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 centur