Cholera

Biochemistry, 2005 Jan 25, 44(3), 873 - 82
Cholera Toxin Entry into Pig Enterocytes Occurs via a Lipid Raft- and Clathrin-Dependent Mechanism; Hansen GH et al.; The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte . The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization . CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation . By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C . By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment . A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed . CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes . Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry . The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits . CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis . In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

Infect Immun, 2004 Dec, 72(12), 6826 - 35
Vesicular transport is not required for the cytoplasmic pool of cholera toxin to interact with the stimulatory alpha subunit of the heterotrimeric g protein; Teter K et al.; Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport . The catalytic A1 polypeptide of CT (CTA1) then crosses the ER membrane, enters the cytosol, ADP-ribosylates the stimulatory alpha subunit of the heterotrimeric G protein (Gsalpha) at the cytoplasmic face of the plasma membrane, and activates adenylate cyclase . The cytosolic pool of CTA1 may reach the plasma membrane and its Gsalpha target by traveling on anterograde-directed transport vesicles . We examined this possibility with the use of a plasmid-based transfection system that directed newly synthesized CTA1 to either the ER lumen or the cytosol of CHO cells . Such a system allowed us to bypass the CT retrograde trafficking itinerary from the cell surface to the ER . Previous work has shown that the ER-localized pool of CTA1 is rapidly exported from the ER to the cytosol . Expression of CTA1 in either the ER or the cytosol led to the activation of Gsalpha, and Gsalpha activation was not inhibited in transfected cells exposed to drugs that inhibit vesicular traffic . Thus, anterograde transport from the ER to the plasma membrane is not required for the cytotoxic action of CTA1.

Int J Med Microbiol, 2004 Oct, 294(4), 217 - 23
Structural biology and structure-based inhibitor design of cholera toxin and heat-labile enterotoxin; Fan E et al.; Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels . Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated . At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Vaccine, 2004 Dec 9, 23(4), 555 - 65
A two-codon mutant of cholera toxin lacking ADP-ribosylating activity functions as an effective adjuvant for eliciting mucosal and systemic cellular immune responses to peptide antigens; Lomada D et al.; Vaccination with peptide antigens is an effective strategy against mucosal viral infections . We tested a two-codon mutant of cholera toxin (CT-2*) lacking ADP-ribosylating activity and toxicity as a mucosal adjuvant for T cell epitope peptides for intranasal immunization of mice . Efficient induction of helper and cytotoxic T lymphocyte responses associated with TH1 cytokine production were observed in the systemic and mucosal compartments including nasal, gut, and vaginal associated lymphoid tissues . Single or multiple dosing with the peptide antigen and CT-2* induced strong memory immunity without tolerance . These results demonstrate CT-2* as a suitable mucosal adjuvant for priming antigen-specific cellular immune responses.

J Org Chem, 2004 Oct 29, 69(22), 7737 - 40
Large cyclic peptides as cores of multivalent ligands: application to inhibitors of receptor binding by cholera toxin; Zhang Z et al.; Large cyclic decapeptides (up to 50-atom ring) were synthesized efficiently on the solid phase with allyl-ester protection of the carboxyl terminus during elongation . Pentavalent ligands, in a "core-linker-finger" modular setup, were assembled by using these cyclic peptide cores to demonstrate large affinity gains for inhibition of surface receptor binding by the cholera toxin B pentamer . The results suggest that the peptide cores retain expanded conformation in solution so that shorter flexible linkers are needed for larger peptide cores to achieve the best inhibitory results.

Cochrane Database Syst Rev . 2004 Oct 18;(4):CD003754.
Reduced osmolarity oral rehydration solution for treating cholera; Murphy C et al.; BACKGROUND: Oral rehydration solution (ORS) is used to treat dehydration caused by diarrheal diseases including cholera . Reduced osmolarity formulations are safe and more effective than standard ORS for treating non-cholera diarrhea . As cholera causes rapid electrolyte loss, it is important to know if these benefits are similar for people with cholera . OBJECTIVES: To compare the safety and efficacy of reduced osmolarity oral rehydration solution (ORS) with standard ORS for treating diarrhea due to cholera . SEARCH STRATEGY: We searched the Cochrane Infectious Disease Group Specialized Register (January 2004), CENTRAL (The Cochrane Library Issue 1, 2004), MEDLINE (1966 to January 2004), EMBASE (1974 to January 2004), and LILACS (1982 to January 2004) . We also contacted organizations and searched reference lists . SELECTION CRITERIA: Randomized controlled trials comparing reduced osmolarity ORS with standard ORS for treating adults and children with acute diarrhea due to cholera . DATA COLLECTION AND ANALYSIS: Two reviewers independently applied eligibility criteria, assessed trial quality, and extracted data . We pooled binary data using relative risks (RR), continuous data using weighted mean difference (WMD) or the standardized mean difference (SMD), and presented the results with 95% confidence intervals (CI) . MAIN RESULTS: For glucose-based reduced osmolarity ORS, seven trials (718 participants) met the inclusion criteria . Biochemical hyponatremia (serum sodium < 130 mmol/L) was more common with reduced osmolarity ORS (RR 1.67, CI 1.09 to 2.57; 465 participants, 4 trials); for severe biochemical hyponatremia (serum sodium < 125 mmol/L) this was not significant (RR 1.58, CI 0.62 to 4.04; 465 participants, 4 trials) . No trials reported symptomatic hyponatremia or death . We found no statistically significant difference in the need for unscheduled intravenous infusion . Analyses separating children and adults showed no obvious trends.Two trials also examined rice-based ORS . In the reduced osmolarity group, duration of diarrhea was shorter (WMD -16.85 hours, CI -21.22 to -12.48; 102 participants, 2 trials) . REVIEWERS' CONCLUSIONS: In people with cholera, reduced osmolarity ORS is associated with biochemical hyponatremia when compared with standard ORS, although there are similar benefits in terms of other outcomes . Although this risk does not appear to be accompanied by serious consequences, the total patient experience in existing trials is small . Under wider practice conditions, especially where patient monitoring is difficult, caution is warranted.

Vaccine, 2004 Oct 22, 22(31-32), 4306 - 15
Intranasal immunization with C . muridarum major outer membrane protein (MOMP) and cholera toxin elicits local production of neutralising IgA in the prostate; Hickey DK et al.; Successful control of sexually transmitted diseases (STDs) through vaccination will require the development of vaccine strategies that target protective immunity to both the female and male reproductive tracts (MRT) . In the male, the immune privileged nature of the male reproductive tract provides a barrier to entry of serum immunoglobulins into the male reproductive ducts, thereby preventing the induction of protective immunity using conventional injectable vaccination techniques . In this study we investigated the potential of intranasal (IN) immunization to elicit anti-chlamydial immunity in BALB/c male mice . Intranasal immunization with Chlamydia muridarum major outer membrane protein (MOMP) admixed with cholera toxin (CT) resulted in high levels of MOMP-specific IgA in prostatic fluids (PF) and MOMP-specific IgA-secreting cells in the prostate . Prostatic fluid IgA inhibited in vitro infection of McCoy cells with C . muridarum . Using RT-PCR we also show that mRNA for the polymeric immunoglobulin receptor (PIgR), which transports IgA across mucosal epithelia, is expressed only in the prostate but not in other regions of the male reproductive ducts upstream of the prostate . These data suggest that using intranasal immunization to target IgA to the prostate may protect males against STDs while at the same time maintaining the state of immune privilege within the MRT.

Vaccine, 2004 Oct 22, 22(31-32), 4163 - 72
Immunological tools for the assessment of both humoral and cellular immune responses in Foxes (Vulpes vulpes) using ovalbumin and cholera toxin B as an antigenic model; Rolland-Turner M et al.; The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed . To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model . Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay . The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization . Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs) . Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs . This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia . Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes . These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.

J Biomol Struct Dyn, 2004 Dec, 22(3), 299 - 313
Disialogangliosides and their interaction with cholera toxin - investigation by molecular modeling, molecular mechanics and molecular dynamics; Jeya Sundara Sharmila D et al.; Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment . The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3 . These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between . The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds . The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out . The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%) . The molecular modeling, molecular mechanics and molecular dynamics of disialoganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialoganglioside GD1A in three different modes . A single mode of binding is permissible for GD1B and GD3 . Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin . This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.

J Immunol, 2004 Oct 15, 173(8), 5103 - 11
In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin; Anjuere F et al.; Although dendritic cells (DCs) regulate immune responses, they exhibit functional heterogeneity depending on their anatomical location . We examined the functional properties of intestinal DCs after oral administration of cholera toxin (CT), the most potent mucosal adjuvant . Two CD11c+ DC subsets were identified both in Peyer's patches and mesenteric lymph nodes (MLN) based on the expression of CD8alpha (CD8+ and CD8- DCs, respectively) . A third subset of CD11c+CD8int was found exclusively in MLN . Feeding mice with CT induced a rapid and transient mobilization of a new CD11c+CD8- DC subset near the intestinal epithelium . This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8int DCs in MLN . MLN DCs from CT-treated mice were more potent activators of naive T cells than DCs from control mice and induced a Th2 response . This increase in immunostimulating properties was accounted for by CD8int and CD8- DCs, whereas CD8+ DCs remained insensitive to CT treatment . Consistently, the CD8int and CD8- subsets expressed higher levels of costimulatory molecules than CD8+ and corresponding control DCs . Adoptive transfer experiments showed that these two DC subsets, unlike CD8+ DCs, were able to present Ags orally coadministered with CT in an immunostimulating manner . The ability of CT to mobilize immature DCs in the intestinal epithelium and to promote their emigration and differentiation in draining lymph nodes may explain the exceptional adjuvant properties of this toxin on mucosal immune responses.

J Water Health, 2003 Mar, 1(1), 45 - 52
A large cholera outbreak in Kano City, Nigeria: the importance of hand washing with soap and the danger of street-vended water; Hutin Y et al.; The aim of this study was to identify the risk factors for cholera during an outbreak in Nigeria . Cases were defined as recent onset of acute diarrhoea with dehydration in a patient hospitalised at the Infectious Diseases Hospital in Kano City . Meningitis patients admitted concurrently at the same hospital were recruited as unmatched controls . Data were collected on age, sex, place of residence, hygienic practices, and on food and water consumption . A total of 5600 cholera cases and 340 cholera deaths were reported between December 1995 and May 1996 (attack rate = 86.3 per 100,000 population) in the state of Kano . Compared to the 77 controls, the 102 cases were more likely to have drunk street-vended water (age-adjusted odds ratio (AAOR) = 3.2; 95% confidence interval (CI): 1.4-7.1) and less likely to have drunk tap water in their homes (AAOR = 0.2; 95% CI: 0.1-0.7) or to have washed hands with soap prior to eating food (AAOR = 0.2; 95% CI: 0.1-0.6) . While no data suggested that the municipal water supply was contaminated, safe water systems and hand hygiene practices might have prevented a high proportion of cases if implemented early during this outbreak.

Chem Biol, 2004 Sep, 11(9), 1205 - 15
Nonspanning bivalent ligands as improved surface receptor binding inhibitors of the cholera toxin B pentamer; Pickens JC et al.; A series of bivalent ligands of varying length were synthesized to inhibit the receptor-binding process of cholera toxin . Competitive surface receptor binding assays showed that significant potency gains relative to the constituent monovalent ligands were achieved independently from the ability of the extended bivalent ligands to span binding sites within the toxin pentamer . Several models that could account for the unexpected improvement in IC(50) values are examined, taking into account crystallographic analysis of each ligand in complex with the toxin pentamer . Evidence is presented that steric blocking at the receptor binding surface may play a role . The results of our study suggest that the use of relatively short, "nonspanning" bivalent ligands, or monovalent ligands of similar topology and bulk may be an effective way of blocking the interaction of multimeric proteins with their cell surface receptors.

Chemistry . 2004 Sep 20;10(18):4395.
Intramolecular carbohydrate-aromatic interactions and intermolecular van der Waals interactions enhance the molecular recognition ability of GM1 glycomimetics for cholera toxin; Bernardi A et al.; The design and synthesis of two GM1 glycomimetics, 6 and 7, and analysis of their conformation in the free state and when complexed to cholera toxin is described . These compounds, which include an (R)-cyclohexyllactic acid and an (R)-phenyllactic acid fragment, respectively, display significant affinity for cholera toxin . A detailed NMR spectroscopy study of the toxin/glycomimetic complexes, assisted by molecular modeling techniques, has allowed their interactions with the toxin to be explained at the atomic level . It is shown that intramolecular van der Waals and CH-pi carbohydrate-aromatic interactions define the conformational properties of 7, which adopts a three-dimensional structure significantly preorganized for proper interaction with the toxin . The exploitation of this kind of sugar-aromatic interaction, which is very well described in the context of carbohydrate/protein complexes, may open new avenues for the rational design of sugar mimics.

Eur J Public Health, 2004 Sep, 14(3), 274 - 9
Spatial and temporal distribution of cholera in Ecuador between 1991 and 1996; Chevallier E et al.; BACKGROUND: The seventh pandemic of cholera affected South America in 1991 after a century of absence . Favoured by local conditions, the epidemic of cholera in Ecuador had a rapid impact . The epidemic of cholera evolved with temporal and geographical variations . METHODS: The temporal and geographical variations of cholera in Ecuador between 1991 and 1996 have been analysed . The Ecuadorian epidemiological surveillance system is a semi-active one based on obligatory weekly declarations . A geographical representation of annual impact rate has been made . Using a smoothing technique by cross-validation, time curves were identified and spatial diffusion was studied by cartography . RESULTS: In 1991 and 1992, cholera in Ecuador evolved in an epidemic mode with two explosive epidemic peaks . Cholera then entered a phase of regression . The disease spread from two main epicentres, one in the South (El Oro, Guayas, Los Rios) and the other in the North (Esmeraldas and Imbabura) . These focal outbreaks spread to neighbouring provinces during the peak outbreaks between 1991 and 1993 . CONCLUSION: This study demonstrated that the epidemic spread from the affected provinces in the South and the North of the country.

QJM, 2004 Oct, 97(10), 681 - 96
Acidosis in a patient with cholera: a need to redefine concepts; Zalunardo N et al.; A patient presented with cholera and a severe degree of ECF volume contraction . Despite large losses of bicarbonate (HCO3-)-containing diarrhoeal fluid, laboratory acid-base values were remarkably close to normal . A detailed analysis emphasizing principles of physiology and a quantitative approach provided new insights and eventually better definitions of metabolic and respiratory acidosis . A shift in focus from HCO3- concentration to HCO3- content in the extracellular fluid (ECF) compartment revealed the presence of metabolic acidosis . Central to this analysis was an emphasis on the haematocrit to enable a more accurate estimate of the degree of ECF volume contraction . The latter also revealed 'contraction' metabolic alkalosis, which masked the underlying metabolic acidosis . The presence of a respiratory acidosis of the tissue type was evident from the raised venous PCO2, which was not surprising once the magnitude of the ECF contraction had been appreciated . 'Bad buffering', as defined by Professor McCance, was the immediate danger and prompted swift action to restore an effective circulation . The haematocrit and the venous PCO2 also contribute valuable information to monitor the response to therapy . Nevertheless, there were still dangers to be discovered when an in-depth analysis suggested that the administration of isotonic saline would introduce an unanticipated danger for the patient.

Structure (Camb), 2004 Sep, 12(9), 1655 - 67
Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 A crystal structure reveals the details; Holmner A et al.; A hybrid between the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin has been described, which exhibits a novel binding specificity to blood group A and B type 2 determinants . In the present investigation, we have determined the crystal structure of this protein hybrid, termed LCTBK, in complex with the blood group A pentasaccharide GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAcbeta, confirming not only the novel binding specificity but also a distinct new oligosaccharide binding site . Binding studies revealed that the new specificity can be ascribed to a single mutation (S4N) introduced into the sequence of Escherichia coli heat-labile enterotoxin . At a resolution of 1.9 A, the new binding site is resolved in excellent detail . Main features include a complex network of water molecules, which is well preserved by the parent toxins, and an unexpectedly modest contribution to binding by the critical residue Asn4, which interacts with the ligand only via a single water molecule.

J Immunol, 2004 Sep 1, 173(5), 3310 - 9
The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues; Eriksson AM et al.; Although highly effective, the use of GM1-receptor binding holotoxins as nasal mucosal adjuvants has recently been cautioned due to the risk for their accumulation in the brain and other nervous tissues . Therefore we have explored the efficacy of the CTA1-DD adjuvant for its ability to enhance nasal immune responses in mice . We found that despite the lack of a mucosal binding element, the B cell-targeted CTA1-DD molecule was an equally strong adjuvant as cholera toxin (CT) . The potency of CTA1-DD was not a result of endotoxin contamination because more than a 50-fold higher dose of LPS was needed to achieve a similar enhancement . Moreover, the adjuvant effect was TLR4-independent and absent in mutant CTA1-E112K-DD, lacking enzymatic activity . The CTA1-DD adjuvant augmented germinal center formations and T cell priming in the draining lymph nodes, and contrary to CT, promoted a balanced Th1/Th2 response with little effect on IgE Ab production . CTA1-DD did not induce inflammatory changes in the nasal mucosa, and most importantly did not bind to or accumulate in the nervous tissues of the olfactory bulb, whereas CT bound avidly to the nervous tissues . We believe that the nontoxic CTA1-DD adjuvant is an attractive solution to the current dilemma between efficacy and toxicity encountered in CT-holotoxin adjuvant or Escherichia coli heat-labile toxin-holotoxin adjuvant strategies and provides a safe and promising candidate to be included in future vaccines for intranasal administration.

Hybrid Hybridomics, 2004 Aug, 23(4), 258 - 61
Production and purification of monoclonal and polyclonal antibodies against cholera toxin; Chou SF; The aim of this study was to produce monoclonal and polyclonal antibodies against cholera toxin (CT) . Hyperimmune ICR mice produced polyclonal antibodies (PAbs) after injection with 0.5 mL of pristane and were injected with NS-1 myeloma cells 2 weeks later . Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs) . After these mice were immunized four times and given a final boost, their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500 . The fused cells were then selected in the hypoxanthine, aminopterin, and thymidine (HAT)-RPMIX medium . Anti-CT antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium . Eleven murine hybridoma producing anti-CT MAbs were obtained and designated CT-A2, CT-B4, CT-B11, CT-C7, CT-D7, CT-E8, CT-F4, CT-F2, CT-F8, CT-E3, CT-E6 . Isotypes of MAbs were identified as IgM heavy chain and all were lambda light chain . Hitrap rProtein A and Hitrap IgM purification columns were used for the purification of PAbs and MAbs, respectively.

Public Health, 2004 Sep, 118(6), 387 - 94
John Snow, William Farr and the 1849 outbreak of cholera that affected London: a reworking of the data highlights the importance of the water supply; Bingham P et al.; OBJECTIVES: This paper examines why Snow's contention that cholera was principally spread by water was not accepted in the 1850s by the medical elite . The consequence of rejection was that hundreds in the UK continued to die . METHODS: Logistic regression was used to re-analyse data, first published in 1852 by William Farr, consisting of the 1849 mortality rate from cholera and eight potential explanatory variables for the 38 registration districts of London . RESULTS: Logistic regression does not support Farr's original conclusion that a district's elevation above high water was the most important explanatory variable . Elevation above high water, water supply and poor rate each have an independent significant effect on district cholera mortality rate, but in terms of size of effect, it can be argued that water supply most strongly 'invited' further consideration . CONCLUSIONS: The science of epidemiology, that Farr helped to found, has continued to advance . Had logistic regression been available to Farr, its application to his 1852 data set would have changed his conclusion.

Am J Physiol Cell Physiol, 2004 Nov, 287(5), C1453 - 62 Epub 2004 Aug 04.
Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton; Badizadegan K et al.; Intestinal epithelial lipid rafts contain ganglioside GM1 that is the receptor for cholera toxin (CT) . The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) . In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase . Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion . Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of adenylyl cyclase . CT bound to GM1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins . In this actin-rich fraction of lipid rafts, CT and actin colocalize on the same membrane microdomains, suggesting a possible functional association . Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi and reduces the levels of cAMP generated in the cytosol . Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts while CT remains stably raft associated . On the basis of these observations, we propose that the CT-GM1 complex is associated with the actin cytoskeleton via the lipid rafts and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of adenylyl cyclase.

Am Nat, 2004 Jun, 163(6), 901 - 13 Epub 2004 May 18.
Disentangling extrinsic from intrinsic factors in disease dynamics: a nonlinear time series approach with an application to cholera; Koelle K; Alternative explanations for disease and other population cycles typically include extrinsic environmental drivers, such as climate variability, and intrinsic nonlinear dynamics resulting from feedbacks within the system, such as species interactions and density dependence . Because these different factors can interact in nonlinear systems and can give rise to oscillations whose frequencies differ from those of extrinsic drivers, it is difficult to identify their respective contributions from temporal population patterns . In the case of disease, immunity is an important intrinsic factor . However, for many diseases, such as cholera, for which immunity is temporary, the duration and decay pattern of immunity is not well known . We present a nonlinear time series model with two related objectives: the reconstruction of immunity patterns from data on cases and population sizes and the identification of the respective roles of extrinsic and intrinsic factors in the dynamics . Extrinsic factors here include both seasonality and long-term changes or interannual variability in forcing . Results with simulated data show that this semiparametric method successfully recovers the decay of immunity and identifies the origin of interannual variability . An application to historical cholera data indicates that temporary immunity can be long-lasting and decays in approximately 9 yr . Extrinsic forcing of transmissibility is identified to have a strong seasonal component along with a long-term decrease . Furthermore, noise appears to sustain the multiple frequencies in the long-term dynamics . Similar semiparametric models should apply to population data other than for disease.

J Biomater Sci Polym Ed, 2004, 15(5), 661 - 9
IgG responses to intranasal immunization with cholera-toxin-immobilized polymeric nanospheres in mice; Kaneko T et al.; IgG responses to antigen-nanosphere hybrids were studied in mice . Cholera toxin (CT) was covalently immobilized onto the surface of polymeric nanospheres (NS) with a nanophase-separated structure consisting of a polystyrene core and a poly(methacrylic acid) graft corona . Reaction conditions favoring the dehydroxide condensation reaction of the amino group of the CT with the carboxyl group of NS effectively immobilized CT onto their surface . When CT-immobilized nanospheres (CT-NS) were suspended in aqueous solution and administrated to mice either intranasally or intramuscularly, serum IgG titers elevated with increasing time and reached a maximum level at 8 weeks after immunization . On the other hand, intranasal administration of CT alone induced an even higher serum IgG titer than that of CT-NS at 4 weeks . However, the titer gradually decreased thereafter . Thus, polymeric NS may be an effective substrate to covalently immobilize antigen on their surface, steadily inducing a high level of IgG production in response to the intranasal administration.

Org Biomol Chem, 2004 Jul 21, 2(14), 2113 - 24 Epub 2004 Jun 30.
Synthesis and cholera toxin binding properties of multivalent GM1 mimics; Arosio D et al.; Dendrimers based on the 3,5-di-(2-aminoethoxy)-benzoic acid branching unit were used to attach multiple copies of a GM1 mimic for inhibition of cholera toxin binding . Systems up to octavalent were synthesized along with relevant reference compounds that contained in one case the ligand in a monovalent format and in another case the scaffold but not the ligand . Using a surface plasmon resonance inhibition assay the prepared inhibitors showed good inhibition . While the monovalent GM1 mimic showed the expected inhibition in the 200 microM range the multivalent scaffolds led to increased binding . The tetravalent compound was shown to be 440-fold more potent than its monovalent counterpart . The octavalent analog, however, was the most potent compound as determined using an ELISA assay.

Rev Saude Publica, 2004 Jun, 38(3), 351 - 7 Epub 2004 Jul 08.
{Use of an artificial neural network for detecting excess deaths due to cholera in Ceará, Brazil}; Penna ML; OBJECTIVE: To evaluate recurrent neural networks as a predictive technique for time-series in the health field . METHODS: The study was carried out during a cholera epidemic which took place in 1993 and 1994 in the state of Ceara, northeastern Brazil, and was based on excess deaths having 'poorly defined intestinal infections' as the underlying cause (ICD-9) . The monthly number of deaths with due to this cause between 1979 and 1995 in the state of Ceara was obtained from the Ministry of Health's Mortality Information System (SIM) . A network comprising two neurons in the input layer, twelve in the hidden layer, one in the output layer, and one in the memory layer was trained by backpropagation using the fist 150 observations, with 0.01 learning rate and 0.9 momentum . Training was ended after 22,000 epochs . We compare the results with those of a negative binomial regression . RESULTS: ANN forecasting was adequate . Excessive mortality (number of deaths above the upper limit of the confidence interval) was detected in December 1993 and October/November 1994 . However, negative binomial regression detected excess mortality from March 1992 onwards . CONCLUSIONS: The artificial neural network showed good predictive ability, especially in the initial period, and was able to detect alterations concomitant and a subsequent to the cholera epidemic . However, it was less precise that the binomial regression model, which was more sensitive to abnormal data concomitant with cholera circulation.

Clin Immunol, 2004 Jul, 112(1), 35 - 44
Vaccination with dendritic cells pulsed in vitro with tumor antigen conjugated to cholera toxin efficiently induces specific tumoricidal CD8+ cytotoxic lymphocytes dependent on cyclic AMP activation of dendritic cells; Sun JB et al.; We investigated the development of CD8+ tumor-specific cytotoxic lymphocytes (CTL) and protection against tumor growth after vaccination with bone marrow-derived dendritic cells (DC) pulsed with a model protein ovalbumin conjugated to cholera toxin (OVA-CT) in B6 mice using E.G7 tumor cells expressing OVA(257-264) peptide (SIINFEKL) as target cells in vitro and in vivo . Vaccination with OVA-CT-pulsed DC concurrently induced strong CTL in vitro activity and anti-E.G7 tumor protection in vivo in WT, NK-depleted and CD4-deficient mice as well as in IL-12-/- and IFN-gamma-/- mice but not in CD8-deficient mice . Importantly, activation of CTL by OVA-CT-pulsed DC was dependent on CT-induced activation of adenylate cyclase and increased cAMP production by DC associated with increased expression of MHC class I and co-stimulatory molecules (CD80, CD86 and CD40) . These results show that vaccination with DC pulsed with antigens (Ag) conjugated to CT induces a strong CTL response and suggest that conjugation of tumor Ag to CT for DC vaccination represents a promising approach for tumor vaccination and immunotherapy.

Int J Pharm, 2004 Jul 8, 278(2), 379 - 90
Cholera toxin B subunit conjugated bile salt stabilized vesicles (bilosomes) for oral immunization; Singh P et al.; Bile salt stabilized vesicles, bilosomes appear to be a promising and potential carrier system for oral delivery of peptides and proteins . Bilosomes containing bovine serum albumin (BSA), a model antigen, were prepared and conjugated with cholera toxin B subunit (CTB) in order to enhance their affinity towards M cells of Peyer's patches . Stability studies were undertaken to ascertain the effect of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 7.5) and different concentrations of bile salts . Intactness and biological activity of CTB were checked by hemagglutination test . A single oral dose of CTB-conjugated bilosomes produced almost equivalent response compared to parenteral administration of antigen with Freund's complete adjuvant (FCA) . However, in contrast to FCA, oral administration of bilosomes is convenient and devoid of any adverse effects that are observed with parenteral administration of FCA . Serum IgG titers after single administration were significantly better (P < 0.05) than oral administration of antigen with other systems for 3 consecutive days, suggesting an effective stimulation of systemic immune response . Mucosal IgA titers obtained advocated a possible application of CTB-conjugated bilosomes as oral vaccine delivery system.

Clin Exp Immunol, 2004 Jul, 137(1), 201 - 8
Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease; Stanford M et al.; Behcet's disease (BD) specific peptide (p336-351) was identified within the human 60 kD heat shock protein (HSP60) . Oral p336-351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB) . This strategy was adopted in a phase I/II clinical trial by oral administration of p336-351-CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD . The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations . Oral administration of p336-351-CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen . After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10-18 months after cessation of all treatment . Control of uveitis and extra-ocular manifestations of BD was associated with a lack of peptide-specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN-gamma and TNF-alpha production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed . The efficacy of oral peptide-CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

Vaccine, 2004 Jun 23, 22(19), 2444 - 51
Can oral cholera vaccination play a role in controlling a cholera outbreak?
Calain P, Chaine JP, Johnson E, Hawley ML, O'Leary MJ, Oshitani H, Chaignat CL.
Control measures to limit the spread of a cholera outbreak in Pohnpei Island (Micronesia), included mass vaccination with the single-dose live-attenuated oral cholera vaccine CVD 103-HgR as a potential adjunct measure . The outbreak provided a unique opportunity to evaluate the practicality of use and effectiveness of this vaccine . Under field conditions encountered in Pohnpei, crude vaccine efficacy was estimated at 79.2% (95% CI: 71.9-84.6%) in the target population . Retrospective analysis suggests that mass vaccination with oral cholera vaccines can be a useful adjunct tool for controlling outbreaks, particularly if implemented early in association with other standard control measures.

Biophys J, 2004 Jun, 86(6), 3700 - 8
Cholera toxin assault on lipid monolayers containing ganglioside GM1; Miller CE et al.; Many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components . Using neutron reflectivity, we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B-subunit (CTB(5)) . Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported . The density of the lipid layer was found to decrease slightly upon protein binding . However, the A-subunit of the whole toxin is clearly located below the B-pentameric ring, away from the monolayer, and does not penetrate into the lipid layer before enzymatic cleavage . Using Monte Carlo simulations, the observed monolayer expansion was found to be consistent with geometrical constraints imposed on DPPE by multivalent binding of GM(1) by the toxin . Our findings suggest that the mechanism of membrane translocation by the protein may be aided by alterations in lipid packing.

EMBO Rep, 2004 Jun, 5(6), 596 - 601 Epub 2004 May 21.
Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells; Feng Y et al.; Cholera toxin (CT) follows a glycolipid-dependent entry pathway from the plasma membrane through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) where it is retro-translocated into the cytosol to induce toxicity . Whether access to the Golgi apparatus is necessary for transport to the ER is not known . Exo2 is a small chemical that rapidly blocks anterograde traffic from the ER to the Golgi and selectively disrupts the Golgi apparatus but not the TGN . Here we use Exo2 to determine the role of the Golgi apparatus in CT trafficking . We find that under the condition of complete Golgi ablation by Exo2, CT reaches the TGN and moves efficiently into the ER without loss in toxicity . We propose that even in the absence of Exo2 the glycolipid pathway that carries the toxin from plasma membrane into the ER bypasses the Golgi apparatus entirely.

