Anti-cholera Toxin Antibody Research Articles

Electrospun Carbon Nanofibers as an Electrochemical Immunosensing Platform for Vibrio cholerae Toxin: Aging Effect of the Redox Probe.

An electrochemical immunosensor for Vibrio cholerae toxin (VCT) has been developed using electrospun carbon nanofibers (CNFs) as the electrode platform. To fabricate the immunosensor, the anti-cholera toxin antibody (Ab) was covalently immobilized on the electrode platforms using the carbodiimide chemistry for the amide bond formation. Every step of the formation of the immunosensor and the subsequent binding of the VCT subunit antigen (Ag) was electrochemically interrogated. The immunosensor gave excellent reproducibility and sensitivities: limits of detection (ca. 1.2 × 10–13 g mL–1), limits of quantification (ca. 1.3 × 10–13 g mL–1), and a wide linear range for the anti-cholera detection of 8 orders of magnitude (10–13 to 10–5 g mL–1). One of the key findings was the enhanced sensitivity of the VCT detection using aged rather than the freshly prepared redox probe, described here as Redox Probe Aging-Induced Sensitivity Enhancement (“Redox-PrAISE”). The Redox-PrAISE was found more useful in the real application of these immunosensors, showing comparable or even better sensitivity for eight real cholera-infested water samples than the conventional clinical culture method. This immunosensor shows promise for the potential development of point-of-care diagnosis of VCT. Importantly, this study highlights the importance of considering the nature of the redox probe on the electrochemical sensing conditions when designing impedimetric immunosensors.

Age-related immunogenicity and reactogenicity of live oral cholera vaccine CVD 103-HgR in a randomized, controlled clinical trial

Aging is accompanied by a decline in immune function which can lead to decreased responses to vaccines. Attenuated recombinant Vibrio cholerae O1 vaccine strain CVD 103-HgR elicits a rapid serum vibriocidal antibody (SVA) response and protects against cholera diarrhea in volunteer challenge studies but has not been studied in older adults. We evaluated CVD 103-HgR (PXVX0200) in adults age 46–64, compared them to previously studied adults age 18–45, and studied age-related immunogenicity across adults 18–64 years of age. Volunteers were randomized to receive a single dose of 1 × 109 CFU of PXVX0200 or placebo. Immunogenicity endpoints included SVA and anti-cholera toxin (CT) antibody levels on days 1, 11, 29, 91 and 181 and lipopolysaccharide (LPS) and CT-specific IgA and IgG memory B cells on days 1, 91 and 181. Safety was assessed by comparing solicited signs and symptoms on days 1–8 and other adverse events through day 181. 2979 volunteers received vaccine, including 291 age 45–64. Day 11 seroconversion occurred in 90.4% of older adults vs 93.5%% of younger adults and met the endpoint of demonstrating non-inferiority between the two groups. Significant increases in LPS-specific IgG and IgA and CT-specific memory IgG memory B cells were seen at days 91 and 181. There appeared to be a continuous age-related decline in SVA seroconversion and geometric mean titers, but not memory B cell responses, across the 18–64 year age range. Most reactogenicity was mild and was more common in the placebo group. PXVX0200 appears safe and immunogenic in older adults.Clinical Trials Registration: clinicaltrials.gov NCT02100631.

Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin.

Aptamers are envisioned to serve as powerful synthetic substitutes to antibodies in a variety of bioanalytical assay formats. However, lateral flow assays (LFAs) remain dominated by antibody-based strategies. In this study, a LFA for the detection of cholera toxin as a model analyte is developed and optimized using a synthetic aptamer and a naturally occurring receptor as biorecognition elements and directly compared with solely aptamer and aptamer and antibody-based alternative approaches. The aptamer (CT916) recently selected by our group, GM1 receptors and an anti-cholera toxin antibody were evaluated. Relying solely on molecules that can easily be synthesized while aiming for high sensitivity, we applied a novel combination of capture aptamer and GM1 cell receptor-labeled liposomes for cholera toxin detection, achieving a limit of detection (LOD) of 2 ng ml-1 (3σ)/10 ng ml-1 (visual) in ∼15 min. To put our novel aptasensor into perspective, we developed a competitive lateral flow assay, exploiting the competition of cholera toxin in solution with immobilized cholera toxin for binding of aptamer-coated gold nanoparticles (AuNPs) (LOD = 51 ng ml-1 (3σ)/100 ng ml-1 (visual), assay time ∼10 min). As dual simultaneously binding aptamers were not available, we designed aptamer antibody pair-based lateral flow assays using aptamer-coated AuNPs which yielded a LOD of 5 ng ml-1 (by the 3σ rule)/10 ng ml-1 (visual) in a 10 min assay and an even better LOD of 0.6 ng ml-1 (3σ)/1 ng ml-1 (visual), with an ∼20 min total assay time. All set-ups are highly specific and provide an excellent alternative for cholera toxin detection in places where professional knowledge and sophisticated equipment are not readily available and cost efficient, simple, and rapid tests are needed, while the combination of GM1 cell receptor-labeled liposomes and aptamers is clearly the most promising.

