucoid‐type Pseudomonas aeruginosa and gellan gum from Pseudomonas elodea, the role of exopolysaccharides as a barrier against drug penetration was examined. The penetration of positively charged hydrophilic drugs such as aminoglycosides and polypeptides was markedly inhibited by the gels tested, but that of β‐lactams, quinolones, and macrolides was not inhibited. The penetration of gentamicin was strongly influenced by the gel concentration, the solution to be used, and the presence of Ca2+. These results suggest that the microenvironment at the infection site could greatly influence drug penetration through biofilms in vivo.
Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo
Authors: Guth Beatriz Ernestina Cabilio , Aguiar Eliana Gonçalves , Griffin Patricia Marie , da Silva Ramos Sonia Regina Testa , Gomes Tânia Aparecida Tardelli
Published: November 2013

Fifty‐eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat‐stable (ST‐I) enterotoxin and in strains also producing heat‐labile toxin (LT‐I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST‐I‐producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST‐I, and 88% carried CS6 antigen.
The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor‐Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii
Authors: Jerrells Thomas R. , Geng Peigin
Published: November 2013

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage‐like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat‐killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat‐killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH‐1 subset of T cells is predominant in immunity to R. tsutsugamushi.
Elevated Plasma Levels of P‐Selectin (GMP‐140/CD62P) in Patients with Plasmodium falciparum Malaria
Authors: Facer Christine A. , Theodoridou Agapi
Published: November 2013

The plasma concentration of soluble P‐selectin (GMP‐140/CD62P/PADGEM), a selectin produced by activated platelets and endothelial cells, was quantitated in a group of adults and East African negro children presenting with either non‐severe or severe (cerebral) malaria caused by Plasmodium falciparum. Sixty percent of adults with non‐severe malaria had immunoreactive levels of P‐selectin above 200 ng/ml (the maximum recorded for any normal healthy adult in the assay) and 86 % of all African children with malaria had concentrations above normal irrespective of their clinical categorization, and most exceeded the maximum limits of the assay (> 640 ng/ml). There was no correlation between P‐selectin levels and parasitemia. These results raise the possibility that elevated soluble P‐selectin in malaria may have an important beneficial antiinflammatory function.
Characterization of Immunological Activity of a Low Toxicity Antitumor Lipopolysaccharide from Bordetella pertussis
Authors: Ohnishi Megumi , Kimura Sadao , Yamazaki Masatoshi , Abe Shigeru , Yamaguchi Hideyo
Published: November 2013

Immunological properties of a low toxicity lipopolysaccharide (BP‐LPS) extracted from Bordetella pertussis (Tohama strain) which was reported to have high antitumor activity against murine tumors were examined and compared with those of LPS extracted from other enterobacteria. The activation or stimulation of murine macrophages and lymphocytes by these LPS, including TNF induction, was found to be similar. However, BP‐LPS was clearly less active in its stimulation of murine and human neutrophils as estimated by neutrophil‐adherence assay and by their TNF production than E. coli LPS. Furthermore, BP‐LPS also suppressed the activation of human neutrophils by Escherichia coli LPS. A comparative study with 7 LPS preparations indicated that their toxicity in terms of animal body weight loss correlated with their ability to induce human neutrophil adherence. The inability of BP‐LPS to activate neutrophils may thus have some bearing on its low toxicity.
Detection of Human Cytomegalovirus DNA in Breast Milk by Means of Polymerase Chain Reaction
Authors: Hotsubo Tomoyuki , Nagata Nobuo , Shimada Masayoshi , Yoshida Koichi , Fujinaga Kei , Chiba Shunzo
Published: November 2013

Three hundred and twenty‐five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.
Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice
Authors: Cellini Luigina , Allocati Nerino , Angelucci Domenico , Iezzi Teresa , Campli Emanuela Di , Marzio Leonardo , Dainelli Benedetto
Published: November 2013

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD550 = 5 Mc Farland) of Helicobacter pylori with bacillary (2‐5×109 CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non‐culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.
Influence of Molecular Sizes of Cryptococcus neoformans Capsular Polysaccharide on Phagocytosis
Authors: Yasuoka Akira , Kohno Shigeru , Yamada Hiroshi , Kaku Mitsuo , Koga Hironobu
Published: November 2013