Int J Med Microbiol, 2004 Apr, 293(7-8), 491 - 4
Retrograde transport of cholera toxin into the ER of host cells; Lencer WI; Cholera toxin moves from the plasma membrane to the ER of host cells to cause disease . Here we discuss recent studies on the mechanism of transport from plasma membrane to the ER and on the reactions that unfold and retrotranslocate a portion of the toxin to the cytosol where toxicity is induced.

Mol Biol Cell, 2004 Aug, 15(8), 3631 - 41 Epub 2004 May 14.
Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways; Massol RH et al.; Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER) . In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity . Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways . Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity . Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected . These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.

Eur J Immunol, 2004 May, 34(5), 1272 - 81
Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor; Eriksson K et al.; We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1-dominated B cell and CD4(+) T cell responses . In this study we show that vaccination with DC pre-pulsed ex vivo with CT-conjugated OVA (OVA-CT) gives rise to OVA-specific CD8(+) T cells that produce IFN-gamma and are cytotoxic for OVA-expressing E.G7 tumor cells both in vitro and in vivo . The induction of specific CD8(+) CTL by OVA-CT-treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC . Vaccination of mice with OVA-CT-pulsed DC resulted in rejection of already established MHC class I-positive, MHC class II-negative, OVA-expressing E.G7 tumors in an antigen-specific, CD8(+) T cell-dependent fashion and was associated with high numbers of tumor-infiltrating CD8(+) T cells . Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor-binding B subunit and induced strong activation-maturation signals through the biologically active A subunit . These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.

Immunol Lett, 2004 Apr 15, 92(3), 245 - 52
Nitric oxide modulation of the immune response against cholera toxin-adjuvated ovalbumin administered by the intranasal route; Gamba G et al.; Here, we studied the effect of aminoguanidine (AG) treatment, a nitric oxide synthase (NOS)-2 inhibitor, during the immune response against intranasal administration of ovalbumin (OVA) mixed with cholera toxin (CT) in BALB/c mice . NOS-2 mRNA was detected by reverse transcription-PCR (RT-PCR) in samples of lungs and turbinates early post-inoculation of the antigen . Animals intranasally treated with AG, showed an increase in the levels of seric specific IgG and IgM . A higher IgG1/IgG2a ratio against OVA was also observed in sera of same animals . Moreover, high levels of specific IgA were detected in samples of pulmonar washings obtained from treated animals . On the contrary, treated animals showed a lower DTH response while splenocytes obtained from the same animals showed a reduced proliferative capability against OVA compared to controls . Finally, RT-PCR analysis showed increased expression of TGF-beta in turbinates, lungs and cells from pulmonar washings obtained from AG treated mice . Taken together, these data suggest a role of nitric oxide (NO) in modulating the primary immune response against intranasal antigens.

Vaccine, 2004 Mar 29, 22(11-12), 1553 - 63
Maintenance of long-term immunological memory by low avidity IgM-secreting cells in bone marrow after mucosal immunizations with cholera toxin adjuvant; Soenawan E et al.; To understand the mechanisms involved in maintaining long-term immunological memory following mucosal immunizations, we determined the quality of serum hapten-specific immunoglobulins (Ig) and localized Ig-secreting cells (SC) of various isotypes in acute, persistent/resting memory and effector memory phases following oral versus intra-muscular (IM) immunizations . In the acute phase, both oral and IM immunizations induced high avidity Ig . However, in the persistent/resting memory phase, oral immunizations induced low avidity Ig while IM immunizations induced high avidity Ig . Following oral immunizations, in the persistent/resting memory phase, hapten-specific IgM titers in serum and IgM-SC in bone marrow (BM) dominated the immune response, suggesting an important role for IgM in the maintenance of memory.

Biochemistry, 2004 Apr 6, 43(13), 3772 - 82
Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism; O'Neal CJ et al.; Cholera toxin (CT) is a heterohexameric bacterial protein toxin belonging to a larger family of A/B ADP-ribosylating toxins . Each of these toxins undergoes limited proteolysis and/or disulfide bond reduction to form the enzymatically active toxic fragment . Nicking and reduction render both CT and the closely related heat-labile enterotoxin from Escherichia coli (LT) unstable in solution, thus far preventing a full structural understanding of the conformational changes resulting from toxin activation . We present the first structural glimpse of an active CT in structures from three crystal forms of a single-site A-subunit CT variant, Y30S, which requires no activational modifications for full activity . We also redetermined the structure of the wild-type, proenzyme CT from two crystal forms, both of which exhibit (i) better geometry and (ii) a different A2 "tail" conformation than the previously determined structure {Zhang et al . (1995) J . Mol . Biol . 251, 563-573} . Differences between wild-type CT and active CTY30S are observed in A-subunit loop regions that had been previously implicated in activation by analysis of the structure of an LT A-subunit R7K variant {van den Akker et al . (1995) Biochemistry 34, 10996-11004} . The 25-36 activation loop is disordered in CTY30S, while the 47-56 active site loop displays varying degrees of order in the three CTY30S structures, suggesting that disorder in the activation loop predisposes the active site loop to a greater degree of flexibility than that found in unactivated wild-type CT . On the basis of these six new views of the CT holotoxin, we propose a model for how the activational modifications experienced by wild-type CT are communicated to the active site.

J Health Popul Nutr, 2003 Dec, 21(4), 304 - 8
Coverage and costs of mass immunization of an oral cholera vaccine in Vietnam; Vu DT et al.; The objective of this study was to describe a mass-immunization campaign of a locally-produced oral, killed whole-cell cholera vaccine in Hue city, Vietnam . Mass immunization with a 2-dose regimen of the vaccine was conducted in 13 communes in early 1998 . The total, age- and sex-specific vaccine coverage was calculated using data from the vaccination records and the government census . The number of vaccine doses procured, administered, wasted, and left over, and the human and other resources required to prepare and conduct the vaccination campaign were systematically recorded . Government expenditure for planning, procurement, and delivery of the vaccine were documented . In total, 118,555 (79%) of the 49,557 targeted population were fully vaccinated during the mass-vaccination campaign . The total expenditure for the project was US dollar 105,447, resulting in a cost per fully-vaccinated person of US dollar 0.89 . Mass immunization with this locally-produced oral, killed cholera vaccine was found to be feasible and affordable with attainment of high vaccination coverage.

Infez Med, 1997 Sep, 5(3), 189 - 203
{Cholera in Bologna in XIX century . A brief report on the scientific knowledge of the period}; Sabbatani S et al.; not available

Infez Med, 1995 Mar, 3(1), 48 - 54
{Therapy of cholera in 19th century}; Laquidara L; not available

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jan-Feb, (1), 93 - 6
{Cholera prevalence worldwide and in Ukraine}; Mukharskaiia LM et al.; In the course of the 7th pandemic cholera morbidity has been registered in 163 countries of the world . 5 periods in the development of the pandemic hav been established . The pandemic process has a pronounced tendency to growth . The spread and dynamics of cholera morbidity have their specific features on each continent . In Asia the epidemic process is manifested as permanent morbidity . Africa determines the total morbidity level in the 7th pandemic . In America both the import of cholera infection and large local outbreaks due to the formation of the secondary foci are registered . In Europe the infection is mainly brought from different territories, and in a number of cases an epidemic spread of this infection occurs . The paths of the penetration of cholera to Europe and Ukraine are, probably, identical . In Ukraine 3 pandemic periods have been established, corresponding to the periods of pandemic spread.

FEBS Lett, 2004 Mar 12, 561(1-3), 122 - 6
Cholera toxin induces expression of ion channels and carriers in rat small intestinal mucosa; Flach CF et al.; Cholera toxin causes cyclic adenosine monophosphate (cAMP)-induced electrolyte and water secretion in the small intestine . The toxin-induced change in gene expression in rat small intestine was evaluated with microarray technique and the results were confirmed by semiquantitative polymerase chain reaction (PCR) . The transporter CNT2 for nucleosides was upregulated between 6 and 18 h after challenge, whereas the level of GLUT1 transporter for glucose became elevated at 6 h . Both changes probably facilitate uptake of these nutrients in the gut . At 18 h, the major chloride channel in the villus, ClC2, was upregulated . Aquaporin 8 was downregulated at 6 h and two mucin-producing genes were upregulated 18 h after toxin challenge . The expression was back to normal after 72 h, which is the turnover time for intestinal epithelial cells.

Biochem Biophys Res Commun, 2004 Feb 27, 315(1), 235 - 9
High-level expression of codon optimized foot-and-mouth disease virus complex epitopes and cholera toxin B subunit chimera in Hansenula polymorpha; Song H et al.; A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the N-terminal end of a 6x His-tagged cholera toxin B subunit (CTB) gene with the similar synonymous codons preferred by the methylotropic yeast Hansenula polymorpha . The fusion gene was synthesized based on a polymerase chain reaction (PCR) and subsequently overexpressed in H . polymorpha . The chimeric protein was successfully secreted into the culture medium (up to 100mg/L) and retained the antigenicity associated with CTB and FMDV antibodies by Western blot analysis . The chimera after purification through Co(2+)-charged resin column bound specifically to GM1 ganglioside receptor and thus retained the biological activity of CTB . This study has important implications in the construction of CTB chimera for mucosal vaccines against FMDV.

Pharmacol Biochem Behav, 2004 Mar, 77(3), 509 - 15
Cholera toxin B decreases bicuculline seizures in prenatally morphine- and saline-exposed male rats; Schindler CJ et al.; Prenatal morphine exposure on gestation days 11-18 alters bicuculline-induced seizures in rats during development and in adulthood . Adult, morphine-exposed male progeny exhibit an increased latency to bicuculline seizures, which can be reversed by administration of the opioid receptor antagonist naloxone . In chronically morphine-treated adult mice, cholera toxin B (CTX-B) can reverse the effects of chronic morphine administration . Therefore, the present study investigated whether prenatally morphine-exposed rats show a similar response to CTX-B as chronically morphine-treated adult rodents . Prenatally morphine-, saline- and unexposed male progeny were tested for seizure susceptibility with a 7.5-mg/kg intraperitoneal injection of bicuculline in adulthood . CTX-B or saline was injected subcutaneously at 24, 12, and 0.5 h before bicuculline injection . CTX-B decreased the occurrence of bicuculline-induced seizures in both prenatally saline- and morphine-exposed but not unexposed rats . Furthermore, three, but not one, saline injections administered at 12-h intervals prior to bicuculline administration reversed the increase in seizure latency in prenatally morphine-exposed adult males, suggesting an altered responsiveness of the stress system . The present study demonstrates that CTX-B can decrease the occurrence of bicuculline seizures in prenatally stressed rats and that increased seizure latencies in prenatally morphine-exposed male rats may be related to stress responses.

Med Sci (Paris), 2004 Feb, 20(2), 236 - 40
{The cholera epidemics and the development of public health in Meiji Japan . 2 . Strength and weakness of public health politics}; Chemouilli P; We present here the beginnings of public health politics in Meiji Japan (1868-1912) . Due to a two century isolation of Japan, public health concepts developed in the West from the end of the 18th century were foreign in premodern Japan . Due to its isolation, Japan was also relatively preserved from some acute infectious diseases such as cholera . In this paper, we investigate the role of cholera epidemics in the emergence of public health concepts in the peculiar context of Meiji Japan . We show that chronic diseases such as tuberculosis and leprosy were neglected for a long time and that the Meiji government set priority on acute infectious diseases that were considered as long as they disturbed public order . Nevertheless, some physicians and government officials considered issues of welfare and poverty . We also review some emerging concepts of social medicine . We try to show that in Japan as well as in western nations public health politics were not exempt of contradictions and paradoxes and a permanent tension existed between coercitive policies and conceptions of welfare and rights to health.

J Cell Sci, 2004 Mar 15, 117(Pt 8), 1421 - 30 Epub 2004 Mar 02.
Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus; Pang H et al.; Cholera toxin is associated with caveolae and raft domains in various cell types and previous studies have shown that cholera toxin can be internalized by caveolae/raft-dependent endocytosis as well as by other pathways . We undertook the study of cholera toxin endocytosis in CaCo-2 and HeLa cells . CaCo-2 cells do not express detectable levels of caveolin and, relative to HeLa cells, also present significantly reduced expression of ganglioside GM1, the cholera toxin receptor, that remains Triton X-100 insoluble . Amongst the HeLa cell population, caveolin expression is constant, however, GM1 expression is highly variable . Cholera toxin is internalized to the Golgi apparatus via a caveolae/raft-dependent pathway sensitive to methyl-beta-cyclodextrin and genistein in high-GM1-expressing HeLa cells but not in low-GM1 HeLa cells or in CaCo-2 cells . Limited cholera toxin endocytosis to endosomes sensitive to neither methyl-beta-cyclodextrin nor genistein is also observed in all cells and corresponds to a non-caveolae/raft endocytic pathway . Increasing cell-associated GM1 by adding GM1 to the cell media of both HeLa and CaCo-2 cells selectively enhances the methyl-beta-cyclodextrin-, genistein-sensitive delivery of cholera toxin to the Golgi apparatus but not to endosomes . GM1 expression levels are therefore a selective determinant of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus and variable expression of GM1 between cells can impact on the endocytosis and choice of pathway followed by cholera toxin.

Bioorg Med Chem, 2004 Mar 1, 12(5), 907 - 20
3,5-Substituted phenyl galactosides as leads in designing effective cholera toxin antagonists; synthesis and crystallographic studies; Mitchell DD et al.; With the aim of developing high-affinity mono and multivalent antagonists of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) we are using the galactose portion of the natural receptor ganglioside GM1 as an anchoring fragment in structure-based inhibitor design efforts . In order to establish a better structure-activity relationship for guiding these studies, we designed and prepared a small focused library of twenty 3,5-substituted phenylgalactosides based on two previous leads . The compounds were tested for their ability to block CTB(5) binding to immobilized ganglioside receptor and compared to the two previous leads . The crystal structures of the most promising compounds bound to either CTB(5) or LTB(5) were then determined in order to understand the basis for affinity differences . The most potent new compound yielded a six-fold improvement over our benchmark lead m-nitrophenyl-alpha-d-galactopyranoside (MNPG), and a two-fold improvement in IC(50) over a newer MNPG derivative . These results support the notion that the m-nitrophenyl moiety of MNPG and its derivatives is an important element to retain in future optimization efforts . Additionally, a consensus binding-pocket for the alkylmorpholine or piperazine moiety present in all of the designed antagonists was established as an important area of the GM1 binding site to target in future work.

Vis Neurosci, 2003 Sep-Oct, 20(5), 481 - 93
Retinal projections in the cat: a cholera toxin B subunit study; Matteau I et al.; The B fragment of cholera toxin (CTb) is a highly sensitive anterograde tracer for the labelling of retinal axons . It can reveal dense retinofugal projections to well-known retinorecipient nuclei along with sparse but distinct input to target areas that are not commonly recognized . Following a unilateral injection of CTb into the vitreous chamber of seven adult cats, we localized the toxin immunohistochemically in order to identify direct retinal projections in these animals . Consistent with previous findings, the strongest projections were observed in the superficial layers of the superior colliculus, the dorsal and ventral lateral geniculate nuclei, the pretectal nuclei, the accessory optic nuclei, and the suprachiasmatic nucleus of the hypothalamus . However, we also found labelled terminals in several other brain areas, including the zona incerta, the medial geniculate nucleus, the lateral posterior-pulvinar complex, the lateral habenular nucleus, and the anterior and lateral hypothalamic regions . The morphological characteristics of the retinal axon terminals in most of the identified novel target sites are described.

Curr Top Med Chem, 2004, 4(5), 509 - 19
Modulation of the immune response by the cholera-like enterotoxins; Plant A et al.; Cholera toxins and heat labile enterotoxin from E . coli differ from most soluble proteins in eliciting systemic immunity both against themselves and unrelated admixed antigens, rather than tolerance following administration to a mucosal surface . Several reports have also demonstrated preferential induction of Th2-type responses when these molecules are used as adjuvants . Conversely, these proteins and their non-toxic derivatives, including the B sub-units are also able prevent and alleviate autoimmune diseases in naive and systemically immune hosts demonstrating wide-ranging effects on the immune system . The recent observation that amelioration of autoimmune disease is associated with the generation of regulatory T cells which inhibit pathogenic Th1 responses may also help to consolidate these two apparently contradictory outcomes of exposure to the cholera-like enterotoxins . Furthermore, the observation that EtxB is able to alleviate autoimmune disease in the absence of conjugation to autoantigen highlights its potential for use in the clinical setting where the target antigen is often unknown . Direct effects on T cells, B cells and APC have been demonstrated in vitro which have provided insights into how these molecules may elicit these diverse effects . Further investigation is required for elucidation of the mechanisms of action of adjuvanticity and tolerance induction by these molecules to realise their potential for use in vaccines and therapies for autoimmune disease in humans.

Med Sci (Paris), 2004 Jan, 20(1), 109 - 14
{The cholera epidemics and the development of public health in Meiji Japan . 1 . Modernity, cholera, and health thought}; Chemouilli P; We present here the beginnings of public health politics in Meiji Japan (1868-1912) . Due to a two century isolation of Japan, public health concepts developed in the West from the end of the 18th century were foreign in premodern Japan . Due to its isolation, Japan was also relatively preserved from some acute infectious diseases such as cholera . In this paper, we investigate the role of cholera epidemics in the emergence of public health concepts in the peculiar context of Meiji Japan . We show that chronic diseases such as tuberculosis and leprosy were neglected for a long time and that the Meiji government set priority on acute infectious diseases that were considered as long as they disturbed public order . Nevertheless, some physicians and government officials considered issues of welfare and poverty . We also review some emerging concepts of social medicine . We try to show, that in Japan as well as in Western nations, public health politics were not exempt of contradictions and paradoxes and a permanent tension existed between coercitive policies and conceptions of welfare and rights to health.

Int J Health Serv, 2003, 33(4), 819 - 30
The politics of underdevelopment: metered to death-how a water experiment caused riots and a cholera epidemic; Pauw J; Water privatization programs in South Africa, part of a government policy aimed at making people pay for the full cost of running water ("total cost recovery"), was developed by private water companies and the World Bank to finance improved water supplies and build the country's economy . Instead the programs are causing more misery than development . Millions of poor people have had their water supply cut off because of inability to pay, forcing them to get their water from polluted rivers and lakes and leading to South Africa's worst cholera outbreak--which the government paid millions of dollars to control . Residents in some townships are rebelling, and many of the private multinational water companies are reassessing their involvement in South Africa.

J Clin Invest, 2004 Feb, 113(3), 334 - 9
The human, societal, and scientific legacy of cholera; Greenough WB 3rd; The recent history of research on cholera illustrates the importance of establishing research and care facilities equipped with advanced technologies at locations where specific health problems exist . It is in such settings, where scientific research is often considered difficult due to poverty and the lack of essential infrastructure, that investigators from many countries are able to make important advances . On this, the 25th anniversary of the founding of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), this article seeks to recount the Centre's demonstration of how high-quality research on important global health issues, including cholera, can be accomplished in conditions that may be considered by many as unsuitable for scientific research.

J Am Chem Soc, 2004 Feb 4, 126(4), 1047 - 54
Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry; Turnbull WB et al.; The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known . Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity . Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands . Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin . This analysis also provides an estimate of 25.8 kJ mol(-1) for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2 . The high affinity and selectivity of the GM1-cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.

Infect Immun, 2004 Feb, 72(2), 1019 - 28
Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection; Berry LJ et al.; Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium . Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells . These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion . The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion . During the extracellular EB stage of the C . trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture . The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells . Thus, an ideal vaccine to protect against C . trachomatis genital tract infection should induce both antibody (immunoglobulin A {IgA} and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes . In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes . This immunization protocol resulted in enhanced clearance of C . muridarum (C . trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

Pac Health Dialog, 2002 Sep, 9(2), 190 - 2
Cholera control on Guam, 2000; Haddock RL et al.; During April, 2000, the island of Pohnpei began experiencing an outbreak of cholera and during June and July of the same year four cases of cholera representing 3 separate introduction events were identified on Guam . Two of these events were associated with eating reef fish imported from Pohnpei . Following the imposition of a narrowly-focused ban on the importation of inshore seafood and processed food products from Pohnpei, no additional local or imported cases of cholera were detected on Guam.

J Leukoc Biol, 2004 May, 75(5), 756 - 63 Epub 2004 Jan 02.
Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent; Lavelle EC et al.; Cholera toxin (CT) is a potent vaccine adjuvant when administered via parenteral, mucosal, or transcutaneous routes . It also inhibits innate inflammatory responses induced by pathogen-derived molecules, such as lipopolysaccharide (LPS) . We demonstrated previously that CT promotes the induction of regulatory type 1 T cells (Tr1) as well as T helper type 2 cells (Th2) . T cells from mice immunized with antigen in the presence of CT produced high levels of interleukin (IL)-10 and IL-5 and low levels of IL-4 and interferon-gamma (IFN-gamma) . Here, we demonstrate that immunization with antigen in the presence of CT induced a population of antigen-specific CD4(+) T cells that produced IL-10 in the absence of IL-4, in addition to cells that coexpressed IL-4 and IL-10 or produced IL-4 only . CT-generated Tr1 cells inhibited antigen-specific proliferation as well as IFN-gamma production by Th1 cells, and this suppression was cell contact-independent . It is interesting that coincubation with Th1 cells significantly enhanced IL-10 production by the Tr1 cells . As IL-10 can promote the differentiation of Tr1 cells, we investigated cytokine production by dendritic cells (DC) following exposure to CT . Previous data showed that CT can modulate the expression of costimulatory molecules and inhibit the production of chemokines and cytokines, including IL-12 and tumor necrosis factor alpha and enhance IL-10 production . Here, we show that CT synergizes with LPS to induce IL-6 and IL-1beta in addition to IL-10 production by immature DC . Therefore, CT may promote the induction of Th2 and Tr1 cells in part via selective modulation of DC cytokine production and costimulatory molecule expression.

J Biomol Struct Dyn, 2004 Feb, 21(4), 591 - 614
Monosialogangliosides and their interaction with cholera toxin - investigation by molecular modeling and molecular mechanics; Sharmila DJ et al.; Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment . Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1 . The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1 . The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes . Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin . This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.

Gut, 2004 Jan, 53(1), 50 - 7
Intracellular potentiation between two second messenger systems may contribute to cholera toxin induced intestinal secretion in humans; Banks MR et al.; BACKGROUND: Cholera toxin (CT) acts on intestinal epithelial cells both directly and indirectly via activation of a secretory neural reflex . The reflex may release acetylcholine as one of its final neurotransmitters . This opens up the possibility of a third mechanism of action for CT, namely a synergistic interaction between two secretagogues acting on different second messenger systems within the epithelial cell . AIMS: To establish evidence for cholinergic innervation to human ileal epithelial cells and to investigate whether CT potentiates the action of acetylcholine on human intestinal epithelial cells . METHODS: Transverse sections of human ileum were examined for mucosal cholinergic nerves and M3 muscarinic receptors using antibodies raised to choline acetyltransferase and M3 receptors . Short circuit current (Isc) responses and ion flux movements were elicited from T84 epithelial cell monolayers set up in Ussing chambers . RESULTS: Immunohistochemistry of native human ileal mucosa revealed the presence of both cholinergic nerves and muscarinic M3 receptors located to the basolateral domain of epithelial cells . Secretory responses of T84 cell monolayers to acetylcholine were greatly potentiated in the presence of CT . This effect, substituting forskolin for CT, was mirrored by increases in basolateral 86Rb and apical 125I efflux . Charybdotoxin plus apamin reduced both Isc and 86Rb efflux evoked by acetylcholine, in the presence of forskolin . CONCLUSIONS: Human ileal mucosa receives a direct cholinergic innervation to its epithelial cells . Secretory effects of acetylcholine on epithelial cells are augmented in the presence of CT . Such a synergistic response is dependent on optimum opening of basolateral potassium channels by acetylcholine and apical chloride channels by CT . The interaction may contribute to the mechanism of action of cholera toxin induced secretory diarrhoea.

Bull Exp Biol Med, 2003 Sep, 136(3), 307 - 10
Ultrastructural changes in smooth muscle cells of the small intestine in suckling rabbits with experimental cholera; Bardakhch'yan EA et al.; Ultrastructural study of the small intestine in suckling rabbits with experimental cholera revealed involvement of the inner and outer smooth muscle layers into the pathological process . Smooth muscle cells were characterized by vacuolar and fatty degeneration and focal colliquative necrosis . Apoptosis played little role in gastrointestinal motility disturbances . The presence of considerable amounts of fluid in intestinal loops reflects peristaltic dysfunction due to generalized damage to smooth muscle cells.

Trends Biochem Sci, 2003 Dec, 28(12), 639 - 45
The intracellular voyage of cholera toxin: going retro; Lencer WI et al.; Cholera toxin (CT) and related AB(5)-subunit toxins move from the plasma membrane through the trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells . The toxins exploit a specific glycolipid pathway rather than a protein pathway . They bind glycolipids that associate with lipid rafts at the cell surface, which carry the toxins retrograde to the Golgi and ER . In the ER, the A1-chain of the CT unfolds and enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation . Upon entering the cytosol, the A1-chain rapidly refolds, avoids the proteasome and induces toxicity.

Infect Immun, 2003 Dec, 71(12), 6850 - 6
An enzymatically active a domain is required for cholera-like enterotoxins to induce a long-lived blockade on the induction of oral tolerance: new method for screening mucosal adjuvants; Bagley KC et al.; The cholera-like enterotoxins (CLETS), cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), are powerful mucosal adjuvants . Here we show that these toxins also induce a long-lived blockade (of at least 6 months) on the induction of oral tolerance when they are coadministered with the antigen ovalbumin . Strikingly, only enzymatically active CLETS induced this blockade on the induction of oral tolerance . In this regard, the enzymatically inactive mutants of CT and LT, CTK63 and LTK63, and their recombinant B pentamers, rCTB and rLTB, failed to block the induction of oral tolerance, demonstrating a stringent requirement for an enzymatically active A domain in this phenomenon . Together with the results of other recent studies, these results indicate that the enzymatic activity of CLETS, most likely cyclic AMP elevation, is responsible for their adjuvant effects . The results of this study also indicate that measuring the ability of putative mucosal adjuvants to block the induction of oral tolerance may be a superior method for measuring mucosal adjuvanticity.

FEBS Lett, 2003 Nov 20, 554(3), 439 - 42
BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes; Winkeler A et al.; Cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) from where it is translocated to the cytosol in a process depending on ATP and luminal ER proteins . To test whether the molecular chaperone BiP (heavy chain binding protein), which is an ER-luminal ATPase, was one of the required proteins the export of CT was analyzed using ER-derived CT-loaded microsomes . The resubstitution of extracted export-incompetent microsomes with purified BiP was sufficient to restore the export of CT . As BiP protected CT from aggregation it is proposed that BiP maintains CT in a soluble, export-competent state.

J Pain, 2001 Apr, 2(2), 118 - 27
Intrathecally administered cholera toxin blocks allodynia and hyperalgesia in persistent pain models; Caudle RM et al.; In persistent pain, the spinal cord concentration of the opioid peptide dynorphin increases dramatically, yet the function of dynorphin remains unknown . If prodynorphin expression could be manipulated in vivo, it might be possible to determine what role dynorphin plays in persistent pain . Previous work in our laboratory showed that prodynorphin expression is regulated through the cyclic adenosine monophosphate pathway . Therefore, we attempted to enhance prodynorphin expression in the spinal cord of rats by stimulating adenylate cyclase with cholera toxin; however, contrary to our hypothesis, intrathecally administered cholera toxin did not enhance prodynorphin expression . Rather, cholera toxin suppressed the increase in prodynorphin produced by inflammation . Cholera toxin also inhibited the allodynia and hyperalgesia associated with inflammation and nerve injury . Interestingly, the antiallodynic and antihyperalgesic actions of cholera toxin were reversed with the opioid receptor antagonist, naloxone . These findings suggest that cholera toxin enhances or unmasks an endogenous opioid pathway to produce its antiallodynic and antihyperalgesic effects . Furthermore, these data indicate that the suppression of the inflammation-induced increase in spinal cord prodynorphin is caused by the opioid-mediated decrease in the nociceptive stimulus.

Trop Doct, 2003 Oct, 33(4), 215 - 6
Use of cholera beds in the delivery room: a simple and appropriate method for direct measurement of postpartum bleeding; Strand RT et al.; Abundant obstetric bleeding is a predominant cause of maternal death, with the immediate postpartum period being the most critical time . Visual estimation of postpartum haemorrhage (PPH) often leads to severe underestimation and delay in treatment . Various methods have been developed in order to measure blood loss accurately, but none has proved appropriate in poor settings . The aim of this study was to present a method which is appropriate for measuring postpartum blood loss in a setting with limited resources . Parturient women (n = 814) with active management of third stage of labour in Luanda, Angola were studied . Vaginal bleeding immediately after birth and during the first 2 hours postpartum was collected using a combination of a plastic sheet and a bucket belowa cholera bed, in which the women rested during postpartum observation . Monitoring postpartum blood loss in the same way as cholera patients are monitored for loss of stool fluid was found to be a useful and practical way of measuring haemorrhage of parturient women after childbirth . The method described here is simple and appropriate, which makes it a good alternative to more costly methods in detecting and quantifying PPH.