Safety and immunogenicity of single-dose live oral cholera vaccine strain CVD 103-HgR in healthy adults age 18–45

The attenuated recombinant Vibrio cholerae O1 vaccine strain CVD 103-HgR, re-developed as PXVX0200, elicits a rapid serum vibriocidal antibody (SVA) response and protects against cholera diarrhea in volunteer challenge studies. We performed a phase 3, placebo controlled, double blind, multi-center study to further assess the safety, immunogenicity, and lot-to-lot consistency of PXVX0200. Adult volunteers 18–45 years of age were randomized 8:1 to receive a single dose of 1 × 109 CFU of PXVX0200 from three production lots or saline placebo. Immunogenicity endpoints included SVA and anti-cholera toxin (CT) antibody levels on days 1, 11, 29, 91 and 181. Safety was assessed by comparing solicited signs and symptoms on days 1–8, unsolicited adverse events through day 29 and serious adverse events through day 181. A total of 3146 participants were enrolled, including 2795 vaccine and 351 placebo recipients. The SVA seroconversion rates at day 11 were 94% and 4% in the PXVX0200 and placebo recipients, respectively (P < .0001). Cumulative SVA seroconversion occurred among 96% of vaccine recipients. PXVX0200 SVA GMTs peaked on day 11 and remained significantly higher than placebo through day 181 while the fold-rise over baseline in PXVX0200 anti-CT antibody was significantly greater than placebo at every post-vaccination time point. Most reactogenicity was mild and resolved within 1–3 days with headache and diarrhea more frequently reported in PXVX0200 recipients. There were no differences in unsolicited adverse events and no study-related serious adverse events. Immunogenicity and safety endpoints were equivalent between the three production lots. PXVX0200 is immunogenic and well tolerated across multiple production lots.Clinical Trials Registration: Clinicaltrials.gov NCT02094586.

Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody

An original impedimetric immunosensor was developed based on carbon nanotube (CNT) deposits with controlled thicknesses for enhanced electroactive surface areas leading to improved sensor performances. Cholera monitoring was chosen as the model immune system for this setup. These CNT deposits were characterized using confocal laser microscopy and electrochemical methods. To form the sensor device, the CNT deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly(pyrrole-NTA)) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B Subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. Each step of the formation of the immunosensor and the subsequent binding of the analyte antibody anti-cholera toxin were investigated with cyclic voltammetry and Electrochemical Impedance Spectroscopy. After optimization, the resulting impedimetric cholera sensor shows excellent reproducibility, increased sensitivities, a very satisfying detection limit of 10−13gmL−1 and an exceptional linear range for anti-cholera detection of 8 orders of magnitude (10−13–10−5gmL−1) and a sensitivity of 24.7 ± 0.4Ω per order of magnitude.

Safety and immunogenicity of a live oral recombinant cholera vaccine VA1.4: a randomized, placebo controlled trial in healthy adults in a cholera endemic area in Kolkata, India.

