Cholera

Anat Embryol (Berl), 1997 Jun, 195(6), 539 - 55
Development of the retinotectal system in the pigeon: a cytoarchitectonic and tracing study with cholera toxin; Manns M et al.; The optic tectum of the pigeon (Columba livia) is marked by morphological dorso-ventral and left-right differences . Both features seem to be related to functional specializations, but the responsible developmental mechanisms are unclear . Since the visual system becomes functional only after hatching, the developmental processes might be extended into the post-hatching period . The development of the asymmetries in the tectofugal system, however, depends on an asymmetric light stimulation acting already before hatching . As a first attempt to resolve this discrepancy, we examined the ontogeny of the retinotectal system by labeling the developing retinal projection with cholera toxin subunit B, in conjunction with an analysis of the cytoarchitectonic differentiation of the optic tectum . The data demonstrate that the first fibers to penetrate all retinoreceptive tectal layers could be observed from embryonic day 15 onwards, indicating that visual information could in principle be already processed before hatching . The afferent projection already exhibited the adult lamination pattern directly at the beginning of the invasion of the tectal layers; a surprising finding, since at that time the lamination pattern of the tectal layers did not have an adult appearance . The differentiation of the outer retinoreceptive laminae started only when the whole optic tectum was occupied by retinal fibers, 4 days after hatching, and was finished a week later . The dorso-ventral differences in the thickness of layers 4 and 5 were not apparent before the first week after hatching . The late appearance of these differences indicates that their maturation may be influenced by retinal input.

J Immunol, 1997 Jun 1, 158(11), 5321 - 9
Oral immunization with simian immunodeficiency virus p55gag and cholera toxin elicits both mucosal IgA and systemic IgG immune responses in nonhuman primates; Kubota M et al.; Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 microg) of recombinant SIV p55gag (p55) with or without cholera toxin (CT, 50 microg) as a mucosal adjuvant . The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 microg) and CT . Further, high levels of p55-specific IgG and IgA Abs were present in external secretions from both groups . The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group . When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent . CD4+ T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs . These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4+ T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4 . These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4+ Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.

Eur J Pharmacol, 1997 May 20, 326(2-3), 223 - 8
Nitric oxide counteracts 5-hydroxytryptamine- and cholera toxin-induced fluid secretion and enhances the effect of oral rehydration solution; Beubler E et al.; The effects of pharmacological modulation of the nitric oxide (NO) pathway on intestinal fluid transport were studied in a model of ligated jejunal loops of anaesthetized rats in vivo . Close intraarterial infusion of 5-hydroxytryptamine (5-HT) (0.16 microg/min) induced net fluid secretion . Intravenous infusion of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (0.55 mg/kg per min) reversed net fluid absorption in controls to net secretion and significantly enhanced 5-HT-induced fluid secretion . 5-HT-induced net fluid secretion was inhibited by intravenous infusion of L-arginine (8.88 mg/kg per min), sodium nitroprusside (22.2 microg/kg per min), or 3-morpholino sydnonimine (SIN-1) (22.2 microg/kg per min) . Intraluminal instillation of cholera toxin (0.5 microg/ml) induced net secretion, which was significantly enhanced by L-NAME and reduced by L-arginine . Another series of experiments was performed using a model of luminally perfused jejunal loops . Cholera toxin (10 microg/ml) induced profuse net fluid secretion also in this model . L-Arginine and sodium nitroprusside significantly enhanced net fluid absorption compared to controls and abolished the secretory effect of cholera toxin . Luminal perfusion with oral rehydration solution enhanced net absorption of fluid in controls and reversed cholera toxin-induced secretion to absorption . Intravenous infusion, but not intraluminal administration, of L-arginine significantly enhanced the antisecretory effect of oral rehydration solution . These results give further support to the existence of an intestinal NO-mediated proabsorptive tone, which also downregulates fluid secretion elicited by different enterotoxins or mediators of secretion . Intravenous administration of exogenous sources of NO counteracts intestinal fluid accumulation and augments the antisecretory effect of oral rehydration solution, findings which may lead to therapeutic consequences.

Mol Microbiol, 1997 May, 24(3), 489 - 97
Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit; Backstrom M et al.; The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins . A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB . These were used to investigate the influence of specific residues on receptor-binding specificity . A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids . In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed . In addition, a mutant LTB with a single Gly-33-->Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides . We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.

Scand J Gastroenterol, 1997 May, 32(5), 478 - 84
Regional differences in the effect of mucosal glucose and amino acids on ion transport in normal and cholera toxin-stimulated porcine small intestine; Grondahl ML et al.; BACKGROUND: This study explores regional differences in the response to mucosal D-glucose and L-amino acids in both normal intestine and intestine stimulated with cholera toxin . METHODS: Proximal, mid and distal small intestines from 6- to 8-week-old pigs were bathed in Ussing chambers with a buffer containing 15 mM serosal glucose, and the effect of adding a cocktail giving luminal chamber concentrations of 15 mM D-glucose and 20 mM of each L-alanine, L-proline, L-lysine, L-phenylalanine, and L-glutamine on transmucosal Na+ and Cl- transport was measured . RESULTS: In all segments of both normal and cholera toxin-treated intestine, electrogenic Na+ and electroneutral NaCl absorption were promoted . No significant differences in the net increase of Na+ and Cl- absorption between normal and cholera toxin-stimulated intestine were present . Under both conditions no segmental differences were present in the stimulated Cl- absorption, describing identical capacity for stimulated electroneutral NaCl absorption . In contrast the electrogenic Na+ absorption was, compared to the proximal part, doubled in the mid and distal parts under both conditions . CONCLUSIONS: We conclude that mucosal D-glucose and L-amino acids stimulate electroneutral NaCl and electrogenic Na+ absorption to the same degree in normal and cholera toxin-treated small intestine . There is no segmental difference in stimulated electroneutral NaCl absorption, while electrogenic Na+ absorption is highest in mid and distal parts.

Mol Endocrinol, 1997 May, 11(5), 538 - 49
Luteinizing hormone/choriogonadotropin-dependent, cholera toxin-catalyzed adenosine 5'-diphosphate (ADP)-ribosylation of the long and short forms of Gs alpha and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha*; Rajagopalan-Gupta RM et al.; Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, Gs) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and Gs or any other G protein(s) . We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G alpha proteins were associated with the LH/CG receptor in ovarian follicular membranes . Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G alpha protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by {32P}NAD . Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner . Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of Gs alpha and the 40-kDa protein as Gi alpha . A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of Gs alpha and Gi . These results suggest that a portion of Gi along with the long and short forms of Gs alpha are associated physically with the LH/CG receptor in ovarian follicular membranes.

Gastroenterology, 1997 May, 112(5), 1529 - 35
Glucose-stimulated sodium transport by the human intestine during experimental cholera; Schiller LR et al.; BACKGROUND & AIMS: Net sodium absorption from oral rehydration solution is increased by both glucose-sodium cotransport and solvent drag . The aim of this study was to measure the relative importance of glucose-sodium cotransport and solvent drag in the stimulation of net sodium absorption by oral rehydration solution . METHODS: Total intestinal perfusion was used in normal subjects with and without intrajejunal cholera toxin using three test solutions containing 100 mmol/L sodium and either 100 mmol/L mannitol (control), 100 mmol/L glucose, or no additional solute (hypotonic solution) . The increase in sodium absorption greater than control with hypotonic solution represented sodium absorption stimulated by solvent drag; the further increase in sodium absorption induced by glucose, greater than that noted with the hypotonic solution, represented sodium absorption stimulated by cotransport . RESULTS: Without cholera toxin, solvent drag and cotransport promoted sodium absorption at rates of 62 and 33 mmol/h, respectively . With cholera toxin, solvent drag and cotransport promoted sodium absorption at rates of 44 and 71 mmol/h, respectively . CONCLUSIONS: Net sodium absorption caused by cotransport increased more than twofold after exposure of the intestine to cholera toxin (P < 0.003) . This could be mediated by increased cotransport, a change in the stoichiometry of cotransport, or an increase in chloride permeability.

Synapse, 1997 May, 26(1), 46 - 54
Enhanced reward-related responding following cholera toxin infusion into the nucleus accumbens; Kelley AE et al.; In recent years, considerable focus has been directed to understanding how drugs of abuse affect neuronal function at the molecular level . For example, repeated administration of stimulants or opiates can induce long-lasting alterations in gene expression, transcription factors, and signal transduction pathways . Our laboratory previously showed that intraaccumbens infusion of cholera toxin (CTX), which alters the Gs protein such that production of cyclic Adenosine Monophosphate (AMP) is upregulated, causes pronounced, long-lasting motor activation and sensitization to stimulants . In the present experiments, the effect of intraaccumbens infusion of cholera toxin on reward-related responding was investigated . The conditioned reinforcement (CR) paradigm was employed, which measures an animal's instrumental response to obtain presentation of a stimulus previously paired with a primary reward . When this stimulus supports acquisition of a new operant response (lever-pressing), it is termed a conditioned reinforcer (CR) . In the first experiment, the effects of bilateral intraaccumbens infusion of CTX (100 ng/1 microliter) were examined on previously-established responding . CTX treatment resulted in enhanced responding for the CR . This enhancement developed over several days and reached its peak 3 days following infusion . In the second experiment, the influence of CTX was examined on acquisition of responding for the CR . The group treated with CTX (100 ng) discriminated between the CR and control (NCR) lever earlier than the vehicle-infused group, and showed greater levels of responding on the CR lever . In the third experiment, it was determined that infusion of CTX (300 ng bilaterally) into the anterior dorsal striatum did not affect levels of responding, although a later test with cocaine in these animals (25 mg/kg, intraperitoneally) (i.p.) indicated that they were capable of potentiated responding . These data are interpreted as evidence that the G(S) protein-cyclic AMP second messenger system within the nucleus accumbens is directly involved in reward-related behavior.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4610 - 4
A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes; Bergerot I et al.; Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens . Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders . However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease . We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals . We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice . The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis . Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration . These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Biochim Biophys Acta, 1997 Apr 24, 1356(2), 237 - 48
T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-alpha; Szamel M et al.; Activation and translocation of protein kinases C is a key event in the regulation of T lymphocyte activation, proliferation and function . Stimulation of human peripheral blood lymphocytes with the monoclonal antibody BMA 031 raised against the T cell antigen receptor led to a bimodal activation of protein kinases C . The immediate activation and translocation of the protein kinase C isoform PKC-alpha was followed by activation and translocation of the protein kinase C-beta isoenzyme after 90 min of stimulation . Pretreatment of the cells with cholera toxin for 90 min completely abolished activation of protein kinase C-alpha . In sharp contrast, activation and translocation of protein kinase C-beta was not influenced by the bacterial toxin, suggesting that activation and translocation of different protein kinase C isoenzymes are regulated by distinct mechanisms of transmembrane signalling coupled to the T cell antigen receptor/CD3 complex . The expression of high affinity IL-2 receptors was completely inhibited by cholera toxin, while IL-2 synthesis and secretion were not influenced in BMA 031-stimulated human lymphocytes . Extensive control experiments have shown that the effects of cholera toxin were not mediated by its B subunit, and were independent of elevation of intracellular cAMP concentration, suggesting that cholera toxin interfered with a signalling pathway leading to activation of protein kinase C-alpha, which could be responsible for the inhibition of IL-2 receptor expression . This hypothesis was substantiated by the finding that upon introduction of antibodies against protein kinase C-alpha, IL-2 receptor gene expression was completely suppressed . The results suggest, that protein kinase C-alpha might be the major protein kinase C isoenzyme of a signal transduction cascade regulating IL-2 receptor expression in stimulated human lymphocytes.

J Clin Invest, 1997 Apr 15, 99(8), 1999 - 2004
Role of platelet-activating factor in Chinese hamster ovary cell responses to cholera toxin; Thielman NM et al.; Cholera toxin (CT)-induced intestinal secretion and Chinese hamster ovary cell (CHO) elongation involves cyclic adenosine monophosphate and protein synthesis-dependent prostaglandin formation . We previously reported inhibition of CT-induced intestinal secretion and CHO elongation by platelet-activating factor (PAF) receptor antagonists and secretion of PAF by human intestinal epithelial cells exposed to CT . Herein, we show that PAF is involved after cAMP and that PAF, like CT, mediates prostaglandin E2 synthesis in CHO cells . CT-induced CHO elongation was blocked by specific PAF receptor antagonists, BN52021 and SR27417 . SR27417 blocked dibutyryl cAMP-induced CHO elongation, but did not alter CHO elongation caused by PGE2 . Neither CT-stimulated cAMP accumulation nor PGE2 production was inhibited by SR27417 . Both PGE2 and PAF caused significant CHO elongation, but the latter did not stimulate significant cAMP production . In addition, PAF, like CT and dibutyryl cAMP, stimulated significant PGE2 production . Finally, the protein synthesis inhibitor cycloheximide, which completely blocks the effect of CT on prostaglandin synthesis, also blocked that of PAF, suggesting that PAF also mediates protein synthesis-dependent prostaglandin formation . We conclude that PAF is involved in CHO cytoskeletal responses to CT after the accumulation of cAMP and, like CT, PAF stimulates protein synthesis-dependent prostaglandin accumulation.