MMWR Morb Mortal Wkly Rep, 2003 Nov 14, 52(45), 1093 - 5
Cholera epidemic after increased civil conflict--Monrovia, Liberia, June-September 2003; Centers for Disease Control and Prevention (CDC); Since 1989, civil war in Liberia has resulted in the displacement of hundreds of thousands of persons . In June 2003, as rebel forces approached the capital city of Monrovia (2003 estimated population: one million), an estimated 300,000 internally displaced persons (IDPs) settled in private homes with family members, public buildings, and other sites . Because of fighting during June-July, the normal collection of health data by the Liberian Ministry of Health (MoH) was interrupted . In June, cases of cholera were confirmed by international nongovernment organizations . To estimate the magnitude of the outbreak, in August, the World Health Organization (WHO) conducted a retrospective review of data collected by health organizations during June--August 2003 but not reported to MoH . Additional data were collected from an emergency surveillance system that began operation on August 25 . This report summarizes the results of that analysis, which indicated that as of September 22, a cholera epidemic was ongoing in Monrovia . During the week ending October 20, a total of 1,252 cases of suspected cholera were reported (WHO, MoH, unpublished data, 2003) . As of November 12, the epidemic was continuing . The epidemic began in June and was associated temporally with increased fighting and the movement of IDPs . Because cholera transmission was probably attributable to an acute shortage of clean water, poor sanitation, and crowded living conditions, international and Liberian organizations attempted to supply IDP settlements with sufficient potable water and began chlorinating wells . To stop cholera transmission and avoid additional illness and death, further preventive measures are needed.

J Vet Sci, 2000 Jun, 1(1), 49 - 52
Relation between lymphocyte subpopulations of peripheral blood and immune responses of modified live hog cholera virus vaccine in pigs treated with an ionized alkali mineral complex; Park BK et al.; Thirty-nine healthy pigs (28-32 days old) were purchased from a commercial swine farm and housed at swine pens of the College . The animals were vaccinated intramuscularly (1 ml) with an attenuated live hog cholera virus (HCV, LOM strain) and then boostered at 5 weeks after the first vaccination . The animals were divided into 4 experimental groups: 0.05% (w/w) PowerFeel-supplemented diet (T-1, n = 10); 3% (w/w) SuperFeed-supplemented diet (T-2, n = 10); diluted PowerFeel solution (1 : 500, v/v) as drinking water (T-3, n=9); control (n=10) . PowerFeel is an original form of ionized alkali mineral complex (IAMC) and SuperFeed is a commercial product of IAMC . The subpopulation of lymphocyte in blood was assayed by a flow cytometry and HCV-specific antibody was determined by an indirect immunofluorescence assay . In IMAC-treated groups, the proportions of subpopulation expressing MHC-class II, CD2+, CD4+, CD8+, and surface IgM+ B lymphocytes were significantly decreased at 5-weeks after the first vaccination . Significant decreases were also observed in the proportions of MHC-class II, CD2+ and CD8+ lymphocyte at 3-weeks after the booster injection . The humoral immune responses in T-1 and T-2 groups were greater than those in T-3 or control group . These results suggest that IAMC-supplemented diets may have an HCV-specific immunostimulatory effect in pigs.

Expert Rev Mol Med, 2002 Oct 01, 2002, 1 - 16
Immune modulation by the cholera-like enterotoxins; Salmond RJ et al.; The role of cholera toxin and heat-labile enterotoxin in the pathogenesis of diarrhoeal disease has been well documented for many years . In addition to these deleterious effects, a wealth of data is accumulating that suggests that these toxins and their subunits might be used to modulate immune responses in a variety of beneficial ways . In this regard, the toxins can boost immune responses to unrelated antigens, leading to the possibility of their use in the generation of improved vaccines to a variety of pathogens . Furthermore, recent evidence suggests that recombinant preparations of the nontoxic B subunits of the toxins have distinct immunomodulatory activities, with potential applications to the treatment of autoimmune and inflammatory diseases . This article reviews our current understanding of the mechanisms of immune modulation by these fascinating proteins.

J Indian Med Assoc, 2003 Jun, 101(6), 379 - 80, 386
Role of oral rehydration therapy in controlling epidemic of cholera and watery diarrhoea; Sarkar K; Oral rehydration therapy (ORT) is basically oral administration of liquid containing various electrolytes in specific proportions to prevent and treat dehydration . This treatment facilitates safe and optimal absorption of water and essential electrolytes such as sodium chloride, sodium bicarbonate and potassium chloride in dehydrated patients . Successful ORT was experienced in cholera patients in Kolkata and Dhaka which was followed by the development of oral rehydration salt (ORS) . This procedure can be safely implemented at home . ORT reduced mortality rate both in cholera and non-cholera watery diarrhoea . The various health authorities must support preparedness before pre-positioning of adequate stocks of ORS packets for emergency situations . Health workers should have been the knowledge to prepare ORS solutions.

Eur J Immunol, 2003 Nov, 33(11), 3205 - 12
Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB translocation; Kawamura YI et al.; Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated . We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor . When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4+ T cell responses . In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by up-regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses . On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT . Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4 . Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion . These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.

Vaccine, 2003 Nov 7, 21(31), 4527 - 31
Mass psychogenic illness following oral cholera immunization in Ca Mau City, Vietnam; Khiem HB et al.; INTRODUCTION: Targeted cholera immunization of high-risk populations in Vietnam is conducted based on routine surveillance data . Following mass immunization of schoolchildren in Ca Mau City using an oral bivalent killed cholera vaccine, adverse reactions were noted . METHODS: Salient data were collected in a systematic fashion including the review of medical records; interview of the school principal, teachers, students, parents and doctors; and review of the storage and handling of the vaccine . FINDINGS: On 18 December 2001, 234 children at a primary school in Ca Mau City received the cholera vaccine . Within 1h of immunization, three children in one of the classrooms complained of trembling, nausea and headache and were brought to the library and soon other children followed . Out of 234, 97 (42%) pupils were affected and brought to the Municipal Health Center or Ca Mau Provincial Hospital . Those who were affected were younger (mean age=9.6 years; 95% CI=9.4-9.7) compared to those who were not affected (mean age=10 years; 95% CI=9.7-10.3; t-test=-2.4; P-value=0.02) . The proportion of affected females among those who had received the vaccine (49/114 or 43%) was similar to the proportion in males (48/120 or 40%; RR=1.07; 95% CI=0.79-1.46) . The most frequent presenting complaint was cold extremities (60%) followed by headache (27%) . All affected children recovered and were discharged in a few hours . None reported any sequelae or relapse . Once the situation was recognized, the cholera immunization campaign was continued . Laboratory tests of vaccine samples from the same batch detected no abnormality or contaminating agent . DISCUSSION: The findings suggest that the children at primary school number 1 suffered from a mass psychogenic illness . This incident was unusual in that a similar number of boys and girls were affected, in contrast to the frequently reported preponderance of female cases . Furthermore the underlying cause was very quickly diagnosed, medical interventions were kept to a minimum, and no relapse was observed . Future vaccination campaigns have to assure that the families are informed in advance.

Infect Immun, 2003 Nov, 71(11), 6234 - 42
Role of cyclooxygenase enzymes in a murine model of experimental cholera; Gessell-Lee DL et al.; Nonsteroidal anti-inflammatory drugs (e.g., indomethacin) inhibit and reduce the fluid secretion responses elicited by cholera toxin (CT), but it has not been conclusively determined which cyclooxygenase (COX) isoform is involved in CT's action . This study evaluated the role of the COX enzymes and their arachidonic acid metabolites in experimental cholera . Swiss-Webster mice were dosed with celecoxib and rofecoxib and challenged with CT in ligated small intestinal loops, and intestinal segments from mice deficient in COX-1 and COX-2 were challenged with CT . The effects of CT on fluid accumulation, prostaglandin E(2) production, mucosal tissue injury, and markers of oxidative stress were measured . Celecoxib and rofecoxib given at 160 micro g per mouse inhibited CT-induced fluid accumulation by 48% and 31%, respectively, but there was no significant difference among cox-1(-/-) and cox-2(-/-) mice in response to CT compared to wild-type controls . CT elevated tissue levels of oxidized glutathione and lipid peroxides and elicited small intestinal tissue injury in two of five cox-1(-/-) and four of five cox-2(-/-) mice . A role for COX-2 in CT's mechanism of action has previously been suggested by the effectiveness of COX-2 inhibitors in reducing CT-induced fluid secretion, but CT challenge of COX-1 and COX-2 knockout mice did not corroborate the pharmacological data . The results of this study show that CT induced oxidative stress in COX-deficient mice and suggest a tissue-protective role for arachidonic acid metabolites in the small intestine against oxidative stress.

Yeni Tip Tarihi Arastirmalari, 2001, 7, 45 - 63
{Preventive measures taken in Adrianople during the 1893-1894 cholera epidemic, as reflected in a local newspaper}; Gokce N; Cholera is derived from the Latin words colos (large intestine) and reo (to flow) and means flowing of the liquids through the stomach and the intestines . The first cholera epidemic was experienced in India and spread out to other countries . Although it had been known for ages, it was not recognized until the 16th century . Seven serious cholera epidemics have broken out in the world since the 19th century . In spite of all precautions taken by the Ottoman government, the sixth world cholera epidemic that started in Asia in 1891 and caused the loss of 40 thousand people, reached Istanbul in 1893 . Later, it spread to Iznik, Salonika and Anatolia . Just as it appeared in Europe, precautions started to be taken in Adrianople . At first, special care was taken for city hygiene and a commission was formed to inspect the cleanliness of the city . Many brochures and articles were published on the protection against the illness, in order to inform the citizens of the cholera epidemic . Preachers spoke of cholera in their sermons . To protect Adrianople against the epidemic, entrance into and exit out of the city were patrolled and passengers coming from Europe or Istanbul to Adrianople were kept waiting for three days at the quarantines built in Cisri Mustafa Pasha and Catalca.

Avian Dis, 2003 Jul-Sep, 47(3), 777 - 80
An outbreak of fowl cholera in ring-necked pheasants (Phasianus colchicus); Einum P et al.; A total of 120 ring-necked pheasants from a 3000-bird flock in Zeeland, MI, died over a 3-day period . Clinical signs included sudden death, diarrhea, and limping . At necropsy, hepatomegaly with multifocal cream-colored foci randomly distributed throughout the parenchyma was observed in diseased birds . Additionally, the spleen was enlarged up to three times its normal size and had a marbled appearance . Microscopically, there was multifocal splenic and hepatic necrosis with intralesional rod-shaped bacteria . Pasteurlla multocida serotype 3/4 was isolated from liver and spleen . In this paper, we report an outbreak of acute fowl cholera in ring-necked pheasants.

Bioorg Med Chem Lett, 2003 Nov 3, 13(21), 3831 - 4
Ganglioside GM1 mimics: lipophilic substituents improve affinity for cholera toxin; Arosio D et al.; Ganglioside GM1 mimics including (R)-2-hydroxy-3-cyclohexylpropionic acid or (R)-2-hydroxy-3-phenylpropionic acid as replacements for NeuAc are stronger cholera toxin binders than the parent ligand 2, which includes (R)-2-hydroxy-propionic acid.

Can Bull Med Hist, 1999, 16(2), 317 - 39
{The moral lessons of miasma: the "Memorandum on cholera" of Joseph-Charles Taché}; Curtis B; In the spring of 1866, the Canadian government feared that a cholera epidemic was imminent . A conference of medical experts was held at Ottawa to determine the nature of the disease on the basis of "authentic facts" but the participants were unable to agree among themselves . Faced with the necessity of advising Canadians on how to live in time of plague, and yet unable to define clearly the nature of cholera, in his Memorandum on Cholera the deputy-minister of Agriculture, J.-C . Tache, translated medical preoccupations into matters of the government of oneself and others, in terms heavily accented by his fundamentalist Catholic religious beliefs.

Probl Sotsialnoi Gig Istor Med, 2003 Jul-Aug, (4), 41 - 2
{Social-economic expenditures for the liquidation of cholera in a big city}; Ziatdinov VB; While making a cost-assessment related with one cholera disease case, the authors suggest to take account of the damage inflicted on the health of patients with other pathologies (apart from cholera) by the failure to render them the ambulatory and hospital medical care, which results from that the medical staff at large focus primarily on curing the cholera patients and on that the hospital beds (otherwise used for somatic patients) are preferably allocated for patients with cholera at epidemic.

Medizinhist J, 2003, 38(1), 35 - 56
{The traveling laboratory: Robert Koch investigates cholera 1883/84}; Gradmann C; Based on the example of Robert Koch's cholera expedition of 1883/84, this paper analyses travelling as a mode of doing research that was characteristic of an applied science such as hygiene . A laboratory for hygiene is dependent on a certain environment which becomes noticeable through its absence . Examples are its dependence on the cultural acceptance of pathological dissections or a suitable climate in which gelatine-based solid culture media do not liquefy . Travelling abroad gave Koch and his collaborators privileged access to the public at home . Laboratory work was given the heroic image of a crusade against epidemics, and what had been complex research was presented as a simple discovery . Travelling abroad fulfilled Koch's own youthful ambitions and simultaneously opened up his career as a figure of public life . In addition it provided him with the experience of doing science in a profitable and enjoyable way.

Mol Cells, 2003 Aug 31, 16(1), 106 - 12
Intraperitoneal delivery of cholera toxin B subunit enhances systemic and mucosal antibody responses; Park SJ et al.; Although cholera toxin (CT) is a potent mucosal adjuvant, its activity in systemic immunity is relatively undocumented . In the present study, we investigated its adjuvant effect on systemic and mucosal antibody responses following intraperitoneal immunization of mice with BSA . CT increased levels of anti-BSA specific IgG1, IgM, and IgA antibodies in the peritoneum and serum, as well as IgA and IgG1 antibodies in the intestinal fluids . The B subunit of CT (CTB) was as potent as CT itself, with potency comparable to that of incomplete Freund's adjuvant . CTB also increased the number of BSA-specific Ig secreting cells in the spleen and mesenteric lymph node, and stimulated expression of B7.2 but not of MHC class II molecules on peritoneal macrophages, particularly in the presence of IFN-gamma . Our results imply that intraperitoneally administered CTB enhances systemic and mucosal antibody responses, in part at least via effects on macrophages.

Mol Biol Cell, 2003 Dec, 14(12), 4783 - 93 Epub 2003 Sep 17.
Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm; Fujinaga Y et al.; Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease . A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity . Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs . The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER . In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER . Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.

Clin Exp Immunol, 2003 Oct, 134(1), 38 - 45
Coupling of oral human or porcine insulin to the B subunit of cholera toxin (CTB) overcomes critical antigenic differences for prevention of type I diabetes; Petersen JS et al.; Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models . Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials . Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin . As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses . Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy . Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred . Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

Gut, 2003 Oct, 52(10), 1419 - 23
Efficacy and tolerability of racecadotril in the treatment of cholera in adults: a double blind, randomised, controlled clinical trial; Alam NH et al.; BACKGROUND: The enkephalins, endogenous opiate substances, act as neurotransmitters along the entire digestive tract where they elicit intestinal antisecretory activity without affecting intestinal transit time or motility . Racecadotril, through inhibition of enkephalinase, reinforces the physiological activity of endogenous enkephalins and, therefore, shows intestinal antisecretory activity . AIM: We conducted the study to determine the role of racecadotril as an adjunct to the standard treatment of cholera in adults . METHODS: The study was a double blind, randomised, placebo controlled clinical trial involving 110 adult male cholera patients who received either racecadotril or placebo in addition to standard cholera treatment . The major outcome measures (stool output, oral rehydration solution (ORS) intake, requirements for unscheduled intravenous fluid infusion, and duration of diarrhoea) were compared between the groups . RESULTS: Of 110 patients enrolled, 54 received racecadotril and 56 received placebo . Admission clinical characteristics were comparable between the groups . There was no significant difference in (mean (SD)) total stool output (racecadotril v placebo 315 (228) v 280 (156) g/kg), total ORS intake (390 (264) v 369 (240) ml/kg), or duration of diarrhoea (35 (15) v 32 (13) hours) between the groups . Clinical success, defined as resolution of diarrhoea within 72 hours of initiation of study intervention, was similar in both groups (racecadotril v placebo 96% v 89%) . The number of patients receiving unscheduled intravenous infusions was not significantly different between the groups (racecadotril v placebo 22% v 14%) . No drug related adverse effect was noted . CONCLUSION: The study demonstrated that racecadotril therapy, although found to be safe, does not provide additional benefit in the treatment of severe cholera in adults.

Exp Brain Res, 2003 Dec, 153(4), 514 - 21 Epub 2003 Sep 05.
A subpopulation of dorsal raphe nucleus neurons retrogradely labeled with cholera toxin-B injected into the inner ear; Kim DO et al.; Previous studies have shown that: (1) raphe neurons respond to acoustic and vestibular stimuli, some with a latency of 10-15 ms; (2) alterations of the raphe nuclei alter the acoustic startle reflex; (3) the dorsal raphe nucleus (DRN) is the major source of serotonergic neurons; and (4) approximately 57% of the DRN neurons are nonserotonergic . In the present study, cholera toxin subunit-B (CTB) was injected into cat cochleas, and the brain tissue was examined after a survival period of 5-7 days . Aside from neurons which were known to project to the inner ear, i.e., olivocochlear and vestibular efferent neurons, a surprising new finding was made that somata of a subpopulation of DRN neurons were intensely labeled with CTB . These CTB-labeled neurons were densely distributed in a dorsomedian part of the DRN with some in a surrounding area outside the DRN . The present results suggest that a novel raphe-labyrinthine projection may exist . A future study of anterograde labeling with injections of a tracer in the DRN will be needed to establish the existence of a raphe-labyrinthine projection more thoroughly . A raphe-labyrinthine descending input, together with an ascending input from the inner ear to the DRN through intervening neurons, such as the juxta-acousticofloccular raphe neurons (JAFRNs) described by Ye and Kim, may mediate a brain stem reflex whereby a salient multisensory (including auditory and vestibular) stimulus may alter the sensitivity of the inner ear . As a mammal responds to a biologically important auditory-vestibular multisensory event, the raphe projections to the inner ear and other auditory and vestibular structures may enhance the mammal's ability to localize and recognize the sound and respond properly . The raphe-labyrinthine projection may also modulate the inner ear's sensitivity as a function of the sleep-wake arousal state of an organism on a slower time course.

Bull Hist Med, 2003 Summer, 77(2), 298 - 331
Coleridge's choleras: cholera morbus, Asiatic cholera, and dysentery in early nineteenth-century England; Rousseau GS et al.; Samuel Taylor Coleridge suffered from a variety of bowel disorders throughout his life; though a large part of his ailment was caused by his famous opium habit, he continuously sought an organic origin, and on at least two separate occasions, in 1804 and 1831-32, he ascribed his disorders to attacks of "cholera." With Asiatic cholera apparently first reaching England in late 1831, there was considerable argument among both physicians and the general public as to whether it was a distinctly new disease, or merely a severer variation of traditional English cholera, known as "cholera morbus." Coleridge took a particular interest in these discussions . In this paper, we attempt to establish the exact nature of his attacks of illness, and point to the complexities of describing and framing new diseases and bowel disorders in the early nineteenth century.

Eur J Epidemiol, 2003, 18(7), 617 - 21
Sociomedical indicators in the cholera epidemic in Ferrara of 1855; Scapoli C et al.; The historical report on the cholera epidemic of 1855, conserved in Ferrara City's archives allowed us to verify the probable relation between the environment and epidemic in a broad sense, using log-linear analysis and multiple logistic regression . Two thousand and thirty-three cases were analyzed and the quantitative/qualitative variables available from the report were analyzed in relationship with mortality and morbidity rates, considered as response variables . From the analysis of the quantitative variables, it emerges that the variables having a significant influence on the morbidity/mortality rates are the number of individuals and the average number of inhabitants per house . From the analysis of the qualitative variables, it emerges that all the descriptive variables of the state of the streets and houses express a strong association with mortality and morbidity . With the present analysis, data available--a detailed 'street by street' morbidity and mortality recording from cholera in 1855 in Ferrara--were analyzed with modern means and the overall picture that emerge is that in the better kept houses in the better parts of the town had less cholera morbidity and especially mortality.

Scand J Immunol, 2003 Sep, 58(3), 342 - 9
The cholera toxin B subunit directly costimulates antigen-primed CD4+ T cells ex vivo; Wang M et al.; The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination . However, the mechanisms behind its adjuvant effects remain unclear . Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells . Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier . However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior . This adjuvant effect was dose-dependent, with maximum at 30-300 ng/ml rCTB . At higher doses of CTB this effect declined because of the induction of apoptosis . Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells . Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-gamma(IFN-gamma) by four to fivefold . Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e . rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen . Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner . This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.

Gastroenterology, 2003 Sep, 125(3), 765 - 74
An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice; Izzo AA et al.; BACKGROUND & AIMS: Cholera toxin (CT) is the most recognizable enterotoxin causing secretory diarrhea, a major cause of infant morbidity and mortality throughout the world . In this study, we investigated the role of the endogenous cannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) in CT-induced fluid accumulation in the mouse small intestine . METHODS: Fluid accumulation was evaluated by enteropooling; endocannabinoid levels were measured by isotope-dilution gas chromatography mass spectrometry; CB(1) receptors were localized by immunohistochemistry and their messenger RNA (mRNA) levels were quantified by reverse-transcription polymerase chain reaction (PCR) . RESULTS: Oral administration of CT to mice resulted in an increase in fluid accumulation in the small intestine and in increased levels of the endogenous cannabinoid, anandamide, and increased expression of the cannabinoid CB(1) receptor mRNA . The cannabinoid receptor agonist CP55,940 and the selective cannabinoid CB(1) receptor agonist arachidonoyl-chloro-ethanolamide inhibited CT-induced fluid accumulation, and this effect was counteracted by the CB(1) receptor antagonist SR141716A, but not by the CB(2) receptor antagonist SR144528 . SR141716A, per se, but not the vanilloid VR1 receptor antagonist capsazepine, enhanced fluid accumulation induced by CT, whereas the selective inhibitor of anandamide cellular uptake, VDM11, prevented CT-induced fluid accumulation . CONCLUSIONS: These results indicate that CT, along with enhanced intestinal secretion, causes overstimulation of endocannabinoid signaling with an antisecretory role in the small intestine.

Anal Chem, 2003 Jun 1, 75(11), 2633 - 9
Development of an "electroptode" immunosensor: indium tin oxide-coated optical fiber tips conjugated with an electropolymerized thin film with conjugated cholera toxin B subunit; Konry T et al.; We demonstrate that it is possible to create surface-conductive fiber optics, upon which may be electropolymerized a biotinylated polypyrrole thin film, which may then be used to affinity coat the fiber with molecular recognition probes . This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide . Thereafter, biotin-pyrrole monomers are electropolymerized onto the conductive metal oxide surface and then exposed to avidin . Avidin-biotin interactions were used to modify the fiber optics with biotin-conjugated cholera toxin B subunit molecules, for the construction of an immunosensor to detect cholera antitoxin antibodies . The biosensor was tested for sensitivity, nonspecificity, and overall practicality.

J Virol, 2003 Sep, 77(18), 9823 - 30
Mucosal immunization with virus-like particles of simian immunodeficiency virus conjugated with cholera toxin subunit B; Kang SM et al.; To enhance the efficiency of antigen uptake at mucosal surfaces, CTB was conjugated to simian immunodeficiency virus (SIV) virus-like particles (VLPs) . We characterized the immune responses to the Env and Gag proteins after intranasal administration . Intranasal immunization with a mixture of VLPs and CTB as an adjuvant elicited higher levels of SIV gp160-specific immunoglobulin G (IgG) in sera and IgA in mucosae, including saliva, vaginal-wash samples, lung, and intestine, as well as a higher level of neutralization activities than immunization with VLPs alone . Conjugation of CTB to VLPs also enhanced the SIV VLP-specific antibodies in sera and in mucosae to similar levels . Interestingly, CTB-conjugated VLPs showed higher levels of cytokine (gamma interferon)-producing splenocytes and cytotoxic-T-lymphocyte activities of immune cells than VLPs plus CTB, as well as an increased level of both IgG1 and IgG2a serum antibodies, which indicates enhancement of both Th1- and Th2-type cellular immune responses . These results demonstrate that CTB can be an effective mucosal adjuvant in the context of VLPs to induce enhanced humoral, as well as cellular, immune responses.

Org Biomol Chem, 2003 Mar 7, 1(5), 785 - 92
Mimics of ganglioside GM1 as cholera toxin ligands: replacement of the GalNAc residue; Bernardi A et al.; Two new cholera toxin (CT) ligands (4 and 5) are described . The new ligands were designed starting from the known GM1 mimics 2 and 3 by replacement of their GalNAc residue with the C4 isomer GlcNAc . As predicted by molecular modelling, the conformational properties of the equivalent pairs 2-4 and 3-5 are very similar and their affinity for CT is of the same order of magnitude . NMR experiments have also proved that 5 occupies the GM1-binding site of the toxin and have revealed its bound conformation.

J Immunol, 2003 Sep 1, 171(5), 2384 - 92
Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation; Lavelle EC et al.; It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag . Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag . Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma . Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells . Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5 . It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells . Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC . CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1 . Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.

Soc Sci Med, 2003 Oct, 57(8), 1397 - 407
Vulnerability to coastal cholera ecology; Collins AE; The battle to completely control cholera continues . Multiple strains, high levels of morbidity in some regions of the world, and a complex of influences on its distribution in people and the environment are accompanied by only rough resolution prediction of outbreaks . Uncertainty as to the most effective array of interventions for one of the most researched infectious diseases thwarts further progress in providing cost-effective solutions . Progress on the research front consistently points towards the importance of disease ecology, coastal environments, and the sea . However, evaluation of the link between cholera in people and environment can only be effective with analysis of human vulnerability to variable coastal cholera ecologies . As there are some clear links between the organism, cholera incidence and the sea, it is appropriate that cholera research should examine the nature of coastal population vulnerability to the disease . The paper reviews the cholera risks of human-environment interactions in coastal areas as one component of the evaluation of cholera management . This points to effective intervention through integrative knowledge of changing human and environmental ecologies, requiring improved detection, but also an acceptance of complex causality . The challenge is to identify indicators and interventions for case specific ecologies in variable locales of human vulnerability and disease hazard . Further work will therefore aim to explore improved surveillance and intervention across the socio-behavioural and ecological spectrum . Furthermore, the story of cholera continues to inform us about how we should more effectively view emergent and resurgent infectious disease hazards more generally.

Anal Chem, 2003 May 15, 75(10), 2256 - 61
Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin; Ahn-Yoon S et al.; An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes . Cholera is a diarrheal disease, often associated with water or seafood contamination . Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer . CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT . In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies . The intensity of the band could be visually estimated or measured by densitometry, using computer software . The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample . The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays . The assay could be completed within 20 min . These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.

Int Arch Allergy Immunol, 2003 Aug, 131(4), 256 - 63
Experimental parameters differentially affect the humoral response of the cholera-toxin-based murine model of food allergy; Kroghsbo S et al.; BACKGROUND: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies . However, for studying subtle immunomodulating factors or factors effective during response initiation, this maximal response-based model is less suitable due to a lack of sensitivity . Therefore, in attempts to identify essential parameters to fine-tune the immune response towards a submaximal level, potentially more sensitive, we were interested in characterizing the individual effects of the parameters in the CT-based model: CT dose, antigen type and dose, and number of immunizations . METHODS: BALB/c mice were orally sensitized weekly for 3 or 7 weeks with graded doses of CT and various food antigens (soy-trypsin inhibitor, ovalbumin or ovomucoid) . Antigen-specific IgG1, IgG2a, IgA and IgE were monitored by ELISA . RESULTS: The CT dose exerted a clear dose-dependent effect on the antigen-specific antibody response whereas the antigen dose tended to affect the kinetics of the developing response . Both the intensity and kinetics of the antibody response depended on the type of antigen and number of immunizations . CONCLUSIONS: The critical parameters of the CT-based murine allergy model differentially control the intensity and kinetics of the developing immune response . Adjustment of these parameters could be a key tool for tailoring the response to submaximal levels rendering the model potentially more sensitive for evaluating the effect of subtle immunomodulating factors that would be lost in the maximal response-based model .

Biotechnol Lett, 2003 Jul, 25(13), 1087 - 92
Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast; Sun M et al.; A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C . reinhardtii by the biolistic method . The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark . The CTBVP1 fusion protein was expressed in C . reinhardtii chloroplast and accounted for up to 3% of the total soluble protein . The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins . These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.

Eur Neuropsychopharmacol, 2003 Aug, 13(4), 281 - 8
Effects of spinally and supraspinally injected 3-isobutyl-1-methylxanthine, cholera toxin, and pertussis toxin on immobilization stress-induced antinociception in the mouse; Chung KM et al.; The effects of intracerebroventricular (i.c.v.) and intrathecal (i.t.) 3-isobutyl-1-methylxanthine (IBMX), cholera toxin (CTX) and pertussis toxin (PTX) administration on immobilization-induced antinociception were studied in ICR mice . Antinociception was assessed by the tail-flick assay . Immobilization of the mouse increased inhibition of the tail-flick response for at least 1 h . The pretreatment with i.t . IBMX (0.01-1 ng), but not i.c.v . IBMX, significantly attenuated immobilization-induced inhibition of the tail-flick response . The pretreatments with i.c.v . PTX (0.05-0.5 microg) as well as i.t . CTX, but neither i.c.v . CTX (0.05-0.5 microg) nor i.t . PTX, potentiated the inhibition of the tail-flick response induced by immobilization stress . Our results suggest that spinally located phosphodiesterase appears to be involved in the production of immobilization stress-induced antinociception . In addition, inactivation of supraspinally located PTX-sensitive G-proteins and spinally located CTX-sensitive G-proteins may modulate immobilization stress-induced antinociception.

Clin Infect Dis, 2003 Aug 1, 37(3), 398 - 405 Epub 2003 Jul 22.
Cholera, diarrhea, and oral rehydration therapy: triumph and indictment; Guerrant RL et al.; Cholera drove the sanitary revolution in the industrialized world in the 19th century and now is driving the development of oral rehydration therapy (ORT) in the developing world . Despite the long history of cholera, only in the 1960s and 1970s was ORT fully developed . Scientists described this treatment after the discovery of the intact sodium-glucose intestinal cotransport in patients with cholera . This new understanding sparked clinical studies that revealed the ability of ORT to reduce the mortality associated with acute diarrheal disease . Despite the steady reductions in mortality due to acute dehydrating diarrheal diseases achieved by ORT, the costly morbidity due to these diseases remains, the result of a failure to globalize sanitation and to control the developmental impact of diarrheal diseases and their associated malnutrition . New advances in oral rehydration and nutrition therapy and new methods to recognize its costs are discussed in this review.