BackgroundA live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.MethodA lyophilized dose of 1.9×109 CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.ResultThe vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1st dose) and 21 days (i.e. after 2nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.ConclusionThis study demonstrates that VA 1.4 at a single dose of 1.9×109 is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.Trial RegistrationClinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582

Electrogenerated trisbipyridyl Ru(II)-/nitrilotriacetic-polypyrene copolymer for the easy fabrication of label-free photoelectrochemical immunosensor and aptasensor: Application to the determination of thrombin and anti-cholera toxinantibody

A bifunctional copolymer was electrogenerated, which allows efficient bioreceptor immobilization and transduction of the biorecognition event. This copolymer was formed using pyrenebutyric acid Nα′,Nα-bis(carboxymethyl)-L-lysine amide (NTA–pyrene) and [tris-(2,2′-bipyridine) (4,4′-bis(4-pyrenyl-1-ylbutyloxy)-2,2′-bipyridine] ruthenium(II) hexafluorophosphate (Ru(II)–pyrene) complex. The pyrene groups, present in both compounds, undergo oxidative electropolymerization on platinum electrodes. The resulting copolymer contains NTA moieties, which were used as a versatile immobilization system for biotin- and histidine-tagged biomolecules, while Ru(II)–pyrene was employed as a photoelectrochemical transducing molecule. The efficiency of this copolymer for biomolecule anchoring was investigated with biotin- and histidine- tagged glucose oxidases, biotin-tagged cholera toxin and a histidine-tagged thrombin aptamer. The constructed enzyme electrodes exhibited an amperometric response toward glucose at 0.6V vs SCE, demonstrating the anchoring of this enzyme via two coordination systems. An immunosensor configuration based on the immobilization of biotin-tagged cholera toxin was applied to the detection of anti-cholera antibody while the aptasensor based on the immobilization of histidine-tagged thrombin aptamer was tested for thrombin determination. The biorecognition events were monitored via the evolution of the photocurrent intensity generated by the polymerized Ru(II)–pyrene in the presence of visible light and a sacrificial donor (ascorbate). The binding of the targets hinders the diffusion of the sacrificial donor, inducing thus a photocurrent decrease. The constructed immunosensor presents a specific label-free photoelectrochemical response to anti-cholera antibody without labeling step, the detection limit being 0.2μgmL−1. The label-free photoelectrochemical response of the aptasensor varies linearly with thrombin concentrations up to 10pmolL−1, the detection limit being 1×10−13molL−1.

Retroviral Vectors Induce Epigenetic Chromatin Modifications and IL-10 Production in Transduced B Cells via Activation of Toll-like Receptor 2

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.).

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB-sCOE), and the sCTB-sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB-sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB-COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB-COE fusion protein to its receptor, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-ELISA, the expression level of CTB-COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB-COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

Retinopetal Neurons Located in the Diencephalon of the Japanese Monkey (Macaca fuscata)

After a monocular injection of the cholera toxin B subunit (CTB) into the vitreous chamber of one eye, the retrogradely labeled retinopetal neurons were studied in the diencephalon of the Japanese monkey. The retrogradely transported tracer was visualized using the peroxidase antibody technique and an anti-cholera toxin antibody. The CTB-labeled nerve cell bodies were scattered in the periventricular nucleus of the hypothalamus, lateral hypothalamic area, and midline nuclei of the thalamus on both sides. In addition, a few retrogradely labeled nerve somata were observed in the most rostral portion of the lateral geniculate nucleus on the contralateral side.

Electropolymerized biotinylated poly (pyrrole–viologen) film as platform for the development of reagentless impedimetric immunosensors

We describe herein the synthesis and electrochemical polymerization of a viologen monomer functionalized by a biotin group and the use of its redox properties for the impedimetric sensing of protein binding. The electrochemical oxidation of the pyrrole group at 0.85 V in CH 3CN led to a biotinylated polypyrrole film allowing the successive anchoring of an avidin layer and a biotinylated cholera toxin layer. EIS performed at −0.45 V/SCE without redox probe in solution showed the specific immobilization of the anti-cholera toxin antibody.

BRAIN RESPONSES TO BLADDER FILLING MEASURED BY FUNCTIONAL MAGNETIC RESONANCE IMAGING REFLECT CLINICAL SEVERITY OF URGE INCONTINENCE