J Exp Med, 1997 Apr 7, 185(7), 1203 - 10
Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity; Yamamoto S et al.; Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties . We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis . Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops . Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses . Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs . OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response . These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Parasite Immunol, 1997 Apr, 19(4), 183 - 90
Intranasal administration of Schistosoma mansoni adult worm antigen in combination with cholera toxin induces a Th2 cell response; Akhiani AA et al.; Mice immunized with soluble adult worm antigen (SWAP) in combination with cholera toxin (CT) displayed significantly larger numbers of IgG1, IgM and IgA secreting cells in the spleen and in the lungs as compared to mice which had received SWAP only . The ratio of SWAP-specific IgG1 to IgG2a antibody-secreting spleen cells was also significantly higher in the SWAP-CT group . Analysis of cytokine responses revealed that SWAP-stimulated spleen and lung cells from the SWAP-CT group produced lower levels of IFN-gamma but higher levels of IL-4 and IL-5 as compared to cells from the SWAP group . These findings indicate that intranasal administration of SWAP-CT induces a Th2 cell response in the spleen and in the lungs . Our findings also suggest that CT was responsible for induction of this Th2 cell response, since intranasal administration of SWAP alone induced a Th1 type response in the spleen and in the lungs.

Gen Pharmacol, 1997 Apr, 28(4), 607 - 10
Effects of spinally and supraspinally injected 3-isobutyl-1-methylxanthine, cholera toxin, and pertussis toxin on cold water swimming stress-induced antinociception in the mouse; Suh HW et al.; 1 . The cold (4 degrees C) water swimming stress (CWSS) for 3 min significantly increased the inhibition of the tail-flick response in ICR mice . 2 . Pertussis toxin (PTX, 0.05-0.5 microgram) in mice pretreated intrathecally (IT) for 6 days attenuated the inhibition of the tail-flick response induced by CWSS . However, intracerebroventricular (ICV) pretreatment with PTX at the same doses did not affect CWSS-induced inhibition of the tail-flick inhibition . 3 . 3-Isobutyl-1-methylxanthine (IBMX, 0.01-1 ng) in mice pretreated IT for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by CWSS . However, IBMX in mice ICV pretreated ICV at the same doses was not effective in attenuating the CWSS-induced inhibition of the tail-flick response . 4 . Neither IT nor ICV pretreatment with cholera toxin (CTX, 0.05-0.5 microgram) for 24 hr affected the inhibition of the tail-flick response induced by CWSS . 5 . The ICV or IT injection of PTX, CTX, or IBMX did not affect the basal tail-flick response latency . 6 . It is concluded that spinal, but not supraspinal, PTX-sensitive G-proteins and cAMP phosphodiesterase may be involved in the antinociception produced by CWSS . However, neither spinal nor supraspinal CTX-sensitive G-proteins appear to be involved in mediating the antinociception induced by CWSS.

Am J Physiol, 1997 Apr, 272(4 Pt 1), C1123 - 33
Cholera and pertussis toxins increase acidification of endocytic vesicles without altering ion conductances; Van Dyke RW; Acidification of endocytic vesicles, driven by the vacuolar H+ pump, is affected by parallel ion transporters . Because adenosine 3',5'-cyclic monophosphate (cAMP) and heterotrimeric G proteins may alter ion transporters, I tested whether cholera and pertussis toxins affected acidification of rat liver endosomes . Fluorescein-labeled dextran-loaded "10-min" endosomes from cholera toxin-treated rats exhibited ATP-dependent rates of acidification in the presence and absence of Cl- or K+ that were approximately 60-120% (P < 0.05) faster than rates from control endosomes . This increase was greater for "older" "20-min" endosomes and less for 'early" "2-min" endosomes . Ion transport functions of 10-min and 20-min toxin-exposed endosomes were similar to those of 2-min control endosomes . Cholera toxin also increased ATP-dependent steady-state intravesicular H+ concentration by 38-218% (P < 0.05) . Pertussis toxin increased endosome acidification rates by 20-54% (P < 0.05) . Both toxins increased liver cAMP content, and endosomes prepared from perfused livers exposed to 0.75 mM dibutyryl cAMP exhibited similar increases in acidification rates . These studies indicate that both cholera and pertussis toxins markedly alter the function of rat liver endosomes . The mechanism is unlikely to reflect major changes in vesicle ion transporters but rather may indicate either an increase in the number of H+ pumps per endosome and/or changes in fusion, remodeling, and maturation of early endocytic vesicles in response to cAMP.

Synapse, 1997 Apr, 25(4), 335 - 44
Robust sensitization to amphetamine following intra-VTA cholera toxin administration; Byrnes JJ et al.; Studies were conducted regarding the hypothesis that enhanced cAMP formation in the ventral tegmental area (VTA) affects the magnitude of the behavioral responses elicited by psychostimulant drugs . In the first paradigm, spontaneous and amphetamine-elicited locomotor activity was measured at various times following injection of cholera toxin (CTX), a known activator of adenylate cyclase, into the VTA . Adult male rats showed enhanced amphetamine-stimulated locomotor activity when tested 1 or 3 days after treatment with 0.5 microgram CTX into the VTA . Spontaneous activity was markedly increased 1 and 3 days following treatment with the higher dose of 1.0 microgram CTX into the VTA, and amphetamine was still capable of eliciting an increased level of locomotor activity above this high baseline . Using a paradigm in which repeated amphetamine injections were given on an intermittent schedule following injection of CTX into the VTA, it was observed that a single low dose of amphetamine (0.5 mg/kg) given 1 day after CTX (0.5 microgram) injection into the VTA led to a markedly potentiated locomotor activity response to subsequent treatment with amphetamine . Evaluation of this protocol (initial amphetamine dose 24 h after CTX injection, and challenge treatment of amphetamine at various times thereafter) showed that the sensitization was long-lasting and could be observed after an initial dose of amphetamine as low as 0.1 mg/kg . A sensitized response was also expressed when the challenge dose was given directly into the nucleus accumbens . These data suggest that injection of CTX into the VTA enhances the induction of locomotor sensitization to amphetamine.

Pharmacol Biochem Behav, 1997 Mar, 56(3), 499 - 505
Cholera toxin effects on body temperature changes induced by morphine; Basilico L et al.; The present study evaluates the influence of cholera toxin and its B-subunit on thermic responses to morphine in the rats . The holotoxin (1 microg/rat) and the B-subunit (5 microg) were administered ICV and three days later rats were challenged ICV with morphine and tested for changes of body temperature . Cholera toxin, but not its B-subunit, modified the time course of the hyperthermic response induced by a low dose of morphine (2.5 microg), converted the hypothermia due to a higher dose of morphine (18 microg) to a consistent hyperthermia and only partially reduced the greater hypothermia induced by 36 microg of morphine . Cholera toxin-induced modifications of thermic responses to morphine were paralleled with a decreased Gs(alpha) immunoreactivity and a reduced ability for the toxin to catalyse the "in vitro" ADP-ribosylation of Gs(alpha) in hypothalamic membranes . In contrast, at the same time when morphine-induced effects on body temperature were assessed, no changes in pertussis toxin-mediated ADP-ribosylation of Gi(alpha)/Go(alpha), or basal adenylate cyclase activity, or binding of mu-opioid receptor selective ligand {3H}-DAMGO were observed in hypothalamic areas from rats treated with cholera toxin . These findings suggest that adaptative events secondary to prolonged activation of Gs(alpha) play a role in the modifications of thermic responses to morphine induced by CTX.

Pflugers Arch, 1997 Mar, 433(5), 638 - 47
Differential stimulation of intestinal mucin secretion by cholera toxin and carbachol; Epple HJ et al.; Cholinergic stimulation triggers the secretion of apically stored, preformed mucin from goblet cells but the pathway of cAMP-stimulated mucin secretion is not known . In this study the effect of cholera toxin on mucin secretion in the human colonic goblet cell line HT-29/B6 was investigated and compared to the action of carbachol . PAS staining of mucin blotted onto nitrocellulose served to quantify the secretion of total mucin . Metabolic labelling was used to evaluate the secretion of newly synthesized mucin . The mucinous nature of the detected material was confirmed with an immunoblot employing a well-characterized polyclonal antibody reacting with MUC2-mucin . Cholera toxin caused a 116-fold increase of intracellular cAMP and strongly stimulated the secretion of both preformed and newly synthesized mucin for more than 20 h . Carbachol only triggered the release of preformed mucin immediately after addition . The secretory response to cholera toxin could be partly inhibited by the protein kinase A inhibitor H8 and the microtubule inhibitor colchicine . The action of carbachol was not affected by these agents . In conclusion, we demonstrate a direct cAMP-dependent effect of cholera toxin on mucin secretion by intestinal goblet cells . In contrast to carbachol, the action of cholera toxin involves de novo synthesis of mucin molecules and microtubule-mediated secretion . There seem to be distinct secretion pathways for muscarinic or cAMP-dependent stimulation of mucin secretion.

Neurosci Lett, 1997 Feb 14, 223(1), 45 - 8
Amphetamine-induced sensitization and release of dopamine in slices from the ventral tegmental area of rats is enhanced following administration of cholera toxin into the ventral tegmental area; Byrnes JJ et al.; Administration of cholera toxin (CTX) into the ventral tegmental area (VTA) markedly potentiates the development of behavioral sensitization to amphetamine . Experiments were conducted to determine whether this phenomenon is associated with altered dopamine release from the VTA and nucleus accumbens (NAC) . Adult, male rats received bilateral injections of CTX (0-1 microgram) or its vehicle into the VTA . Half of the animals then received four injections of amphetamine (0.5 mg/kg, i.p.) given every other day, while the other half received no additional treatments . In both groups, locomotor responses to amphetamine (0.5 mg/kg, i.p.) were measured on experimental day 18 . One day later, amphetamine-induced {3H}dopamine release was measured in tissue slices of the VTA and NAC . Amphetamine-induced locomotor activity was augmented in rats receiving 0.5 or 1.0 microgram intra-VTA CTX pretreatment and the repeated amphetamine regimen . Amphetamine-induced {3H}dopamine release was increased from VTA but not NAC slices obtained from animals showing this behavioral sensitization . Thus, intra-VTA CTX treatment facilitates sensitization to low doses of repeated amphetamine which appears to be associated with the increased ability of this psychostimulant to release dopamine in the VTA.

J Biol Chem, 1997 Feb 14, 272(7), 4591 - 9
Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line; Orlandi PA; A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide . The latter is an ADP-ribosyltransferase, which activates the alpha-subunit of the stimulatory G protein of adenylyl cyclase . In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized . Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells . In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin . Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS . Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase . This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes . This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER) . When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI . Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI . Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes . These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.

Int J Epidemiol, 1997 Feb, 26(1), 212 - 9
A cost-benefit analysis of programmatic use of CVD 103-HgR live oral cholera vaccine in a high-risk population; Cookson ST et al.; BACKGROUND: Cholera spread to Latin America in 1991; subsequently, cholera vaccination was considered as an interim intervention until long-term solutions involving improved water supplies and sanitation could be introduced . Three successive summer cholera outbreaks in northern Argentina and the licensing of the new single-dose oral cholera vaccine, CVD 103-HgR, raised questions of the cost and benefit of using this new vaccine . METHODS: This study explored the potential benefits to the Argentine Ministry of Health of treatment costs averted, versus the costs of vaccination with CVD 103-HgR in the relatively confined population of northern Argentina affected by the cholera outbreaks . Water supplies and sanitation in this area are poor but a credible infrastructure for vaccine delivery exists . RESULTS: In our cost-benefit model of a 3-year period (1992-1994) with an annual incidence of 2.5 case-patients per 1000 population and assumptions of vaccine efficacy of 75% and coverage of 75%, vaccination of targeted high risk groups would prevent 1265 cases . CONCLUSION: Assuming a cost of US$602 per treated case and of US$1.50 per dose of vaccine, the total discounted savings from use of vaccine in the targeted groups would be US$132,100 . The projected savings would be altered less by vaccine coverage (range 75-90%) or efficacy (60-85%) changes than by disease incidence changes . Our analysis underestimated the true costs of cholera in Argentina because we included only medical expenditures; Indirect losses to trade and tourism had the greatest economic impact . However, vaccination with CVD 103-HgR was still cost-beneficial in the base case.

Immunol Cell Biol, 1997 Feb, 75(1), 47 - 53
Mucosal unresponsiveness to aflatoxin B1 is not broken by cholera toxin; Oliver AR et al.; Rabbits immunized via chronically isolated ileal loops with aflatoxin B1 (AFB) conjugated to porcine thyroglobulin (TG) mixed with the mucosal adjuvant cholera toxin (CT) produced very small mucosal antibody responses to AFB . Strong mucosal and systemic antibody responses to CT and TG were generated by this immunization protocol, suggesting that the observed unresponsiveness was specific to AFB . Parenteral immunization with AFB-TG produced strong serum IgG anti-AFB responses, indicating that the conjugate preparation was immunogenic and that the rabbits possess the requisite systemic B and T cell repertoires to recognize and respond to AFB . This mucosal unresponsiveness was distinct from oral tolerance, as animals immunized mucosally with AFB-TG mixed with CT produced vigorous serum IgG anti-AFB responses upon subsequent parenteral immunization with AFB-TG . In vitro mitogen stimulation of lymphocytes isolated from Peyer's patches and mesenteric lymph nodes of unimmunized rabbits revealed the presence of AFB-specific B cells at levels comparable with these found in the spleen . These observations indicate that unresponsiveness to AFB is hapten-specific, restricted to the mucosa, and refractory to the adjuvancy of CI.