J Comp Neurol, 2003 Aug 25, 463(3), 317 - 40
Pattern of retinal projections in the California ground squirrel (Spermophilus beecheyi): anterograde tracing study using cholera toxin; Major DE et al.; The retinofugal pathways in the California ground squirrel, Spermophilus beecheyi, were mapped after intravitreal injections of cholera toxin B-subunit . The results of the current study are consistent with work in other mammals and provide new details relevant to the organization and evolution of the visual system . All retinorecipient nuclei received bilateral input, with a contralateral predominance . The suprachiasmatic nucleus is heavily innervated, and sparse terminals were noted in other hypothalamic areas . In addition to the interstitial, medial, lateral, and dorsal terminal nuclei, a few fibers of the accessory optic tract may enter the ventral lateral geniculate and the nucleus of the optic tract, though this innervation may not derive from the same ganglion cells innervating the accessory optic nuclei . Retinal terminals are found in the intergeniculate leaflet and the "dorsal cap" of the ventral lateral geniculate . Retinal fibers pass rostrally from the dorsal cap toward the anterodorsal thalamus, confirming a projection described in the tree shrew and monkeys . Retinal termination patterns in the dorsal lateral geniculate reveal a hexilaminate organization of alternating ipsilateral and contralateral input . Variations in terminal morphology suggest that sublayers receive input from distinct ganglion cell types and that laminar comparisons can be made with primates . Finally, terminal patterns in the superior colliculus reveal a dense, highly ordered columnar organization supporting functional properties of tectal receptive fields . All the visual structures in the ground squirrel are large and well differentiated, making the sciurid visual system an accessible rodent model for comparing visual processing with that in other diurnal vertebrates .

J Vet Sci, 2002 Dec, 3(4), 315 - 9
Host immune responses against hog cholera virus in pigs treated with an ionized alkali mineral complex; Park BK et al.; To determine the immune responses in pigs to hog cholera virus after treatment with an ionized alkali mineral complex (IAMC), 40 healthy pigs (28-32 days old) from a commercial swine farm were purchased and housed into 4 groups (n=10 each) . All pigs were vaccinated intramuscularly (1 ml) with an attenuated live hog cholera virus (HCV, LOM strain) at 28-32 days old and challenged with a virulent hog cholera virus at 8 weeks after vaccination . Each group was treated with PowerFeel sprayed diet as 0.05% (w/w) in a final concentration (T-1, n=10), a diet mixed with SuperFeed as 3% (w/w) in a final concentration (T-2, n=10), or a diluted PowerFeel solution (1:500, v/v) as drinking water (T-3, n=10), respectively . A group (n=10) served as a non-treated control . Proportions of expressing CD2+ and CD8+ cells increased significantly (p<, 0.05) at 8-week post-application . Mean antibody titers of each group against HCV gradually increased to higher levels after vaccination and with challenge of the virulent virus . In conclusion, the IAMC-treated diets can be helpful for the improvement of growth in pigs with proper vaccination program, while the IAMC-treated diets have no effects on the clinical protection against hog cholera.

Infect Immun, 2003 Jul, 71(7), 4093 - 101
Cholera holotoxin assembly requires a hydrophobic domain at the A-B5 interface: mutational analysis and development of an in vitro assembly system; Tinker JK et al.; Cholera toxin (CT) and related Escherichia coli enterotoxins LTI and LTIIb have a conserved hydrophobic region at the AB(5) interface postulated to be important for toxin assembly . Hydrophobic residue F223 in the A subunit of CT (CTA) as well as residues 174, L77, and T78 in the B subunit of CT (CTB) were replaced individually with aspartic acid, and the resulting CTA and CTB variants were analyzed for their ability to assemble into holotoxin in vivo . CTA-F223D holotoxin exhibited decreased stability and toxicity and increased susceptibility to proteolysis by trypsin . CTB-L77D was unable to form functional pentamers . CTB-I74D and CTB-T78D formed pentamers that bound to GM(1) and D-galactose but failed to assemble with CTA to form holotoxin . In contrast, CTB-T78D and CTA-F223H interacted with each other to form a significant amount of holotoxin in vivo . Our findings support the importance of hydrophobic interactions between CTA and CTB in holotoxin assembly . We also developed an efficient method for assembly of CT in vitro, and we showed that CT assembled in vitro was comparable to wild-type CT in toxicity and antigenicity . CTB-I74D and CTB-T78D did not form pentamers or holotoxin in vitro, and CTA-F223D did not form holotoxin in vitro . The efficient system for in vitro assembly of CT described here should be useful for future studies on the development of drugs to inhibit CT assembly as well as the development of chimeric CT-like molecules as potential vaccine candidates.

Vaccine, 2003 Jul 4, 21(23), 3335 - 41
Immunogenicity of DNA vaccines that direct the coincident expression of the 120 kDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin; Bagley KC et al.; Passive antibody studies unequivocally demonstrate that sterilizing immunity against lentiviruses is obtainable through humoral mechanisms . In this regard, DNA vaccines represent an inexpensive alternative to subunit vaccine for mass vaccination programs designed to induce such responses to human immunodeficiency virus type I (HIV-1) . At present, however, this vaccine modality has proven relatively ineffective at inducing humoral responses . In this report, we describe the immunogenicity of DNA vaccines that direct the coincident expression of the cholera toxin catalytic domain (CTA1) with that of the human immunodeficiency virus type I gp120 through genes either encoded in individual plasmids or in a single dicistronic plasmid . In BALB/cJ mice, coincident expression of CTA1 in either a separate plasmid or in the dicistronic plasmid in the DNA vaccines induced serum IgG responses to gp120 that were at least 1000-fold greater, and remained elevated longer than, the analogous responses in mice vaccinated with a DNA vaccine that expressed gp120 alone . In addition, mice vaccinated with CTA1 and gp120 produced significantly more gp120-specific IFN-gamma ELISPOTs than mice vaccinated with the gp120 DNA vaccine . Combined, these data show that the adjuvant properties of cholera toxin can be harnessed in DNA vaccine modalities.

Vaccine, 2003 Jul 4, 21(23), 3143 - 8
Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes; Arakawa T et al.; Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control . To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT) . Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response) . Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes . The observed transmission-blocking effect was dependent on immune sera dilution . This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria.

Vaccine, 2003 Jun 20, 21(21-22), 3081 - 92
Mucosal co-administration of cholera toxin and influenza virus hemagglutinin-DNA in ponies generates a local IgA response; Soboll G et al.; We have previously demonstrated that equine influenza virus hemagglutinin (HA) DNA vaccination protects ponies from challenge infection, and induces protective IgGa and IgGb responses . However, this approach does not induce a nasal IgA response . The objective of this study was to examine the value of cholera toxin (CT) administration as an adjuvant for intranasal HA DNA vaccination, and to measure protection 3 months after DNA vaccination . After an immunogenic dose of CT was determined, ponies were immunized on two occasions by intranasal administration of HA DNA and cholera toxin, or HA DNA alone . Ponies in both groups received two additional HA DNA particle mediated vaccinations at skin and mucosal sites . Antibody responses, and protection from challenge infection 3 months after the last vaccination were studied and compared to an influenza virus naive control group . Ponies in both vaccination groups produced virus-specific IgG antibodies in serum following vaccination and showed clinical protection from challenge infection 3 months after the last vaccination . Co-administration of CT plus HA DNA vaccination induced a nasal IgA response . In addition, analysis of antibody titers in nasal secretions indicated local production of nasal IgGb, which was amplified by CT administration.

Rev Saude Publica, 2003 Jun, 37(3), 292 - 6 Epub 2003 Jun 03.
{Validation of clinical and epidemiological diagnosis criterion for confirming cholera}; Pedrosa Fde A et al.; OBJECTIVE: To validate the clinical and epidemiological diagnosis criterion for confirming cholera suspect cases . METHODS: The study comprised 2,687 and 716 patients admitted with diarrhea to a public hospital in Macei , Brazil, in 1992 and 1997 respectively . Culture of V . cholerae O1 (Koch, 1884) in TCBS-agar was performed and the clinical and epidemiological criterion was applied and compared to the gold standard . The statistical analysis was carried out in two age-groups: less than five years old and five years old or more . RESULTS: There were studied 833 patients, of which 517 in 1992 and 316 in 1997; 72 aged less than five years and 761 aged five years or more . In those aged less than five years in 1992, sensitivity was 40% and specificity 84.6% . For this same age-group in 1997, sensitivity was 28.6% and specificity 62.5% . In patients aged five years or more in 1992, sensitivity was 99% and specificity 1.2% . For this same age-group in 1997, sensitivity was 86.9% and specificity 8.7% . CONCLUSION: The higher sensitivity of the clinical and epidemiological cholera diagnosis criterion in patients aged five years or more found in the study support its application during epidemics periods . In periods of lower incidence, all cases need to be confirmed using laboratory methods.

Bioconjug Chem, 2003 May-Jun, 14(3), 614 - 8
A novel method for the rational construction of well-defined immunogens: the use of oximation to conjugate cholera toxin B subunit to a peptide-polyoxime complex; Chen J et al.; Cholera toxin B subunit (CTB), capable of binding to all mucous membranes in its pentameric form, is a potential carrier of mucosal vaccines . In our previous work we reported that the N-terminus of CTB, a threonine, could in principle undergo oxidation and oximation to form conjugates with a cascade of immunogenic peptides . In this study, we set up a model by chemically coupling CTB to a polyoxime that possessed five copies of influenza virus-derived peptides displayed in comblike form . The construct was reconstituted into pentameric form when eluted from a Superdex column after conjugation, and the pentameric nature of this CTB-viral peptide complex was confirmed by SDS-PAGE . GM(1)-ELISA assay showed that the binding properties of CTB-viral peptide complex were increased 4-5-fold over native CTB.

Scand J Immunol, 2003 May, 57(5), 432 - 8
The cholera toxin B subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes; Bregenholt S et al.; When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration . Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration . Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes . In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold . Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression . Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model . In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration . The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

Vaccine, 2003 Jun 2, 21(19-20), 2394 - 403
Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi children 18-36 months of age; Qadri F et al.; A phase II safety and immunogenicity study of an oral-formalin inactivated enterotoxigenic Escherichia coli (ETEC) vaccine containing six colonization factors (CFA/I, CS1, CS2, CS3, CS4, CS5) and 1mg of recombinant cholera toxin B subunit (the CF-BS-ETEC vaccine) was carried out in an urban slum of Dhaka city in Bangladesh . The study was carried out in a double blinded, placebo controlled design in 158 children, 18-36 months of age . Children were given two doses of the CF-BS-ETEC vaccine or the placebo which consisted of E . coli K12 . The vaccine was well tolerated.The immune response was studied in 60 children (30 each in the placebo and vaccine group) . Significant vaccine specific IgA antibody-secreting cell (ASC) responses were seen 7 days after ingestion of the first and second dose of the vaccine . The responses to CFA/I (P<or=0.001), CS2 (P=0.021), CS4 (P=0.009) and rCTB (P<or=0.001) were elevated in the vaccines in comparison to the pre-immune values and in comparison to those seen in the placebo recipients (P=0.018 to <0.001) . Vaccines but not placebo recipients also showed significantly increased IgM ASC responses to all three CF antigens that were tested (P=0.012 to <0.001) and IgG-ASCs to rCTB (P<0.001) . Peak ASC levels were reached after one dose of the vaccine with no further increase or decrease after the second dose.The vaccine recipients also responded with IgA plasma antibodies to CFA/I, CS1, CS2, CS4 and rCTB after one or two doses of the vaccine (P=0.01 to <0.001) . Subjects in the placebo group failed to mount responses to any of the antigens . The vaccine also induced responses in mucosal IgA antibodies in feces to CFA/I, CS2 and rCTB (61, 88 and 69% responder frequency, respectively) and the magnitude of the response was elevated in comparison to the pre-immune levels (P=0.031 to <0.001) and to the levels of the control group (P=0.003 to <0.001).This study thus shows that the CF-BS-ETEC vaccine is well tolerated in children, 18-36 months of age and gives rise to significant systemic and mucosal IgA antibody responses.

FEMS Immunol Med Microbiol, 2003 May 25, 36(3), 169 - 73
Orogastric vaccination of guinea pigs with Helicobacter pylori sonicate and a high dose of cholera toxin lowers the burden of infection; Durrani Z et al.; Guinea pigs were vaccinated orogastrically with Helicobacter pylori cell sonicate (CS) and 10 microg or 100 microg cholera toxin (CT) or CT only . Nai;ve animals were used as a control . In both experiments, vaccination primed the local IgG and IgA response, irrespective of the CT dose . After challenge, only the group of animals immunised with CS and 100 microg CT had a significantly lower number of H . pylori in the antral region of the stomach, but vaccination did not prevent H . pylori infection . This protective effect was not associated with a switch in IgG subclass, which remained predominantly IgG2 . The levels of specific antibodies in serum and the gastric mucosa which were similar to naive unprotected animals . In conclusion, the ability of mucosal adjuvants such as CT to induce a protective immune response may be host dependent and findings in the Helicobacter-mouse model should be interpreted with caution.

Infez Med, 1998, 6(4), 233 - 248
{Il cholera morbus nel Comune di Bologna nel 1886}; Sabbatani S et al.; Not available

J Virol, 2003 May, 77(10), 5589 - 97
Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins; Albu DI et al.; We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants . It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n . immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes . Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120 . However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6 . Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.

J Med Virol, 2003 Jun, 70(2), 329 - 35
Live varicella vaccine polarizes the mucosal adjuvant action of cholera toxin or its B subunit on specific Th1-type helper T cells with a single nasal coadministration in mice; Sasaki K et al.; This study was undertaken to determine whether the specific Th1- or Th2-cell response to varicella-zoster virus was induced predominantly by a mucosal adjuvant, cholera toxin, in mice . A commercially available live varicella vaccine (Oka strain) and cholera toxin or its B subunit were administered simultaneously via the nasal route . Delayed-type hypersensitivity to the Oka vaccine was induced, but the systemic neutralizing antibody response was low . The delayed-type hypersensitivity evoked after a single administration was relatively higher than that on administration three times . When spleen cells from mice immunized once with the vaccine and cholera toxin or its B subunit were restimulated with the live vaccine in vitro, there was greater thymidine uptake and production of interleukin- 2 (IL-2) than controls, but only a low level of IL-4 production . The production of IL-2 induced by the B subunit of cholera toxin was less than that by cholera toxin and a mutant of Escherichia coli enterotoxin on co-immunization with the vaccine in mice . Cholera toxin and its B subunit have been reported to induce predominantly a specific Th2-type T-cell response to various antigens . However, the Oka vaccine is an antigen that polarizes the activation of specific Th1/Th2-type T cells by cholera toxin or its B subunit to the Th1-type side . Cholera toxin and its B subunit are thus useful mucosal adjuvants for inducing cellular immunity to the Oka vaccine similar to Escherichia coli enterotoxin .

Biochemistry, 2003 Apr 15, 42(14), 4028 - 34
The binding potential between the cholera toxin B-oligomer and its receptor; Cai XE et al.; Employing atomic force microscopy as an in situ molecular force probe, we have measured the binding strength between cholera toxin B-pentamer (ctB) and its membrane receptor, ganglioside G(M1) . By application of the basic principle of the reaction rate theory, key parameters of the ligand-receptor binding potential can be obtained from our data . The potential has a well with a spatial span of about 2.5 A and a depth of at least six times the thermal energy at room temperature . The very short range nature of the binding potential leads to the specificity of the ctB-G(M1) coupling.

Infect Immun, 2003 Apr, 71(4), 2272 - 5
Mucosal immunization with a genetically engineered pertussis toxin S1 fragment-cholera toxin subunit B chimeric protein; Lee SF et al.; A chimeric protein consisting of a divalent pertussis toxin (PT) S1 fragment linked to the cholera toxin (Ctx) A(2)B fragment was constructed . The chimera induced a mucosal immunoglobulin A (IgA) and a serum IgG immune response to PT and CtxB in BALB/c mice following intranasal immunization . The immune sera neutralized PT in vitro . In the mouse model of Bordetella pertussis respiratory infection, the chimera-immunized animals showed a significant reduction in bacterial lung counts (P = 0.01) from that of the sham control group . Thus, a divalent S1 fragment CtxA2B chimera is an immunogenic antigen and can elicit a protective immunity.

Infect Immun, 2003 Apr, 71(4), 1740 - 7
Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development; Eriksson K et al.; Cholera toxin (CT) is a strong mucosal adjuvant for codelivered antigens, whereas its nontoxic B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens . We investigated the effects of CT and CTB on the immunogenicity of in vitro-treated antigen-pulsed dendritic cells (DC) following intravenous injection into mice . Prior to infusion, DC were pulsed for 90 min with either free ovalbumin (OVA), OVA mixed with CT or CTB, or chemical conjugates of OVA with CT and CTB (OVA-CT and OVA-CTB) . DC pulsed with OVA or with OVA and CTB gave rise to modest antibody and T-cell responses . Conjugation of OVA with CTB enhanced both the subsequent B-cell and T-cell responses to OVA and preferentially induced Th2 responses . CT was shown to be a strong adjuvant when it was coadministered to DC with OVA and was even stronger when it was coadministered with OVA-CTB and primed for a mixed Th1-Th2 response . The antibody and T-cell responses were further enhanced if OVA was coupled to CT, implying that CT can utilize a combined carrier and adjuvant function vis-a-vis linked antigens for DC vaccination . The immunopotentiating capacity of CT- and CTB-linked antigen was associated with both upregulated secretion of interleukin-1beta by the pulsed DC and increased expression of CD80 and CD86 on the DC surface . These results imply that CT and CTB can be used to both markedly increase and partially direct the DC vaccine-induced immune response with respect to Th1 and Th2 responses, which has obvious implications for DC-based vaccine development.

Neuroscience, 2003, 117(4), 831 - 45
Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit; Wu CC et al.; The present study examined the efficiency of fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylinodocarbocyanine perchlorate and cholera toxin B subunit in tracing the crossed tectal projection to the nucleus rotundus of the thalamus (tectorotundal pathways) of paraformaldehyde-fixed and living chick embryos . The tracers were injected into the optic tectum under three experimental conditions (carbocyanine postfix, carbocyanine in vivo, and cholera toxin B subunit in vivo) and the anterograde transport of the nucleus rotundus was monitored and compared.In the carbocyanine postfix method, small crystals of carbocyanine dye were inserted into the tectum of paraformaldehyde-fixed embryos . A 6-month post-insertion period was required to label the crossed tectorotundal pathway . Results showed that tectal neurons did not begin to innervate the ipsilateral nucleus rotundus until embryonic day 9 and the contralateral nucleus rotundus until embryonic day 17 . This slow progression of labeling through the crossed tectal projection resulted in significant contrast of the labeling between the ipsilateral and contralateral nuclei rotundus.In the carbocyanine in vivo method, a small volume of carbocyanine dye solution was injected into the tectum of living embryos . A 8- to 12-h survival period was sufficient enough to label the tectorotundal pathway . By embryonic day 8, the labeled axons terminated in the ipsilateral nucleus rotundus and the crossed tectorotundal projection was first detected by embryonic day 10 . Similarly, in the cholera toxin B subunit in vivo method, a small volume of cholera toxin B subunit solution was injected into the tectum of living embryos . After a 6- to 10-h survival period, heavily labeled axons were found to innervate bilaterally the nucleus rotundus by embryonic day 8 . This appeared to be the earliest schedule for detecting the crossed tectorotundal projection, compared with that of both the postfix and in vivo methods of carbocyanine dye.Based on the differences in the detectability of the crossed tectorotundal projection between the postfix and in vivo methods, the present data suggest that the former method is of limited purpose for labeling tectal collaterals during embryogenesis . Moreover, given the rapid transport rate and absence of photobleaching, which is often seen when using carbocyanine dye, the cholera toxin B subunit in vivo method appears to be the tracer of choice for investigating embryonic pathways.

Vaccine, 2003 Apr 2, 21(15), 1613 - 9
Induction of cytotoxic T lymphocyte responses by cholera toxin-treated bone marrow-derived dendritic cells; Jang MH et al.; Cholera toxin (CT), a powerful mucosal adjuvant, is a potent inducer of Th2-type responses via activation of co-stimulatory molecules for the induction of IgA antibody responses . Less appreciated is the ability of CT to induce and regulate cytotoxic T lymphocyte (CTL) responses . In order to help for clarifying mechanisms underlying the CTL-inducing ability of CT, we have examined the effects of CT on dendritic cells (DCs) that could lead to the induction of cytotoxic CD8(+) T cells . When bone marrow-derived DCs (BM-DCs) were cultured with CT in vitro, B7-1 but not B7-2 molecules were significantly enhanced and allogenic CTL responses were induced . Also, increased numbers of IFN-gamma-secreting CD8(+) T cells were elicited when CT-treated BM-DCs were co-cultured with allogenic CD8(+) CTLs . Antibody blockade of B7-1 on CT-treated BM-DCs suppressed allogenic CTL responses, further indicating the importance of CT-induced B7-1 molecules on DCs for the acquisition of cytolytic function by CTL precursors . CD40 signaling was proven not necessary for the CT-induced CTL response since CT-treated CD40(-/-) BM-DCs developed CTL responses equivalent to those detected in CT-treated BM-DCs derived from normal mice . Our results suggest that CT-treated DCs are effective inducers of CD8(+) CTL, and this induction is mediated through CT's ability to enhance B7-1 expression on DCs.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Jan-Feb, (1), 96 - 102
{Features of the epidemiology of modern cholera}; Ziatdinov VB; In this review information on the spread of cholera in the world, embracing the period of the XIX and XX centuries, is presented . The detailed description of the pandemic character of cholera, including all seven pandemics, is given . The epidemiological situation in cholera is analyzed and the conclusion is made that the main regularity of the epidemic process in modern EI Tor cholera are the endemic character of the disease and its epidemic character due to the prolonged and continuous course of pandemic VII . The work points out that the epidemic process on the global level, as well as on the regional and local levels, is regulated mainly by growing activation of the international and internal movement of the population.

Eur J Immunol, 2003 Jan, 33(1), 224 - 32
Prevention of mucosally induced uveitis with a HSP60-derived peptide linked to cholera toxin B subunit; Phipps PA et al.; Oral administration of the uveitogenic peptide (aa 336-351) derived from human HSP60 induced clinical and histological manifestations of uveitis in 65.8% (48/73) of Lewis rats . Uveitis was significantly decreased to 16.7% (11/66) in parallel experiments with the peptide linked to recombinant cholera toxin B subunit (rCTB), also given by mouth (chi(2)=34.2, p<0.0001) . The protective efficacy between tolerized and immunized animals was 74.7% . Adoptive transfer of mesenteric lymph node cells from tolerized rats prevented the development of uveitis . A significantly higher proportion of regulatory CD4(+)CD45RC(low)RT6(+) subset of Th2 memory cells were found in the mesenteric lymph nodes (p<0.005) and spleens (p</=0.05) of tolerized rats without uveitis, as compared with immunized rats and uveitis . In situ hybridization studies of mesenteric lymph nodes and/or the uveal tract showed significant increases in IL-10 and TGF-beta mRNA but decreases in IFN-gamma and IL-12 mRNA in tolerized, as compared with immunized animals . Thus, the mechanism of tolerance, preventing the development of uveitis may involve a regulatory subset of memory cells and a shift from Th1 to Th2 and Th3 cytokines . We suggest that mucosally induced uveitis can be prevented by oral administration of the peptide-rCTB conjugate.

Brain Res, 2003 Feb 28, 964(2), 218 - 27
Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury; Shehab SA et al.; In order to investigate whether cholera toxin B subunit (CTb) is transported by unmyelinated primary afferents following nerve injury, we transected the sciatic nerves of six rats, and injected the transected nerves (and in three cases also the intact contralateral nerves) with CTb, 2 weeks later . The relationship between CTb and two neuropeptides, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), was then examined in neurons in the ipsilateral L4 and L5 dorsal root ganglia, using immunofluorescence staining and confocal microscopy . We also immunostained sections of spinal cord and caudal medulla for CTb, NPY and VIP . Following nerve section, VIP immunoreactivity was increased in laminae I-II of the spinal cord while NPY immunoreactivity was increased in laminae III-IV of the spinal cord and in the gracile nucleus . On the contralateral side, CTb labelling was detected in laminae I and III-V of the dorsal horn of the L4 and L5 spinal segments, as well as in the gracile nucleus . CTb labelling was seen in the same areas on the lesioned side, but with a dramatic increase in lamina II . No VIP or NPY immunoreactivity was observed in L4 and L5 dorsal root ganglia on the side of the intact nerve, but on the lesioned side VIP was detected in many small neurons and NPY in numerous large neurons . In agreement with the report by Tong et al . {J . Comp . Neurol . 404 (1999) 143}, we found that while CTb labelling in the dorsal root ganglion on the side of the intact nerve was mainly in large neurons, on the lesioned side CTb was present in dorsal root ganglion neurons of all sizes . The main finding of the present study was that almost all of the VIP- (96%) and NPY- (98%) positive neurons in the dorsal root ganglia on the lesioned side were also CTb-labelled . After nerve injury VIP is upregulated in fine afferents that terminate in laminae I and II, and most of these probably have unmyelinated axons . Since the cell bodies of these neurons were labelled with CTb that had been injected into the transected sciatic nerve, this suggests that many of these fine afferents, which do not normally transport CTb, are capable of doing so after injury.

J Commun Dis, 2001 Dec, 33(4), 282 - 5
Epidemiology of cholera--a five year study; Bajaj JK et al.; A total of 286 strains of Vibro Cholerae were isolated and tested over a period of five years . The strains were identified by standard methods and confirmed by slide agglutination tests with polyvalent, Ogawa and Inaba antisera . The non-agglutinating strains were tested with O-139 antisera . The maximum number of cases were found in the age group of 0-10 years . The number of females affected was more than the males . V . cholerae O-139 was isolated in the year 1998 and then again in 2000 . V . cholerae serotype Inaba was found only in the year 1999 . All of the other isolates belonged to the serotype Ogawa . The periodic shift between O1 and O-139 serogoups is reminiscent of the shifts from the Ogawa to the Inaba serotypes periodically witnessed among V . cholerae, possibly mediated by the immune pressure in the population.

Sheng Wu Gong Cheng Xue Bao, 2002 Sep, 18(5), 605 - 8
{Cloning of the major antigen region of E2 gene of hog cholera virus and expression in Escherichia coli}; Zhang YG et al.; The major antigen region of E2 gene of Hog Cholera Prevalent Strain (Guangxi Yuling Strain) and Chinese Hog Cholera Lapinised Virus (C-strain) derived from hog and rabbit spleen tissue, was amplified by reverse transcription polymerase chain reaction(RT-PCR) and the nested Polymerase Chain Reaction (nPCR) . After the amplified fragments were cloned into the expression vector pPROEX-HTb, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained . The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis . SDS-PAGE indicated that both of the reciepient germs transducted and induced by the recombinant plasmids pPROEX-GXYL and pPROEX-C could express the major antigen region of E2 gene . Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of CSFV.

Vaccine, 2003 Mar 7, 21(11-12), 1165 - 73
Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant; Isaka M et al.; To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant . At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant . However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung . These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous . Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd . These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before .

Wei Sheng Wu Xue Bao, 1999 Dec, 39(6), 554 - 8
{Molecular clone and sequence analysis of cDNA fragments of hog cholera virus strain C}; Li H et al.; The 5' nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half--nested PCR . The PCR products were cloned into pGEM-T vector . Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators . The obtained sequences on 5' noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann's lab . The result showed that the homology between HCV strains C sequenced in this report and in Moormann's lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69% . It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann's lab et al.

Wei Sheng Wu Xue Bao, 2001 Jun, 41(3), 320 - 8
{Sequencing of E2 gene and comparison analysis of four strains hog cholera virus (HCV)}; Wang Q et al.; Four cDNA fragments of envelope glycoprotoin E2 gene of SM strain, HCLV strain, F03 strain and F07 strain were amplified respectively with RT-PCR method . The amplified E2 fragments of four HCV strains were all 1273 bp in length by agarose gel electrophoresis . Four E2 fragments were cloned respectively into pGEM-T easy vector . 1273 bp cDNA fragment of Four HCV E2 gene were sequenced and 381 residues amino acid sequence of E2 glycoprotein were deduced . The signal peptide sequence (WLLLVTGA) located in the N-terminal residues 683-690 and the transmenbrane region (TMR) sequence located in the C-terminal residues 1031-1063 of Four E2 gene were highly conserved and hydrophobic region . The conserved sequence among the E2 protein of pestiviruses, RYLASLH which located in the N-terminal residues 753-759 of domains B and C, was hydrophilic, and none of them were variable among the pestiviruses, and the greatest values of antigenic in all E2 antigenic domain . This suggest that the conserved sequence RYLASLH are involved in E2 epitopes . The number and locations of 15 cysteine residues in E2 are conserved among pestiviruses, suggesting that the structure of this glycoprotein is similar in pestiviruses and the first six cysteines are critical for the correct folding of E2 and essential for all identified epitopes . It is showed epitopes . between HCLV strains and field strains are not variable obviously by analysising the varintion of some main amino acid residuse substitutions of E2 major antigenic domains.

Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 614 - 21
{Nucleotide sequence analysis of E2 major protective antigen encoding region of 12 strains of hog cholera virus(HCV)}; Wang Q et al.; cDNA fragments, of HCV envelope glycoprotein E2 major gene of 11 field strains isolated in China in different time and 1 French reference strain(Thiveral) were amplified respectively with RT-PCR method and sequenced . The fragments amplified located by the 5' 2485 to 2708 of E2 major domains B and C and encoded 75 amino acid residues of E2 glycoprotein . All the products amplifield by RT-PCR from 12 strains in the study were same size of 224 bp . Comparing 12 sequences with other 9 references strains sequence reported before using software DNAstar, it was found that Hog Cholera Virus could be classified in two groups by analysis of phylogenetic tree . Strains Brescia, Gpe, Ald, Thiverval, C, CW, HCLV, HCVSM, BJCY1/96, BJTX3/96, BJSY2/96, HeNXH2/98, HeNZZ1/82 and GDGZ1/95 were assigned to group A and were 85.7%-100% for nucleotide sequence and 83.8%-100% for amino acid sequence in homology; but strains HCVF98, HCVF94, HeBHH2/95, LN1/84, SZGM1/85, SCCD1/79 and Alfort were assigned to group B and were 84.3%-100% for nucleotide sequence and 85.1%-100% for amino acid sequence in homology; and 21 strains of HCV were 78.1%-100% for nucleotide sequence and for 78.4%-100% amino acid sequence in homology . Homology were 99.1% for nucleotide sequence and 100% for amino acid sequence between strains HCLV in our study and strains C reported by Rijn's in the Netherlands . It's showed our method of sequencing is reliable . There were obvious differences between the two groups in sequences of envelope glycoprotein E2 major gene, especially in the amino acid substitutions of sites 713 and 729 respectively, and it is showed the two groups of HCV field strains might vary genetically in some extents . The results of other report of challenging of the partial field strains showed that the Chinese stock vaccine virus(HCLV) has good immunity.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 32 - 7
{Cloning and sequence analysis of E0 gene of hog cholera virus lapinized Chinese strain and virulent shimen strain}; Wang J et al.; According to the published nucleotide sequences of genome of hog cholera virus, one pair of specific primers were designed and synthesized . From the spleen of rabbits which were infected with HCLV and HCV Shimen strain infected pig blood, the two E0 genes were amplified by RT-PCR . The amplified fragments were cloned into pGEM-T vector and sequenced . Sequence analysis showed nucleotide sequence and deduced amino acid sequence homologies of the E0 gene between HCLV and Shimen strain were 95.0% and 94.3% . There was 13 amino acid substitutions between them . One N-glycosylation site was missing from E0 gene of the Shimen strain . The nucleotide sequence homologies of the Shimen strain with the ALD, GPE-, Brescia and Alfort strains were 97.4%, 96.5%, 92.2% and 86.5%, respectively, the deduced amino acid sequence homologies were 97.4%, 96.0%, 95.2% and 92.5%, respectively . The nucleotide sequence homologies of E0 gene of the HCLV strain with the ALD, GPE-, Brescia and Alfort strains were 95.6%, 94.9%, 91.3% and 85.5%, respectively, the deduced amino acid sequence homologies were 93.4%, 92.5%, 91.6% and 90.7%, respectively . E0 gene from HCV was shown to be similar to a family of fungal and plant ribonuclease . The catalytically important residues were 28 to 40 and 71 to 89 of HCV E0 gene.

Vaccine, 2003 Feb 14, 21(9-10), 856 - 61
Maturation of human dendritic cells induced by the adjuvant cholera toxin: role of cAMP on chemokine receptor expression; Gagliardi MC et al.; Cholera toxin (CT) is a very effective adjuvant for mucosal vaccination . It binds to cells through its B subunit and induces intracellular increase of cAMP through the A subunit . We previously showed that CT induces maturation of human dendritic cells (DCs) and this may account for its adjuvant property . Here, we investigated the role of the A subunit on DCs maturation by using forskolin, a cAMP inducer . The results show that although cAMP does not stimulate full maturation of DCs it induces upregulation of the chemokine receptors CXCR4 and CCR7.

J Immunol, 2003 Feb 1, 170(3), 1586 - 92
Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses; Anjuere F et al.; Topical application of cholera toxin (CT) onto mouse skin can induce a humoral immune response to CT as well as to coadministered Ags . In this study, we examined the nontoxic cell-binding B subunit of CT (CTB) as a potential adjuvant for cutaneous immune responses when coadministered with the prototype protein Ag, OVA . CTB applied onto skin induced serum Ab responses to itself with magnitudes comparable to those evoked by CT but was poorly efficient at promoting systemic Ab responses to coadministered OVA . However, transcutaneous immunization (TCI) with either CT or CTB and OVA led to vigorous OVA-specific T cell proliferative responses . Furthermore, CTB potentiated Th1-driven responses (IFN-gamma production) whereas CT induced both Th1 and Th2 cytokine production . Coadministration of the toxic subunit CTA, together with CTB and OVA Ag, led to enhanced Th1 and Th2 responses . Moreover, whereas TCI with CT enhanced serum IgE responses to coadministered OVA, CTB suppressed these responses . TCI with either CT or CTB led to an increased accumulation of dendritic cells in the exposed epidermis and the underlying dermis . Thus, in contrast to CT, CTB appears to behave very differently when given by the transcutaneous as opposed to a mucosal route and the results suggest that the adjuvanticity of CT on Th1- and Th2-dependent responses induced by TCI involves two distinct moieties, the B and the A subunits, respectively.

Med Trop (Mars), 2002, 62(4), 361 - 7
{Management of a cholera epidemic by a humanitarian organization}; Piarroux R; Far from disappearing with modern progress, cholera outbreaks have never been more common than in the last decade . Because of its close relationship with poverty, malnutrition, poor access to health care, warfare, and sometimes natural disaster, cholera is a major priority for action by doctors from humanitarian organization . In this article a brief overview about cholera is followed by an account of one non-governmental organization's approach to management of a cholera outbreak . The author describe the circumstances surrounding the alert and the fact-finding mission focused on documenting the presence of an epidemic, measuring its magnitude, and predicting its outcome . The next phase of management involved response planning with development of a control strategy and raising money for intervention, which is often difficult for epidemics not reported in the media . The control modalities were then deployed in the field including assisting patient management, preventing transmission, training personnel, supervising activities, coordinating tasks performed by different actors, and finally program evaluation . This experience shows that successful cholera management programs can be conducted in a professional manner by teams composed essentially of volunteers under the supervision of a knowledgeable staff able to adapt general control techniques to the specific conditions encountered in the field.

Vaccine, 2003 Jan 17, 21(5-6), 376 - 85
A modified cholera holotoxin CT-E29H enhances systemic and mucosal immune responses to recombinant Norwalk virus-virus like particle vaccine; Periwal SB et al.; In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine . This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses . We observed that a low dose of NV-VLP (5 microg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 microg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses . Higher counts of antigen specific IgA secreting cells were observed in the Peyer's Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H . Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant . Delivery of the vaccine by the oral route results in antigen specific CD4(+) and CD8(+) T cells in PP and spleen . Addition of CT-E29H results in an increase of antigen specific CD4(+) cell population in PP and both CD4(+) and CD8(+) populations in the spleen . These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response . Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.

J Nutr, 2003 Jan, 133(1), 333S - 335S
Tropical enteritis: nutritional consequences and connections with the riddle of cholera; Rosenberg IH; One of the important consequences of the infection-nutrition interaction is mediated by malabsorption associated with chronic inflammation in the intestine, enteritis . Studies made possible after development of the peroral intestinal biopsy technique in the 1950s indicated the wide prevalence of enteropathy, particularly in tropical developing countries with poor levels of sanitation . Some consider this so-called subclinical tropical malabsorption to be the base of an iceberg, whose tip is tropical sprue, a severe form of malabsorption leading to nutritional deficiency that had been reported in colonial expatriates in tropical countries for 200 y . Some of the first demonstrations of the prevalence of tropical enteritis in Asia were made in quest of the pathologic lesion of cholera, and further examination of the water and electrolyte, as well as nutrient, malabsorption in cholera led serendipitously to the discovery of the oral rehydration solution for the treatment of diarrheal disease.

J Immunol, 2003 Jan 1, 170(1), 454 - 62
Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit; Boyaka PN et al.; Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins . Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes . Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses . To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed . Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT . Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity . The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-gamma and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs . Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-gamma . The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells . Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.

J Immunol, 2003 Jan 1, 170(1), 55 - 63
Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant; Gardby E et al.; In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA . Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses . The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice . This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes . Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice . Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches . By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28 . However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor . Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events . Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.

J Assoc Res Otolaryngol, 2002 Dec, 3(4), 457 - 78 Epub 2002 Apr 09.
Organization of olivocochlear neurons in the cat studied with the retrograde tracer cholera toxin-B; Warr WB et al.; We employed cholera toxin-B (CTB), an efficient retrograde tracer, to examine olivocochlear (OC) neurons in the cat . Our primary goals were (1) . to determine whether the cat has two types of lateral OC (LOC) neurons as is found in certain rodents and (2) . to document the morphology, number, and caudorostral distribution of OC neurons, bilaterally . Adult cats received injections of CTB through the round window of the left cochlea, and, after 3-6 days, the brains were sectioned transversely and CTB was revealed immunocytochemically in every section . In three cats, OC neurons were mapped, counted differentially according to cell group, and the numbers of each plotted bilaterally from caudal to rostral . In one cat, measurements were made on labeled LOC and medial OC (MOC) neurons . The results indicate that LOC neurons can be divided into two groups based on their proximity to the lateral superior olive (LSO): a more populous group of small neurons that have intimate contact with the LSO, designated marginal-LOC neurons, and a less populous, morphologically heterogeneous group, lying more distantly from the LSO, designated para-LOC neurons . Para-LOC neurons lying dorsal and rostral to LSO were significantly larger than marginal-LOC . We hypothesize that the cat marginal-LOC neurons and most probably the larger para-LOC neurons correspond to rodent intrinsic and shell LOC neurons, respectively, which have focal versus diffuse projections beneath the inner hair cells . Concerning MOC neurons, we confirm and extend previous observations on the clustering of these neurons near the rostral tip of the medial superior olivary nucleus and also show that MOC neurons differ in size according to cell group . Finally, we compare the present counts of OC neurons (mean total 1607, consisting of 1058 LOC neurons and 549 MOC neurons innervating one cochlea) and their proportional distribution ipsilaterally and contralaterally with those reported previously . Our estimate of the number of LOC neurons is somewhat higher than those previously obtained either by retrograde labeling with horseradish peroxidase or by counting unmyelinated axons in the olivocochlear bundle . In contrast, our estimate of the number of MOC neurons is very similar to those previously reported.

Exp Neurol, 2002 Nov, 178(1), 139 - 46
Widespread dispersal of cholera toxin subunit b to brain and spinal cord neurons following systemic delivery; Alisky JM et al.; We have discovered novel transport properties of cholera toxin subunit b beyond well-known anterograde and retrograde axonal transport . Injection of 1500 microg of CTb intraperitoneally or intravenously in young adult mice resulted in generalized enhanced labeling of motor nuclei at all levels of the brain stem and spinal cord (oculomotor, trochlear, abducens, facial, trigeminal, vagal, hypoglossal, cervical, and lumbar) . There was also extensive labeling of trigeminal and spinal primary afferent fibers, bulk labeling of the area postrema, and finally numerous labeled neurons in the periventricular and supraoptic hypothalamic nuclei . Generalized labeling of motor, sensory, and hypothalamic neurons could also be produced on a more limited scale from intramuscular injections of 500 microg of CTb in the tongue . Neuronal uptake of peripherally administered CTb may be useful as a research tool, or, when fused to therapeutic peptides, enzymes, growth factors, or gene therapy vectors, may have application in amyotrophic lateral sclerosis, diabetic neuropathy, motor neuronopathic lysosomal storage diseases, and other neurodegenerative disorders.

EMBO Rep, 2002 Dec, 3(12), 1222 - 7 Epub 2002 Nov 21.
Role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation; Rodighiero C et al.; Cholera toxin travels from the cell surface of affected mammalian cells to the endoplasmic reticulum (ER), where the A1 chain is released and retro-translocated across the ER membrane into the cytosol . We have tested whether, as in other cases, retro-translocation requires poly-ubiquitination . We show that an A1 chain mutant that lacks lysines and has a blocked N-terminus, and therefore cannot be ubiquitinated, remains active in vivo . The A1 chain is not degraded in the cytosol, as demonstrated by the fact that proteasome inhibitors do not stimulate its activity . When additional lysines are introduced into the A1 chain, moderate degradation by the proteasome is observed . The unfolded A1 chain rapidly refolds in vitro . These results show that poly-ubiquitination is not required for retro-translocation of all proteins across the ER membrane and indicate that the reason why the toxin escapes degradation in the cytosol may be both its paucity of lysines and its rapid refolding.

Parasite Immunol, 2002 Aug, 24(8), 423 - 7
Diminished immunopathology in Schistosoma mansoni infection following intranasal administration of cholera toxin B-immunodominant peptide conjugate correlates with enhanced transforming growth factor-beta production by CD4 T cells; Hernandez HJ et al.; In Schistosoma mansoni infection, CD4 T cells specific for parasite egg antigens mediate perioval granuloma formation in the liver and intestines . Mice of the CBA strain develop a severe form of disease and a significant proportion of their CD4 T cell response is directed against the major egg antigen Sm-p40 and its immunodominant T cell epitope peptide 234-246 . Here, we show that intranasal (i.n.) treatment of infected CBA mice with a fusion protein of the cholera toxin B subunit (CTB) with peptide 234-246 (CTB::pep) results in significant down-modulation of hepatic granulomatous inflammation and fibrosis . Moreover, egg antigen-stimulated dispersed hepatic granuloma cells, as well as mesenteric lymph node CD4 T cells from the CTB::pep-treated mice, produced significantly more transforming growth factor (TGF)-beta than that produced by treated or untreated controls . The data demonstrate that i.n . administration of a single immunodominant peptide conjugated to CTB can lead to down-regulation of the hepatic immunopathology associated with schistosomiasis, and that this down-regulation is, at least in part, mediated by TGF-beta.

J Am Chem Soc, 2002 Nov 6, 124(44), 12991 - 8
Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin; Zhang Z et al.; The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors . The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry . Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand . The largest of these ligands has a molecular weight of 10.6 kDa . These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin . A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand . This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand . Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation . This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state . These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

J Cell Biol, 2002 Oct 28, 159(2), 207 - 16 Epub 2002 Oct 28.
Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1; Tsai B et al.; The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain . This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol . The polypeptide is initially unfolded by binding to the reduced form of PDI . We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released . Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction . Ero1 has a preference for the PDI-toxin complex . We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1 . Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.

East Afr Med J, 2002 Mar, 79(3), 150 - 5
Combating cholera epidemics by targeting reservoirs of infection and transmission routes: a review; Nyamogoba HD et al.; OBJECTIVES: To determine the parameters which can be investigated for prevention and effective control of cholera . DATA SOURCES: Literature search on compact disk-read only memory (CD-ROM), medline and internet, using the key words: cholera outbreaks, and cholera transmission . A few reviews were manually reviewed . STUDY SELECTION: Relevant studies or articles on cholera outbreaks and transmission worldwide, with special reference to Kenya is included in the review . DATA EXTRACTION: From individual study or articles . DATA SYNTHESIS: Information on cholera epidemics worldwide and in Kenya is synchronized under the headings; Introduction, History and predisposing factors, Current situation, Bioecology and transmission patterns, and, Use of molecular epidemiological and geographic information system (GIS) techniques in mapping out the bioecology, reservoirs and transmission routes of cholera . CONCLUSION: Cholera can be prevented and controlled more effectively at environment level . This requires a multi-disciplinary approach including poverty alleviation.

Infect Immun, 2002 Nov, 70(11), 6166 - 71
Transfer of the cholera toxin A1 polypeptide from the endoplasmic reticulum to the cytosol is a rapid process facilitated by the endoplasmic reticulum-associated degradation pathway; Teter K et al.; The active pool of internalized cholera toxin (CT) moves from the endosomes to the Golgi apparatus en route to the endoplasmic reticulum (ER) . The catalytic CTA1 polypeptide is then translocated from the ER to the cytosol, possibly through the action of the ER-associated degradation (ERAD) pathway . Translocation was previously measured indirectly through the downstream effects of CT action . We have developed a direct biochemical assay for CTA1 translocation that is independent of toxin activity . Our assay is based upon the farnesylation of a CVIM motif-tagged CTA1 polypeptide (CTA1-CVIM) after it enters the cytosol . When expressed from a eukaryotic vector in transfected CHO cells, CTA1-CVIM was targeted to the ER, but was not secreted . Instead, it was translocated into the cytosol and degraded in a proteosome-dependent manner . Translocation occurred rapidly and was monitored by the appearance of farnesylated CTA1-CVIM in the detergent phase of cell extracts generated with Triton X-114 . Detergent-phase partitioning of CTA1-CVIM resulted from the cytoplasmic addition of a 15-carbon fatty acid farnesyl moiety to the cysteine residue of the CVIM motif . Our use of the CTA1-CVIM translocation assay provided supporting evidence for the ERAD model of toxin translocation and generated new information on the timing of CTA1 translocation.

Toxicon, 2002 Oct, 40(10), 1487 - 94
Pro-inflammatory effects of cholera toxin: role of tumor necrosis factor alpha; Viana CF et al.; Cholera toxin has been traditionally described as the one that does not induce inflammation . It has, however, potent adjuvant and immuno-modulatory activities . Since the adjuvanticity of other compounds is linked to their capacity to induce inflammation, in the present study the pro-inflammatory activity of cholera toxin was investigated . We studied this activity in the following rat models of inflammation: paw edema and neutrophil migration into the peritoneal cavity, and evaluated cholera toxin's effect on tumor necrosis factor alpha (TNF-alpha) production by mouse macrophages . We, also, explored the effects of dexamethasone (DEXA) and of two inhibitors of TNF-alpha production, thalidomide (TAL) and pentoxifylline, on paw swelling . Cholera toxin-induced significant and dose-dependent paw edema, which peaked 48 h after toxin challenge (Cholera toxin(2.5 microg): 2.39 +/- 0.22 ml) . Cholera toxin B subunit did not show edematogenic activity . DEXA, TAL and pentoxifylline significantly reduced cholera toxin-induced edema (DEXA(0.5 mg/kg): 42.6% of inhibition; TAL(45 mg/kg): 36% of inhibition; pentoxifylline (45 mg/kg): 61% of inhibition) . Neither cholera toxin nor its B subunit induced neutrophil migration into peritoneal cavities . Cholera toxin stimulated the release of TNF-alpha by macrophages (cholera toxin(10 microg): 11.46 +/- 0.44 UI/ml) . These data provide evidences that cholera toxin exhibits significant pro-inflammatory activity . It also indicates the role of TNF-alpha upon the pathophysiology of this event based on the inhibitory action of DEXA, TAL and pentoxifylline, and on TNF-alpha secretion induced by cholera toxin .

FEBS Lett, 2002 Oct 2, 529(1), 49 - 53
Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin; Sandvig K et al.; Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action . Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER . The toxins can also be used as vectors to carry other proteins into the cells . Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.

Anal Chem, 2002 Sep 15, 74(18), 4763 - 73
Probing antigen-antibody binding processes by impedance measurements on ion-sensitive field-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors; Zayats M et al.; Impedance measurements on ISFET devices are employed to develop new immunosensors . The analysis of the transconductance curves recorded at variable frequencies, upon the formation of antigen-antibody complexes on the ISFET devices, allows determination of the biomaterial film thicknesses . Complementary surface plasmon resonance measurements of analogous biosensor systems, using Au-coated glass slides as support, reveal similar film thicknesses of the biomaterials and comparable detection limits . A dinitrophenyl antigen layer is immobilized on the ISFET gate as a sensing interface for the anti-dinitrophenyl antibody (anti-DNP-Ab) . The anti-DNP-Ab is analyzed with a sensitivity that corresponds to 0.1 microg mL(-1) . The assembly of the biotinylated anti-anti-DNP-Ab and avidin layers on the base anti-DNP-Ab layer is characterized by impedance measurements . The development of an ISFET-based sensor for the cholera toxin is described . The anti-cholera toxin antibody is immobilized on the ISFET device . The association of the cholera toxin (CT) to the antibody is monitored by the impedance measurements . The detection limit for analyzing CT is 1.0 x 10(-11) M.

Moyo, 1980 Apr, 12(1), 3 - 7
Simple and effective treatment of acute dehydrating diarrhoeas including cholera; Mkandawire AC; PIP: General treatment for diarrhea, including infantile and cholera-related diarrhea, is discussed . Prompt and complete replacement of water and electrolytes lost through diarrhea must be the aim of all treatment . To do this, a solution of electrolytes and glucose is given either orally or intravenously, depending on the severity of the diarrhea-caused dehydration . To avoid misdiagnosis, a brief physical examination and history must be taken beforehand . Symptoms useful in distingushing mild from severe dehydration are listed . Severe dehydration includes extreme weakness, even unconsciousness in children, a weak and fast pulse, sunken eyes, and cool skin . Intravenous treatment should be started at once in such cases . Method of administration and dosage quantities for different weights of patient are given . The necessary ingredients for oral rehydration therapy should be available at all treatment centers, including rural dispensaries . Rehydration treatment should be continued until diarrhea stops . A normal diet should also be continued if possible .

QA Brief, 1994 Winter, 3(3), 32 - 5
QA methods improve quality of cholera / acute diarrhea care in local health districts in Ecuador; Hermida J et al.; PIP: The Ministry of Health (MOH) in Ecuador in 1993 received Quality Assurance Program (QAP) technical assistance in initiating a demonstration project aimed at applying quality assurance methods to the case management of cholera and acute diarrhea . The purpose of the project was to demonstrate the feasibility of attaining objective improvements in the quality of care of cholera/acute diarrhea patients at the health district level, to train management teams to use quality assurance (QA) methods and techniques, and to institutionalize QA as part of routine management after the end of the project . The health area of Babahoyo with its 80-bed hospital and eleven health centers, and the health area of La Troncal which includes twelve health centers were selected to participate in the project's activities . Both health areas are located in the coastal region of Ecuador and serve the mostly poor peasant population . This article reports on QA experiences working in nineteen ambulatory health centers of the two health areas . The intervention model provides the health areas with a technical approach and basic tools which will allow health workers and managers to systematically examine the quality and quantity of their work, identifying weaknesses and attaining improvements .

Vaccine, 2002 Oct 4, 20(29-30), 3443 - 55
Characterization of protective immune responses induced by nasal influenza vaccine containing mutant cholera toxin as a safe adjuvant (CT112K); Watanabe I et al.; Immune responses induced by a nasal influenza vaccine with a mutant cholera toxin (CT112K), known to be a safe adjuvant, were characterized in BALB/c mice to confirm the most suitable regimen of this vaccine for humans . Mice received a primary intranasal administration of the adjuvant (0.1 micro g)-combined PR8 vaccine (0.1 micro g) and a secondary administration of the PR8 vaccine alone (0.1 micro g) 4 weeks later . Two weeks after the secondary immunization, the mice were infected with a nonlethal or a lethal dose of PR8 viruses . Nasal and lung wash virus titers 1 or 3 days after infection indicated that complete protection could be provided by secondary immune responses, which had an immediate effect of preventing infection 2 weeks after the secondary immunization . In this two-dose regimen, high levels of secondary IgA, IgG and IgM antibody-forming cell (AFC) responses were induced in the nasal-associated lymphoid tissue and the spleen . In parallel with the AFC responses, high levels of nasal wash anti-PR8 HA IgA, and lung and serum IgG antibody (Ab) responses were induced 2 weeks after the secondary immunization . The two-dose regimen also induced accelerated delayed-type hypersensitivity responses, which exhibited almost the same peak height as that in the case of the primary response . In addition, the two-dose regimen induced a low memory cell activity of cytotoxic T lymphocytes, detected by in vitro culture of spleen cells . Thus, the immediate effect of preventing infection was mainly provided by the secondary Ab responses . Moreover, the levels of nasal wash IgA Abs correlated well with cross-protection against infection with variant viruses in the upper respiratory tract (RT) . These results suggest that the major protective factors among Ab and T cell-mediated immune responses, which are induced by the two-dose regimen using CT112K-combined vaccines, are the cross-reactive IgA Abs in the upper RT and the less cross-reactive IgG Abs in the lower RT, and that the two-dose regimen is a suitable vaccination condition for humans.

Afr Health . 1998 Nov;21(1):43.
Cholera in Turkana.
{The cholera epidemic of 1833 and mortality in Mexico City}
Velasco MD.

PIP: The author examines the impact of the 1833 cholera epidemic in Mexico City, Mexico, on social, economic, and political aspects of life in that city . She finds that some five percent of the population died during the epidemic, and enumerates them by age and sex .

Estud Demogr Urbanos Col Mex, 1992 Jan-Apr, 7(1), 77 - 93
{Cholera in Mexico City during the nineteenth century}; Marquez Morfin L; PIP: The author draws on epidemiological and historical records for this description of the demographic impact of the fatal cholera epidemics of 1833 and 1848-1850 on the population of Mexico City, Mexico . Consideration is given to political, economic, and social factors that influenced the spread of the disease .

Bol Asoc Demogr Hist, 1992, 10(2), 87 - 111
{Morbidity and mortality from the cholera epidemic of 1833-1835 in Andalusia}; Rodriguez Ocana E; PIP: The author examines the demographic impact of the Asiatic cholera epidemic from 1833 to 1835 in Andalusia, Spain, using archival medical reports . Data on mortality by region, sex, and age are included, and some worldwide comparisons among cities that experienced the 1830s epidemic are made .

Brain Res, 2002 Oct 4, 951(2), 209 - 17
Enhancement of NGF- and cholera toxin-induced neurite outgrowth by butyrate in PC12 cells; Suzuki-Mizushima Y et al.; It has been shown that sodium butyrate (NaBu) does not elicit neurite outgrowth of PC12, one of the most widely used cell lines as a model of neuronal differentiation . In this study, the effects of NaBu on nerve growth factor (NGF)- and cholera toxin-induced neurite outgrowth in PC12 cells were examined . NaBu dose-dependently enhanced neurite formation induced by both agents . The maximum responses obtained at 0.5 mM NaBu were nearly twice those of the inducers alone . Propionate and valerate were also effective, but acetate and caproate were ineffective . Among the butyrate analogs with a moiety of three to five carbon atoms tested, isobutyrate, isovalerate, vinylacetate and 3-chloropropionate enhanced neurite outgrowth promoted by both inducers . However, neither alpha-, beta-, and gamma-aminobutyrates nor alpha-, beta-, and gamma-hydroxybutyrates were effective . All of the effective short-chain fatty acids and their analogs increased the level of histone acetylation, while ineffective ones did not . Furthermore, Helminthosporium carbonum toxin (HC toxin), a structurally dissimilar inhibitor of histone deacetylase, mimicked the effect of butyrate . These results suggest that NaBu enhances neurite outgrowth induced by NGF and cholera toxin in PC12 cells through a mechanism involving an increase in the level of histone acetylation.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12901 - 6 Epub 2002 Sep 12.
ENSO and cholera: a nonstationary link related to climate change?
Rodo X, Pascual M, Fuchs G, Faruque AS.
We present here quantitative evidence for an increased role of interannual climate variability on the temporal dynamics of an infectious disease . The evidence is based on time-series analyses of the relationship between El Nino/Southern Oscillation (ENSO) and cholera prevalence in Bangladesh (formerly Bengal) during two different time periods . A strong and consistent signature of ENSO is apparent in the last two decades (1980-2001), while it is weaker and eventually uncorrelated during the first parts of the last century (1893-1920 and 1920-1940, respectively) . Concomitant with these changes, the Southern Oscillation Index (SOI) undergoes shifts in its frequency spectrum . These changes include an intensification of the approximately 4-yr cycle during the recent interval as a response to the well documented Pacific basin regime shift of 1976 . This change in remote ENSO modulation alone can only partially serve to substantiate the differences observed in cholera . Regional or basin-wide changes possibly linked to global warming must be invoked that seem to facilitate ENSO transmission . For the recent cholera series and during specific time intervals corresponding to local maxima in ENSO, this climate phenomenon accounts for over 70% of disease variance . This strong association is discontinuous in time and can only be captured with a technique designed to isolate transient couplings.

Infect Immun, 2002 Oct, 70(10), 5533 - 9
Cholera toxin and heat-labile enterotoxin activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a cyclic AMP-dependent pathway; Bagley KC et al.; Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown . There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo . Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro . We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent . ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3',5'-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT . In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC . CT, LT, dibutyryl-cyclic-3',5'-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide . Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.

Soc Sci Med, 2002 Sep, 55(6), 1015 - 24
The spatial epidemiology of cholera in an endemic area of Bangladesh; Ali M et al.; This paper defines high-risk areas of cholera based on environmental risk factors of the disease in an endemic area of Bangladesh . The risk factors include proximity to surface water, high population density, and low educational status, which were identified in an earlier study by the authors . Cholera data were analyzed by spatially referenced extended household units for two time periods, 1983-1987 and 1992-1996 . These periods were chosen because they had different dominant cholera agents . From 1983-1987 classical cholera was dominant and from 1992-1996 El Tor was dominant . By defining high-risk areas based on risk factors, this study builds a spatial risk model for cholera . The model is then evaluated based on the locations of observed cholera cases . The study also identifies the determinants of death due to cholera for the two different time periods dominated by the different cholera agents . The modeled risk areas that were based on the risk factors were found to correspond with actual distributions of cholera morbidity and mortality . The high-risk areas of the dominant cholera agents are relatively stable over time . However, from 1983-1987 El Tor cholera, which was not the dominant agent during that period, was not associated with high-risk areas, suggesting that the El Tor habitat may have changed over time . The case fatality rate for cholera was related to proximity to a diarrhea treatment hospital in the study area.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Mar, 30(1), 31 - 3
{Preparation of oral microspheres carrying V . cholera vaccine and its target's distribution}; Zhang W et al.; To prepare oral biodegradable microspheres carrying V . cholera vaccine, the major outer membrane protein (OMP, MW = 41 kd) as a common antigen of cholera Vibriae was obtained from the classical strain Inaba 569 B, and the OMP was encapsulated in the biodegradable delivery system comprising Poly (DL-Lactide)-Co-Poly(ethylene glycol)microspheres . The average size of the microspheres was less than 5 microns, the amount of OMP encapsulated in microspheres was 15.3% . It was found that microspheres were taken up in Peyer's patches and then distributed in spleen, liver and mesenteric lymph nodes after oral administration.

J Neurosci Res, 2002 Sep 1, 69(5), 669 - 80
Characterization of cholera toxin B subunit-induced Ca(2+) influx in neuroblastoma cells: evidence for a voltage-independent GM1 ganglioside-associated Ca(2+) channel; Fang Y et al.; The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108-15 neuroblastoma cells with the GM1-specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca(2+) influx together with neuritogenesis . Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1 . An L-type voltage-operated Ca(2+) channel (VOCC) was previously proposed as the mediator of this phenomenon . This investigation, employing fura-2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca(2+) channel not of the L type . It was opened by Ctx B; was permeable to Ca(2+) and Ba(2+) but not Mn(2+); and was blocked by Ni(2+), Cd(2+), and La(3+) . Although most dihydropyridines inhibited Ctx B-induced Ca(2+) influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC . In addition, Ca(2+) influx induced by Ctx B was not mediated by cGMP-dependent or G-protein-coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp-cGMPS or the G-protein/receptor uncoupling agent suramin, respectively . Finally, Ca(2+) influx was unlikely to be due to inhibition or reversal of Na(+)-Ca(2+) exchanger via Ctx B induction of Na(+) uptake, insofar as no effect was seen on blocking Na(+) channels, inhibiting Na(+)-K(+)-ATPase, or eliminating extracellular Na(+) . The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca(2+) influx . This in turn induces signaling for the onset of neuritogenesis .

