Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2.

Shiga toxin 2 (Stx2)-specific mAb-producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat-labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)-specific whereas five were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of Stx2B-specific mAbs against Stx2 was greater than that of Stx2A-specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs may be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

Gastric emptying and duodenal motility upon intake of a liquid meal with monosodium glutamate in healthy subjects.

Glutamate is thought to serve as a special signal for gut functions. We investigated the effects of monosodium l-glutamate (MSG) on gastric emptying and duodenal motility. Ten healthy male volunteers underwent rapid magnetic resonance imaging (MRI) of the abdomen. Coronal images were successively acquired after ingestion of liquid meal (200 kcal in 200 mL: 9 g protein, 28.4 g carbohydrate, 5.6 g fat, 370 mg Na(+)) with and without 0.5% MSG. During the acquisition of MRI, participants breathed freely. In all participants, the gastric residual volume gradually decreased to 80.1 ± 14.2% without MSG and to 75.9 ± 14.3% with MSG after 60 min (P = 0.45 between the groups, n = 10). In two of 10 participants, gastric emptying slowed down significantly, whereas in the remaining eight participants, gastric residual volume decreased to 84.0 ± 13.1% without MSG, and to 73.0 ± 14.6% with MSG after 60 min (P = 0.015, n = 8). There was no difference in the shape of the stomach between groups. In four of the eight participants responding positively to MSG, the duodenum wall was sufficiently identified to quantify the motions. The inclusion of MSG enhanced duodenal motility, judging from changes in (1) the magnitude of the duodenal area, (2) the center of gravity, and (3) the mean velocity of the wall motions. The third parameter most significantly indicated the excitatory effect of l-glutamate on duodenum motility (~ three- to sevenfold increase during 60 min, P < 0.05, n = 4). These results suggest that MSG accelerates gastric emptying by facilitating duodenal motility, at least in subjects with positive responses to MSG.

Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.

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 lacZα 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 61.1 mg 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.

Assessment of gastric emptying and duodenal motility upon ingestion of a liquid meal using rapid magnetic resonance imaging.

Gastric emptying is achieved by co-operation between gastric and duodenal motor activity. Therefore, evaluation of gastric emptying and its associated mechanisms would benefit clinical therapy as well as medical research. Healthy volunteers underwent rapid magnetic resonance imaging (MRI) of the abdomen along the coronal plane after ingestion of a liquid meal. The gastric fundal and duodenal areas were quantified semi-automatically by self-developed segment software. The average gastric fundal area determined by the serosal end in 40 sequential images was reduced to ∼81% 30 min after and to ∼70% 60 min after ingestion of a liquid meal. The average duodenal area also decreased to ∼86% after 30 min and to 83% after 60 min. In contrast, changes in the centre of gravity increased to about fivefold after 30 min and to about threefold after 60 min. The mean velocity of the duodenal wall mimicked changes in the centre of gravity. The application of metoclopramide, a dopamine D(2) receptor antagonist, accelerated gastric emptying, presumably due to facilitated duodenal activity even immediately after liquid meal ingestion. The ingestion of water caused fast gastric emptying in 30 min, accompanied by high duodenal motility, but it ceased after 60 min, presumably reflecting complete gastric emptying. A rapid MRI scan visualized the association between gastric emptying and duodenal motility that could be modified by calories and dopaminergic neurotransmission. Changes in the centre of gravity and mean velocity of the duodenal wall appear to quantify the motility obtained from cine MRI accurately.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

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 naïve 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 B22 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.

A nasal vaccine comprising B-subunit derivative of Shiga toxin 2 for cross-protection against Shiga toxin types 1 and 2.

Enterohemorrhagic Escherichia coli (EHEC) produces Stx1 and Stx2 causing severe diseases. Their B-subunits (StxBs) exhibit low immunogenicity and the anti-StxB antibodies neutralizing both Stxs has not been prepared yet. By intranasal vaccination with His-tagged-StxB (Stx1B-His or Stx2B-His) plus a mutant heat-labile enterotoxin (mLT) in mice, their serum and lung fluid reacted with appropriate StxB. Mice vaccinated with Stx2B-His plus mLT had antibodies reacting Stx1B and showed the resistance to toxemia of Stx1 and Stx2. This is the first demonstration to get anti-Stx2B serum neutralizing both Stxs. These suggest that the nasal vaccination with Stx2B-His and mLT is effective for preventing toxemias by EHEC.

A mutant of Escherichia coli enterotoxin inducing a specific Thl-type of T cells to varicella-zoster vaccine enhances the production of IL-12 by IFNgamma-stimulated macrophages.

A mutant of Escherichia coli enterotoxin induces specific Thl-type T cells to varicella-zoster vaccine. The mutant increased IL-12p40, TNFalpha and nitric oxide production by IFNgamma-stimulated bone marrow macrophages but cholera toxin did not. Anti-TNFalpha antibodies blocked its stimulation of IL-12p40 production but iNOS inhibitor did not. IL-12p40 and IL-12p35 production was stimulated at the level of mRNA formation by the mutant. Cholera toxin suppressed IL-12beta1 expression by spleen T cells stimulated with anti-CD3 antibodies but the mutant did not. These findings indicate that the mutant may induce Thl-type response to the vaccine through its IL-12 and TNFalpha induction by macrophages.

Induction of thymus-derived gammadelta T Cells by Escherichia coli enterotoxin b subunit in peritoneal cavities of mice.

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both gammadelta and alphabeta T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM1 ganglioside-binding ability. Early after administration a small number of gammadelta T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing gammadelta T cells were detected. gammadelta T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of gammadelta T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like alphabeta T cells. gammadelta T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal gammadelta and alphabeta T cells in a manner dependent upon its ability to bind to GM1 ganglioside. gammadelta T cells induced by the B subunit are Th2-type cells derived from the thymus. These gammadelta T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of alphabeta T cells.