FEBS Lett, 1997 Jan 20, 401(2-3), 104 - 8
Reduction of protein disulfide bonds in an oxidizing environment . The disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum; Majoul I et al.; Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge {Majoul et al . (1996) J . Cell Biol . 133, 777-789}, although the redox state in the ER favors disulfide formation . We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3 . Protein disulfide isomerase (PDI) exerts only a minor accelerating effect . Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction . These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.

J Immunol, 1997 Jan 15, 158(2), 834 - 41
Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo; Porgador A et al.; To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT) . Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization . Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen . To test whether CTL induced by i.n . immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth . Animals immunized i.n . with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v . injection with OVA-pulsed dendritic cells . Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.

FEBS Lett, 1997 Jan 13, 401(1), 95 - 7
Engineering of cholera toxin A-subunit for carriage of epitopes at its amino end; Sanchez J et al.; The cholera toxin A-subunit (CTA) was genetically engineered at its amino end and tested for carriage of epitopes by fusion of the STa heat-stable enterotoxin analogue CAELCCNPAC . Efficient holotoxin formation by complementation in trans with cholera toxin B-subunit (CTB) indicated no decrease in affinity for CTB but evidence of reduced toxicity suggests steric interference by the decapeptide with the active site . The holotoxin was stable, able to bind to GM1 and was recognized by anti-STa and anti-CTA antibodies . The use of a full-length CTA might have been a key step for successful genetic fusions . Based on these findings, it seems worthwhile pursue the development of CTA for construction of recombinant mucosal immunoadjuvants.

Lik Sprava, 1997 Jan-Feb, (1), 68 - 72
{A methodological approach to determining electrolyte disorders in cholera patients}; Kyrychenko PD; A total of 198 cholera patients were studied for blood concentrations of electrolytes; the above patients were treated at Mykolaiv Cholera Hospital during an outbreak in 1995 . It is advisable that blood plasma concentration of electrolytes be represented as mmol/kg of the mass of a cholera patient's body instead of mmol/l, to indicate disturbances in electrolytic balance . It is a matter of principle for the assessment of the patient's state to be done, first of all, before initiating the rehydration therapy treatments . Determinants of electrolytes in cholera patients got decreased not only in severe course of the illness but also in moderately severe one . Of all the electrolytes studied in blood plasma, it is in K+ and Cl- that deviations from the norm were at their greatest . Since electrolytic balance is a sensitive indicator of homeostasis of human organism it is useful to calculate the volume of salt solutions for the primary rehydration according to blood plasma concentration of Cl- in mmol/kg of mass of the patient's body . Lowering of electrolyte concentration in erythrocytes occurred only in severe course of cholera involving K+ only.

Adv Exp Med Biol, 1997, 419, 93 - 7
Enhanced degradation of stimulatory G-protein (Gs alpha) by cholera toxin is mediated by ADP-ribosylation of Gs alpha protein but not by increased cyclic AMP levels; Shah BH; Cholera toxin (CT) catalyses ADP-ribosylation of the alpha-subunit of stimulatory protein (Gs) leading to stimulation of adenylyl cyclase and elevated intracellular cAMP . Persistent treatment (24-48 h) of C6 glioma cells with cholera toxin (100 ng/ml) caused marked downregulation of Gs alpha (75-80%) which could not be mimicked by dibutyryl cAMP (1 mM) and forskolin (10 microM) over the same time periods suggesting that CT-mediated Gs alpha downregulation is independent of cAMP production . However, CT increased the expression of Gq/11 alpha proteins at 24 and 48 h of treatment . The increase in mRNA levels of Gq/11 alpha proteins preceded the increase in Gq/11 proteins . Such stimulatory effects of CT were mimicked by forskolin and dibutyryl-cAMP . These results suggest that CT-mediated downregulation of Gs alpha is independent of cAMP but CT upregulates the expression of Gq/11 alpha proteins in a cAMP-dependent manner.

Yi Chuan Xue Bao, 1997, 24(1), 78 - 86
{A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene}; Cao C et al.; A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene . The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene . Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition . The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter . This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V . cholerae and Escheria coli respectively . The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

Life Sci, 1997, 60(7), PL107 - 13
An examination of the relationship between mu-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins; Goode TL et al.; The hypothesis that mu-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than mu-opioid agonists having higher antinociceptive efficacy was tested . Supraspinal antinociceptive efficacy for the three mu-opioid agonists morphine, {D-Ala2, NMePhe4, Gly5-ol}-enkephalin (DAMGO) and sufentanil in the mouse 55 degrees C warm-water tail-flick test was evaluated 18-24 h after intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA) . The beta-FNA pretreatment (0.2-2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy . The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v . (0.1-3.0 micrograms) pertussis toxin (PTX) or cholera toxin (CTX) . The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy . CTX augmented sufentanil-induced antinociception . Morphine- and DAMGO-induced antinociception were unaffected by CTX . These data suggest that: (i) highly efficacious mu agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory Gi-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious mu agonists have greater capacity to utilize CTX-sensitive stimulatory Gs-proteins than do mu-agonists with lower efficacy.

Am J Physiol, 1997 Jan, 272(1 Pt 1), E7 - 17
Effect of in vivo injection of cholera and pertussis toxin on glucose transport in rat skeletal muscle; Ploug T et al.; Cholera toxin (CTX) and pertussis toxin (PTX) were examined for their ability to inhibit glucose transport in perfused skeletal muscle . Twenty-five hours after an intravenous injection of CTX, basal transport was decreased approximately 30%, and insulin- and contraction-stimulated transport was reduced at least 86 and 49%, respectively, in both the soleus and red and white gastrocnemius muscles . In contrast, PTX treatment was much less efficient . Impairment of glucose transport appeared to develop 10-15 h after CTX administration, which coincided with development of hyperglycemia despite hyperinsulinimia, increased plasma free fatty acid levels, increased adenosine 3',5'-cyclic monophosphate (cAMP) concentrations in muscle, but no difference in plasma catecholamines . Twenty-five hours after CTX treatment, GLUT-4 protein in both soleus and red gastrocnemius muscles was decreased, whereas no change in GLUT-1 protein content was found . In contrast, GLUT-4 mRNA was unchanged, but transcripts for GLUT-1 were increased > or = 150% in all three muscles from CTX-treated rats . The findings suggest that CTX via increased cAMP impairs basal as well as insulin- and contraction-stimulated muscle glucose transport, at least in part from a decrease in intramuscular GLUT-4 protein.

J Clin Microbiol, 1997 Jan, 35(1), 284 - 5
Cholera from raw seaweed transported from the Philippines to California; Vugia DJ et al.; In March 1994, a California woman without any recent travel developed acute, profuse, watery diarrhea . Her astute physician diagnosed cholera after ordering the appropriate stool culture, and the patient improved on an oral antibiotic . Epidemiologic investigation implicated seaweed from the Philippines that was transported by a friend to California and subsequently eaten raw as the vehicle of infection.

J Comp Neurol, 1996 Dec 9, 376(2), 265 - 77
Intrinsic association fiber system of the piriform cortex: a quantitative study based on a cholera toxin B subunit tracing in the rat; Datiche F et al.; By using retrograde and anterograde transport of the B subunit of cholera toxin (CTb), we examined quantitatively the association fiber systems, i.e., the collaterals of pyramidal cell axons, that reciprocally connect both the rostral and the caudal parts of the piriform cortex (PC) . Well-defined CTb injections were obtained in layers Ib or II-III of the rostral and the caudal parts of the PC . Using precision counting, we determined the proportion of cellular profiles in layers II and III that gave rise to association fibers and thus demonstrated a predominance of rostrocaudal fibers over the caudorostral ones . Our data also support a precise laminar organization of the PC in which the rostrocaudal fibers originated mainly from layer II and the caudorostral fibers primarily from layer III . Cholera toxin injections into layer Ib produced a peak of labeled profiles 2 mm from the site, indicating that a large proportion of the association fibers from layer II travel for at least 2 mm and then synapse in layer Ib . At either end of the PC, the association projections with respect to olfactory processing, propagation of the activity within the PC, and the possible role of intrinsic fibers in olfactory memory.

Zentralbl Veterinarmed A, 1996 Dec, 43(10), 611 - 8
Effect of cholera toxin on glucose absorption and net movements of water and electrolytes in the intestinal loop of sheep; Hyun HS et al.; This study was designed to evaluate the effect of cholera toxin on glucose absorption and net movement of water and electrolytes in the jejunal loop of sheep . Intraluminal perfusion was performed at the rate of 1 ml/min with isotonic 10 mM glucose solution . Osmolality was adjusted by adding NaCl, and the outflow solution was collected every 10 min . After a 30 min control period, cholera toxin was applied intraluminally for 30 min at doses of 30, 60, and 120 micrograms/loop . In the control period, water, sodium and chloride were absorbed, while potassium and bicarbonate were secreted . Cholera toxin reversed the net absorption of water, sodium and chloride to net secretions, and this secretory response to cholera toxin was dose-dependent . Bicarbonate secretion was stimulated dose-dependently by cholera toxin . Potassium secretion was also increased at all doses, though this response was not dose-dependent . The net glucose absorption was decreased dose-dependently by cholera toxin . In conclusion, these results indicate that cholera toxin stimulates water and electrolyte secretion, and inhibits glucose absorption in the jejunal loop of sheep.

Int Immunol, 1996 Dec, 8(12), 1849 - 56
Cholera toxin B subunit binding to an antigen-presenting cell directly co-stimulates cytokine production from a T cell clone; Li TK et al.; Cholera toxin (CT) is a powerful immunomodulator with strong adjuvant activity . Much of this activity is retained by the binding component alone, cholera toxin B subunit (CTB) . Little is known about the mechanism of the immunomodulatory activity of CTB . In this study, both CT and CTB were found to dramatically enhance IL-4 production from a T cell clone stimulated with antigen and the B cell hybridoma LB as antigen-presenting cell (APC) . Enhancement of cytokine production was seen following pretreatment of the APC with CT or CTB, while pretreatment of the T cells had no effect . Furthermore, stimulatory activity on the APC was stable to fixation with paraformaldehyde, demonstrating that the activity was mediated by a surface molecule on the APC . CT-pretreated APC also enhanced IL-4 production from anti-CD3 mAb-stimulated T cells, indicating that CT was providing a co-stimulatory signal . CT treatment of LB cells did not alter the expression of class II MHC molecules, CTLA-4 counter-receptors, LFA-1 or ICAM-1 . When mAb were raised against the CT-pretreated APC, the only antibodies that were found to inhibit IL-4 production were those specific for CTB itself . The antibodies blocked even when the CT or CTB were already bound to the APC, arguing that co-stimulation was provided by a direct interaction with CTB . Blocking experiments suggested that APC-associated CTB molecules are interacting with non-GM1 receptors on the T cells . This novel finding of CTB-mediated T-B interaction provides one of the first potential mechanisms for the adjuvant activity of CTB.

Endocrinology, 1996 Dec, 137(12), 5392 - 9
Transformation of rat thyroid follicular cells stably transfected with cholera toxin A1 fragment; Zeiger MA et al.; Activating mutations of the alpha subunit of the G protein G(s) (G(s)alpha) have been identified in thyroid adenomas and well-differentiated thyroid carcinomas . To examine the role of activating mutations of G(s)alpha in thyroid neoplasia, we transfected rat follicular thyroid (FRTL-5) cells with a transgene in which the cholera toxin A1 subunit (CTA1) is expressed under the control of the rat thyroglobulin gene promoter (TG) . This transgene recapitulates effects of the activating mutation of G(s)alpha by its ability to ADP-ribosylate and thereby inhibit GTPase activity of endogenous G(s)alpha molecules . To assess the effect of G(s)alpha activation on cell growth, TGCTA1, or control, pM AM neotransfected FRTL-5 cells (10(4)-10(6)) were injected s.c . into nude mice . TGCTA1-transfected FRTL-5 cells grow in nude mice, whereas control cells do not . Tumor histology revealed increased mitotic activity, infiltration of skeletal muscle, perineural invasion, and plugging of lymphatic spaces . In addition, nude mice injected with TGCTA1 transfected cells or xenografted with the tumors developed metastases to lung . These results indicate that activation of G(s)alpha and constitutive production of cAMP in FRTL-5 cells can result in TSH-independent cellular proliferation and neoplastic transformation.

Zentralbl Veterinarmed A, 1996 Nov, 43(9), 543 - 52
Effect of 5-HT2 and 5-HT3 receptor antagonists on cholera toxin-induced fluid hypersecretion in the pig jejunum; Grondahi ML et al.; 5-Hydroxytryptamine is a mediator in cholera toxin-induced hypersecretion in the small intestine . The aim of this study was to determine the effect of the 5-hydroxytryptamine receptor antagonists ketanserin, granisetron, ondansetron and tropisetron on cholera toxin-induced hypersecretion in the pig jejunum . Hypersecretion was induced by cholera toxin in ligated jejunal loops . The antagonists were administered subcutaneously at a dose of 100 micrograms/kg . Furthermore, the effect of intraluminally instilled ondansetron was studied . None of the antagonists altered basal absorption or caused fluid hypersecretion . Cholera toxin caused a dose-dependent electrolyte and fluid hypersecretion . The apparent maximal effect, 6.8 +/- 0.4 mg fluid x mg dry loop-1, was reduced by ondansetron, granisetron and tropisetron by about 40%, 30%, and 20%, respectively, whereas ketanserin had no effect . Intraluminal ondansetron reduced the effect of cholera toxin by about 50% . These results demonstrate that 5-hydroxytryptamine3 antagonists administered subcutaneously reduce the cholera toxin-induced hypersecretion in the pig jejunum . Finally, the results support species differences with respect to the antagonistic effect of the tested drugs in cholera toxin-induced hypersecretion.