The role of capsular polysaccharides (CPS) of Cryptococcus neoformans in phagocytosis by murine alveolar macrophages was investigated in four strains of C. neoformans serotype A, YC‐11, YC‐5, YC‐27 and YC‐13. Phagocytosis rates increased markedly after adding 10% mouse serum, compared to fetal calf serum. The reverse relation between capsular thickness of C. neoformans and phagocytosis by alveolar macrophages was observed except in YC‐27, which had thin capsules and high virulence. The phagocytosis rate in mice serum was 17.3% in YC‐11 (capsule thickness 2.8‐3.5 μm), 39.8% in YC‐5 (capsule size 0.8‐1.5 μm), 20.3% in YC‐27 (capsule size 0.6‐1.1 μm), and 62.8% in YC‐13 (capsule not detected microscopically). The CPS of YC‐11, YC‐5, and YC‐27 analyzed by gel‐filtration using CL‐2B showed high molecular fractions near the void volume. However, the CPS of YC‐13 showed only low molecular fractions. The widely eluted CPS of YC‐11 was separated into 3 fractions and each fraction was added in the phagocytosis assay of YC‐13. Phagocytosis was markedly suppressed particularly by the addition of a higher molecular fraction. These results suggest that phagocytosis of C. neoformans by alveolar macrophages is influenced by the molecular sizes of the CPS.
Characterization of a Chlamydia pneumoniae Strain Isolated from a 57‐Year‐Old Man
Authors: Miyashita Naoyuki , Kubota Yoshifumi , Kimura Masashi , Nakajima Masamitsu , Niki Yoshihito , Soejima Rinzo , Matsumoto Akira
Published: November 2013

The isolation of Chlamydia pneumoniae, especially from elderly persons, is generally not easy. Recently, we succeeded in isolating a chlamydial strain, which was designated KKpn‐15, from a 57‐year‐old man suffering from acute bronchitis. It was compared with well established strains of C. pneumoniae, C. trachomatis and C. psittaci, and its biological properties, such as the morphology of elementary bodies (EBs) and inclusions, and the immunochemistry of EB proteins, were investigated. Based on the results obtained in the present study, it was confirmed that the new chlamydial strain, KKpn‐15, is a member of the C. pneumoniae strain and that the organisms of KKpn‐15 are useful as an antigen for the serodiagnosis and epidemiology of C. pneumoniae infection.
Studies on Serological Cross‐Reaction in Sequential Flavivirus Infections
Authors: Makino Yoshihiro , Tadano Masayuki , Saito Mika , Maneekarn Niwat , Sittisombut Nopporn , Sirisanthana Virat , Poneprasert Boosom , Fukunaga Toshihiko
Published: November 2013

Acute‐ and convalescent‐phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre‐existing flavivirus antibodies were tested for cross‐reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent‐phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross‐reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross‐reactive. Of three JE cases with pre‐existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross‐reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.
Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli
Authors: Sarker Mahfuzur R. , Akimoto Shigeru , Ono Tsuneko , Kinouchi Takemi , Ohnishi Yoshinari
Published: November 2013

Clones containing the Bacteroides fragilis leuB‐complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB‐complementing determinant in pOT865 was localized to a region of 1.5‐kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42‐kDa protein. These results suggest that the cloned segment contains the structural gene for β‐isopropylmalate dehydrogenase (leuB).
Immunogenicity of the Shigella flexneri Serotype Y (SFL124) Vaccine Strain Expressing Cloned Glucosyl Transferase Gene of Converting Bacteriophage SfX
Authors: Huan Pham Thi , Taylor Rona , Lindberg Alf A. , Verma Naresh K.
Published: November 2013

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O‐antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild‐type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O‐antigen immunity in the host defense against shigellosis.
Isolation of Coxiella burnetii from Dairy Cattle and Ticks, and Some Characteristics of the Isolates in Japan
Authors: Ho To , Htwe Khin Khin , Yamasaki Naomi , Zhang Guo Quan , Ogawa Motohiko , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty‐nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.
Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency
Authors: Nagahata Hajime , Higuchi Hidetoshi , Nochi Hiromi , Tamoto Koichi , Noda Hiroshi , Kociba Gary J.
Published: November 2013

