辻 孝雄

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing a-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial a-galactose-immobilized agarose resin, but not to beta-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing beta-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through a-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZa fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1. G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3 h later showed a significant dose-dependent reduction in frequency (p < 0.05) and duration (p < 0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n = 26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p = 0.004), mean duration of intravenous fluid administration (p = 0.03), mean duration of diarrhea from day of admission (p < 0.01) and mean duration of rotavirus clearance from stool from day of admission (p = 0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients. (C) 2012 Elsevier Ltd. All rights reserved.

Dendritic cell (DC) activation is commonly used as a measure of the immunomodulatory potential of candidate exogenous and endogenous molecules. Residual lipopolysaccharide (LPS) contamination is a recurring theme and the potency of LPS is not always fully appreciated. To address this, polymyxin B (PmB) is often used to neutralise contaminating LPS. However, the limited capacity of this antibiotic to successfully block these effects is neglected. Therefore, this study aimed to determine the minimum LPS concentration required to induce murine bone marrow-derived dendritic cell (BMDC) maturation and cytokine secretion and to assess the ability of PmB to inhibit these processes. LPS concentrations as low as 10 pg/ml and 20 pg/ml induced secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha respectively, while a concentration of 50 pg/ml promoted secretion of IL-12p40. A much higher threshold exists for IL-12p70 as an LPS concentration of 500 pg/ml was required to induce secretion of this cytokine. The efficacy of PmB varied substantially for different cytokines but this antibiotic was particularly limited in its ability to inhibit LPS-induced secretion of IL-6 and TNF-alpha. Furthermore, an LPS concentration of 50 pg/ml was sufficient to promote DC expression of costimulatory molecules and PmB was limited in its capacity to reverse this process when LPS concentrations of greater than 20 ng/ml were used. There is a common perception that LPS is heat resistant. However, heat treatment attenuated the ability of low concentrations of LPS to induce secretion of IL-6 and IL-12p40 by BMDCs, thus suggesting that heat-inactivation of protein preparations is also an ineffective control for discounting potential LPS contamination. Finally, LPS concentrations of less than 10 pg/ml were incapable of promoting secretion of IL-6 independently but could synergise with heat-labile enterotoxin (LT) to promote IL-6, indicating that reducing contaminating endotoxin concentrations to low pg/ml concentrations is essential to avoid misleading conclusions regarding candidate immunomodulators.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are pyrogenic superantigenic toxins that are involved in human diseases including food poisoning and toxic shock syndrome. Although the superantigenic activity of SEs has been well characterized, its role and mechanism in clinical symptoms of food poisoning remain poorly understood. In this study, house musk shrews (Suncus murinus), a small emetic animal model, were used to study the role of SEs in clinical manifestations of food poisoning. Administration of SEA induced a potent emetic response in vivo and showed significant superantigenic activity in vitro in house musk shrews. However, SEA revealed no diarrheagenic activity. SEA directly injected into the intestinal loops of house musk shrews failed to induce fluid exudation and consequent dilation of the intestinal segments. Rabbit intestinal loop experiments were further carried out to confirm the results and also showed that SEA induced no fluid exudation and consequent dilation. Furthermore, the SEA-producing S. aureus also failed to induce fluid exudation in the administered loops of these animal models. These results indicate that SEA has potent superantigenic and emetic activities, but does not have a diarrheagenic activity. (C) 2012 Elsevier GmbH. All rights reserved.

1）新研修医が研修開始直後に必要となる手技を習得できることを目標としたスキルスラボを利用したトレーニングをオリエンテーション期間内に導入した．<br>2）新2年目研修医がトレーニングの内容を計画し，新研修医を指導し，3年目以上の上級医は，新2年目研修医の指導を支援する「屋根瓦方式」の指導体制の構築を目指した．<br>3）本トレーニングを開催したことにより研修医間の関係が良好なものとなり，新研修医に安心感を与えるといった波及効果がみられた．

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

Enterotoxigenic Escherichia coli (ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that E. coli of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 angstrom. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites. (C) 2011 Elsevier Inc. All rights reserved.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naive human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The 822 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems. (C) 2011 Elsevier Ltd. All rights reserved.

Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40%) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60 degrees C and 100 degrees C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2%) were positive for ST II (estB) and two (5%) positive for aHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmidencoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1 angstrom resolution, respectively. The crystals belonged to space group P2(1)2(1)2(1).

Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZ alpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modi. cations and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity. (C) 2009 Elsevier Inc. All rights reserved.

We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (IT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The IT and STIa region was located 13.5 kb apart and was surrounded by three IS Is and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.

2003年6月に東京都および千葉県で発生した,仕出し弁当を原因とした食中毒事例は,6種類の毒素原性大腸菌を原因とした非常に珍しい事例であった.検査は,DHL寒天平板上に発育した大腸菌を対象にColony-sweep PCR法でスクリーニングする方法を導入した結果,非常に効率的に検査を実施できた.患者糞便84検体について下痢原性大腸菌の各種病原因子をスクリーニングした結果,いずれかの毒素遺伝子が陽性となった検体は56検体(66.7%)であった.内訳は,LT遺伝子35検体(41.7%),STp遺伝子21検体(25.0%),STh遺伝子11検体(13.1%)であった.このうち11検体では,2種類の毒素遺伝子が陽性であった.これらの検体からETECの分離を試みた結果,患者糞便84検体中48検体(57.1%)から毒素原性大腸菌(ETEC)が検出された.1検体から1種類のETECのみが検出された例では,O25:NM(LT)が21検体,O27:H20(STp)が12検体,O148:H28(STh)が8検体,O25:NM(STh)およびO27:H7(STp)が各1検体であった.一方,1人の糞便から複数タイプのETECが検出されたものが5検体あった.その血清型および毒素型はO25:NM(LT)&O27:H20(STp)が3検体,O27:H20(STp)&O148:H28(STh)あるいはO25:NM(LT)&O78:NM(STh)が各1検体であった.(著者抄録)