Eur J Neurosci, 1996 Nov, 8(11), 2320 - 7
Cholera and pertussis toxins reveal multiple regulation of cAMP levels in the rabbit carotid body; Cachero TG et al.; It is known that hypoxia (PO2 approximately equal to 66-18 mm Hg), acting via unknown receptors, increases carotid body cAMP levels in Ca(2+)-free solutions, indicating that low PO2 activates adenylate cyclases independently of the action of the released neurotransmitters . The aim of the present work was to investigate the involvement of G proteins in the genesis of the basal level of cAMP and on the increase in cAMP induced by low PO2 . In carotid body homogenates, cholera toxin- and pertussis toxin-induced {32P}ADP-ribosylation of two protein bands of approximately equal to 42 and 45 kDa, and approximately equal to 39 and 40 kDa respectively; in both cases, prior incubation of the carotid bodies with the toxins reduced {32P}ADP-ribosylation by > 90% . In intact carotid bodies, cholera toxin treatment increased cAMP levels more in normoxic than in hypoxic organs, indicating that hypoxia releases neurotransmitters acting on receptors negatively coupled to adenylate cyclases . Cholera toxin-treated carotid bodies incubated in Ca(2+)-free solution had identical cAMP levels in normoxia and in hypoxia . In pertussis toxin-treated normoxic carotid bodies the cAMP level was close to control, but in pertussis toxin-treated hypoxic carotid bodies cAMP rose to a level similar to those seen in normoxic cholera toxin-treated organs, indicating that low PO2 releases neurotransmitters acting on receptors positively coupled to adenylate cyclases . Pertussis toxin-treated carotid bodies incubated in Ca(2+)-free solution lost their capacity to increase cAMP in response to hypoxia, indicating that a G protein sensitive to pertussis toxin is needed for this response . This implies that the carotid bodies express a pertussis toxin-sensitive G protein positively coupled to adenylate cyclases, or that a Gs protein requiring the cooperative action of Go/Gi donated beta gamma subunits mediates the increase in cAMP level produced by hypoxia.

J Neurochem, 1996 Nov, 67(5), 2134 - 40
Rate of retrograde transport of cholera toxin from the plasma membrane to the Golgi apparatus and endoplasmic reticulum decreases during neuronal development; Sofer A et al.; Various glycolipid-binding toxins are internalized from the cell surface to the Golgi apparatus . Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity . We now demonstrate that rhodamine (Rh)-CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG-2 cells and that the A subunit is essential for retrograde transport to the ER . In addition, the rate of internalization of Rh-CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature . The Golgi apparatus was labeled in almost all 1-day-old neurons after < 1 h of incubation with Rh-CT but was labeled in < 10% of 14-day-old neurons after 1 h . During the first 14 days in culture, there was a 15-fold increase in the number of 125I-CT-binding sites per cell, indicating that the decrease in the rate of internalization of Rh-CT is not due to reduced levels of cell surface GM1 in older neurons . These results imply that the rate of retrograde transport of CT from the plasma membrane to the Golgi apparatus and ER is regulated during neuronal development and differentiation.

Ann N Y Acad Sci, 1996 Oct 31, 795, 361 - 5
Interleukin-12 alters helper T-cell subsets and antibody profiles induced by the mucosal adjuvant cholera toxin; Marinaro M et al.; We have shown that systemic administration of rmIL-12 could trigger Th1-type responses to a protein antigen delivered orally with CT as mucosal adjuvant . The most striking finding was that IL-12 could retain its regulatory effects when orally administered and could redirect the immune response to the oral vaccine toward a Th1-type . However, regulation by orally administered IL-12 differed from parenteral treatment with IL-12 since only the latter treatment affected mucosal S-IgA responses . These findings have important implications for the development of mucosal vaccines that induce the desired immune response.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12339 - 43
Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum; Sandvig K et al.; Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence . Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells . Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased . Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER . The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation . The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.

Lik Sprava, 1996 Oct-Dec, (10-12), 146 - 8
{Experience with the elimination of a cholera focus in a psychiatric clinic and the measures for its prevention}; Shikulov VA et al.; The epidemic process of cholera in a mental-hospital setting is to a great extent influenced by specific factors . In view of a danger of cholera being brought into a mental in-patient facility the authors insist, based on their own experience, that in summer and autumn seasons in South regions and urban settlements with unstable, in respect of cholera, epidemic situation, not only patients with intestinal disfunction be examined for cholera but all those individuals to be managed at above facility . It is all-important for a mental hospital to have a plan at their disposal of primary antiepidemic measures to be instituted in case dangerous infections will pose too difficult a problem to deal with, with pharmacy being envisaged, provided with all the stores required.

J Vet Diagn Invest, 1996 Oct, 8(4), 414 - 9
Evaluation of nucleic acid amplification methods for the detection of hog cholera virus; Harding MJ et al.; A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues . The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered . Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods . A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA . However, the sensitivity was increased to 100% following a second PCR test . In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied . Novel nucleic acid sequences were generated for 9 HCV strains . Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

Cell Immunol, 1996 Sep 15, 172(2), 224 - 8
Evidence for protein kinase C pathway in the response of human peripheral blood mononuclear cells to cholera toxin; Krakauer T; Cholera toxin (CT) is a potent mucosal adjuvant and is widely used for vaccine studies in animal models . However, there have been few studies that describe the immunomodulating effects of CT on cells of the human immune system . In this study, the immunomodulatory properties of CT on human peripheral blood mononuclear cells (PBMC) were examined to gain insights to its effects on cells of the human immune system . CT induced production of immunostimulating (IL-1 beta and IL-6) and immunosuppressive (IL-10) cytokines by PBMC . However, the dose-response curve of its cytokine-inducing activity did not correlate well with the concentrations of intracellular cAMP generated by varying doses of CT . the CT mode of action on human PBMC, regarding induction of these cytokines, was clarified by the use of inhibitors of adenyl cyclase, protein kinase A (PKA), and protein kinase C (PKC) . 2',3'-Dideoxyadenosine, which inhibits adenyl cyclase activity, reduced IL-1, IL-6, and IL-10 levels by 29, 15, and 28% respectively . HA1004, an inhibitor of PKA, reduced the IL-1 and IL-6 levels by 29 and 27%, respectively . The PKC inhibitor, H7, completely blocked the induction of all three cytokines by CT, suggesting a cAMP-independent mode of action for CT on human PBMC . These observations suggest that CT induces immunomodulating cytokines from human PBMC via the PKC pathway.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 140 - 4
Fluorescence analysis of galactose, lactose, and fucose interaction with the cholera toxin B subunit; Mertz JA et al.; The cholera toxin B subunit (CTB) recognizes ganglioside GM1 receptors on target cells to facilitate entry of the toxin's A1 polypeptide into the host cytoplasm . GM1 binding to the CTB homopentamer occurs cooperatively with the most prominent interactions involving the terminal galactose residue of the ganglioside . Here, it is shown that association of galactose, lactose, or fucose (6-deoxy-galactose) with CTB is readily monitored using fluorescence spectroscopy . In many respects, however, the formation of CTB complexes with these small sugar analogues of GM1 greatly differs from the formation of complexes with the ganglioside itself . Each of these monosaccharides has a much weaker affinity for CTB than does GM1 and none of the sugars appear to be bound cooperatively . Moreover, GM1 binding conveys a stabilizing effect to CTB which is not seen upon binding of galactose or lactose . These data indicate that CTB-GM1 interactions involving sites other than the terminal galactose of the ganglioside serve prominently in the proper placement of CT on the target cell surface.

Immunology, 1996 Sep, 89(1), 54 - 8
Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin; Penney I et al.; Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections . Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum . Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum . CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum . Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.

Soc Sci Med, 1996 Sep, 43(6), 1007 - 24
"I'm not dog, no!": cries of resistance against cholera control campaigns; Nations MK et al.; Popular reactions toward government efforts to control the recent cholera epidemic in Northeast Brazil are evaluated . Intensive ethnographic interviews and participant-observation in two urban slums (favelas), reveal a high level of resistance on the part of impoverished residents towards official cholera control interventions and mass media campaigns . "Non-compliance" with recommended regimens is described more as a revolt against accusatory attitudes and actions of the elite than as an outright rejection of care by the poor . "Hidden transcripts" about "The Dog's Disease," as cholera is popularly called, voices a history of social and economic inequity and domination in Northeast Brazil . Here, cholera is encumbered by the trappings of metaphor . Two lurid cultural stereotypes, pessoa imunda (filthy, dirty person) and vira lata (stray mutt dog) are used, it is believed, to equate the poor with cholera . The morally disgracing and disempowering imagery of cholera is used to blame and punish the poor and to collectively taint and separate their communities from wealthy neighborhoods . The authors argue that metaphoric trappings have tragic consequences: they deform the experience of having cholera and inhibit the sick and dying from seeking treatment early enough . Controlling cholera requires eliminating "blaming the victim" rhetoric while attacking the social roots of cholera: poverty, low earning power, female illiteracy, sexism, lack of basic sanitation and clean water supplies, medical hegemony, etc . For health interventions to be effective, it is necessary to take into account people's "hidden transcripts" when designing action programs.

Kidney Int, 1996 Sep, 50(3), 952 - 61
Deficient IgA1 immune response to nasal cholera toxin subunit B in primary IgA nephropathy; de Fijter JW et al.; Twelve IgA nephropathy (IgAN) patients and 18 controls were immunized with novel protein antigens, cholera toxin subunit B (CTB) via the nasal route and keyhole limpet hemocyanin (KLH) subcutaneously . Antibody secreting cells and antibody response in body fluids were determined by ELISPOT assay and ELISA, respectively . Analysis of variance showed, in contrast to controls (P < 0.001), no CTB-specific IgA response in the nasal washes of patients with IgAN . Significantly lower numbers of CTB-specific antibody-secreting cells in peripheral blood (P < 0.001) and CTB-specific antibodies in plasma (P < 0.005) were found in IgAN, both restricted to the IgA1 subclass . The proportions of CTB-specific IgA1-secreting cells in bone marrow aspirates correlated significantly with the corresponding ratios in plasma, with significantly lower values (P < 0.005) in IgAN as compared to controls . These results support the existence of a "mucosa-bone marrow axis" in humans, but no dysregulation of this axis was found in IgAN . The deficient mucosal IgA immune response to CTB observed in this study after primary mucosal immunization indicates that patients with IgAN have a defective immune response when challenged intranasally . These patients may depend on more frequent and/or prolonged antigen encounter at mucosal sites before efficient mucosal immunity is established . Repeated seeding of antigen-specific cells to secondary lympoid organs could result secondarily in the relative hyperresponsiveness found in IgAN upon reactivation by parenteral immunization.

EMBO J, 1996 Aug 15, 15(16), 4246 - 53
Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin; Bastiaens PI et al.; The subcellular localization and corresponding quaternary state of fluorescent labelled cholera toxin were determined at different time points after exposure to living cells by a novel form of fluorescence confocal microscopy . The compartmentalization and locus of separation of the pentameric B subunits (CTB) from the A subunit (CTA) of the toxin were evaluated on a pixel-by-pixel (voxel-by-voxel) basis by measuring the fluorescence resonance energy transfer (FRET) between CTB labelled with the sulfoindocyanine dye Cy3 and an antibody against CTA labelled with Cy5 . The FRET efficiency was determined by a new technique based on the release of quenching of the Cy3 donor after photodestruction of the Cy5 acceptor in a region of interest within the cell . The results demonstrate vesicular transport of the holotoxin from the plasma membrane to the Golgi compartment with subsequent separation of the CTA and CTB subunits . The CTA subunit is redirected to the plasma membrane by retrograde transport via the endoplasmic reticulum whereas the CTB subunit persists in the Golgi compartment.