Clin Infect Dis, 2002 Sep 15, 35(6), 713 - 20 Epub 2002 Aug 23.
A legacy in 20th-century medicine: Robert Allan Phillips and the taming of cholera; Savarino SJ; The legacy of Captain Robert Allan Phillips (1906-1976) was to establish effective, evidence-based rehydration methods for the treatment of cholera . As a Navy Lieutenant at the Rockefeller Institute for Medical Research (New York, New York) during World War II, Phillips developed a field method for the rapid assessment of fluid loss in wounded servicemen . After the war, he championed the establishment of United States Naval Medical Research Unit (NAMRU)-3 (Cairo; 1946) and NAMRU-2 (Taipei; 1955), serving at the helm of both units . Phillips embarked on cholera studies during the 1947 Egyptian cholera epidemic and brought them to maturity at NAMRU-2 (1958-1965), elucidating the pathophysiologic derangements induced by cholera and developing highly efficacious methods of intravenous rehydration . His conception of a simpler cholera treatment was realized in the late 1960s with the development of glucose-based oral rehydration therapy, a monumental breakthrough to which many other investigators made vital contributions . Today, these simple advances have been integrated into everyday medical practice across the globe, saving millions of lives annually.

Cell Immunol, 2002 Feb, 215(2), 141 - 50
Study of the physical meaning of the binding parameters involved in effector-target conjugation using monoclonal antibodies against adhesion molecules and cholera toxin; Garcia-Penarrubia P et al.; In earlier work, we established a mathematical model to characterize the binding properties of cytotoxic cells to target cells . These properties can be described by the values of the maximum effector and target conjugate frequencies, alpha(max) and beta(max), respectively, and the dissociation constant of the conjugates formed, K(D) (Garcia-Penarrubia, P., Cabrera, L., Alvarez, R., and Galvez, J., J . Immunol . Methods 155 (1992) 133) . Here, we address the problem of exploring the physical meaning of these parameters and their relationships with cytotoxicity . With this purpose, conjugation between a human leukemic NK cell line (NKL) and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios (R) . The results have been compared to those obtained after target cells treatment with monoclonal antibodies recognizing adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) (which are able to block some of the receptors implicated in conjugation), as well as with cholera toxin (CTX) that can modify the state of affinity of some adhesion molecules such as LFA-1 (CD11a/CD18) . The results show that: (1) blocking adhesion receptors CD54 and CD58 on the surface of target cells leads to a significant decrease of alpha(max) and beta(max), indicating that these parameters are related to the density of expression of receptors implicated in effector-target adhesion; (2) treatment of effector cells with CTX induced an increase of K(D), demonstrating that this parameter is associated with the effector-target affinity of the system; and (3) parallel experiments of conjugation and cytotoxicity showed that effector-target affinity and saturability influence the cytotoxic activity of the effector population.

Links . 1992 Spring;9(2):7, 25.
The deadliest wake-up call . Cholera; Dajer T; PIP: It is clear that cholera has caused an uproar in Latin America, but the meaning of the epidemic is not so certain . In January 1991, cases of cholera began to spring up along the coast north of Lima . A year later, 1/2 million people in the continent had fallen ill from the disease and over 4000 had died from it . There is not effective antibiotic of vaccine against it . Sanitation is the only "cure" for cholera, a disease which can dehydrate and kill an otherwise strong and healthy person in 6 hours . There are 4 ways of looking at the significance of the epidemic: 1) Who cares? 4000 deaths -- less and 1% of all cholera cases -- is a minuscule figure . Last year in Africa, over 11,000 cholera deaths went unnoticed by the press . 2) The Lost 80s . Economically disastrous for Latin America, the 1980s saw billions of dollars being siphoned out of the continent by multinational banks, money that should have gone into public works and water systems . 3) Just Nature Doing Her Thing . The outbreak of cholera in Latin America is simply the most recent stop of a worldwide cholera epidemic which began in Indonesia in 1961 . And 4) Kids Don't Count . In 1991, some 200,000 mostly poor Latin American children died of common diarrheal diseases . Only when bad water began affecting adults was rehydration therapy emphasized . None of these 4 explanations, however, fully explains the commotions surrounding the cholera outbreak . Cholera is one of those diseases that can brand a country as backwards . At bottom, the epidemic represents an embarrassment to the ruling classes of the Latin America .

Links, 1991 Fall, 8(3), 20 - 3
Cholera in the time of civil war . Liberia; Barth J; PIP: 170 years of strife in Liberia between freed slaves from the Americas (Americo-Liberians) and indigenous tribes erupted in 1980 when a member of the indigenous ethnic group the Khran overthrew and killed the president . Contrary to what he promised, an equitable distribution of goods and services did not occur, human rights abuses continued, and civil service jobs went to Khran members . Many Americo-Liberians left . The US government recognized his government in 1986 . US government and corporate interest in Liberia included a Voice of American transmitter, a navigation station, and rubber plantations . A civil war followed . An African American teacher ran a cholera ward during the war at Island Hospital where staff was not used to treating the lower class . She also started an orphanage next to the hospital, initially, for the well children from the ward . She operated the ward because the physician had left and cholera treatment included double-dosing patients with chloramphenicol and tetracycline . Moreover, patients also received an anti-parasitic . Further, kwashiorkor was common in Liberia . Only soldiers received intravenous therapy, so cholera patients were not rehydrated . The teacher and 3 student nurses began administering oral rehydration fluids orally every 5 minutes and saved lives . Despite evidence that the patients indeed had cholera, the US embassy physician refused to admit it for a long time . Eventually, the embassy sent medicine stored for weeks to the ward . A long time after widespread diarrhea started, the British Broadcasting Corporation began a radio program about oral rehydration . Yet many people did not understand its message without seeing a demonstration of mixing oral rehydration salts . Moreover, many people treated diarrhea with a diuretic medication which only complicated it . Nevertheless, the teacher and her efforts stopped cholera . She also helped a woman set up a home for handicapped children who survived the war .

Microb Pathog, 2002 Jun, 32(6), 273 - 7
Positive and negative regulation of water channel aquaporins in human small intestine by cholera toxin; Hamabata T et al.; Analysis of osmotic water permeability of aquaporin (AQP) 1, AQP3 and AQP4, which are expressed in human small intestine, in the presence or absence of cholera toxin (CT) was performed using a Xenopus oocyte expression system . When treated with CT, water permeability of AQP4 was facilitated while that of AQP3 was suppressed . AQP1 did not show any significant change of water permeability when treated with CT . An adenylyl cyclase accelerator forskolin showed similar effects as CT did, suggesting that changes of the water permeability of AQP4 and AQP3 were due to an increase of intracellular cAMP concentration . A possibility that these AQPs are responsible molecules for causing acute secretory diarrhoea as in cholera is considered.

J Am Chem Soc, 2002 Jul 31, 124(30), 8818 - 24
Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E . coli heat-labile enterotoxin; Merritt EA et al.; Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites . One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family . We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin . This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose . The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs . We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation . This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.

Health Place, 2002 Sep, 8(3), 201 - 10
Identifying environmental risk factors for endemic cholera: a raster GIS approach; Ali M et al.; The bacteria that cause cholera are known to be normal inhabitants of surface water, however, the environmental risk factors for different biotypes of cholera are not well understood . This study identifies environmental risk factors for cholera in an endemic area of Bangladesh using a geographic information systems (GIS) approach . The study data were collected from a longitudinal health and demographic surveillance system and the data were integrated within a geographic information system database of the research area . Two study periods were chosen because they had different dominant biotypes of the disease . From 1992 to 1996 El Tor cholera was dominant and from 1983 to 1987 classical cholera was dominant . The study found the same three risk factors for the two biotypes of cholera including proximity to surface water, high population density, and poor educational level . The GIS database was used to measure the risk factors and spatial filtering techniques were employed . These robust spatial methods are offered as an example for future epidemiological research efforts that define environmental risk factors for infectious diseases.

J R Soc Health, 2002 Jun, 122(2), 89 - 94
Cholera: a continuous epidemic in Africa; Naidoo A et al.; Cholera continues to plague many parts of the world, but has largely been concentrated in Africa, which contributes more than 80% of the total cases worldwide . Natural disasters, like the 2000 floods in Mozambique and the volcanic eruption in the Democratic Republic of the Congo in 2002, generally lead to new outbreaks of the disease . The refugee problem in many countries throughout the world also causes potential threats for disease outbreaks . Case fatality rates are high, and we are not anywhere near curbing new cholera epidemics, especially in Africa . It is thus imperative to renew discussions about the nature of this deadly disease, its treatment, measures for prevention and control, modes of transmission, its physical, social and economic impact, and potential solutions.

Infect Immun, 2002 Aug, 70(8), 4621 - 7
Effects of oral vaccination and immunomodulation by cholera toxin on experimental Helicobacter pylori infection, reinfection, and gastritis; Raghavan S et al.; Therapeutic vaccination is an attractive strategy to control infection and disease caused by Helicobacter pylori . In mice infected with H . pylori we have studied the protective effect of oral immunization with an H . pylori lysate preparation given together with the mucosal adjuvant cholera toxin (CT), both against the initial infection and against a later reinfection challenge . We have also examined the effects of treatment with the CT adjuvant alone on H . pylori infection and reinfection . Specific immunization with lysate was found to result in a sixfold reduction of the extent (bacterial load) of the primary infection and also to provide similar levels of protection against reinfection . However, these effects were associated with severe postimmunization gastritis . In contrast, oral treatment with CT alone at the time of initial infection, while unable to suppress the initial infection, gave rise to a 20-fold reduction in bacterial load upon reinfection without causing any associated gastric inflammation . Both the infected animals that were specifically immunized and those that were treated with CT only displayed increased in vitro proliferative responses of mononuclear cells to H . pylori antigens . Antibody levels in response to H . pylori were on the other hand only marginally increased after treatment with CT, whereas they were markedly elevated after immunization with lysate plus CT, with a rise in both (Th2-driven) immunoglobulin G1 (IgG1) and, especially, (Th1-driven) IgG2a antibodies . The results illustrate the complex balance between protection and harmful inflammation after postinfection vaccination against H . pylori as studied in a mouse model.

Neurosci Lett, 2002 Jul 26, 327(3), 161 - 4
Cholera toxin B subunit labeling in lamina II of spinal cord dorsal horn following chronic inflammation in rats; Ma QP et al.; We have investigated the effect of inflammation on the labeling pattern of cholera toxin B subunit (CTB)-conjugated horseradish peroxidase, an A-fiber marker, by an intra-sciatic nerve injection of the tracer . Following chronic inflammation in one hind paw in rats, there was substantial CTB labeling in lamina II of the spinal dorsal horn, which is normally absent . However, there was no change in the labeling pattern of wheat germ agglutinin or fluoride resistant acid phosphatase/thiamine monophosphatase, two C-fiber markers . The CTB labeling in lamina II after peripheral nerve injury has been interpreted as central sprouting of A-fibers or uptake of the tracer by injured C-fibers . Our results suggest that chronic inflammation and nerve injury may share some common mechanisms in generating allodynia and hyperalgesia.

Mol Cell Biochem, 2002 Apr, 233(1-2), 19 - 26
Selection of a WEHI-3B leukemia cell subclone resistant to inhibition by cholera toxin; Pessina A et al.; The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT . In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation . To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT . This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells) . Stimulation ofWEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside . Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHl-3B/CT/REV . These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation) . Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.

Pediatr Infect Dis J, 2002 Apr, 21(4), 322 - 30
Introductory evaluation of an oral, killed whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Egyptian infants; Savarino SJ et al.; BACKGROUND: We conducted the first trial to assess the safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli plus cholera toxin B-subunit vaccine in children <2 years old . METHODS: Three doses of vaccine or killed E . coli K-12 control were given at 2-week intervals to 64 Egyptian infants, 6 to 18 months old, in a randomized, double blind manner . Adverse events were monitored for 3 days after each dose . Blood was collected before immunization and 7 to 10 days after each dose to assess vaccine-specific serologic responses . RESULTS: There was no statistically significant intergroup difference in the percentage of subjects reporting the primary safety endpoint (diarrhea or vomiting) after the first (31%, vaccine; 30%, control) or third (14%, vaccine; 18%, control) dose, whereas there was a trend toward greater reporting in the vaccine group after Dose 2 (36%, vaccine; 18%, control; P = 0.052) . The percentage of children showing IgA seroconversion after any dose was higher in the vaccine than the control group for recombinant cholera toxin B-subunit (97% vs . 46%), colonization factor antigen I (61% vs . 18%) and coli surface antigen 4 (39% vs . 4%) (P < 0.001 for each comparison) . IgG seroconversion rates in the vaccine and control groups were 97 and 21% to recombinant cholera toxin B-subunit (P < 0.001), 64 and 29% for colonization factor antigen I (P < 0.01), 53 and 21% for coli surface antigen 2 (P < 0.05) and 58 and 4% for coli surface antigen 4 (P < 0.001), respectively . The third vaccine dose was followed by augmented IgG antitoxin titers . CONCLUSION: The oral enterotoxigenic E . coli vaccine was safe and immunogenic in this setting in Egyptian infants.

Dis Colon Rectum, 2002 Jun, 45(6), 819 - 25
Effect of mucosal immunomodulation with fed cholera toxin on healing of experimental colonic anastomosis; Kaplan M et al.; PURPOSE: The aim of this study was to investigate in rats whether preoperative orogastric administration of low doses of cholera toxin would influence the mechanical strength of experimental colonic anastomosis on the basis of the gut mucosal immunomodulation effect of this antigen . METHODS: The cholera toxin group (n = 14) was fed 10 microg of cholera toxin in phosphate-buffered saline three times before surgery at 10-day intervals, whereas the controls (n = 14) received phosphate-buffered saline only . Twenty-four hours after the last dose of cholera toxin (or placebo in control group), the animals underwent left colonic transection and anastomosis . Seven days after colonic transection-anastomosis, the bursting pressure of the anastomotic segment was recorded in situ . Perianastomotic and extra-anastomotic tissue samples were obtained for measurements of tissue transforming growth factor-beta, interleukin-6, and interferon-gamma levels with enzyme-linked immunosorbent assay . RESULTS: Cholera toxin administration resulted in a significantly higher bursting pressure than in the control group (165.78 +/- 12.37 vs . 138.4 +/- 7.87 mmHg; P < 0.001) . Compared with the control group, the heightened mechanical strength of colonic anastomosis provided by cholera toxin was associated with significant increases in the perianastomotic tissue levels of transforming growth factor-beta (199.34 +/- 24.85 vs . 70.66 +/- 10.63 pg/ml; P < 0.001) and interleukin-6 (439.31 +/- 95.14 vs . 289.57 +/- 96.59 pg/ml; P = 0.001), whereas interferon-gamma was significantly lower (174.04 +/- 44.82 vs . 219.00 +/- 31.35 pg/ml; P < 0.05) . This cytokine pattern induced by cholera toxin in the wound milieu was also found to be similar in the extra-anastomotic colon . CONCLUSION: The mechanical strength of uncomplicated experimental colonic anastomosis increased significantly with gut mucosal immunomodulation with repeated low preoperative doses of cholera toxin . This enhanced healing had significant positive correlation with the colonic tissue level of transforming growth factor-beta and inverse correlation with interferon-gamma . If the relevant dose regimen is identified and its safety is assured in humans, gut mucosal immunomodulation might provide an efficient, safe, and inexpensive tool to improve surgical outcome in colorectal surgery, particularly in high-risk situations.

Microbiol Immunol, 2002, 46(4), 249 - 55
Inhibition by apple polyphenols of ADP-ribosyltransferase activity of cholera toxin and toxin-induced fluid accumulation in mice; Saito T et al.; The effects of crude polyphenol extracted from immature apples on the enzymatic and biological activities of a cholera toxin (CT) were investigated . When the apple polyphenol extract (APE) was examined for properties to inhibit CT-catalyzed ADP-ribosylation of agmatine, it was found that APE inhibited it in a dose-dependent manner . The concentration of APE to inhibit 50% of the enzymatic activity of CT (15 microg/ml) was approximately 8.7 microg/ml . The APE also diminished CT-induced fluid accumulation in two diarrhea models for in vivo mice . In the ligated ileum loops, 25 microg of APE significantly inhibited fluid accumulation induced by 500 ng of CT . In a sealed mouse model, even when APE was administered orally 10 min after a toxin injection, fluid accumulation was significantly inhibited at a comparable dosage . Lineweaver-Burk analysis demonstrated that APE had negative allosteric effects on CT-catalyzed NAD: agmatine ADP-ribosyltransferase . We fractionated the APE into four fractions using LH-20 Sephadex resin . One of the fractions, FAP (fraction from apple polyphenol) 1, which contains non-catechin polyphenols, did not significantly inhibit the CT-catalyzed ADP-ribosylation of agmatine . FAP2, which contains compounds with monomeric, dimeric, and trimeric catechins, inhibited the ADP-ribosylation only partially, but significantly . FAP3 and FAP4, which consist of highly polymerized catechin compounds, strongly inhibited the ADP-ribosylation, indicating that the polymerized structure of catechin is responsible for the inhibitory effect that resides in APE . The results suggest that polymerized catechin compounds in APE inhibit the biological and enzymatic activities of CT and can be used in a precautionary and therapeutic manner in the treatment of cholera patients.

Immunology, 2002 Jun, 106(2), 237 - 45
Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo; Sadeghi H et al.; The pentameric B-subunit of cholera toxin (CTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes . In this study a series of fusions was constructed between the genes encoding CTB and the B-chain of human insulin (InsB) . The resulting fusion proteins were expressed in Escherichia coli and isolated as cytoplasmic inclusion bodies that were then dissolved and assembled in vitro . GM1 enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses showed that the protein construct in which InsB was fused to the C-terminus of a CTB monomer (CI) assembled into structures that both bound to the receptor GM1 ganglioside and reacted with monoclonal antibodies to CTB and insulin . Fusion of InsB to the N-terminus of CTB resulted in protein that could not assemble into pentameric CTB . In vitro assays showed that the CI fusion protein was 300-fold more potent than native insulin at inducing interleukin-2 (IL-2) production by an insulin-specific T-cell hybridoma . When administered orally, the CI fusion protein induced efficient immunological suppression of ovalbumin-specific T-cell responses in mice co-immunized parenterally with insulin and ovalbumin . These results demonstrate the stability, GM1 receptor-binding activity and antigenic authenticity of the CI fusion protein as well as its ability to elicit insulin-specific T-cell responses in vitro . In addition, we demonstrate that the CI fusion protein induces efficient immunosuppression after oral administration, raising the possibility of using such constructs in the treatment of type-1 diabetes.

Nutrition, 2002 Jun, 18(6), 458 - 62
Effects of an alanyl-glutamine-based oral rehydration and nutrition therapy solution on electrolyte and water absorption in a rat model of secretory diarrhea induced by cholera toxin; Lima AA et al.; OBJECTIVES: Recurring diarrhea and persistent diarrhea are commonly associated with malnutrition and long-term functional deficits . A beneficial approach would be to develop an alanyl-glutamine (AlaGln)-based oral rehydration and nutrition therapy (ORNT) . We investigated the effect of an AlaGln-ORNT solution on electrolyte and water absorption in a rat model of secretory diarrhea induced by cholera toxin . METHODS: Phenolsulfonphthalein (50 microg/mL) was used as a non-absorbable marker for calculation of net water and electrolyte transport . Solutions tested were Ringer's solution, a glutamine-based ORNT (Gln-ORNT) solution, and an AlaGln-ORNT solution . Cholera toxin (1 microg/mL) was injected into lumen of rat small intestinal segments and incubated for 18 h before the initiation of the perfusion . RESULTS: Cholera toxin induced significant secretion of electrolyte and water in the control Ringer's solution . AlaGln-ORNT and Gln-ORNT solutions reduced the sodium secretory effect of cholera toxin by 128% and 36%, respectively . The net water secretion also was reduced by 95% and 60%, respectively, with the AlaGln-ORNT and Gln-ORTN solutions . CONCLUSIONS: These results showed that AlaGln-ORNT solution can enhance water and electrolyte intestinal absorption even better than glutamine or glucose and thus provide a potential novel approach for ORNT to break the vicious cycle of diarrhea and malnutrition . Clinical trials are now needed in children and adults with diarrhea and malnutrition.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 17 - 22
{Cholera in Kazan . Organization and implementation of cholera control interventions}; Onishchenko GG et al.; Data on emergent epidemiological analysis of the cholera outbreak in Kazan are presented . A version of the cholera focus emergence was confirmed, namely water route of transmission as a result of bathing in a water reservoir where sewage waters had penetrated . The outbreak had local and acute character . The complex of cholera control interventions aimed at localization and liquidation of the focus proved to be effective.

Neuropeptides, 2001 Oct-Dec, 35(5-6), 197 - 203
Pretreatment with cholera or pertussis toxin differentially modulates morphine- and beta-endorphin-induced antinociception in the mouse formalin test; Chung KM et al.; The present study was designed to examine the possible involvement of supraspinal CTX- and PTX-sensitive G-proteins in an opioid-induced antinociception in the formalin test . Morphine (1 microg) and beta-endorphin (1 microg) given i.c.v . displayed near-maximal inhibitory effects against the formalin response in the first (0-5 min) and the second (20-40 min) phases . CTX (0.1-0.5 microg) pretreated i.c.v . produced antinociceptive effects in both phases of the formalin responses . Its effect was more pronounced in the first phase . However, PTX (0.05-0.5 microg) injected i.c.v produced the antinociceptive effect only in the first, but not the second, phase . Both CTX (0.5 microg) and PTX (0.5 microg), at the dose which had no intrinsic effect, significantly reversed the beta-endorphin-induced antinociceptive effect observed during the second, but not the first, phase . However, the antinociceptive effect by morphine failed to be affected by the same dose of treatment with CTX or PTX . Our results indicate that, at the supraspinal level, CTX- and PTX-sensitive G-proteins appear to be involved in the modulation of antinociception induced by supraspinally administered beta-endorphin, but not morphine, in the formalin pain model .

Indian J Med Sci, 2001 Aug, 55(8), 429 - 33
Epidemiological investigation of cholera outbreak in a periurban slum colony in Chandigarh; Thakur JS et al.; An investigation was carried out in a periurban slum colony in Chandigarh in September 1999 following a report of two microscopically confirmed cases of cholera admitted in Govt . Medical College Hospital, Chandigarh . Rapid survey in the colony covering a population of 1404 found that there were 14 cases of diarrhoea in the colony with attack rate of 9.97/1000 population . Majority (70%) of cases were females and 62% cases were under five years of age . Health education, ORS packets and medications were distributed to cases . Water sampling was also done and it was found that water from one of the hand pump was positive for V . cholerae 01 biotype El Tor serotype Ogawa . Closing of that hand pump and chlorination of drinking water in other parts of slum was recommended to higher health authorities, which was done immediately . Surveillance for diarrhoeal diseases was found to be poor . Provision of safe drinking water, improving sanitation and strengthening of disease surveillance is necessary for control of cholera and other diarrhoeal diseases in slum areas.

J Health Popul Nutr, 2002 Mar, 20(1), 85 - 92
Cholera in Brazil during 1991-1998: socioeconomic characterization of affected areas; Waldman EA et al.; The paper describes the trends in, and spatial patterns of, the incidence of cholera in Brazil from 1991 to 1998 . During this period, 161,432 cases and 1,296 deaths from cholera were reported . The poorest (North and Northeast) regions of the country had the highest morbidity and mortality rates . The remaining regions had self-limited outbreaks . Seventy-eight percent of affected municipalities had populations of fewer than 30,000, and about 65% of them lived in rural areas . The affected municipalities of the North and Northeast regions had consistent indications of deprivation: average Human Development Index was 0.41, infant mortality rate 90.3%, average life expectancy 59.4 years, and adult illiteracy rate 46.5% . The epidemiological profile of the disease in Brazil highlights intra- and inter-regional socioeconomic differentials in the country and indicates the importance of planning and implementing public-health interventions and specific policies aimed at reducing health inequalities.

Vet Immunol Immunopathol, 2002 Jul, 86(3-4), 177 - 82
Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep; Chen D et al.; Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs . In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep . The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep . Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5) . The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio . In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT . Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups . IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group . Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.

Mol Biol Cell, 2002 May, 13(5), 1750 - 64
Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection; Richards AA et al.; Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway . We now show that the initial entry step is blocked by brefeldin A and by incubation at 20 degrees C . Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway . This intracellular trafficking pathway is also brefeldin A sensitive . Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP . In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action . Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.

Org Lett, 2002 May 16, 4(10), 1807 - 8
Synthesis and cholera toxin binding properties of a lactose-2-aminothiazoline conjugate; Vrasidas I et al.; {structure: see text} During the search for improved monovalent ligands for cholera toxin (CT), a new lactose-2-aminothiazoline conjugate was discovered . In a fluorescence binding assay the compound was found to be one of the strongest relatively simple CT ligands to date with a K(d) of 23 microM.

Rev Sci Tech, 2002 Aug, 21(2), 287 - 303
Classical swine fever (hog cholera) in wild boar in Europe; Artois M et al.; Classical swine fever (CSF) is of increasing concern in Europe where wild boar appear to play an important epidemiological role . In most parts of the continent, demographic trends are on the increase, due to improvement in game management . As a result of higher densities, populations become more susceptible to various infectious diseases, among which CSF is cause for particular concern . Wild boar do not appear to be a classic reservoir in most cases, but nevertheless may perpetuate foci of infection over the long term, constituting a real threat for the pig farming industry . Since the infection does not appear to spread easily in natural populations of free-ranging wild boars, control of the disease may be feasible . However, most of the appropriate measures, such as banning hunting, are not considered acceptable . Consequently, the expertise of wildlife disease specialists is required to help solve the problem when it occurs.

J Virol, 2002 May, 76(9), 4536 - 46
Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines; Arrington J et al.; Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals . Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon {IFN-gamma}) to multiple viral antigens when codelivered with DNA vaccines . In addition, Th2 cytokine responses (interleukin 4 {IL-4}) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent . The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios . Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells . The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes . Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations . Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.

Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 621 - 5
{A fusion protein of rotavirus VP6 and cholera toxin B subunit: expression in Escherichia coli and analysis of biological activities}; Guo TX et al.; Rotavirus infection is a major cause of dehydrating diarrhea in infants worldwide . The non-toxic cholera toxin B subunit(CTB), known as an immunomodulatory carrier, might help to stimulate mucosal immune response when coupled to rotavirus antigens in oral immunization . Here we report for the first time the construction of a translational fusion of CTB gene 5' to the VP6 gene of a human rotavirus A(field strain T114), and expression of the CTB-VP6 fusion protein in E . coli BL21(DE3) . The expressed fusion protein has a molecular weight of 56 kD, as expected, and accounts for about 15% of the total E . coli protein . Western blottings with the hyperimmune serum against rotavirus strain WA and the antibody against cholera toxin indicated that the fusion protein retains the antigenicity identical to the native CTB and VP6 . The GM1-ELISA analysis proves that the renatured CTB-VP6 has strong affinity for GM1 ganglioside.

Int J Med Microbiol, 2002 Feb, 291(6-7), 571 - 5
Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses; Gagliardi MC et al.; Cholera toxin (CT) is a potent mucosal adjuvant . When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens . Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property . These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action . CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.

Int J Med Microbiol, 2002 Feb, 291(6-7), 531 - 5
New insights into the structure-function relationships and therapeutic applications of cholera-like enterotoxins; Hirst TR et al.; Cholera toxin and E . coli heat-labile enterotoxin are structurally homologous proteins comprised of an enzymatically active A-subunit and five B-subunits that bind with high affinity to GM1-ganglioside receptors found on the surface of mammalian cells . The B-subunits have long been thought of simply as trafficking vehicles that trigger entry and subsequent delivery of the 'toxic' A-subunit into cells . Indeed, such is the capacity of the B-subunits to enter cells, that they have been developed as generic carriers for attachment and delivery of a variety of peptides into mammalian cells . However, the B-subunits also appear to possess discrete 'signalling functions', that induce both transcription factor and cell activation . These are thought to be directly responsible for the potent immunomodulatory properties of the B-subunits, and have resulted in their use as adjuvants and as agents to suppress inflammatory immune disorders . The relationship between the signalling properties of the B-subunits and their capacity to act as trafficking vehicles has remained unclear . In an effort to understand the structural requirements for these two functions, a set of mutant B-subunits, with amino acid substitutions at position His-57, have been generated and studied . Importantly, such mutant B-subunits retain an ability to bind with high affinity to GM1 and to traffic into cells, but have entirely lost their capacity to activate immune cell populations . Thus, while binding via GM1 appears to be sufficient to trigger cellular uptake it is not sufficient to activate signal transduction . The His-57 region is therefore speculated to be actively engaged in triggering signalling events, possibly via cognate interaction with other cell surface molecules.

Indian J Pathol Microbiol, 2001 Apr, 44(2), 123 - 4
Rapid diagnosis of cholera by coagglutination test; Hanumanthappa AR et al.; In this study the coagglutination test for the rapid diagnosis of cholera is evaluated in comparison with the conventional culture method . A total of 553 stool specimens were processed from cases of acute gastro-enteritis . The sensitivity and specificity of coagglutination test was 92.77% and 95.65% respectively . The coagglutination test is found to be simple, reliable and rapid method for the diagnosis of cholera.

Microbes Infect, 2002 Feb, 4(2), 237 - 45
Cholera and climate: revisiting the quantitative evidence; Pascual M et al.; Cholera dynamics in endemic regions display regular seasonal cycles and pronounced interannual variability . We review here the current quantitative evidence for the influence of climate on cholera dynamics with reference to the early literature on the subject . We also briefly review the incipient status of mathematical models for cholera and argue that these models are important for understanding climatic influences in the context of the population dynamics of the disease . A better understanding of disease risk related to the environment should further underscore the need for changing the socioeconomic conditions conducive to cholera.