In order to clarify the enteropathogenicity of Plesiomonas shigelloides, we investigated a cytotoxin produced by the P-1 strain isolated from patients suffering from diarrhea. The cytotoxicity of the culture filtrate of the strain reached a maximum in culture at 37 °C after 12 h shaken in BHI medium. The cytotoxin in the cultures was purified by (NH4)2SO4 precipitation, and Sephacryl S-100, Mono Q HR, and Superdex 200 HR column chromatographies. An approximate 340-fold purification was achieved, with a recovery of about 1.4%, from the culture supernatant. The cytotoxin is heat-stable, and is a complex of three major proteins (LPS-binding proteins with molecular weights of 32, 40, and 48 kDa), with lipopolysaccharide (LPS) giving a total a molecular weight of more than 600 kDa. The ratio of protein to LPS in the cytotoxin was 6–5. The cytotoxic activity was reduced by about 80% by proteinase K treatment or when incubated with anti-cholera toxin antibody (Anti-CT). Western blotting of the cytotoxin with Anti-CT demonstrated the presence of two anti-cholera toxin-reactive protein (ACRP) bands with molecular weights of 40 kDa (a major single protein band) and 48 kDa. The N-terminal amino acid sequence (20 residues) of the 40 kDa protein was 75% identical to Pasteurella multocida cell membrane proteins. The cytotoxin gave a positive reaction in the suckling mouse assay whereas LPS alone hardly exhibited any cytotoxic or enterotoxigenic activity. In conclusion, P. shigelloides produces a cytotoxin that consists of a complex of protein and LPS with the former component exhibiting both cytotoxicity and enteropathogenicity. This cytotoxin has the potential to have an important role in the enteropathogenicity of P. shigelloides.

Characterization of thin poly(pyrrole-benzophenone) film morphologies electropolymerized on indium tin oxide coated optic fibers for electrochemical and optical biosensing

We report that electropolymerization parameters (duration and voltage) modulate the properties and morphological structures of photoactive polymers which influence both the efficiency of bioreceptor attachment to the polymer surface, and the resulting biosensor assay performance. We have compared two biosensor technologies, optical and electrochemical, for the detection of anti-cholera toxin antibodies using our singular conductive optic fibers, albeit at this stage in separate experiments. Both biosensor systems were more sensitive than conventional detection and pave the way for the possibility to one day make simultaneous optical and electrochemical sensing using the same biosensor.

Structure of an anti‐cholera toxin antibody Fab in complex with an epitope‐derived D‐peptide: a case of polyspecific recognition

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence-the only difference is a tyrosine residue in position 7-there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies.

CD8 Raft Localization Is Induced by Its Assembly into CD8αβ Heterodimers, Not CD8αα Homodimers

The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.

Identification of Protein Expression Signatures Associated with Helicobacter pylori Infection and Gastric Adenocarcinoma Using Recombinant Antibody Microarrays

Antibody microarray based technology is a powerful emerging tool in proteomics, target discovery, and differential analysis. Here, we report the first study where recombinant antibody fragments have been used to construct large scale antibody microarrays, composed of 127 different antibodies against mostly immunoregulatory antigens. The arrays were based on single framework recombinant antibody fragments (SinFabs) designed for high on-chip stability and functionality and were used for the analysis of malignant and normal stomach tissue samples from Helicobacter pylori-positive and -negative patients. Our results demonstrate that distinct tumor- as well as infection-associated protein expression signatures could be identified from these complex tissue proteomes, as well as biomarkers such as IL-9, IL-11, and MCP-4, previously not found in these diseases. In a longer perspective, this study may improve the understanding of H. pylori-induced stomach cancer and lead to development of improved diagnostics.

Photoelectrochemical Immunosensor for Label-Free Detection and Quantification of Anti-cholera Toxin Antibody

We demonstrate herein a newly developed photoelectrochemical immunosensor for the determination of anti-cholera toxin antibody by using a photosensitive biotinylated polypyrrole film. The latter was generated by electro-oxidation of a biotinylated tris(bipyridyl) ruthenium(II) complex bearing pyrrole groups. The photoexcitation of this modified electrode potentiostated at 0.5 V vs SCE, in the presence of an oxidative quencher, pentaaminechloro cobalt(III) chloride (15 mM), led to a cathodic photocurrent. As a result of the affinity interactions, a layer of biotinylated cholera toxin was firmly bound to the functionalized polypyrrole film via avidin bridges. The resulting modified electrodes were tested as immunosensors for the detection of the corresponding antibody from 0 to 200 microg mL(-)(1). The antibody concentration was measured through the decrease in photocurrent intensity resulting from its specific binding onto the polymeric coating, the detection limit being 0.5 microg mL(-)(1).