Int J Epidemiol, 1996 Aug, 25(4), 872 - 8
Using a knowledge, attitudes and practices survey to supplement findings of an outbreak investigation: cholera prevention measures during the 1991 epidemic in Peru; Quick RE et al.; BACKGROUND: To assess the effectiveness of the cholera prevention activities of the Peruvian Ministry of Health, we conducted a knowledge, attitudes, and practices (KAP) survey in urban and rural Amazon communities during the cholera epidemic in 1991 . METHODS: We surveyed heads of 67 urban and 61 rural households to determine diarrhoea rates, sources of cholera prevention information, and knowledge, attitudes, and practices regarding ten cholera prevention measures . RESULTS: Twenty-five per cent of 482 urban and 11% of 454 rural household members had diarrhoea during the first 3-4 months of the epidemic . Exposure to mass media education was greater in urban areas, and education through interpersonal communication was more prevalent in rural villages . Ninety-three per cent of rural and 67% of urban respondents believed they could prevent cholera . The mean numbers of correct responses to ten knowledge questions were 7.8 for urban and 8.2 for rural respondents . Practices lagged behind knowledge and attitudes (mean correct response to ten possible: urban 4.9, rural 4.6) . Seventy-five per cent of respondents drank untreated water and 91% ate unwashed produce, both of which were identified as cholera risk factors in a concurrently conducted case-control study . CONCLUSIONS: The cholera prevention campaign successfully educated respondents, but did not cause many to adopt preventive behaviours . Direct interpersonal education by community-based personnel may enhance the likelihood of translating education into changes in health behaviours . Knowledge, attitudes, and practices surveys conducted with case-control studies during an epidemic can be an effective method of refining education/control programmesPIP: The authors conducted a knowledge, attitudes, and practices (KAP) survey in urban and rural Amazon communities during the 1991 cholera epidemic to assess the effectiveness of the Peruvian Ministry of Health's cholera prevention activities . Diarrhea rates, sources of cholera prevention information, and knowledge, attitudes, and practices regarding 10 cholera prevention measures were determined by surveying the heads of 67 urban and 61 rural households . 25% of 482 urban and 11% of 454 rural household members had diarrhea during the first 3-4 months of the epidemic . Exposure to mass media education was greater in urban areas, while education through interpersonal communication prevailed in rural villages . 93% of rural and 67% of urban respondents believed they could prevent cholera . Rural respondents were slightly more knowledgeable than urban respondents about cholera . Overall, however, practices did not reflect their knowledge and attitudes; 75% of respondents drank untreated water and 91% ate unwashed produce .

Biol Pharm Bull, 1996 Aug, 19(8), 1032 - 7
Potentiation by higenamine of the aconitine-induced positive chronotropic effect in isolated right atria of mice: the effects of cholera toxin, forskolin and pertussis toxin; Kimura I et al.; Aconitine and higenamine are the major cardioactive compounds obtained from processed aconite . The chronotropic interaction between these two compounds was investigated in isolated right atria of mice . Both aconitine and higenamine potentiated the action of the other . Practolol (1 nM), a selective beta 1-adrenergic antagonist, but not butoxamine (1 microM), a beta 2-adrenergic antagonist, blocked the potentiation by higenamine (5 nM) of the aconitine-induced positive chronotropic effect and, at high concentrations (30 and 300 nM) also shifted the aconitine concentration-response curves to the right . The potentiating interaction between aconitine and higenamine was reversed by pretreating with cholera toxin (CTX) and forskolin . In CTX (100 nM, 1 h)- and forskolin (30 and 100 nM)-treated atria, higenamine significantly depressed the aconitine-induced response, which was abolished by pertussis toxin (PTX, 150 micrograms/kg, i.p., 3 d) . Neither CTX (50 and 100 nM) nor forskolin (15-100 nM) significantly affected the aconitine-induced positive chronotropic effect, while PTX (150 micrograms/kg) depressed it . These results suggest that the potentiating interaction between aconitine and higenamine involves "cross-talk" between the beta 1-adrenergic signalling pathway and Gi-protein.

Clin Immunol Immunopathol, 1996 Aug, 80(2), 147 - 54
Reversible effects on B and T cells of the gut-associated lymphoid tissues in rats malnourished during suckling: impaired induction of the immune response to intra-Peyer patches immunization with cholera toxin; Flo J et al.; To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry . After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01) . These alterations remained even after 3 weeks of refeeding with stock diet . The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur . At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus . When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery . When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001) . These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids . When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low . When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values . These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7196 - 201
Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit; Sun JB et al.; Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective . Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder . We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals . We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis . Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP . Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production . Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP . These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

Trans R Soc Trop Med Hyg, 1996 Jul-Aug, 90(4), 378 - 82
Epidemiological features of epidemic cholera (El Tor) in Zimbabwe; Bradley M et al.; Epidemics of cholera have been frequent in southern Africa since the reintroduction of the disease to the continent in 1970 . In late 1992, following a severe drought and an influx of refugees from Mozambique, cholera reappeared in Zimbabwe for the first time since 1985 and rapidly spread through the rural areas of the country . Data relating to symptomatic cholera infection collected during 2 large outbreaks on the eastern border of the country showed that host age and sex were important factors relating to symptomatic infection, as were population density and access to water . Epidemic profiles for the 2 study areas differed in that one of the profiles exhibited a distinct second phase epidemic . This unusual pattern was compared qualitatively with the output of a series of simple mathematical models to examine the contribution of different epidemiological processes to the pattern of disease observed . Model output suggested a complex disease process, in which the dynamics may have been influenced by spatial components . Statistical analysis of these unusual data showed that the observed pattern was independent of the effects of host age or sex, and provided compelling evidence of a marked spatial component of the second phase epidemic.

Soc Sci Med, 1996 Jul, 43(1), 93 - 9
Epidemiology and health policy--a world of difference? A case-study of a cholera outbreak in Kaputa district, Zambia; Van Bergen JE; The relationship between epidemiology and health policy is an area of considerable debate . This article demonstrates the use of epidemiological methods to make health policy formulation more 'policy significant' and 'down to earth' . A case-study of a cholera outbreak in Kaputa district, Zambia is used as an illustration . A closer liaison between epidemiology and social sciences is advocated . Epidemiological data should be supplied as feed back to the study population to facilitate community-based action for health.

Gut, 1996 Jun, 38(6), 853 - 8
Developmental differences in the expression of the cholera toxin sensitive subunit (Gs alpha) of adenylate cyclase in the rat small intestine; Sanderson IR et al.; BACKGROUND: The stimulatory guanosine triphosphate (GTP) binding protein alpha subunit (Gs alpha) of adenylate cyclase is the target protein for cholera toxin . AIMS/METHODS: The expression of this signal transducer was analysed in the small intestine of developing rats by RNA transfer (northern blot) analysis by immunoblotting, and by ADP-ribosylation of membrane proteins . RESULTS: Intestinal Gs alpha mRNA (about 1.9 kb) was increased in the neonate compared with the adult rat . Two isoforms of Gs alpha proteins, a 45,000 and a 52,000 form, were expressed in the small intestinal epithelial cell and both were ADP-ribosylated by cholera toxin . A significant increase in the larger isoform (52,000) and in its ribosylation was noted in the 2 week old suckling compared with post-weaned older animals . The protein content or ribosylation of the smaller form (45,000) did not significantly change with age . CONCLUSION: These data show that a developmental decline of intestinal Gs alpha expression seems to be, in part, regulated at the mRNA level . An increased Gs alpha expression in the immature intestine may help to explain a previously reported, dose dependent increased adenylate cyclase response and an increase in fluid secretion to cholera toxin in neonates compared with adults.

Am J Physiol, 1996 Jun, 270(6 Pt 1), G1001 - 9
Role of 5-HT in cholera toxin-induced mucin secretion in the rat small intestine; Moore BA et al.; We examined the role of 5-hydroxytryptamine (5-HT) in cholera toxin (CT)-induced mucin secretion in the proximal and distal regions of the rat small intestine . Neither the 5-HT2 receptor antagonist ketanserin nor the cyclooxygenase inhibitor indomethacin was capable of inhibiting choleraic mucin secretion . However, in the presence of the mixed 5-HT3/4 receptor antagonist tropisetron at doses that block both receptor subtypes, the secretory response was reduced to baseline levels in the proximal and distal small intestine . The selective 5-HT3 receptor antagonist ondansetron had no significant effect . These findings suggest that choleraic mucin secretion is mediated primarily through the activation of a 5-HT4-like receptor . Mucin secretion in response to the exogenous application of 5-HT occurs via two pathways: one is mediated by a 5-HT4-like receptor and is capsaicin sensitive but tetrodotoxin (TTX) insensitive, and one lacks the capsaicin-sensitive 5-HT4-mediated response but is TTX sensitive . Both converge on a common pathway that is cholinergic . No significant differences were observed between proximal and distal intestinal segments.

Bull Pan Am Health Organ, 1996 Jun, 30(2), 134 - 43
Epidemic cholera in Latin America, 1991-1993: implications of case definitions used for public health surveillance; Koo D et al.; This report presents the various cholera case definitions used by the affected countries of Latin America, shows the numbers of cholera cases and deaths attributable to cholera (as reported by Latin American countries to PAHO through 1993), and describes some regional trends in cholera incidence . The information about how cholera cases were defined was obtained from an October 1993 PAHO questionnaire . In all, 948429 cholera cases were reported to PAHO by affected Latin American countries from January 1991 through December 1993, the highest annual incidences being registered in Peru (1991 and 1992) and Guatemala (1993) . The case-fatality rate over the three-year period, and also in 1993, was 0.8% . A general downward trend in the incidence of cholera was observed in most South American countries, while the incidence increased in most Central American countries . A good deal of variation was noted in the definitions used for reporting cholera cases, hospitalized cholera cases, and cholera-attributable deaths . Because of these variations, broad intercountry comparisons (including disease burden calculations and care quality assessments based on case-fatality rates) are difficult to make, and even reported trends within a single country need to be evaluated with care . The situation is likely to be complicated in the future by the arrival of V . cholerae O139 in Latin America, creating a need to distinguish between it and the prevailing O1 strain . For purposes of simplicity, wide acceptance, and broad dissemination of case data, the following definitions are recommended: Confirmed case of O1 cholera: laboratory-confirmed infection with toxigenic V . cholerae O1 in any person who has diarrhea . Confirmed case of O139 cholera: laboratory-confirmed infection with toxigenic V . cholerae O139 in any person who has diarrhea . Clinical case of cholera: acute watery diarrhea in a person over 5 years old who is seeking treatment . Death attributable to cholera: death within one week of the onset of diarrhea in a person with confirmed or clinically defined cholera . Hospitalized patient with cholera:a person who has confirmed or clinically defined cholera and who remains at least 12 hours in a health care facility for treatment of the disease.

Infect Immun, 1996 Jun, 64(6), 2158 - 66
Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection; Debard N et al.; SAG1 protein of Toxoplasma gondii was evaluated as a protective antigen in mucosal immunization with cholera toxin as an adjuvant . CBA/J mice intranasally immunized with a combination of SAG1 and cholera toxin exhibited significantly fewer cysts in the brain after oral infection with the 76K strain of T . gondii than control mice . This acquired protection lasted at least 5 months . Protected mice developed high levels of serum anti-SAG1 immunoglobulin G antibodies as well as an enhanced systemic cellular response, as assessed by the proliferation of splenocytes in response to SAG1 restimulation in vitro . This cellular proliferation was associated with an increase of interleukin-2 and interleukin-5 synthesis and with barely detectable gamma interferon production . Splenic immune T cells were shown to convey modest protection to recipients against development of brain cysts following oral infection with T . gondii . Significant production of anti-SAG1 immunoglobulin A was induced in intestinal secretions of protected mice . These results indicate that intranasal immunization with SAG1 and cholera toxin can induce mucosal and systemic immune responses and affords partial and long-lasting resistance against the establishment of chronic toxoplasmosis.

Biochemistry, 1996 May 21, 35(20), 6375 - 84
Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance; Kuziemko GM et al.; The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR) . SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1 . The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1 . The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays . Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude . This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface . The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane . Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface.

Zentralbl Veterinarmed B, 1996 May, 43(3), 167 - 77
Expression and characterization of part of hog cholera virus non-structural proteins; Bakkali Kassimi L et al.; In a preceding paper, the molecular cloning and partial nucleotide sequence of the Alfort strain of hog cholera virus (HCV) was described . To study the genetic organization of the 3'-end of the HCV genome, which encodes some of the non-structural proteins, a cDNA fragment (S2.20) of 849 nucleotides was subcloned into the bacterial expression vector pGEX-3X and expressed in Escherichia coli as a S2.20-glutathione-S-transferase fusion protein (S2.20-GST) . This protein was used to produce HCV-specific monoclonal antibodies . Using Western immunoblotting, these antibodies could be used to identify a specific gene product of the HCV Alfort strain . Three proteins, with relative molecular weights of 76, 107 and 145 kDa, were detected . These proteins were also observed for eight other HCV strains . With the bovine viral diarrhoea virus (BVDV) NADL strain and the border disease virus (BDV) Aveyron strain, only one protein, with a relative molecular weight of 72 kDa, was detected . With the BVDV New York strain, two proteins, with relative molecular weights of 70 and 100 kDa, were recognized . The significance of these findings with respect to pestivirus genomic organization is discussed.

Eur J Gastroenterol Hepatol, 1996 May, 8(5), 443 - 8
The effect of L-glutamine on salt and water absorption: a jejunal perfusion study in cholera in humans; van Loon FP et al.; OBJECTIVES: To assess the efficacy of an L-glutamine solution on jejunal salt and water absorption in cholera patients . DESIGN: A randomized double-blind jejunal perfusion study . SETTING: International Centre for Diarrhoeal Disease Research, Bangladesh . PATIENTS: Nineteen adults with acute cholera . INTERVENTIONS: Perfusion of balanced salt solutions alternated with defined glucose salt solution and glutamine glucose salt or alanine glucose salt solutions . MAIN OUTCOME MEASURES: Net jejunal water and sodium secretion . RESULTS: Perfusion of glutamine in the presence of glucose significantly reduced net water secretion (JnetH2O = -2.6 +/- 1.3 ml/h/cm) and also reduced net sodium secretion (JnetNa = -213 +/- 153 mumol/h/cm) . Similar results were observed during the perfusion of solutions that contained alanine in addition to glucose (JnetH2O = -4.2 +/- 1.1 ml/h/cm and JnetNa = -444 U +/- 142 mumol/h/cm, respectively) or glucose alone (JnetH2O = -4.3 +/- 1.7 ml/h/cm and JnetNa = -452 +/- 212 mumol/h/cm, respectively) . In addition, a higher basal secretion was associated with a greater stimulation of water absorption (F = 17, P < 0.001) . CONCLUSION: Glutamine in the presence of glucose significantly reduces net water secretion and also reduces sodium secretion; higher basal secretion is associated with greater water absorption . As glutamine is able to stimulate water absorption to the same degree as glucose and alanine, and because it has the theoretical advantage of providing fuel for the mucosa, the inclusion of glutamine as the sole substrate in oral rehydration solution warrants further study.