Chem Biol, 2002 Feb, 9(2), 215 - 24
Anchor-based design of improved cholera toxin and E . coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes; Pickens JC et al.; The action of cholera toxin and E . coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1 . We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure . In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG . Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC) . Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives . The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.

J Cell Sci, 2002 Feb 15, 115(Pt 4), 817 - 26
Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle; Majoul I et al.; Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively . Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins . The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested . We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase, whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase . The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface . The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide . Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors, suggesting that this novel phenomenon is based on a conserved regulatory mechanism.

Infect Immun, 2002 Mar, 70(3), 1260 - 71
Mutational analysis of ganglioside GM(1)-binding ability, pentamer formation, and epitopes of cholera toxin B (CTB) subunits and CTB/heat-labile enterotoxin B subunit chimeras; Jobling MG et al.; Variants of cholera toxin B subunit (CTB) were made by bisulfite- and oligonucleotide-directed mutagenesis of the ctxB gene . Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC) . Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM(1) in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies . Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB . RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 + 54, 91, and 95 . Tyrosine-12 was identified as a novel residue important for GM(1) binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM(1) in vitro . In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM(1) binding, based on the crystal structure of CTB and the oligosaccharide of GM(1), were not appreciably altered in their ability to bind GM(1) in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC . Hybrid B genes made by fusing ctxB and the related Escherichia coli heat-labile enterotoxin eltB genes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM(1) as well as wild-type CTB . This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs) . Each MAb had a unique pattern of reactivity with the panel of CTB variants . Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope . The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers . Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.

Biochemistry, 2002 Feb 12, 41(6), 1742 - 51
Analysis of cholera toxin-ganglioside interactions by flow cytometry; Lauer S et al.; Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane . We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes . A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants . In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions . From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1) . From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1) . These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1) . We determined the equilibrium surface cross-linking constant {(1.1 +/- 0.1) x 10(-12) cm(2)} and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking . We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits) . Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface . Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1) . These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.

J Immunol, 2002 Feb 15, 168(4), 1730 - 7
Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide; Burkart V et al.; The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system . We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines . Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC . Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin . In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS . Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin . The suppression of TNF-alpha production occurred at the level of mRNA formation . Tolerance induction by CTB was dose and time dependent . The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta . IFN-gamma also antagonized the actions of CTB on macrophages . In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response . These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.

Arkh Patol, 2001 Nov-Dec, 63(6), 30 - 4
{Ultrastructure of renal cortex in suckling rabbits with experimental cholera}; Bardakhch'ian EA et al.; Development of experimental cholera in suckling rabbits is associated with appearance of alterations in glomerular filtration and tubular reabsorption of renal cortex . Ultrastructural changes of nephrons appear in the adhesion period and progress 24 hours later . In this case, particular vulnerability of the kidneys is associated with insufficient development of principal stages both in the cavity and membrane digestion in the gut, therefore, the kidney plays a role of one of the components of the protein-splitting system in the organism.

Arch Hist Filoz Med, 2001, 64(1), 9 - 16
{Contribution to fight against cholera in an instance of epidemic in Osieciny, Eastern Kujawy in 1852}; Rejmanowski T; Description of activities of Lieutenant-Colonel Tomasz Zielinski who was not a physician, but being an officer of the Russian army during the Turkish campaign in 1831, had acquainted himself with methods of fighting against cholera and he used them with quite a good result in Osieciny in 1852.

Vaccine, 2001 Dec 12, 20(5-6), 756 - 62
Highly purified mutant E112K of cholera toxin elicits protective lung mucosal immunity to diphtheria toxin; Ohmura M et al.; We demonstrated that the mutant of cholera toxin (mCT) E112K which was LPS-free supported the induction of protective immunity in mucosal (e.g . lung lavage) and systemic (e.g . serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice . Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K or native CT (nCT) was co-administered nasally with DT . The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands . Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT . The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4+ T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT . The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective . Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity . These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g . lung lavages and the lungs).

Int J Infect Dis, 2001, 5(3), 133 - 8
Health education and cholera in rural Guinea-bissau; Einarsdottir J et al.; OBJECTIVE: The study was undertaken to explore local ideas about cholera and the diffusion of official health educational messages for cholera prevention and to assess whether such messages contributed to changed behavior in the population . METHODS: During the ongoing cholera epidemic in 1994 in Guinea-Bissau, West Africa, a roster of all adult residents in a rural community was established . From this roster of 458 adults, 53 of 60 randomly chosen residents were interviewed for qualitative data on cholera and its prevention . RESULTS: Local preventive rituals performed contributed to high awareness of the epidemic . Radio and word-of-mouth communication were the most important sources of information on cholera, whereas posters and television did not effectively reach the population . All persons with cholera rapidly sought care . Thirty-four (64%) of 53 participants recalled at least one preventive measure; specifically, treatment of water with lemon was mentioned by 21 (40%) of respondents . None of the respondents could explain how cholera is transmitted to humans . CONCLUSIONS: To improve compliance with recommended preventive measures, these should take local conceptions of diseases into account and be few in number, practical, and effective . The impact of the radio could be increased if those who hear the message are urged to spread the recommendation, especially to women who take care of food, water, and general hygiene in the household.

Anal Chem, 2001 Nov 1, 73(21), 5287 - 95
Electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and GM1-functionalized liposomes; Alfonta L et al.; An ultrasensitive method for the detection of the cholera toxin (CT) using electrochemical or microgravimetric quartz crystal microbalance transduction means is described . Horseradish peroxidase (HRP) and GM1-functionalized liposomes act as catalytic recognition labels for the amplified detection of the cholera toxin based on highly specific recognition of CT by the ganglioside GM1 . The sensing interface consists of monoclonal antibody against the B subunit of CT that is linked to protein G, assembled as a monolayer on an Au electrode or an Au/ quartz crystal . The CT is detected by a "sandwich-type" assay on the electronic transducers, where the toxin is first bound to the anti-CT-Ab and then to the HRP-GM1-ganglioside-functionalized liposome . The enzyme-labeled liposome mediates the oxidation of 4-chloronaphthol (2) in the presence of H2O2 to form the insoluble product 3 on the electrode support or the Au/quartz crystal . The biocatalytic precipitation of 3 provides the amplification route for the detection of the CT . Formation of the insulating film of 3 on the electrode increases the interfacial electron-transfer resistance, Ret, or enhances the electrode resistance, R', parameters that are quantitatively derived by Faradaic impedance measurements and chronopotentiometric analyses, respectively . Similarly, the precipitate 3 formed on the Au/quartz crystal results in a mass increase on the transducer that is reflected by a decrease in the resonance frequency of the crystal . The methods allow the detection of the CT with an unprecedented sensitivity that corresponds to 1.0 x 10(-13) M.

J Cell Sci, 2001 Oct, 114(Pt 20), 3737 - 47
Internalization of cholera toxin by different endocytic mechanisms; Torgersen ML et al.; The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced . Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner . Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake . Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis . Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis . These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization . Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells . In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

Infect Immun, 2001 Dec, 69(12), 7285 - 92
Intranasal immunization with recombinant Ascaris suum 14-kilodalton antigen coupled with cholera toxin B subunit induces protective immunity to A . suum infection in mice; Tsuji N et al.; Animals can be rendered immune to Ascaris parasites by immunization with infectious-stage larvae . The specific parasite gene products that mediate protective responses in ascariasis are unknown . We have identified a cDNA encoding Ascaris suum 14-kDa antigen (As14) and evaluated the vaccinal effect of the Escherichia coli-expressed recombinant protein (rAs14) . GenBank analysis showed that As14 has low similarity at the amino acid level to a Caenorhabditis elegans gene product and to antigens of the filarial nematodes but not to other known proteins . In addition, As14 homologues were found to be expressed in human and dog roundworms . In mice that received intranasal administration of rAs14 coupled with cholera toxin B subunit (rAs14-CTB), there was a 64% reduction of recovery of larvae compared with that in the nontreated group . The vaccinated mice showed a significant increase in the total serum immunoglobulin G (IgG) levels and the mucosal IgA responses . Elevation of the rAs14-specific IgE response was also seen . Measurement of the IgG subclasses showed a higher level of IgG1 and a lower level of IgG2a antibody response in the sera of the immunized mice, suggesting that protection was associated with a type II immune response . As14 is the first protective antigen against A . suum infection to be identified . Our immunization trial results in laboratory animals suggest the possibility of developing a mucosal vaccine for parasitic diseases caused by ascarid nematodes.

Scand J Immunol, 2001 Nov, 54(5), 440 - 7
Nasal administration of Schistosoma mansoni egg antigen-cholera B subunit conjugate suppresses hepatic granuloma formation and reduces mortality in S . mansoni-infected mice; Sun JB et al.; Granulomatous inflammation in schistosomiasis is a delayed-type hypersensitivity reaction mediated by CD4+ T cells specific for parasite egg antigens (Ags) . In an attempt to control T-cell responses leading to excessive harmful inflammation and granuloma formation, especially in the liver, BALB/c mice were intranasally (i.n.) treated with soluble Schistosoma mansoni egg Ags (SEA) conjugated to cholera toxin B subunit (CTB), a mucosa-binding protein with demonstrated capacity to suppress inflammatory T-cell functions after mucosal administration . Treatment with CTB-SEA significantly conjugate a reduced liver granuloma formation in infected mice associated with decreased SEA specific Th1- and Th2-type immune responses by liver leukocytes . Importantly, treatment with CTB-SEA conjugate also significantly reduced the mortality in chronically infected mice . In S . mansoni-infected large-granuloma forming CBA mice, i.n . treatment with purified Sm-p40, the major egg antigen, conjugated to CTB likewise significantly inhibited hepatic egg granuloma formation . A reduction of SEA-driven lymphoproliferation and of interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 production, together with an increase in transforming growth factor (TGF)-beta1 production, were observed in splenic cells from CTB-Sm-p40-treated SEA-sensitized mice, as well as in liver leukocytes from CTB-Sm-p40-treated schistosome-infected mice . These results indicate that mucosal administration of SEA or purified Sm-p40 antigen in conjunction with CTB is highly effective in curtailing immunopathologic manifestations of schistosomiasis in vivo in infected hosts.

In Vivo, 2001 Sep-Oct, 15(5), 381 - 4
Oral immunisation of chickens using cholera toxin B subunit and Softigen as adjuvants results in high antibody titre in the egg yolk; Hedlund GP et al.; Oral immunisation by gavage of laying hens with human immunoglobulin G (IgG) combined with a number of potential adjuvants was performed . The resulting immunospecific egg yolk (IgY) antibodies were quantified by ELISA . The following adjuvants were tested: A Poly(lactide-co-glycolide) (PLG) microspheres, Cholera toxin B-subunit (CTB), CTB conjugated with glutaraldehyde, Dimethyl dioctadecyl ammonium bromide (DDA), and Softigen (pegylated C8/C10 mono/di glyceride) . Hens in a positive control group were immunised with human IgG in saline emulsified with an equal volume of Freund's Incomplete Adjuvant . High titres of immunospecific IgY antibodies against human IgG were recorded in the eggs from the chickens immunised orally with the antigen combined with glutaraldehyde conjugated CTB and in the chickens immunised with the antigen combined with Softigen . The present results show that invasive technique related stress could be eliminated/reduced in polyclonal antibody producing animals.

Brain Res, 2001 Nov 16, 919(1), 20 - 30
Cholera toxin-B subunit blocks excitatory opioid receptor-mediated hyperalgesic effects in mice, thereby unmasking potent opioid analgesia and attenuating opioid tolerance/dependence; Shen KF et al.; In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine's analgesic effects . This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions . By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency . The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng/kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia . These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia . Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors . Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12391 - 6
Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells; Sugimoto Y et al.; We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells {NPC1(-) cells} by using cholera toxin (CT) as a probe . Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells . CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes . Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1 . CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain . A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles . U18666A treatment of "knock in" cells {NPC1(-) cells that stably expressed wild-type NPC1} caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells . Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits . In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells . These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Med Secoli, 1993, 5(3), 379 - 403
{The dominant medical thought against the epidemic: the interpretation of the cholera at its first appearance in Italy (1835-37)}; Balfour RN; For the present study in the history of Italian medical thought original texts published during the first cholera epidemic in Italy (1835-37) by Tommasini, Giacomini, Pirondi, Bufalini, are analyzed . Tommasini, Giacomini and Pirondi, belonging to the Rasorian School, nonetheless develope different positions: Pirondi is a strict Rasorian, Giacomini defines a neuro-vascular theory including a symptomatic analysis, Tommasini syncretizes the Italian irritation theses, expanding Rasorian thought to include qualitative variations . Bufalini, by contrast, develops an epidemiological-materialistic theory.

Nippon Ishigaku Zasshi, 1992 Jan, 38(1), 5 - 24
{Cholera epidemics in Kanagawa}; Otaki T; In 1822 cholera first arrived in Japan, but it did not reach Edo, which is the old name of Tokyo, nor Kanagawa, a prefecture located next to Tokyo . During a second epidemic in 1858, 30,000 or so Japanese died and Kanagawa had a heavy toll . Cholera raged in Japan in 1877, 1879, 1882, 1886, 1890, 1891 and 1895 . In 1877, an American doctor named D.B . Simmons was working at Juzen Hospital (the previous hospital of Yokohama Medical College) in the Noge area in Yokohama, Kanagawa . He and his team tried to cure cholera patients by disinfecting the patients and their wastes with carbolic acid or phenol . They knew that isolating the patients was a good way to prevent the epidemic . As there was no hospital for infectious diseases in Kanagawa, they hurriedly built a small temporary hospital near Juzen Hospital and named it Ota Isolation Hospital, where cholera patients were sent and treated . In 1879 as people suffered again from an epidemic Ota Hospital was replaced by Izumicho Isolation Hospital, which became a hospital for infectious diseases two decades later in 1900 and was called Yokohama Manji Hospital . Manji means to cure all . Wilhermus Hubertus van der Heyden, a Dutch doctor, worked for this hospital . The first regulation of cholera prevention in Japan was issued by the Bureau of Health of the Ministry of Internal Affairs in 1879 . ...

Yeni Tip Tarihi Arastirmalari, 1995, 1, 55 - 65
{Cholera epidemics in the Ottoman Empire during 1910-1913 and relevant events}; Unat EK; From 1910 to 1913 was one of the disastrous periods of the history of the Ottoman Empire . Cholera epidemic was one of the causes of this calamity . The early cases of cholera were diagnosed on July 15 in Erzurum in 1910 . The disease was imported from Russia and started to spread in the country . Cholera appeared in Istanbul on September 1st and spread rapidly . This danger necessitated the mobilization of all civilian and military resources . In spite of these efforts, Istanbul became an important focus of cholera for Turkey, because of intensive human traffic in the capital . Some cholera cases came from Iran to Iraq and from Italy to Libya and disease spread out to the vicinities during this year . This epidemic disappeared in January 1911 . According to the official records, between July 15, 1910 and January 12, 1911 cholera killed 4023 people . In May 1911 cholera reappeared in Samsun and spread within the Ottoman Empire and 18876 persons were infected with cholera and 12143 of them died . In 1912 and 1913, the foundations of the Ottoman Empire were shaken by Balkan War, military defeats, lost territory, unlucky refugees and immigrants, and dreadful calamity of cholera epidemic . There is no reliable official record on the exact numbers of cholera patients and deaths . This great epidemic subsided during the Autumn of 1913 . In the Ottoman Empire, preparation of Kolle's vaccine against cholera was started in 1912 and its was applied during 1913.

Cuad Complut Hist Med Cienc, 1993, 1, 129 - 42
{The cholera epidemic in Spanish legislation, 1833 and 1834: the application of a method}; Peral Pacheco D; The problem of the cholera epidemic in two Royal Orders of 1833 and 1834 is analized . To do so, the author applies a method know as "the topology of discourse" with which he attempts to get to know the underlying conceptual basis in the discursive manifestation of the text under analysis . Two notions, antagonism and complementary are the main instruments employed by this method.

Arch Hist Filoz Med, 1995, 58(3), 265 - 76
{Epidemic cholera in the province of Lublin in the year 1866}; Lotysz M; In the year 1866 an epidemic of cholera broke out in the province of Lublin . Expecting the cholera, the authorities governing then issued quite a number of anti-choleric orders, particularly concerning preventive measures . Although the cholera was then supposed not to be a contagious disease, some of the undertaken preventive measures brought a beneficial effect, and mainly the isolation of sick persons, disinfection or interdiction to eat raw food, brought a beneficial effect . The number of persons affected by cholera reached 4836, thereof 3128 (64%) were Jews . 1686 patients, thereof 1039 (61.1%) Jews died . The Jewish population in that time amounted to 14% of the inhabitants of the province . In the group of sick Jews, 49.9% were children, while among Christian patients the proportion of children was 7.5% . The most seriously affected by the cholera was the district of Radzyn . The losses due to the cholera were 10 times smaller than these during the precedent epidemic in the year 1855.

Nippon Ishigaku Zasshi, 1999 Sep, 45(3 Pt 1495), 373 - 400
On the prevalence of Asiatic cholera in Okinawa, 1879 and "Ryukyu-kiko" Written by Hironobu Tsuchiya; Fukase Y; The greatest prevalence of Asiatic cholera since the adoption of modern statistics was in 1879 . Especially in Okinawa Prefecture, its morbidity rate was the largest in Japan in that year . Dr . Hironobu Tsuchiya, who was appointed as an official of the Department of Inner Affairs, wrote "Ryukyu-kiko" as a private memorandum . He wrote more about the situation of administrative confusion than he did about the condition of Asiatic cholera . This paper also mentions the brief sketch of Tsuchiya's life and the description of the manufacturing methods, effectiveness and use of chemical drugs which are contained in his "Sinyaku-shoko".

Med Ges Gesch, 1997, 16, 125 - 44
The homoepathic management of cholera in the nineteenty century with special reference to the epidemic in London, 1854; Leary B; During the nineteenth century homoeopaths claimed better results than their conventional colleagues did for the treatment of epidemic cholera . Those of the London Homoeopathic Hospital in 1854 have been put forward, for 150 years, as evidence of the efficacy of homoeopathy . The evidence is examined in this paper . Failure to inflict exhausting allopathic treatments must have contributed considerably to the homoeopathic success . However it appears probable that the homoeopathic remedies themselves played an active part in the successful treatment of cholera cases.

Hippokrates (Helsinki), 1995, 12, 62 - 88
{Four cholera epidemics in nineteenth-century London}; Tynkkynen K; Asiatic cholera originated in India and spread to Europe in the early years of the nineteenth-century . In Britain the first cases were diagnosed late in 1831 . The epidemic, reached London in February 1832 . The authorities were poorly prepared for the invasion of a new epidemic and the doctors disagreed bitterly on the measures to be taken . There was little co-operation between the authorities, and the fact that the urban poor mistrusted the medical profession did not improve the situation . All this resulted in several cholera riots . These riots were not, however, as violent as those in several other cities in Europe . The 1832-33 cholera epidemic claimed 4,000 to 7,000 victims in London . It seems probable that several isolated cases of cholera occurred in London in 1852 . It was not, however, until September 1853 that it was officially announced by the British authorities that a cholera epidemic was claiming victims not only in London but also in other parts of the country . During this epidemic numerous Londoners lodged complaints against nuisances in the metropolis . Yet, in most cases, the fines imposed on offenders were rather slight, since the authorities were extremely reluctant to interfere with anyone's trade or business . It was during this epidemic that John Snow, a London doctor, succeeded in tracing the epidemic to a single water pump on Broad Street in the Golden Square area . Snow did not, however, succeed in convincing the majority of his colleagues regarding the erroneous nature of the miasma theory during the epidemic of 1853/54, which cost the lives of some 12,000 people in the city area....

Vet Immunol Immunopathol, 2001 Nov, 83(1-2), 107 - 14
Simulated solar UVB exposure inhibits transcutaneous immunization to cholera toxin via an irradiated skin site in cattle; Morrow CK et al.; Transcutaneous immunization (TCI) is a new needle-free vaccination technology with the potential to reduce the risk of needle-borne disease transmission and carcass damage within the livestock industries . The principal antigen-presenting cell involved in TCI is thought to be the epidermal Langerhans cell . Langerhans cell function is inhibited by cutaneous ultraviolet-B radiation (UVB) exposure . Such exposure may inhibit TCI through sun exposed skin sites due to the phenomenon of local low dose photoimmunosuppression . TCI of cattle to cholera toxin (CT) resulted in the generation of a serum anti-CT-specific IgG(2) response . However, exposure of cattle to a sub-inflammatory dose of simulated solar UVB (2.43 x 10(3)J/m(2)) significantly (P<0.05) inhibited TCI to CT via irradiated skin sites.

Soz Praventivmed, 2001, 46(4), 225 - 32
The changing assessments of John Snow's and William Farr's cholera studies; Eyler JM; This article describes the epidemiological studies of cholera by two major British investigators of the mid-nineteenth century, John Snow and William Farr, and it asks why the assessments of their results by contemporaries was the reverse of our assessment today . In the 1840s and 1850s Farr's work was considered definitive, while Snow's was regarded as ingenious but flawed . Although Snow's conclusions ran contrary to the exceptations of his contemporaries, the major reservations about his cholera studies concerned his bold use of analogy, his thoroughgoing reductionism, and his willingness to ignore what seemed to be contrary evidence . Farr's electric use of current theories, his reliance multiple causation, and his discovery of a mathematical law to describe the outbreak in London in 1849 was much more convincing to his contemporaries . A major change in thinking about disease causation was needed before Snow's work could be widely accepted . William Farr's later studies contributed to that acceptance.

Am J Public Health, 2001 Oct, 91(10), 1574 - 6
Collaboration, cholera, and cyclones: a project to improve point-of-use water quality in Madagascar; Dunston C et al.; In November 1999, CARE Madagascar, Population Services International (PSI), and the Centers for Disease Control and Prevention (CDC) selected 30 poor communities in urban Antananarivo as the target population for launch of the Safe Water System . The system consists of behavior change techniques along with point-of-use treatment and safe storage of water . The project was launched in March 2000, ahead of schedule, because a cholera epidemic struck Madagascar in January . Because of the enormous demand created by the cholera epidemic and by 3 cyclones that followed in the next 3 months, the project grew to national scale in less than a year . The combination of community mobilization and social marketing resulted in increased demand for and use of the Safe Water System.

Arch Dis Child, 2001 Oct, 85(4), 293 - 5
Solar disinfection of drinking water protects against cholera in children under 6 years of age; Conroy RM et al.; BACKGROUND AND AIMS: We have previously reported a reduction in risk of diarrhoeal disease in children who used solar disinfected drinking water . A cholera epidemic, occurring in an area of Kenya in which a controlled trial of solar disinfection and diarrhoeal disease in children aged under 6 had recently finished, offered an opportunity to examine the protection offered by solar disinfection against cholera . METHODS: In the original trial, all children aged under 6 in a Maasai community were randomised by household: in the solar disinfection arm, children drank water disinfected by leaving it on the roof in a clear plastic bottle, while controls drank water kept indoors . We revisited all households which had participated in the original trial . RESULTS: There were 131 households in the trial area, of which 67 had been randomised to solar disinfection (a further 19 households had migrated as a result of severe drought) . There was no significant difference in the risk of cholera in adults or in older children in households randomised to solar disinfection; however, there were only three cases of cholera in the 155 children aged under 6 years drinking solar disinfected water compared with 20 of 144 controls . CONCLUSIONS: Results confirm the usefulness of solar disinfection in reducing risk of water borne disease in children . Point of consumption solar disinfection can be done with minimal resources, which are readily available, and may be an important first line response to cholera outbreaks . Its potential in chorine resistant cholera merits further investigation.

J Immunol, 2001 Oct 1, 167(7), 3677 - 81
Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice; Peng X et al.; In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer's patches (PPs), and mesenteric lymph nodes . Mice were implanted s.c . with a morphine slow release pellet . Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet . After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later . Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days . Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo . Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated . Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants . Morphine also inhibited CT-specific IgA and IgG levels in serum . By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo . The effect of morphine on TGF-beta, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT . Morphine significantly decreased TGF-beta mRNA compared with that in the placebo group, and naltrexone blocked this effect . These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-beta, a putative IgA switch factor, in the gastrointestinal tract.

J Neurochem, 2001 Sep, 78(5), 991 - 9
Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons; Shogomori H et al.; We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism . Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes . In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD . Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively . Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation . In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus . We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.

Infect Immun, 2001 Sep, 69(9), 5716 - 25
Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen-presenting cells; George-Chandy A et al.; Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens . CTB binds with high affinity to GM1 ganglioside cell surface receptors . In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets . Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen . In contrast, no beneficial effects were observed when CTB was simply admixed with antigen . CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells . Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface . These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.

Jpn J Ophthalmol, 2001 Jul-Aug, 45(4), 332 - 8
Immunosuppressive effect of cholera toxin B on allergic conjunctivitis model in guinea pig; Saito K et al.; PURPOSE: To investigate the new method of immunotherapy using cholera toxin B (CTB) in experimental allergic conjunctivitis . METHODS: We used 21 white Hartley guinea pigs . The animals were sensitized by intraperitoneal injection of ovalbumin (100 microg/mL) and albumin hydroxide (5 mg/mL) repeated after an interval of 2 weeks . One week after the second injection, conjunctivitis was induced by topical instillation of ovalbumin (5 mg/mL) . The animals were divided into two groups, CTB group and control group . The CTB group underwent pretreatment of topical instillation of CTB (4 microg/30 mL) and ovalbumin (10 microg/30 mL), three times a day for 3 days, 1 week before the intraperitoneal injection . The control group did not undergo the pretreatment . Clinical examination was performed at 0.5, 6, and 24 hours after the development of conjunctivitis . Histological examination was performed at 6 and 24 hours . RESULTS: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation of ovalbumin . The allergic reaction score was significantly less in the CTB group than in the control group (Mann-Whitney U-test: P <.01) . The clinical reactions subsided after 6 hours . The CTB group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival epithelium, than the control group at 6 and 24 hours . CONCLUSION: Pretreatment with topical CTB and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis.

J Cardiovasc Pharmacol, 2001 Aug, 38(2), 232 - 9
Kappa-opioid receptor agonist inhibits the cholera toxin-sensitive G protein in the heart; Yu XC et al.; To explore the signaling mechanisms of the negative modulation of beta-adrenoceptors by kappa-Opioid receptors (kappa-OR) in the heart, the possibility of the interaction at the level of G protein and receptor was determined . Cholera toxin, an activator of the stimulatory G protein (Gs), elevated electrically induced intracellular Ca2+ ({Ca2+}i) transients and induced ribosylation of the alpha-subunit of Gs (Gsalpha) in rat ventricular myocytes . The effects were significantly attenuated by U50,488H, a specific agonist of kappa-OR, and were abolished by nor-binaltorphimine, a selective kappa-OR antagonist . The content of Gsalpha, however, was not affected by U50,488H . Receptor binding experiments showed that neither Bmax nor Kd of the binding of {3H}CGP-12177, a beta-adrenoceptor antagonist, was affected by U50,488H . The current study provides the first evidence that kappa-OR stimulation inhibits the ribosylation of the alpha-subunit of the Gs protein, thus inhibiting the action of cholera toxin on the protein.

J Biol Chem, 2001 Oct 5, 276(40), 36939 - 45 Epub 2001 Jul 30.
A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity; Rodighiero C et al.; Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit . The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1) . Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity . CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT . The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts) . Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro . Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization . These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.

Biochim Biophys Acta, 2001 Jul 27, 1537(1), 27 - 41
Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine; Peterson JW et al.; Mediators of cholera toxin (CT)-induced fluid secretion include 3',5'-adenosine monophosphate (cAMP), prostaglandin E(2) (PGE(2)), and 5-hydroxytryptamine (5-HT) . Administration of L-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE(2) to stimulate Na+ transport in Ussing chambers . We demonstrated that L-histidine chemically modified the structure of PGE(2) but had no direct effect on cAMP or 5-HT . L-Histidine and imidazole reacted with PGE(2) in vitro in cell-free mixtures incubated at 37 degrees C and pH 7.0 under an atmosphere of N(2) with the formation of PGE(2)-imidazole and PGE(2)-histidine covalent adducts . Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE(2) . A Michael adduct then was formed between C11 of 11-deoxy-Delta(10) PGE(2) (PGA(2)) and the tau nitrogen in the imidazole ring of L-histidine . Importantly, the isolated PGE(2)-imidazole and PGE(2)-histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops . The protection provided by PGE(2)-imidazole, PGE(2)-histidine, and L-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.

Lancet, 2001 Jul 21, 358(9277), 233 - 7
Fear and frustration--the Liverpool cholera riots of 1832; Gill G et al.; Public mistrust in the medical profession is not new . We describe a series of street riots that took place in the city of Liverpool in north-west England in 1832 during a cholera epidemic . The disturbances were directed primarily against the local medical fraternity . The episode is of interest, since the same city recently experienced a similar crisis of confidence between doctors and public . On this occasion the cause was not cholera, but rather the reports from Alder Hey Children's Hospital that organ parts from deceased infants undergoing necropsy had been kept for several years without parental consent.

Ann Otol Rhinol Laryngol, 2001 Jul, 110(7 Pt 1), 646 - 54
Oral administration of collagen conjugated with cholera toxin induces tolerance to type II collagen and suppresses chondritis in an animal model of autoimmune ear disease; Kim N et al.; B10.RIII (H-2r) mice were orally administered cyanogen bromide peptide 11 (CB11) or cholera toxin B (CTB)-conjugated CB11 to induce tolerance in collagen-induced autoimmune ear disease . Oral administration of a high dosage of CB11 provided partial protection from chondritis . However, administration of a tiny amount of CTB-CB11 conjugate effectively suppressed chondritis . Oral administration of CTB-CB11 conjugate did not alter the stimulation of T cells in vitro or the fine specificities of B cells . The oral administration of CTB-CB11 caused a higher level of type II collagen-specific IgG and its subclass . Interestingly, increases of TH1 cytokine (interferon-gamma) in Peyer's patches and of TH1/TH2 cytokines (interleukin-2 and interleukin-4) in lymph nodes were detected in mice that had been fed CTB-CB11 . An increase of CD8+ T cells in the Peyer's patches with a decrease of CD8+ T cells in lymph nodes was seen in mice that had been fed CTB-CB11 . These results suggest that protection from chondritis by oral administration of minute amounts of CTB-CB11 conjugate can be achieved by a mechanism distinct from that of conventional oral tolerance induction.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8536 - 41 Epub 2001 Jul 10.
A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity; Aman AT et al.; GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells . More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes . Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action . We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57 . Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity . The implications of these findings on the mode of action of cholera toxin are discussed.