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)‐binding receptors, luminol‐dependent chemiluminescent (LDCL) responses, and the effect of anti‐bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1‐ to 2.5‐fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A‐binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat‐aggregated bovine IgG (Agg‐bovine IgG), sheep red blood cells (SRBC) sensitized with anti‐SRBC antibody (SRBC‐anti‐SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT‐reducing value of neutrophils from a calf with BLAD when stimulated with Agg‐bovine IgG after pretreatment with anti‐bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre‐incubation with anti‐bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC‐anti‐SRBC Ab and NBT‐reducing activity stimulated with Agg‐bovine IgG after pretreatment with anti‐bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).
Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan
Authors: Kozaki Shunji , Nakaue Shoji , Kamata Yoichi
Published: November 2013

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino‐terminal half (H‐1 fragment) of the heavy chain. This may support the hypothesis that the H‐1 fragment functions in the transport of the light chain in the target cell.
Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer, Integral Membrane and Nucleocapsid Proteins of Feline, Canine and Porcine Coronaviruses
Authors: Motokawa Kenji , Hohdatsu Tsutomu , Hashimoto Hiroshi , Koyama Hiroyuki
Published: November 2013

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU‐2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU‐2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc‐1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein‐deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein‐deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein‐deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.
Laboratory Investigation of Ecological Factors Influencing the Environmental Presence of Burkholderia pseudomallei
Authors: Tong Shide , Yang Song , Lu Zhenzhi , He Weihai
Published: November 2013

Factors like temperature, pH value, water content in soil, and ultraviolet rays that might have influence on the survival of environmental Burkholderia pseudomallei strains were evaluated. Data showed that the optimal temperature and pH value for B. pseudomallei were 24 C to 32 C and 5 to 8, respectively. Water content in soil of less than 10% brought about the death of the bacteria within 70 days, while water content of more than 40% maintained bacteria life for 726 days. The bacteria were easily killed by ultraviolet rays at 465 μW/cm2 for 7.75 min while other permanent soil bacteria were killed at 1,860 μW/cm2 for 31 min. From these results, it could be concluded that proper temperature, enough water in soil, and suitable soil pH might be the three major ecological conditions governing the environmental presence of B. pseudomallei.
Long‐Term Acceptance of Major Histocompatibility Complex‐Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506
Authors: Yamada Akira , Murakami Masaaki , Ijima Kenichi , Yagita Hideo , Okumura Ko , Komatsu Sakuzo , Uede Toshimitsu
Published: November 2013

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H‐2b) neonatal hearts were transplanted to CBA/J (H‐2k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.
Involvement of Vulnibactin and Exocellular Protease in Utilization of Transferrin‐ and Lactoferrin‐Bound Iron by Vibrio vulnificus
Authors: Okujo Noriyuki , Akiyama Toshihito , Miyoshi Shin‐Ichi , Shinoda Sumio , Yamamoto Shigeo
Published: November 2013

In vitro growth experiments were conducted to evaluate the ability of vulnibactin, a siderophore produced by Vibrio vulnificus, to sequester transferrin‐ or lactoferrin‐bound iron for growth. Comparative studies with the strain producing vulnibactin and its exocellular protease‐deficient mutant revealed the involvement of the protease in addition to vulnibactin in effective utilization of iron ion (Fe3+) bound to transferrin and lactoferrin. It appears that the protease causes cleavage of these proteins, thereby making bound iron more accessible to vulnibactin.
Sequence Analysis of the Florfenicol Resistance Gene Encoded in the Transferable R‐Plasmid of a Fish Pathogen, Pasteurella piscicida
Authors: Kim Eun‐Heui , Aoki Takashi
Published: November 2013

The florfenicol resistance gene (pp‐flo) derived from a transferable R‐plasmid of Pasteurella piscicida consisted of 1,122 nucleotides, and the predicted amino acid sequence showed 47.4% identity to that of a non‐enzymatic chloramphenicol resistance gene (cmlA). The pp‐flo gene was located in the downstream region of the sulfonamide resistance gene of the transferable R‐plasmid.
An N‐[(R)‐(‐)‐2‐Hydroxypropionyl]‐α‐L‐Perosamine Homopolymer Constitutes the O Polysaccharide Chain of the Lipopolysaccharide from Vibrio cholerae O144 Which Has Antigenic Factor(s) in Common with V. cholerae O76
Authors: Sano Yasuhiro , Kondo Seiichi , Isshiki Yasunori , Shimada Toshio , Hisatsune Kazuhito
Published: November 2013

Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144). The LPS of O144 contained D‐glucose, D‐galactose, L‐glycero‐D‐manno‐heptose, D‐fructose, D‐quinovosamine (2‐amino‐2,6‐dideoxy‐D‐gluco‐pyranose) and L‐perosamine (4‐amino‐4,6‐dideoxy‐L‐manno‐pyranose). The perosamine, a major component sugar of the LPS from O144, was in an L‐configuration, as is also the case in the LPS from V. cholerae O76 (O76), in contrast to the D‐configuration of the perosamine in the LPS of V. cholerae O1. A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an α(1 → 2)‐linked homopolymer of (R)‐(‐)‐2‐hydroxypropionyl‐L‐perosamine. The serological cross‐reactivity between O144 and O76 was clearly revealed by cross‐agglutination and cross‐agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red‐blood cells that had been sensitized with the LPS from O144 and O76. In contrast, in passive hemolysis tests, the LPS of O144 did not cross‐react serologically with the LPSs from other strains such as V. cholerae O1 (Ogawa and Inaba), V. cholerae O140, Vibrio bio‐serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9. The LPSs from these strains consist of O polysaccharide chains composed of α(1 → 2)‐linked homopolymers of D‐perosamine with various N‐acyl groups, and they share the Inaba antigen factor C of V. cholerae O1 in common. The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V. cholerae O1.
Rapid Identification of Mutans Streptococcal Species
Authors: Igarashi Takeshi , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS‐blue dextran‐PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species‐specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS‐blue dextran‐PAGE also showed species‐specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.
Borrelia tanukii Sp. Nov. and Borrelia turdae Sp. Nov. Found from Ixodid Ticks in Japan: Rapid Species Identification by 16S rRNA Gene‐Targeted PCR Analysis
Authors: Fukunaga Masahito , Hamase Akiko , Okada Keiji , Nakao Minoru
Published: November 2013

Based on the results of RFLP‐ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species‐specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.
Serological Survey of Spotted Fever Group Rickettsia in Wild Rats in Thailand in the 1970s
Authors: Okabayashi Tamaki , Tsutiya Kimiyuki , Muramatsu Yasukazu , Ueno Hiroshi , Morita Chiharu
Published: November 2013

In Thailand, the first human cases of spotted fever group rickettsiosis were reported in 1994, but no serosurveys on wild rats have yet been conducted. We investigated the seroepidemiology in wild rats collected in the 1970s from two regions in Thailand, and found a 62.2% positive rate of antibodies for spotted fever group rickettsia (SFGR) by the indirect immunofluorescence antibody test. Of the antibody‐positive rats, 82.2% had higher titers of antibodies against TT‐118 than those against Rickettsia japonica, which suggests that Thailand is infested mainly with the TT‐118 strain or its antigenically related organisms. The prevalence of antibodies in Bandicota indica was significantly higher than that in other species, which suggests that B. indica is important as a reservoir of SFGR in Thailand.
Different Rifampicin Inactivation Mechanisms in Nocardia and Related Taxa
Authors: Tanaka Yasushi , Yazawa Katsukiyo , Dabbs Eric R. , Nishikawa Kazutaka , Komaki Hisayuki , Mikami Yuzuru , Miyaji Makoto , Morisaki Naoko , Iwasaki Shigeo
Published: November 2013

Mycolic acid‐containing bacteria inactivate rifampicin in a variety of ways such as glucosylation, ribosylation, phosphorylation and decolorization. These inactivations were found to be a species‐specific phenomena in Nocardia and related taxa. Gordona, Tsukamurella and fast‐growing Mycobacterium modified rifampicin by ribosylation of the 23‐OH group of the antibiotic. Such ribosylation was not observed in Rhodococcus and Corynebacterium, but phosphorylation of the 21‐OH group of rifampicin was observed in one strain of Rhodococcus. Nocardia modified the antibiotic by glucosylation (23‐OH group) and phosphorylation, but ribosylation was not observed.
Characterization by Western Blotting of Mouse Intestinal Glycoproteins Bound by Escherichia coli Heat‐Labile Enterotoxin Type I
Authors: Shida Kan , Takamizawa Kotaro , Takeda Tae , Osawa Toshiaki
Published: November 2013