J Biochem (Tokyo), 1996 May, 119(5), 985 - 90
Expression of the cholera toxin B subunit in the Golgi apparatus of Swiss 3T3 cells inhibits DNA synthesis induced by basic fibroblast growth factor; Hashimoto Y et al.; We attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection . Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human beta 1,4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector . The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression . The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluoresence microscopy; and third, detection of GM1 binding activity in cell lysates . Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis . These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and platelet-derived growth factor . Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites . These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding.

Cent Afr J Med, 1996 May, 42(5), 125 - 8
Does a direct cholera threat necessarily improve the knowledge, attitude and practices on the disease?
Nsungu M, Jonga M.
OBJECTIVE: To assess and compare the knowledge, attitude, practices and beliefs on cholera in Mudzi and Wedza districts . Mudzi district shares a long border with Mozambique where cholera was already prevalent before the study, while Wedza district does not share any international border . DESIGN: Cross sectional community based survey, Data was collected through interviews using a structured questionnaire . In villages, The source of water for domestic use as well as the toilets of the interviewed individuals were also inspected . SETTING: Two districts of Mashonaland East Province in Zimbabwe . SUBJECTS: Grade seven pupils, form four students and villagers . MAIN OUTCOME MEASURES: a . The level of knowledge on cholera . b . The prevalence of negative beliefs on the disease . c . The proportion of households using unsafe water . d . The proportion of households not using toilets . RESULTS: 140 and 116 individuals were interviewed in Mudzi and Wedza respectively . The level of knowledge on cholera was very poor in both districts and poorer in Mudzi which shares a border with Mozambique . Twenty pc of the people interviewed had negative beliefs towards the disease; 35pc were using unsafe water; 20pc of households did not have toilets and 4.5 to 7.7pc of the available toilets were not being used . CONCLUSION: Health education activities on cholera should target all districts with the same intensity . Specific strategies should be found in order to address the misconceptions which may hinder the control of cholera.

J Neurosci Res, 1996 May 1, 44(3), 243 - 54
Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: modulation of intracellular calcium by GM1 ganglioside; Wu G et al.; Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity . This trophic effect was linked to elevation of intracellular calcium ({Ca2+}i), and was additive to that of high K+ . Survival was optimized when Ctx B was present for several days during the early culture period . 45Ca2+ and cell survival studies indicated the mechanism to involve enhanced influx of Ca2+ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type . Inhibitors of N-methyl-D-aspartate receptor/channels were without effect . During the early stage of culture Ctx B, together with 25 mM K+, caused {Ca2+}i to rise to 0.2-0.7 microM in a higher proportion of cells than 25 mM K+ alone . A significant change in the nature of GM1 modulation of Ca2+ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced {Ca2+}i below the level attained with 25 mM K+ . GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca2+ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period . This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca2+ homeostasis in cultured neurons of the CNS.

J Cell Biol, 1996 May, 133(4), 777 - 89
Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells; Majoul IV et al.; The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence . We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER . Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min) . Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes . Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments . After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears . CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide . Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.

Eur J Immunol, 1996 May, 26(5), 967 - 75
Dissection of lymphocyte function-associated antigen 1-dependent adhesion and signal transduction in human natural killer cells shown by the use of cholera or pertussis toxin; Poggi A et al.; The effect of the guanosine triphosphate-binding protein (G-protein) inhibitors cholera toxin (Ctx) and pertussis toxin (Ptx) has been analyzed on lymphocyte function-associated antigen 1 (LFA-1)-dependent adhesion and signal transduction in human natural killer (NK) cells . Ctx, but not Ptx, inhibited the LFA-1-dependent adhesion of NK cells to tumor target cells which constitutively express the intercellular cell adhesion molecule-1 (ICAM-1) and to NIH/3T3 mouse fibroblasts stably transfected with human ICAM-1 . This effect was detectable only by the use of the entire Ctx but not of the Ctx B subunit . In addition, Ctx could inhibit both NK cell binding and spreading to purified ICAM-1 protein . NK cell treatment with Ctx modified neither the surface expression of LFA-1 nor its Mg2+ binding site . These findings, together with the absence of any detectable effect of Ctx on the constitutive phosphorylation of LFA-1 alpha, suggests that this toxin modifies the avidity of LFA-1 for ICAM-1 by acting on LFA-1-cytoskeletal protein association . Unlike Ctx, Ptx did not affect NK cell adhesion . The effects of Ctx and Ptx are unlikely to depend on intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP), since a strong increase of cAMP was induced by both toxins . Moreover, this was confirmed by the observation that the LFA-1-dependent adhesion was not inhibited by the adenylate cyclase activator forskolin (FSK), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX), or both, which increase intracellular cAMP levels . Unlike the differential effect on cell adhesion, both the intracellular calcium {Ca2+}i increase and phosphoinositide breakdown mediated via LFA-1 were consistently inhibited in a dose-dependent manner by both Ctx and Ptx . Also in this case, the inhibitory effect did not depend on an increase of intracellular cAMP as indicated by NK cell treatment with FSK, IBMX, or both . Further evidence of the involvement of G-proteins in LFA-1-mediated signal transduction was the inhibitory effect of the GDP analog guanosine-5'-O-2-thiodiphosphate (GDP beta S) on LFA-1-mediated calcium mobilization . Taken together, our data provide evidence that the LFA-1-mediated NK cell adhesion and signal transduction are partially independent phenomena which may be regulated by different G-proteins.

Endocrinology, 1996 May, 137(5), 1823 - 7
Progesterone diminishes the sensitivity of gonadotropin-releasing hormone-stimulated luteinizing hormone (LH) release and protects an LH pool from desensitization: actions opposed by cholera toxin; Janovick JA et al.; In the present study we used characterized perifusion model for the development of homologous desensitization to GnRH in the pituitary gonadotrope . This system relies on immobilized primary pituitary cultures that are perifused with various pulse patterns and concentrations of GnRH; these patterns and concentrations are selected to maintain, inhibit, restore, circumvent the desensitization process . The data indicate that progesterone (P) inhibits LH release in response to pulsatile administration of GnRH; this effect is blocked by the P antagonist, RU486, and is opposed by cholera toxin, an agent that provokes the formation of cAMP . In multiple and rapid pulse administration of 5 nM GnRH, which is usually associated with desensitization, the area under the LH release curve is progressively diminished when cells are preincubated with medium only or with medium containing cholera toxin . In contrast, cells pretreated with P are less responsiveness to GnRH, but maintain a relatively constant level of LH release (area under the curve) . These data suggest a modulatory role for P in the development of the desensitized state and indicate that P can functionally protect a pool of LH from the onset of desensitization.

Brain Res Dev Brain Res, 1996 Apr 30, 92(2), 199 - 210
Cholera toxin binds to differentiating neurons in the developing murine basal ganglia; Shindler KS et al.; Cell-surface expression of gangliosides in the developing mammalian central nervous system is temporally-regulated in a cell-type and regionally specific fashion . Gangliosides may be involved in cell-cell and cell-matrix interactions, and can act synergystically with several growth factors or growth factor receptors . Thus, a role for gangliosides in the regulation of neuronal stem cell proliferation and differentiation has been suggested . We have previously shown that cholera toxin B subunit (CTB), which binds to the ganglioside GM1, binds heterogeneously to dissociated neuroepithelial cells from the developing mouse telencephalon . We stained fixed sections of the ganglionic eminences (GE) of fetal mouse brains and found that CTB labels regions which contain differentiating neurons, but does not stain the rapidly dividing neuroepithelial cells in the ventricular zone . We dissociated cells from the GE on day 14 of gestation (E14), labeled the cells with CTB-FITC, and separated them by flow cytometry . We found the highest level of CTB binding in postmitotic cells which had begun to express markers of neuronal differentiation . When CTB-sorted cells were placed into short-term (48 h) cell culture, high CTB binding continued to correlate with fewer numbers of proliferating cells and larger numbers of differentiating neurons . CTB binding and fluorescence activated cell sorting appear to be useful for separating populations of differentiating neurons from immature, proliferating cells . These studies further lead us to suggest that GM1 plays a role in the differentiation of neurons in the basal ganglia.

Pediatr Res, 1996 Apr, 39(4 Pt 1), 625 - 9
Gut flora allows recovery of oral tolerance to ovalbumin in mice after transient breakdown mediated by cholera toxin or Escherichia coli heat-labile enterotoxin; Gaboriau-Routhiau V et al.; Oral tolerance, the antigen-specific immunologic unresponsiveness after antigen (Ag) feeding, is of physiologic importance in preventing antibody (Ab) responses to dietary proteins . This is important in the young, especially at weaning when numerous dietary Ag are encountered for the first time . Two related enterotoxins responsible for much diarrhea in the infant, cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT), have been shown to abrogate oral tolerance to an unrelated Ag fed simultaneously . The main objective of this study was to determine whether the gut flora can play a role in the CT- or LT-mediated abrogation of oral tolerance to the dietary protein ovalbumin (OVA), on a short-term and long-term basis . Conventional and germ-free mice were fed once or twice with toxin plus OVA . After two intraperitoneal immunizations with OVA, anti-OVA IgG and IgE Ab levels were measured . Because IgG and IgE Ab responses were detected, both CT and LT abrogated oral tolerance to OVA in conventional and germ-free mice . As time progressed (observations over 3 mo), whereas the specific IgG Ab response in the germ-free mice remained similar to that of the bicarbonate-fed controls, a hyporesponsive state was observed in conventional mice . The results showed that, although the gut flora did not prevent the CT- and LT-mediated abrogation of oral tolerance, it did shorten the effect and allow oral tolerance to be recovered.

Can J Surg, 1996 Apr, 39(2), 155 - 8
Surgical treatment of pancreatic cholera: a case report; Kirkpatrick AW et al.; After surgical resection for rectosigmoid carcinoma a 63-year-old man had secretory diarrhea causing severe metabolic acidosis, hypokalemia, hypercalcemia and dehydration . Subsequent investigations revealed a mass measuring 4 x 5 cm in the uncinate process of the pancreas and an elevated vasoactive intestinal polypeptide concentration . The diarrhea responded to treatment with the somatostatin analogue . Sandostatin, and remained under control during a prolonged preoperative period . The patient underwent a Whipple procedure with immediate lessening of his diarrhea . This report illustrates a classic case of vipoma and demonstrates the need to consider this condition in the differential diagnosis of secretory diarrhea, even in the presence of other gastrointestinal lesions . The effectiveness of somatostatin analogues in stabilizing the diarrhea preoperatively is also well illustrated.

J Comp Pathol, 1996 Apr, 114(3), 257 - 63
Viral antigen and B and T lymphocytes in lymphoid tissues of gnotobiotic piglets infected with hog cholera virus; Narita M et al.; Four 4-day-old gnotobiotic piglets infected intranasally with the Kanagawa/74 strain of hog cholera virus (HCV) did not develop severe illness over a period of 3 weeks . Large amounts of HCV were isolated from the lymphoid tissues and serum at necropsy . After the acute phase, hyperplasia of histiocytes and plasmacytopoiesis were observed in two pigs (killed 14 and 21 days after inoculation) . The number of CD4+ and CD8+ T lymphocytes increased significantly and their location was consistent with the site of HCV replication . The results suggest that a CD8+ T-lymphocyte reaction is associated with persistent HCV infection.

Infect Immun, 1996 Apr, 64(4), 1272 - 83
Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the Cholera toxin B subunit; Russell MW et al.; Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma . In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens . Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system . IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization . Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period . IgA antibodies were found in nasal and vaginal washes . Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation . Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions . Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes . Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.

Cell Immunol, 1996 Mar 15, 168(2), 229 - 34
Differential induction of programmed cell death in CD8+ and CD4+ T cells by the B subunit of cholera toxin; Yankelevich B et al.; We have recently demonstrated that a short-term treatment of parental splenocytes with the B subunit of cholera toxin (CT-B) abrogates the development of acute GVHD in F1 hybrid mice transplanted with these cells . In order to obtain better insight into the mechanism of the action of CT-B, we studied the effect of CT-B on survival of purified murine T cells and their subsets . We show that treatment with B subunit stimulates apoptosis in T cells, detectable following incubation in vitro . Although apoptosis was noticed in both CD8+ and CD4+ T cell subsets, the treatment preferentially stimulates programmed cell death (PCD) in CD8+ population . Thus, immunosuppressive action of CT-B in vivo may be in part due to its ability to eliminate CD8+ T cells.

J Neurosci Methods, 1996 Mar, 65(1), 101 - 12
Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains; Angelucci A et al.; We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety . Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue . The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) . Using the protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals . Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections . From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.