Sante, 2001 Apr-Jun, 11(2), 73 - 8
{Cholera in Tamatave (Madagascar) February-July 2000: epidemiological characteristics}; Jaureguiberry S et al.; The seventh pandemic of cholera reached Madagascar in March 1999 . In 15 months of dissemination, 31,095 people were infected, 5.8% of whom died . The first case in Madagascar was recorded in Tamatave (the second largest city and the island's main port, on the east coast) on February 29 2000 . The health services of the district responded efficiently to the crisis . We carried out an epidemiological investigation to determine the characteristics of the outbreak in Tamatave . We retrospectively studied the records of patients consulting for diarrhea at the health centers of Tamatave from February 29 to June 30 2000 . We defined cholera cases according to the recommendations of the World Health Organization for international notification . We recorded the date of diagnosis, sex, age, address, main clinical symptoms, treatment and outcome . Statistical analysis was carried out with Epi Infor 5.01b software . During the 123 days of the study, we recorded 356 cases of cholera, 5 of whom died (attack rate = 2.5 per thousand, case fatality rate = 1.4%) . The median age of the patients was 27 years and 8 months and the sex ratio was 1 . The attack rate was highest for men aged over 50 years . Severe clinical signs were observed in one sixth of the cases . Men aged 15 to 29 years and 30 to 49 years presented more severe disease than women of the same age (relative rates of 3.6 and 5.9 respectively, p < 0.01) . Cholera may become endemic in this province following this outbreak . It may be difficult to stop the spread of cholera within a country but appropriate organization of the medical structure and staff and training in cholera management may decrease the rate of mortality due to this disease.

Acta Paediatr, 2001 May, 90(5), 505 - 10
Efficacy of a packaged rice oral rehydration solution among children with cholera and cholera-like illness; Zaman K et al.; In past studies, patients with cholera and cholera-like diarrhoea treated with rice oral rehydration solution (ORS) had lower purging rates and a shorter illness duration . We evaluated a new packet form of rice ORS (CeraLyte-90) in 167 boys aged 5 to 15 y, with acute, dehydrating cholera and cholera-like diarrhoea in Bangladesh . The patients were randomized to receive either CeraLyte-90 (n = 85) or glucose ORS (n = 82) and were given early feeding and early antibiotics . The efficacy of the two solutions was compared for stool output during the first 8 h, the first 24 h, and total output, duration of diarrhoea, hematocrit, serum electrolytes and requirement for unscheduled intravenous fluids . The clinical and laboratory characteristics of the two groups were comparable on admission, and most of the patients had cholera (88% and 84% in the CeraLyte and glucose groups, respectively) . The mean (+/- SE) stool output was 20% less in the rice ORS group during the first 8 h of treatment (86.2+/-6.6 ml/Kg vs 108.8+/-7.9 ml/Kg, p < 0.05), but the outputs during the other time periods were similar in the two groups, although children in the rice ORS group had slightly more vomiting on day one (p < 0.05) . The mean serum electrolyte concentrations in both groups of children remained within normal range . CONCLUSION: The study documents the safety and efficacy of the new, packaged rice ORS.

Can J Physiol Pharmacol, 2001 Jun, 79(6), 471 - 80
Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A; Thorin-Trescases N et al.; The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein alpha(s) subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation . Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G1 phase without induction of necrosis or apoptosis in VSMC . Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic alpha subunit . The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX . Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation . After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively) . After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106 . In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G1 phase.

J Comp Pathol, 2001 Jan, 124(1), 29 - 35
Atypical cilia in the bronchiolar epithelium of pigs experimentally infected with hog cholera virus; Carrasco L et al.; To study the effect of hog cholera virus on the epithelial cells of the bronchiolar mucosa, 12 pigs were inoculated with a highly virulent strain . Immunohistochemical and ultrastructural examination of the ciliated epithelial cells demonstrated an increase in the number of atypical cilia . The latter showed alterations in the microtubular pattern, possibly resulting from viral interference with the normal metabolism of the epithelial cells.

Avian Dis, 2001 Apr-Jun, 45(2), 461 - 6
Relationship of increased fowl cholera outbreaks in turkeys with high environmental temperatures; Simensen E et al.; The relationship of an increase in fowl cholera outbreaks in turkeys with an increase in environmental temperatures during June, July, August, and September between 1959 and 1992 was analyzed . High environmental temperatures were found to be influential in the development of fowl cholera in turkeys . When the average monthly maximum environmental temperatures for 5 mo of July and 7 mo of August during the 13 yr between 1967 and 1979 were above 30.5 C, there was a significantly (P < 0.05) higher number of fowl cholera outbreaks in turkeys for each month than during the same months when the average maximum temperatures were below 30.5 C . To test the hypothesis that an increase in fowl cholera outbreaks was preceded by an increase in temperature, the pre- and postoutbreak temperatures for 46 selected outbreak clusters occurring between 1959 and 1992 were averaged . Both the average maximum and minimum temperatures for the latter 9 days of the preoutbreak period were highly significantly (P < 0.001) higher than those of the average cluster outbreak day and the following four postoutbreak days . Also, for the nine individual days of the latter pre-outbreak period, the daily average maximum temperature was significantly (P < 0.05) higher for 3 days and partially significantly (P < 0.10) higher for 3 days than that of the average cluster outbreak day, and the daily average minimum temperature was significantly (P < 0.05) higher for 2 days and partially significantly (P < 0.10) higher for 1 day than that for the average cluster outbreak day.

Infect Immun, 2001 Jul, 69(7), 4486 - 92
Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells; Martin M et al.; Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles . In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated . CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb . CT but not LT-IIa or LT-IIb reduced the expression of CD4(+) T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells . CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4(+) T cells . In a coculture system, CT-treated CD4(+) T cells induced significantly less TNF-alpha and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4(+) T cells . These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.

Brain Res Bull, 2001 Mar 15, 54(5), 537 - 42
Differential roles of spinal cholera toxin- and pertussis toxin-sensitive G proteins in nociceptive responses caused by formalin, capsaicin, and substance P in mice; Chung KM et al.; The aim of the present study is to characterize the roles of spinal cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G proteins in the regulation of various nociceptive responses . The effects of intrathecal (i.t.) pretreatments with CTX and PTX on the formalin (subcutaneous)-, capsaicin (i.t.)-, and substance P (SP; i.t.)-induced nociceptive behaviours were examined in mice . Pretreatment with CTX (i.t.; 24 h before) significantly and dose-dependently (0.05-0.5 microg) suppressed both the first and second phases of the formalin-induced nociceptive behaviour . On the other hand, pretreatment with PTX (i.t., 6 days before) at the same doses (0.05-N0.5 microg) did not affect the formalin-induced response . Capsaicin (i.t., 0.5 microg)- and SP (i.t., 0.7 microg)-induced nociceptive behaviours were attenuated by the pretreatment with CTX . In addition, SP-induced nociceptive response was also attenuated by the pretreatment with PTX . However, the capsaicin-induced nociceptive response was not influenced by PTX pretreatment . These findings suggest that, at the spinal cord level, CTX-sensitive G-proteins are involved in the formalin-, capsaicin-, and SP-induced nociceptive behavioural responses, whereas PTX-sensitive G proteins are involved in SP-induced nociceptive response.

J Pharmacol Exp Ther, 2001 Jun, 297(3), 940 - 5
Cholera toxin induces prostaglandin synthesis via post-transcriptional activation of cyclooxygenase-2 in the rat jejunum; Beubler E et al.; The mechanisms of diarrhea in Asiatic cholera have been studied extensively . Cyclic AMP, 5-hydroxytryptamine, prostaglandins, and the function of neuronal structures have been implicated in the pathogenesis of cholera . To elucidate the role of the different isoforms (COX-1 and COX-2) of cyclooxygenase in cholera toxin (CT)-induced fluid secretion and intraluminal prostaglandin E(2) (PGE(2)) release in the rat jejunum in vivo, the effects of the COX-2 inhibitors NS-398 ({N-(2-cyclohexaloxy-4-nitrophenyl)methanesulfonamide}) and DFU {5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone}, and of the COX-1 inhibitor SC-560, were studied . Net fluid transport was measured gravimetrically and PGE(2) by radioimmunoassay . COX-1 and COX-2 mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and COX-2 protein by Western blot analysis in mucosal scrapings . CT caused profuse net fluid secretion in all control rats . The COX-2 inhibitors NS-398 and DFU, but not the COX-1 inhibitor SC-560 or dexamethasone, dose-dependently inhibited CT-induced fluid secretion and PGE(2) release . RT-PCR showed expression of COX-1 and of COX-2 mRNA in control rats . CT did not induce an increase and dexamethasone did not reduce COX-2 mRNA, whereas lipopolysaccharide caused a marked induction of COX-2 mRNA, which was inhibited by dexamethasone . A weak band of COX-2 protein was observed in controls; however, CT enhanced COX-2 levels, which remained unaffected by dexamethasone . It can be assumed that post-transcriptional modulation is responsible for CT-induced increase in COX-2 protein . COX-1 does not seem to be involved . Therefore, PGE(2) produced by COX-2 seems to be responsible for the profuse fluid secretion induced by CT, and COX-2 appears to be a specific target for the treatment of Asiatic cholera.

Anal Biochem, 2001 May 15, 292(2), 171 - 7
In vitro assembly of novel cholera toxin-like complexes; Hatic SO 2nd et al.; Cholera toxin (CT) is responsible for the major pathological features of cholera, but in addition to its cytotoxic properties, CT is a potent mucosal adjuvant when coadministered with antigens at mucosal sites . Discovery of CT adjuvanticity has prompted the generation of CT chimeras with reduced toxicity and improved efficiency for antigen presentation at mucosal sites . To date, chimeric forms of CT have been produced in bacterial strains by coexpressing the CT B subunit and a chimeric form of the CT A subunit consisting of a target protein antigen fused with the A2 polypeptide of CT . In this study, a chimeric protein consisting of green fluorescent protein (GFP) fused with polypeptide A2 was generated to investigate the feasibility of assembling CT holotoxin-like complexes in vitro . The assembly of such holotoxin-like complexes would expand the variety of antigenic compounds that could be incorporated into CT-based vaccines . In this study, GFP-A2/CTB complexes could be generated in vitro using a stepwise denaturation-renaturation process . These findings suggest that it is possible to generate novel mucosal vaccines consisting of macromolecules that are chemically coupled to polypeptide A2 and reconstituted into CT-like complexes in vitro .

Acta Physiol Scand, 2001 Feb, 171(2), 153 - 60
Effect of cholera toxin on passive transepithelial transport of 51Cr-ethylenediaminetetraacetic acid and 14C-mannitol in rat jejunum; Fihn BM et al.; Intestinal fluid secretion, mainly derived from the crypts, induced, for example, by cholera toxin, decreases the passive transport of small hydrophilic molecules into the lumen . However, the effect of the fluid secretion on the passive absorption of these substances and thus on the permeability of the villus absorptive area is not known . Therefore, the transport rates of 51Cr-ethylenediaminetetraacetic acid (EDTA) and 14C-mannitol from lumen to plasma and from plasma to lumen were recorded in jejunal loops of anaesthetized rats during cholera toxin-induced fluid secretion in the absence and presence of glucose in the intestinal lumen and expressed as clearance (microL (min g)(-1)) . The results showed that the cholera toxin induced fluid secretion and abolished the passive absorption of 51Cr-EDTA both in the absence and presence of luminal glucose during a high perfusion rate (0.5 mL min(-1)) . The clearance of mannitol was also inhibited at the low perfusion rate (0.2 mL min-1) with the glucose-free perfusate but only reduced with the glucose perfusate . The results show that mechanisms activated by cholera toxin inhibit the passive absorption of inert hydrophilic substances . This is proposed to be mainly caused by a reduction in the accessibility of the villus epithelium to the luminal content . Furthermore, the secretion seems predominantly to inhibit the passive absorption at the basal parts of the villus while the absorption rate at the villus tips is better preserved . The results also show that the intestinal absorption and secretion of fluid takes place at different locations (villus and crypts, respectively).

Infect Immun, 2001 Jun, 69(6), 4125 - 8
Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans; Jertborn M et al.; The induction of immune responses to rectally administered recombinant cholera toxin B subunit (CTB) in humans was studied . Three immunizations induced high levels of CTB-specific antibody-secreting cells, particular of the immunoglobulin A isotype, in both rectum and peripheral blood . Antitoxin antibody responses in rectal secretions and serum were also found.

Eur J Pharmacol, 2001 Apr 6, 417(1-2), 45 - 9
Neuropeptide FF receptors couple to a cholera toxin-sensitive G-protein in rat dorsal raphe neurones; Roumy M et al.; In rat dorsal raphe neurones, nociceptin (300 nM) reduced the peak {Ca(2+)}(i) transient, triggered by depolarization, by 36.7+/-1.8% (n=46) . This effect of nociceptin decreased to 16.7+/-2.9% (n=18) after pre-treatment of the neurones with pertussis toxin (5 microg/ml, 2-6 h) but was unchanged (37.4+/-2.1%, n=44) after pre-incubation with cholera toxin (5 microg/ml, 2-6 h) . This suggests that, in dorsal raphe neurones, the ORL1 receptor couples to inhibitory (G(i/o)) G-proteins . The neuropeptide FF analogue, {D-Tyr1, (N-Me)Phe(3)}neuropeptide FF (10, 100, 1000 nM), acted as an anti-opioid and reduced the effect of nociceptin (300 nM, 30 s) by 62.0+/-3.3% (n=28) . Following pre-incubation with cholera toxin (5 microg/ml, 2-6 h) {D-Tyr1, (N-Me)Phe3} neuropeptide FF was unable, at the three concentrations tested, to block nociceptin activity . We conclude that, in rat dorsal raphe neurones, neuropeptide FF receptors couple to stimulatory G-proteins (Gs).

Cell Microbiol, 2001 May, 3(5), 311 - 29
Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments; Millar DG et al.; Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems . The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum . Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins . Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles . When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles . However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles . The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment . The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells . CtxB also modulated OVA trafficking and inhibited antigen presentation . These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation . The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

Brain Res Mol Brain Res, 2001 Mar 31, 88(1-2), 83 - 93
The comparative analysis of proenkephalin mRNA expression induced by cholera toxin and pertussis toxin in primary cultured rat cortical astrocytes; Won JS et al.; In rat astrocytes, incubation with cholera toxin (CTX; 0.1 microg/ml) for 8 h increased proenkephalin (proENK) mRNA level (10-fold), which was further increased by dexamethasone (DEX; 1 microM) (2.2-fold as much as CTX alone) . Although pertussis toxin (PTX; 0.1 microg/ml) did not affect the basal proENK mRNA level, DEX significantly increased proENK mRNA level in PTX-treated cells (6-fold) . The inhibition of protein synthesis by cycloheximide (CHX; 15 microM) also increased proENK mRNA level in PTX-treated cells (5.2-fold), but not in CTX-stimulated cells . The treatment with CTX, but not PTX, increased c-Fos and Fra-2 protein levels as well as AP-1, CRE, or ENKCRE-2 DNA binding activity, but neither toxin affected Fra-1, c-Jun, JunB, and JunD protein levels . CHX significantly attenuated CTX-induced increase of c-Fos or Fra-2 protein level and AP-1, CRE, or ENKCRE-2 DNA binding activity, although CHX alone did not affect the basal AP-1, CRE, and ENKCRE-2 DNA binding activities . Phosphorylated CREB level was increased by both CTX and PTX, although the magnitude of phosphorylation of CREB by PTX was much less than that by CTX . In addition, CHX further or persistently increased PTX- or CTX-induced phosphorylated CREB levels in parallel with increases in proENK mRNA . However, DEX did not alter the basal or stimulated phosphorylated-CREB level . These results suggest that the elevation of phosphorylation of CREB rather than AP-1 level may be involved in CTX-induced and CHX-dependent-PTX-induced increase of proENK mRNA level . In addition, AP-1 expression or CREB phosphorylation appears not to be involved the potentiative action of DEX on proENK mRNA expression in CTX- and PTX-treated astrocytes.

Infect Immun, 2001 May, 69(5), 3476 - 82
Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin; Millar DG et al.; Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits . Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB . EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes . These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

Infect Immun, 2001 May, 69(5), 3466 - 71
Induction and distribution of intestinal immune responses after administration of recombinant cholera toxin B subunit in the ileal pouches of colectomized patients; Kilhamn J et al.; The induction and dissemination of mucosal immune responses to recombinant cholera toxin B subunit (rCTB) administered into the ileal pouches of patients, who had been colectomized because of ulcerative colitis, was analyzed . Biopsies from the duodenum and ileal pouch were collected, along with peripheral blood and ileostomy fluids . Two immunizations induced strong CTB-specific immunoglobulin A (IgA) antibody-secreting cell (ASC) responses in the duodenum in five of five patients, whereas weaker and less-frequent ASC responses were noted in the ileal pouch . Intestine-derived CTB-specific IgA ASCs were found in peripheral blood in three of the five patients . The vaccination also induced significant IgA antitoxin titer rises in ileostomy fluid in all of the patients . Increased production of gamma interferon in cell cultures from the ileal pouch was found in four of five patients after the vaccination . These results clearly indicate that rCTB administered into the distal ileum is capable of inducing B-cell responses in the "entire" small intestine and that homing of immunocompetent cells occurs preferentially to the duodenum.

Infect Immun, 2001 May, 69(5), 2853 - 7
Induction of systemic antifimbria and antitoxin antibody responses in Egyptian children and adults by an oral, killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine; Hall ER et al.; We assessed serologic responses to an oral, killed whole-cell enterotoxigenic Escherichia coli plus cholera toxin B-subunit (ETEC-rCTB) vaccine in 73 Egyptian adults, 105 schoolchildren, and 93 preschool children . Each subject received two doses of vaccine or placebo 2 weeks apart, giving blood before immunization and 7 days after each dose . Plasma antibodies to rCTB and four vaccine-shared colonization factors (CFs) were measured by enzyme-linked immunosorbent assay . Immunoglobulin A (IgA) antibodies to rCTB and CFA/I were measured in all subjects, and those against CS1, CS2, and CS4 were measured in all children plus a subset of 33 adults . IgG antibodies to these five antigens were measured in a subset of 30 to 33 subjects in each cohort . Seroconversion was defined as a >2-fold increase in titer after vaccination . IgA and IgG seroconversion to rCTB was observed in 94 to 95% of adult vaccinees, with titer increases as robust as those previously reported for these two pediatric cohorts . The proportion showing IgA seroconversion to each CF antigen among vaccinated children (range, 70 to 96%) and adults (31 to 69%), as well as IgG seroconversion in children (44 to 75%) and adults (25 to 81%), was significantly higher than the corresponding proportion in placebo recipients, except for IgA responses to CS2 in adults . IgA anti-CF titers peaked after one dose in children, whereas in all age groups IgG antibodies rose incrementally after each dose . Independently, both preimmunization IgA titer and age were inversely related to the magnitude of IgA responses . In conclusion, serologic responses to the ETEC-rCTB vaccine may serve as practical immune outcome measures in future pediatric trials in areas where ETEC is endemic.

J Biol Chem, 2001 Jun 22, 276(25), 22838 - 43 Epub 2001 Mar 28.
Involvement of ADP-ribosylation factor 1 in cholera toxin-induced morphological changes of Chinese hamster ovary cells; Morinaga N et al.; ADP-ribosylation factor 1 (ARF1) was originally found as a cofactor in CT-catalyzed ADP-ribosylation of Galpha(s) but is now known to participate in vesicle trafficking . We asked whether ARF1 function in vesicular trafficking is necessary for CT-induced morphological changes in Chinese hamster ovary (CHO) cells, which result from increased intracellular cAMP . Brefeldin A treatment of cells suppressed CT action, confirming a requirement for Golgi integrity . Overexpression of a GFP-ARF1 fusion protein did not affect the morphological changes induced by CT, but changes were reduced in cells overexpressing guanine nucleotide exchange-defective ARF1(T31N) or GTP hydrolysis-deficient ARF1(Q71L) mutants . In cells expressing these mutants, 8-bromo-cAMP induced changes similar to those seen in cells transfected with ARF1 or vector . Inhibition of CT action was specific for mutants of ARF1 and not reproduced by analogous mutants of ARF5 or ARF6 . ARF1(Q71L) was mostly colocalized with betaCOP, but ARF5(Q71L) less so . ARF6(Q67L) did not colocalize with betaCOP and was partially associated with the plasma membrane . These data are consistent with the conclusion that ARF1 influenced CT action in cells by its specific function in the vesicular transport pathway used by CT to travel from plasma membrane to Golgi to ER.

Gastroenterol Clin Biol, 2001 Jan, 25(1), 20 - 3
{Comparative effects of oral rehydratation solutions in experimental cholera in the rat}; Beji Serairi R et al.; The composition of the World Health Organisation (WHO) solution in oral rehydration therapy has remained controversial because of its total osmolarity (303 mosm/L) and higher sodium concentration (90 mEq/L), increasing the risk of hypernatraemia . AIM OF THE STUDY: To compare the efficacy of two reduced-osmolarity oral rehydration solutions (S1: 268 mosm/L and 50 mEq/L Na(+); S2: 240 mosm/L and 60 mEq/L Na(+) ) with the WHO recommended formula taken as the reference solution . Water, electrolytes and glucose fluxes were directly measured in vivo, in isolated ligated loops of rat jejunum (n=12) . Intestinal secretion was induced by exposing jejunum to cholera toxin (CT=20 microg/loop) . RESULTS: All three test solutions similarly reversed cholera toxin-induced net water absorption (3.37 +/- 1.35; 3.31 +/- 0.43 and 3.13 +/- 0.66 microL/min.cm(2) for S1, S2 and WHO solutions respectively) . However, net Na secretion induced by cholera toxin was observed with S1 and S2 while Na absorption occurred with the WHO solution . CONCLUSION: For a same amount of water absorbed, Na absorption from reduced - osmolarity rehydration solutions is lower than with the WHO solution . Our data may contribute to a better rationale for the use of orally administered hydration solutions in man.

Vopr Virusol, 2001 Jan-Feb, 46(1), 42 - 4
{Development of delayed-type hypersensitivity to hog cholera}; Shubina NG et al.; Leukocyte migration inhibition test showed that pigs with classical swine fever develop delayed type hypersensitivity . The highest migration inhibition (65-85%) was observed in animals with the acute form, on day 3 after infection, or directly before death, or in animals immunized with reactogenic strain with clinical signs of disease . In some pigs with acute form the migrating capacity of leukocytes was restored on days 7-8 postinfection . Leukocyte migration inhibition factor is detected in the sera of pigs starting from day 3 after infection with the virulent strain . The degree of delayed type hypersensitivity correlated with the outcome of classical swine fever.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 31 - 5
{Cholera in the Far East of Russia . Communication 2 . Epidemiological characteristics of the outbreak of cholera El tor in Iuzhno-Sakhalinsk}; Onishchenko GG et al.; The outbreak of cholera eltor in Yuzhno-Sakhalinsk has been analyzed . The fact of the import of this infection from China and its transmission mainly by the water route due to the intensive contamination of environmental objects has been substantiated . The effective purification decontamination of sewage water plays decisive role on the system of anticholera measures . The conclusion on the necessity of increasing the effectiveness of measures for the sanitary protection of the territory has been made.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 26 - 31
{Cholera in the Far East of Russia . Communication 1 . Epidemiological characteristics of the outbreak of cholera El tor in Vladivostok}; Onishchenko GG et al.; The materials on the investigation of the outbreak cholera eltor in Vladivostok, caused by the import of infection from China . The leading role of the water route of transmission of this infection is shown due to the contamination of water sources with non-decontaminated sewage water . The complex of antiepidemic measures was carried out, which made it possible to arrest the spread of cholera and liquidate its foci.

Neurosci Res, 2001 Feb, 39(2), 221 - 32
Subnuclear distribution of afferents from the oral, pharyngeal and laryngeal regions in the nucleus tractus solitarii of the rat: a study using transganglionic transport of cholera toxin; Hayakawa T et al.; The central distributions of afferents from the oral cavity, the pharynx, the larynx and the esophagus to the nucleus tractus solitarii (NTS) were examined by using transganglionic anterograde transport of the cholera toxin B subunit (CT-b) . Injections of CT-b into the body of the tongue and the hard palate resulted in heavy labeling of the lateral subnucleus (l-NTS) of the NTS rostral to the area postrema . Injection into the root of the tongue resulted in heavy labeling of the l-NTS, the dorsal half of the medial (m-NTS), the intermediate (im-NTS) and the interstitial (is-NTS) subnuclei rostral to the area postrema . Injections into the soft palate and the pharynx resulted in a similar labeling pattern in the is-NTS, im-NTS and m-NTS to that in the case of the root of the tongue, but this labeling extended rostrocaudally . Heavy labeling of the medial aspect of the l-NTS was found in the case of the soft palate, but the labeling was sparse in the case of the pharynx . Moderate labeling was also found in the commissural subnucleus (co-NTS) . Injection into the larynx resulted in labeling of the is-NTS throughout the NTS, and of the rostral half of im-NTS . Injection into the esophagus resulted in heavy labeling of the central subnucleus, and moderate labeling of the co-NTS and the caudal half of im-NTS . A few but consistent anterogradely labeled terminals were found to appose retrogradely labeled small neurons in the rostral tip of the dorsal motor nucleus of vagus in the cases of injections into the root of the tongue, the soft palate, the pharynx, and the larynx . These results have characterized the viscerotopic representation of afferent projections from the oral and the cervical visceral organs to the subnuclei of the NTS.

Clin Sci (Lond), 2001 Mar, 100(3), 291 - 8
Peroral immunization with Helicobacter pylori adhesin protein genetically linked to cholera toxin A2B subunits; Kim BO et al.; Helicobacter pylori is a major cause of gastric-associated diseases . To evaluate the efficacy of a possible vaccine antigen against H . pylori infection, the chimaeric construct adhesin--CTXA2B, derived from H . pylori adhesin genetically coupled to cholera toxin (CTX) subunits A2 and B (CTXA2B), was expressed in Escherichia coli as an insoluble recombinant chimaeric protein . The protein was then purified by denaturation, renaturation and size-exclusion chromatography . The composition of purified adhesin--CTXA2B was verified by SDS/PAGE and Western blotting with antibodies to antigenic components of adhesin and CTXB, and confirmed as a chimaeric protein with G(M1)-ganglioside binding activity and adhesin epitopes by a G(M1)-ELISA developed using antibodies to adhesin . Oral immunization of mice with adhesin--CTXA2B induced higher levels of mucosal IgA and serum IgG antibodies to H . pylori adhesin and to CTXB than in mice immunized with adhesin or CTXA2B alone . Adhesin--CTXA2B was also demonstrated to be a potential protective antigen in a mouse model of H . pylori infection . The immunization of mice with adhesin--CTXA2B protected 62.5% of mice infected with H . pylori SS1 strain, whereas adhesin immunization was not able to confer protection to mice . This protection may be correlated with high levels of mucosal IgA and serum IgG antibodies against H . pylori adhesin . Taken together, the results indicate that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin-CTXA2B chimaeric protein could be a potential component in future H . pylori vaccine development.

Yi Chuan Xue Bao, 2000, 27(11), 966 - 71
{Induction of protective immune responses in rhesus monkey by immunization with recombinant plasmids of polyvalent epitopes of Plasmodium falciparum using cholera toxin B as adjuvant}; Zhong H et al.; The immunogenicity and protective efficacy of the DNA vaccine which include cholera toxin B subunit (CTB) and polyvalent protective epitopes of Plasmodium falciparum (awte gene) was assessed using rhesus monkeys as animal models . Recombinant plasmids of pCMV-CTB-AWTE were given to five rhesus monkeys three times with two weeks intervals by intramuscle (i.m.) route, immunization dose was 500 micrograms per plasmid per animal . High levels of anti-CTB and anti-malaria epitopes antibodies and P . falciparum epitope specific CTL activity were elicited . The vaccinated groups was challenged with 1.25 x 10(8) of P . cynomolgi parasites . All monkeys of the control group was patent for at least 34 days, the DNA vaccinated groups wasn't infected during the 60 days we detected . The cocktail DNA vaccine which contains multi-stage and multi-epitope antigen gene shows excellent immunogenicity and protective efficacy, the results also suggests that DNA vaccine plays an important role against malaria infection.

Ann N Y Acad Sci, 2000, 915, 339 - 46
Mechanisms of cholera toxin-induced diarrhea; Beubler E et al.; In the pathogenesis of cholera, cyclic adenosine monophosphate, 5-hydroxytryptamine, prostaglandins, and the function of neuronal structures have been implicated . To elucidate the role of different isoforms of cyclooxygenase (COX)-1 and COX-2, selective COX-2 inhibitors were used . The selective COX-2 inhibitors NS-398 and DFU completely suppressed cholera toxin-induced prostaglandin E2 biosynthesis and caused a dose-dependent inhibition of cholera toxin-induced fluid secretion in the rat jejunum in vivo . Constitutive expression of COX-1 but also of COX-2 mRNA was found in mucosal scrapings of the rat jejunum . Cholera toxin had no effect on COX-1 as well as COX-2 mRNA expression . Treatment of rats with dexamethasone did not effect cholera toxin-induced prostaglandin E2 biosynthesis and did not influence the expression of COX-2 mRNA, further substantiating that cholera toxin does not cause an induction of COX-2 mRNA . Treatment of rats with E . coli lipopolysaccharide caused a marked increase in COX-2 mRNA expression that was inhibited by dexamethasone . In conclusion, the results provide evidence that cholera toxin, in addition to other mediators, uses prostaglandin E2 to exert its secretory effect and that in the case of cholera toxin prostaglandins are metabolized via COX-2.

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