Escherichia coli heat‐labile enterotoxin type I (LT‐I)‐binding galactoproteins, which were not recognized by cholera toxin, were detected in intestinal epithelial cells of BALB/c mouse by Western blotting. Inhibitory studies using lectins and modifications of sugar chain suggest that LT‐I recognizes certain mucin‐type sugar chains containing the terminal Galβ1‐3GalNAc sugar sequence in the galactoproteins. The terminal sugar sequence is identical to that of GM1 ganglioside, the well‐documented functional receptor for LT‐I.
A New Primer Pair for Detection of Chlamydia pneumoniae by Polymerase Chain Reaction
Authors: Kubota Yoshifumi
Published: November 2013

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53‐kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.
Characterization of a Toxin‐Deficient Clostridium perfringens Strain, KZ1340
Authors: Shimizu Tohru , Ohtani Kaori , Ba‐Thein William , Inui Satoko , Nakamura Shinichi , Hayashi Hideo
Published: November 2013

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an α, θ‐, and κ‐toxin non‐producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (θ‐toxin) gene, but the plc (α‐toxin) and colA (κ‐toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin‐deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.
Role of Milk Whey in the Transmission of Human Cytomegalovirus Infection by Breast Milk
Authors: Asanuma Hideomi , Numazaki Kei , Nagata Nobuo , Hotsubo Tomoyuki , Horino Kiyotaka , Chiba Shunzo
Published: November 2013

Breast‐fed infants are susceptible to human cytomegalovirus (HCMV) infection via breast milk. In our previous study, HCMV was isolated more frequently from breast milk at later than one month after delivery than from colostrum or early breast milk. To clarify the role of milk cells and whey in vertical infection by breast feeding, we separated breast milk into milk cells and whey and examined each fraction for the presence of HCMV. We collected breast milk from mothers who breast‐fed their infants (aged from 3 days to 2 months). The breast milk was centrifuged and separated into the middle layer (layer of milk whey) and the pellet (containing milk cells). We attempted to isolate HCMV from whey and to detect HCMV immediate early (IE) DNA in both milk whey and cells. HCMV was isolated from 7 out of 35 (20.0%) whey samples and HCMV IE DNA was detected from 15 out of 35 (42.9%) whey and/or milk cells. Detection rates of HCMV IE DNA in the whey layer and milk cells were 39.1% (25 out of 64) and 17.2% (11 out of 64), respectively. HCMV IE DNA was not detected in colostrum, but was detected in breast milk samples one month after delivery. Therefore, cell‐free HCMV shed into milk whey may have a more important role in vertical infection by breast milk than cell‐associated HCMV in the milk.
Isolation and Characterization of Mumps Virus Strains in a Mumps Outbreak with a High Incidence of Aseptic Meningitis
Authors: Saito Hiroyuki , Takahashi Yoshihiro , Harata Seizaburo , Tanaka Keiko , Sano Tsuyoshi , Suto Tsunehisa , Yamada Akio , Yamazaki Shudo , Morita Morihiro
Published: November 2013

In 1993, mumps with a high incidence of aseptic meningitis became prevalent in Akita prefecture, Japan. Three mumps virus isolates obtained from the nonvaccine‐associated cases lacked the BamHI restriction cleavage site of the P gene, like the Urabe strain (Yamada, A. et al, Vaccine 8: 553‐557). However, four additional nucleotide substitutions were found in the determined region of 157 bp. Fourteen of 19 cases from which mumps virus showing the Urabe‐like RFLP profile was detected were complicated with symptomatic meningitis, whereas there were only four cases of meningitis among 23 individuals infected with the wild type showing no Urabe‐like RFLP profile (non‐“Urabe‐like” wild‐type). The incidence of meningitis was over 70% among patients infected with the “Urabe‐like” wild‐type virus. The “Urabe‐like” wild‐type disappeared after February 1994 in the epidemic area and was replaced by the non‐“Urabe‐like” wild‐type. Patients infected with the “Urabe‐like” wild‐type lived in a closed colony, in which there were two instances of transmission between siblings. Thus this outbreak was transient and narrowly localized.
Immunohistochemical Studies on the Transneuronal Spread of Virulent Herpes Simplex Virus Type 2 and Its US3 Protein Kinase‐Deficient Mutant after Ocular Inoculation
Authors: Yamamoto Mitsuaki , Kurachi Ryutaro , Morishima Tsuneo , Kito Junzo , Nishiyama Yukihiro
Published: November 2013