Neurochem Int, 1996 Mar, 28(3), 259 - 69
Differential regulation of melatonin receptors in sheep, chicken and lizard brains by cholera and pertussis toxins and guanine nucleotides; Morgan PJ et al.; G-proteins define both the pharmacological characteristics and the signalling pathways of G-protein-coupled receptors . Melatonin receptors have been shown to belong to this class of receptors through their sensitivity to modulators of G-protein function . This study reveals that 2-125I-iodomelatonin (125I-MEL) binding to different target tissues is differentially affected by agents which disrupt the G-protein cycle . GTP gamma S, pertussis (PTX) and cholera (CTX) toxins each reduce 125I-MEL binding to ovine pars tuberalis (oPT) and lizard brain membranes, whereas chicken brain is affected only by GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) and CTX . In contrast, high affinity binding of 125I-MEL in the ovine hippocampus was not affected by any of these agents . This finding, together with the fact that neural binding sites of the sheep brain were found to have markedly lower molecular mass than those of the oPT on native gel electrophoresis (365 vs 525 kDa), suggests that the neural 125I-MEL binding sites in sheep may not be G-protein coupled . Pharmacologically, however, the binding sites in the hippocampus and oPT could not be distinguished using 11 analogues of melatonin . Therefore, these data support the notion not only of multiple forms of melatonin receptor/G-protein complex, but of high affinity binding sites for 125I-MEL which do not display sensitivity to guanine nucleotides.

J Neurochem, 1996 Mar, 66(3), 1019 - 26
Cholera toxin induces cyclic AMP-independent down-regulation of Gs alpha and sensitization of alpha 2-autoreceptors in chick sympathetic neurons; Boehm S et al.; The role of the stimulatory GTP-binding protein (Gs) in the alpha 2-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons . The alpha 2-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked {3H} noradrenaline release with half-maximal effects at 14.0 +/- 5.5 nM . In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 +/- 1.4 nM without changes in the maximal effect of UK 14,304 . The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population . In cultures treated with either 10 microM forskolin or 100 microM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered . Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin . Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of alpha-subunits of Gs (Gs alpha) from particulate to soluble fractions and led ultimately to a complete loss of Gs alpha from the neurons . In contrast, no effect was seen on the distribution of either alpha-subunits of Gi- or Ga-type G proteins or of beta-subunits . These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of Gs alpha . This down-regulation of Gs alpha is associated with the sensitization of alpha 2-autoreceptors.

Curr Eye Res, 1996 Mar, 15(3), 229 - 36
Cholera toxin enhances taurine uptake in cultures of human retinal pigment epithelial cells; Miyamoto Y et al.; Taurine uptake into cultures of human retinal pigment epithelial (HRPE) cells was monitored for 7 days after seeding . A culture medium containing 16% fetal bovine serum (FBS) was used for 2 days and switched to one with 8% FBS . Uptake of taurine (25 nM) was approximately 1.5 pmol/mg protein/15 min for 3 days, then decreased by 45% and was maintained at a decreased level till the 7th day . When the 16% FBS medium was used for the entire culture period, a similar profile of taurine uptake was observed but decrease of the uptake started on the 3rd day . Treatment of cells with 100 ng/ml cholera toxin (CT) for 24 h between the 6th and 7th days returned taurine uptake to its high level observed at the beginning of the cell culture . A similar CT treatment of cells between the 2nd and 3rd days enhanced taurine uptake significantly but this enhancement was much smaller . CT increased taurine uptake in treatment-time and dose dependent manners . Forskolin (FSK) (10 mM) and 8-Bromocyclic adenosine 3',5'-monophosphate (1 mM) also increased taurine uptake . KT5720 at 1 microM, a selective inhibitor of cAMP-dependent protein kinase (PKA), partially blocked CT-induced enhancement of taurine uptake . The level of cAMP was higher on the 3rd day than the 7th day but its response to 3-isobutyl-1-methyl-xanthine, FSK and CT was similar on both days . A kinetic analysis revealed that CT treatment decreases the apparent Michaelis-Menten constant of the taurine transporter while the drastic reduction of taurine uptake during the cell culture period is due to a decrease in the maximal velocity . The results show that cAMP elevated by CT treatment enhances taurine uptake via an increase in the affinity of the transporter . The decrease of taurine uptake during the culture period seems to be related to a decrease in the amount of the transporter.

Vaccine, 1996 Feb, 14(2), 162 - 6
Field trial of inactivated oral cholera vaccines in Bangladesh: results from 5 years of follow-up; van Loon FP et al.; To determine the protective efficacy (PE) of three doses of oral B subunit-killed whole cell (BS-WC) or killed whole cell-only (WC) vaccines against cholera, a clinical trial was conducted among 62285 children over 2 years and adult women in rural Bangladesh . During 5 years of follow-up, there were 144 cases of cholera in the BS-WC group (PE = 49%; P < 0.001), 150 in the WC group (PE = 47%; P < 0.001), and 283 in the K12 group . Protection by each vaccine was evident only during the first three years of follow-up; long-term protection of young children was observed only against classical but not El Tor cholera; 3-year protection against both cholera biotypes occurred among older persons, but at a higher level against classical cholera.

Pharmacol Toxicol, 1996 Feb, 78(2), 104 - 10
The effect of alpha-trinositol on cholera toxin-induced hypersecretion and morphological changes in pig jejunum; Hansen MB et al.; alpha-Trinositol (D-myo-inositol 1,2,6-trisphosphate, PP56) is a novel antiinflammatory drug . This study elucidates the effect of intravenous alpha-trinositol on basal and acute fluid transport and morphological changes following cholera toxin administration in pig jejunum in vivo . Using isolated jejunal tied-off loops, the fluid hypersecretory (accumulation) effect of different doses of cholera toxin was studied in pigs treated intravenously with saline added different doses (0, 4, 8, 16 and 32 mg x kg-1 x hr-1) of alpha-trinositol . Levels of alpha-trinositol, as well as stereomicroscopical, light microscopical and scanning electron microscopical morphological studies were performed . Cholera toxin evoked a dose-dependent fluid hypersecretion . Treatment with alpha-trinositol caused a dose-dependent inhibition of the cholera toxin-induced fluid hypersecretion and did not affect basal fluid absorption . The 16 mg x kg-1 x hr-1 alpha-trinositol dose gave a maximal inhibition of 36% . Morphological studies showed only minor changes following 6 hr of exposure to 20 micrograms x loop-1 cholera toxin . These changes consisted of dilation of the villus capillaries, an increase of apical membrane blebbing and a reduction of the intercellular space . Treatment with 16 mg x kg-1 x hr-1 alpha-trinositol alone did not induce any morphological changes, and did not alter the morphological changes induced by cholera toxin, which caused fluid hypersecretion and only minor acute morphological changes . In conclusion, alpha-trinositol treatment reduced cholera toxin-induced fluid hypersecretion without altering basal fluid absorption, basal morphology, or cholera toxin-induced morphological changes in pig jejunum in vivo.

Immunology, 1996 Feb, 87(2), 220 - 9
CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin; Hornquist E et al.; We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant . Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased . In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells . All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone . In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice . By contrast, no difference was observed for systemic responses between CD8-/- and normal mice . Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses . The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells . On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice . In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance . Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e . CD44high, LECAM-1low and CD45RBlow . We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.

Epidemiol Infect, 1996 Feb, 116(1), 9 - 13
An outbreak of cholera from food served on an international aircraft; Eberhart-Phillips J et al.; In February 1992, an outbreak of cholera occurred among persons who had flown on a commercial airline flight from South America to Los Angeles . This study was conducted to determine the magnitude and the cause of the outbreak . Passengers were interviewed and laboratory specimens were collected to determine the magnitude of the outbreak . A case-control study was performed to determine the vehicle of infection . Seventy-five of the 336 passengers in the United States had cholera; 10 were hospitalized and one died . Cold seafood salad, served between Lima, Peru and Los Angeles, California was the vehicle of infection (odds ratio, 11.6; 95% confidence interval, 3.3-44.5) . This was the largest airline-associated outbreak of cholera ever reported and demonstrates the potential for airline-associated spread of cholera from epidemic areas to other parts of the world . Physicians should obtain a travel history and consider cholera in patients with diarrhoea who have travelled from cholera-affected countries . This outbreak also highlights the risks associated with eating cold foods prepared in cholera-affected countries.

Infect Immun, 1996 Feb, 64(2), 665 - 7
Persistence of serum and salivary antibody responses after oral immunization with a bacterial protein antigen genetically linked to the A2/B subunits of cholera toxin; Hajishengallis G et al.; Primary oral immunization of mice with a bacterial protein antigen genetically coupled to the A2 and B subunits of cholera toxin induced specific secretory immunoglobulin A and serum immunoglobulin G antibodies that persisted at substantial levels for at least 11 months . A subsequent single booster immunization did not further enhance the antibody responses . Long-term antibody persistence may be especially important in infections caused by common pathogens for which continuous immunity would be advantageous.

Scand J Infect Dis, 1996, 28(1), 87 - 90
Reduced osmolarity oral rehydration salt in Cholera; Faruque AS et al.; In a controlled clinical trial conducted in 34 adults with severe cholera diarrhoea, the use of a relatively dilute oral rehydration salt (ORS) solution (sodium 67, potassium 20, chloride 66, citrate 7, glucose 89 mmol/l, osmolality 249 mOsmol/kg) caused a 29% (p=0.003) reduction in stool output over the first 24 h and a 37% (p=0.001) reduction over the first 48 h compared with 29 controls who received the hyperosmolar WHO/UNICEF ORS . No controls but 3 study-group patients had marked but asymptomatic hyponatraemia (sodium <125 mmol/l) at 24 h . Twenty-four % of controls and 12% of patients receiving the dilute ORS needed unscheduled intravenous therapy for recurrence of dehydration . The ORS intake was twice the 48 h stool volume in controls and 3 times in the study group . The test ORS with a reduced glucose and sodium concentration is more efficient than the WHO/UNICEF ORS in preserving net intestinal fluid balance in severe cholera.

Sudhoffs Arch Z Wissenschaftsgesch, 1996, 80(2), 205 - 19
{The democratic movement, cholera epidemic and public health reform in the Zurich canton (1867)}; Condrau F; In the summer of 1867 Zurich, Switzerland, was struck by a severe Cholera outbreak . Recent research suggests that the Cholera epidemic had such an influence on the municipal policy makers that they pushed through a major reform of the public health system, namely of water supply and sewerage . This paper adopts a theoretical conception of crisis and social change to evaluate the plausibility of this hypothesis . The basic idea is that structural change can be usually understood as the consequence of a major social crisis . Exactly this was the case in Zurich during the 1860s . After a decade of economic stability and progress, a severe crisis simultaneously stuck Zurich's agriculture, textile industry and railway companies . The so called Democratic Movement threatened the existing political system; the old political establishment feared a political revolution . On top of all this, Cholera struck Zurich, precipitating a crisis in the public health system . Suddenly, old concepts and institutions were felt to be outdated . The democrats put through political reforms, the economic downswing ended and a large program to reshape the urban environment was initiated . It can be concluded that the Cholera epidemic alone did not cause the public health reform . But together with other crisis phenomena it played a major role in weakening the stability of the old system, and thus contributed to the society's ability to put in place new political, economic, and social structures.

Brain Res Bull, 1996, 41(6), 391 - 8
Reciprocal and topographic connections between the piriform and prefrontal cortices in the rat: a tracing study using the B subunit of the cholera toxin; Datiche F et al.; In the present study, the reciprocal connections between the piriform cortex and the prefrontal areas are described on the basis of experiments using the anterograde and the retrograde transport of the cholera toxin B subunit (CTb) . Following CTb injections placed in the anterior part of the piriform cortex, retrogradely labeled cells and anterogradely labeled fibers were mainly found in the ventrolateral and lateral orbital areas as well as in the anterior part of the agranular insular cortex . Following injections placed in the posterior part of the piriform cortex, the CTb labeling was primarily observed in the infralimbic area and the posterior part of the agranular insular cortex . Thus, we described a topographical organization of the direct reciprocal connections between the anterior and the posterior parts of the piriform cortex parts and some prefrontal areas . This could support a differential modulation of the olfactory processing along the rostrocaudal dimension of the piriform cortex.

Brain Behav Evol, 1996, 48(6), 307 - 37
Use of the sensitive anterograde tracer cholera toxin fragment B reveals new details of the central retinal projections in turtles; Reiner A et al.; Previous anterograde degeneration and autoradiographic studies have yielded inconsistent results on the extent and target areas of the central retinal projections in turtles . We used the highly sensitive anterograde and retrograde tracer, cholera toxin B fragment (CTB), to re-examine the central retinal projections in Pseudemys scripta elegans . In contrast to the results of the previous anterograde degeneration studies and autoradiographic studies, immunohistochemical detection of CTB-labeled central retinal axons and terminals revealed these projections with great morphological detail and clarity . In addition, the CTB labeling revealed much more extensive projections than previously realized to all known major retinorecipient areas, particularly in the dorsal and ventral lateral geniculate nuclei, the pretectal region, the nucleus of the basal optic root and the optic tectum . Although the contralateral projections were always much more extensive, ipsilateral projections were clearly present (and in some cases abundant) in all previously known retinorecipient areas . In addition, prominent contralateral (and lesser ipsilateral) retinal projections to several novel target, including the basal hypothalamus, the perirotundal nuclei, the suprapeduncular nucleus and the region between the pretectal nuclei and the tegmentum, were observed . Our results suggest that the use of CTB as a tracer to examine central retinal projections in diverse other species will show that central retinal projections are much more widespread and extensive than previously realized.