A simple strategy based on photobiotin irradiation for the photoelectrochemical immobilization of proteins on electrode surfaces

A photoactivable organic polymer was prepared first by electrogeneration of a conductive biotinylated polypyrrole film in acetonitrile electrolyte. The successive anchoring of avidin and photobiotin led to a multilayer configuration. The latter was illuminated with light (wavelength 370–400 nm) in the presence of proteins adsorbed onto its surface. The irradiation allowed the covalent linking of the proteins to the modified electrode. As a result of the photochemical reaction, a monolayer of enzyme (glucose oxidase, GOX or alkaline phosphatase, AP) was covalently bound to the photobiotin-modified surface with retention of their catalytic activities. The surfacic activities were 34 and 1.69 mU cm − 2 for GOX and AP photobiotin electrodes, respectively. These enzyme electrodes were compared to similar configurations obtained through the immobilization of biotinylated glucose oxidase or avidin-conjugated alkaline phosphatase on biotinylated polypyrrole film. Our results suggest that both procedures led to the immobilization of the same enzyme amount, namely a protein monolayer. This novel photo-immobilization methodology was also successfully applied to the anchoring of an anti-cholera toxin antibody which was then detected by a secondary antibody labelled with a peroxidase.

Blood Group, Immunity, and Risk of Infection with Vibrio cholerae in an Area of Endemicity

Individuals with blood group O are more susceptible than other individuals to severe cholera, although the mechanism underlying this association is unknown. To assess the respective roles of both intrinsic host factors and adaptive immune responses that might influence susceptibility to infection with Vibrio cholerae, we prospectively followed a cohort of household contacts of patients with cholera in Bangladesh. In this study, we made the novel observation that persons with blood group O were less likely than those with other blood groups to become infected with V. cholerae O1 (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.85; P = 0.008). Consistent with prior studies, however, household contacts with blood group O were more likely to develop severe illness if infected with V. cholerae O1 (OR, 2.3; 95% CI, 0.98 to 5.59; P = 0.05). While blood group O protected significantly against infection with V. cholerae O1, there was no evidence of protection against V. cholerae O139. A multivariate analysis demonstrated that the association between blood group O and protection from infection with V. cholerae O1 was independent of age, gender, and baseline anti-cholera toxin and vibriocidal antibody titers. Based on this epidemiologic evidence, we propose a hypothesis for understanding the association between blood group O and the risk of infection with V. cholerae O1 and O139 as well as the risk of developing severe symptoms once infected.

Isolation and characterization of a cytotoxin produced by Plesiomonas shigelloides P-1 strain

In order to clarify the enteropathogenicity of Plesiomonas shigelloides, we investigated a cytotoxin produced by the P-1 strain isolated from patients suffering from diarrhea. The cytotoxicity of the culture filtrate of the strain reached a maximum in culture at 37 °C after 12 h shaken in BHI medium. The cytotoxin in the cultures was purified by (NH 4) 2SO 4 precipitation, and Sephacryl S-100, Mono Q HR, and Superdex 200 HR column chromatographies. An approximate 340-fold purification was achieved, with a recovery of about 1.4%, from the culture supernatant. The cytotoxin is heat-stable, and is a complex of three major proteins (LPS-binding proteins with molecular weights of 32, 40, and 48 kDa), with lipopolysaccharide (LPS) giving a total a molecular weight of more than 600 kDa. The ratio of protein to LPS in the cytotoxin was 6–5. The cytotoxic activity was reduced by about 80% by proteinase K treatment or when incubated with anti-cholera toxin antibody (Anti-CT). Western blotting of the cytotoxin with Anti-CT demonstrated the presence of two anti-cholera toxin-reactive protein (ACRP) bands with molecular weights of 40 kDa (a major single protein band) and 48 kDa. The N-terminal amino acid sequence (20 residues) of the 40 kDa protein was 75% identical to Pasteurella multocida cell membrane proteins. The cytotoxin gave a positive reaction in the suckling mouse assay whereas LPS alone hardly exhibited any cytotoxic or enterotoxigenic activity. In conclusion, P. shigelloides produces a cytotoxin that consists of a complex of protein and LPS with the former component exhibiting both cytotoxicity and enteropathogenicity. This cytotoxin has the potential to have an important role in the enteropathogenicity of P. shigelloides.