The transneuronal spread of a virulent wild‐type herpes simplex virus type 2 (HSV‐2) and its US3 protein kinase‐deficient (US3 PK−) mutant was immunohistochemically studied in mice after inoculations into the cornea, anterior chamber, tongue, and masseter muscle. After corneal inoculation, the wild‐type virus was demonstrated in various brain stem areas including the trigeminal tract and nucleus, the reticular formation, and cerebellar nucleus group. Viral antigen‐positive neurons were strictly confined to the ipsilateral spinal trigeminal nucleus in mice corneally infected with the US3 PK− mutant. No viral antigens were detected in the central nervous system (CNS) after inoculation with the mutant into the tongue and masseter muscle. However, when mice were immunosuppressed by treatment with cyclophosphamide, both the corneally infected mutant and wild‐type virus could invade the CNS. The results suggest that the US3 PK− mutant principally retains the capacity to spread in the CNS.
Levels of Porphyromonas gingivalis Fimbriae and Inflammatory Cytokines in Gingival Crevicular Fluid from Adult Human Subjects
Authors: Hirose Kimiharu , Isogai Emiko , Miura Hiroko , Ueda Itsuo
Published: November 2013

The Porphyromonas gingivalis fimbriae level was examined in the gingival crevicular fluid (GCF) from adult human subjects using an immunoblot assay with a monoclonal antibody. The cytokines, interleukin‐1α (IL‐1α), IL‐1β, IL‐6 and tumor necrosis factor‐a (TNF‐α) levels in the GCF were quantified by enzyme‐linked immunosorbent assay (ELISA). The reactivity of the GCF samples with the monoclonal antibody against P. gingivalis fimbriae was related to the IL‐1β, IL‐6 and TNF‐α levels. Moreover, the fimbriae content was associated with the gingival index (GI). In contrast, no significant correlation was seen between the fimbriae content and IL‐1α level. These results suggest that there are possible associations between P. gingivalis fimbriae and IL‐1β, IL‐6 and TNF‐α in the gingival crevicular fluid.
Apoptosis of Intestinal Intraepithelial Lymphocytes Induced by Exogenous and Endogenous Glucocorticoids
Authors: Murosaki Shinji , Inagaki‐Ohara Kyoko , Kusaka Hiroaki , Ikeda Hitoshi , Yoshikai Yasunobu
Published: November 2013

To investigate the effect of glucocorticoids on apoptosis in intestinal intraepithelial lymphocytes (i‐IEL), we examined the changes of i‐IEL followed by in vivo treatment with dexamethasone. The fragmented DNA of i‐IEL were significantly increased at 15 hr after dexamethasone treatment and, subsequently, the number of total i‐IEL were decreased by day 4 after treatment. Although all subsets of i‐IEL including CD8α/α+, CD8α/β+, CD4+ and CD4+ CD8+ i‐IEL were decreased after dexamethasone treatment, CD8α/α+ i‐IEL appeared to be relatively resistant to dexamethasone‐induced apoptosis. Consistent with the in vivo findings, CD8α/α+ i‐IEL exhibited less susceptibility to dexamethasone‐induced cell death in vitro than other subsets. To investigate whether this process occurs under physiological conditions, we examined the kinetics of i‐IEL after treatment with 15‐hr water immersion stress. In mice subjected to water immersion stress, plasma glucocorticoids were remarkably elevated soon after the 15‐hr stress. The increase in the fragmented DNA of i‐IEL and subsequent decrease in the number of i‐IEL were observed in the stressed mice in the same kinetics as seen in the dexamethasone‐treated mice. Similar to dexamethasone‐induced cell death, CD8α/α+ i‐IEL appeared to be relatively resistant to stress‐induced apoptosis compared with other i‐IEL subsets. The expression level of Bcl‐2 was significantly higher in CD8α/α+ i‐IEL than in CD8α/β+ i‐IEL. Our results indicate that i‐IEL are subjected to cell death via apoptosis by exogenous and endogenous glucocorticoids and that different sensitivity to steroid‐induced apoptosis may exist among i‐IEL subsets in relation to their Bcl‐2 expression.