Prog Neuropsychopharmacol Biol Psychiatry, 1996 Jan, 20(1), 99 - 108
Evaluation of cyclic AMP accumulation in EBV-transformed human B-lymphocytes: effects of dopamine agonists, isoproterenol, prostaglandin E1, cholera toxin, forskolin, and phorbol 12-myristate-13 acetate; Natsukari N et al.; 1 . Phorbol 12-myristate-13-acetate (PMA), a protein kinase C activator, elevated cyclic AMP accumulation in EBV-transformed human B-lymphocytes, and potentiated isoproterenol-, prostaglandin- (PGE1), cholera toxin-, and forskolin-stimulated cyclic AMP accumulation . 2 . The dopamine D1 receptor agonist, SKF38393 (10(-7) to 10(-5) MH, had no effect on cyclic AMP accumulation in transformed human B-lymphocytes . 3 . The dopamine D2 receptor agonist, quinpirole (10(-7) to 10(-5) MH did not inhibit cyclic AMP accumulation even when cyclic AMP accumulation was maximized by the addition of PMA and forskolin . 4 . These data suggest that dopamine D1- and D2-receptor coupling to a cyclic AMP generating system is not present at detectable levels in transformed human B-lymphocytes.

Pathology, 1996 Jan, 28(1), 58 - 64
The occurrence of macrophage-like cholera toxin uptake cells in the intestinal villi of suckling rats; Shimodori S et al.; An oral administration of cholera toxin (CT . 10m g) caused diarrhea in infant rats ranging in age from 1 to 14 days . After administration of the toxin a time sequence study was carried out using highly sensitive immunohistochemical procedures . CT was exclusively incorporated into a type of macrophage-like (ML) phagocytic cell . These cells were identified within the intestinal epithelium of rats suffering choleraic diarrhea . After 2 hrs cells taking up the toxin markedly increased in number and were found in both the mucosa and the lamina propria mucosae . After 4 hrs a small number of ML cells containing CT were still present in the mucosal epithelium, but were no longer observed in the lamina propria . Two kinds of monoclonal antibodies against rat macrophages were used to gain a clue as to the cytological characteristics of ML cells . ED1- or ED2-positive macrophages were demonstrable in the lamina propria and submucosa of the small intestines from control rats . In CT-treated rats a considerable number of cells positive for CT and ED1, or CT and ED2 antisera, were found within the epithelial cell layer and the lamina propria of intestinal villi . It is suggested that many ML cells responsive to CT, if not all, are ED1 and ED2 macrophages and are resident in the villous lamina propria where they can migrate to uptake CT in the intestinal lumen . CT B-subunit and heat-labile toxin (LT) B'-subunit from a mutant strain Escherichia coli were given to the rats in order to know the onset mechanism of toxin uptake . It seems likely that the toxin receptor, GM1 ganglioside, participates in the initiation of CT-uptake mechanism . A possible role of the intestinal ML cells was discussed.

J Immunol, 1996 Jan 1, 156(1), 316 - 21
Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells; Leal-Berumen I et al.; Mast cells have been traditionally associated with an acute allergic response . However, their role in regulating chronic inflammatory processes must also be considered in view of evidence that mast cells synthesize and release a number of cytokines . In this study, we have examined the effect of cholera toxin (CT) on peritoneal mast cell IL-6 and TNF-alpha production . Highly purified, freshly isolated, rat peritoneal mast cells from Brown Norway rats were cultured in the presence of CT or its B subunit (CTB) alone or in combination with anti-IgE or bacterial LPS . Histamine release was measured after 10 min; IL-16 and TNF-alpha production was assessed in supernatants after 18 h . We found that CT or CTB alone did not affect histamine release; however, mast cell IL-6 production was significantly enhanced by CT but not by CTB . In contrast, constitutive production of TNF-alpha was inhibited by CT . The effects of CT were similar to our previous observations of the actions of prostaglandin E2 on mast cells . We also examined the effects of CT in combination with other mast cell activating agents . CT had no significant effect on anti-IgE-induced histamine release . An additive effect on IL-6 production was observed in the context of LPS . Forskolin, an agent known to increase intracellular cAMP levels, also induced a significant increase in IL-6 production, whereas TNF-alpha production was decreased . These data have important implications for our understanding of the regulation of mast cell cytokine production and the effects of CT on local cytokine production.

Biochem J, 1995 Dec 15, 312 ( Pt 3), 769 - 74
Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C; Zeng L et al.; Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period . When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine . Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid . Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA . Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA . Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity . Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells . It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells . The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.

Glia, 1995 Dec, 15(4), 471 - 9
Prostaglandin E1, E2, and cholera toxin increase transcription of the brain creatine kinase gene in human U87 glioblastoma cells; Kuzhikandathil EV et al.; The creatine kinase isoenzymes play an important role in maintaining ATP levels in some cell types during times of high energy demand . We have previously shown in primary cell cultures from rat brain that glial cells express much higher levels of brain creatine kinase (CKB) mRNA than neurons . In a separate earlier study we observed that transcription of CKB mRNA in glial cells can be stimulated by a forskolin-mediated increase in cAMP via a pathway involving protein kinase A (PKA) . In this report, we show that the level of CKB mRNA in human U87 glioblastoma cells can be increased by either prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), or cholera toxin (an activator of G alpha s proteins) . The induction of CKB mRNA occurs rapidly (with maximal induction after 6 h), is at the level of transcription, and is mediated specifically through PKA . In addition, the results indicate that both PGE1 and PGE2 use the same or related signal transduction pathways to increase CKB transcription . These results suggest that in glial cells CKB mRNA can be regulated by extracellular signals acting through G-protein-coupled receptors . This study may contribute to an understanding of the mechanisms underlying the previously-reported, early postnatal increase in CKB enzyme activity in rat brain . The results are also discussed with regard to the potential involvement of the expression of prostaglandins and CKB during hypoxia and ischemia.

Vet Microbiol, 1995 Dec, 47(3-4), 395 - 400
Cytocidal infection of hog cholera virus in porcine bone marrow stroma cell cultures; Shimizu M et al.; Porcine bone marrow stroma cell (BMSC) cultures producing cells of granulocyte-lineage were established . Hog cholera (HC) virus ALD and Alfort strains replicated in the porcine BMSC cultures showing distinct cytopathic effect (CPE) . The differentiation of granulocyte-lineage cells in the cultures ceased after infection with HC virus . Polyclonal antibody against the ALD strain inhibited completely the development of CPE of the both ALD and Alfort strains . Monoclonal antibodies (mAbs) specific to the ALD strain inhibited CPE of the ALD strain, while CPE of the Alfort strain was not affected by those mAbs, suggesting that CPE induced in the BMSC cultures is due to HC virus.

Br J Anaesth, 1995 Dec, 75(6), 810 - 6
The role of an anaesthetist in a field hospital during the cholera epidemic among Rwandan refugees in Goma; Ginosar Y et al.; In 1849 John Snow, already the leading anaesthetic practitioner and innovator of his day, made a historic contribution to the epidemiology of infectious disease by his famous study of the distribution of cholera around the area of Broad Street in London . We report on our experience as anaesthetists in a field hospital, dispatched as part of the international rescue effort to Goma, Zaire, to help combat the effects of cholera among the Rwandan refugeesPIP: The experience of a medical team dispatched to field hospitals in Goma, Zaire, as part of an international rescue effort to combat the effects of a cholera epidemic among Rwandan refugees illustrates the importance of planning and collaboration . The mission included 5 pediatricians, 4 general practitioners, 2 anesthetists, 2 general surgeons, and 1 orthopedic surgeon . The team's goal was to function as a walk-in clinic for sick and dying refugees and as a regional hospital providing laboratory, x-ray, and surgical facilities . Through a strategy of cooperation with primary care clinics, the team was able to prioritize the treatment of the sickest patients in need of intravenous therapy . Anesthetists had a prominent role in both performing major and central venous cannulations and instructing other physicians in the procedure . Surgery was confined to pregnancy-related procedures and trauma cases . In the 2 weeks of this mission, 1200 patients were treated, most of whom were in severe shock . Where possible, bolus spinal anesthesia was used to minimize the need for postoperative respiratory support . Recommended, in future missions, are baseline electrolyte measurements, a laryngeal mask airway, portable ventilators, and monitoring of end-tidal carbon dioxide .

J Exp Med, 1995 Dec 1, 182(6), 1931 - 42
A role for stem cell factor and c-kit in the murine intestinal tract secretory response to cholera toxin; Klimpel GR et al.; The role of stem cell factor (SCF) and its receptor (c-kit) in the intestinal secretory response to cholera toxin (CT) was investigated using a ligated intestinal loop model in mice having mutations in the dominant white spotting (W) locus and the steel (Sl) locus . W/Wv mice, which express an aberrant form of the c-kit protein, failed to give an intestinal secretory response after luminal CT challenge . In contrast, W/Wv mice and their control littermates had equivalent intestinal secretory responses to Escherichia coli heat-stable enterotoxin (STa) . Sl/Sld mice, which express only a soluble truncated form of SCF, also gave a significantly reduced intestinal secretory response to CT when compared to the secretory response of their littermate controls . The unresponsiveness of W/Wv mice to CT was restricted to the intestinal tract since these mice had foot pad swelling responses to CT challenge that were equivalent to their littermate controls . Restoration of mast cells in W/Wv mice by bone marrow transplantation of control littermate bone marrow did not reverse the CT-unresponsiveness of the intestinal tract . Histological evaluation of the gastrointestinal tract from W/Wv mice showed a normal distribution of enterochromaffin cells (ECC) . CT challenge of either ligated intestinal loops from C57B1/6 mice or a mouse intestinal epithelial cell line (MODE-K) resulted in elevated levels of mRNA for SCF . MODE-K cells exposed to CT also had enhanced expression of c-kit . Finally, fluid obtained from CT-challenged ligated intestinal loops from C57B1/6 mice contained significant levels of SCF . Taken together, the above results suggest that CT-induced intestinal secretory responses are dependent upon SCF-c-kit interactions . These interactions appear to be induced as a consequence of CT stimulation of the intestinal tract and may also play a role in the development or functionality of the enteric nervous system.

Mol Cell Biochem, 1995 Nov 22, 152(2), 103 - 12
The different inhibiting effect of cholera toxin on two leukemia cell lines does not correlate with their toxin binding capacity; Giuliani A et al.; The murine leukemia cell lines L1210 and WEHI-3B show a very different sensitivity to the cholera toxin (CT) . The in vitro growth of L1210 is completely inhibited by 10(-8) M CT, while WEHI-3B growth shows the same inhibition at 10(-11) M . The analysis of membrane ganglioside pattern of the two cell lines shows that in L1210 cells the major component is the GM1a ganglioside while the monosialogangl oside fraction from WEHI-3B is entirely composed of gangliosides of the 'b' series among which GM1b is the more represented . The total cholera toxin binding capacity of the ganglioside extract from L1210 cells is more than hundred fold higher than that of WEHI-3B and this difference is also confirmed by the number of CT receptors/cell and by the binding of FITC-B subunit of CT on the cells . These surprising data are in conflict with the poor sensitivity to CT evidenced by L1210 compared to WEHI-3B cells . In order to clarify this discrepancy we investigated the cAMP accumulation, the cell viability and the clonogenicity of these two leukemia cell lines following the treatment with CT and forskolin (FRSK) . The treatment of WEHI-3B cells with CT induces a dramatic increase of intracellular cAMP which highly correlates with cell death and the decrease of clonogenicity and this result is partially obtained by the treatment with FRSK . L1210 cells do not evidence significant cAMP accumulation neither with CT nor with FRSK treatment . These data suggest that the different inhibiting effect of CT on WEHI-3B and L1210 cells does not correlate with their different pattern of gangliosides and the related toxin binding capacity . Further they indicate that the growth inhibition of WEHI-3B cells is closely related with a cAMP-dependent cell killing mechanism, while the inhibition of L1210 growth (produced by high concentration of CT) is mediated by a cAMP independent mechanism.

Gene, 1995 Nov 20, 165(2), 163 - 71
Characterization of an internal permissive site in the cholera toxin B-subunit and insertion of epitopes from human immunodeficiency virus-1, hepatitis B virus and enterotoxigenic Escherichia coli; Bckstrom M et al.; We previously described the construction of novel hybrid proteins based on the B-subunit of cholera toxin (CTB) {Backstrom et al., Gene 149 (1994) 211-217}, in which a neutralizing B-cell epitope from the third variable (V3) loop in the envelope glycoprotein gp120 from human immunodeficiency virus type 1 (HIV-1) was inserted within a surface-exposed region between amino acids (aa) 55 and 64 . The resulting protein retained properties of native CTB and could induce strong anti-CTB antibody (Ab) responses, but the inserted gp120 epitope was only modestly immunogenic . In this study, the potential use of this internal permissive site in CTB for the insertion of heterologous epitopes has been further investigated . Six additional plasmids were constructed encoding HIV::CTB hybrid proteins with ten to fourteen aa from the V3 loop of gp120 genetically inserted at different positions between aa 52 and 65, with deletions of different CTB aa . Plasmids encoding proteins with peptides inserted between aa 53 and 64 in CTB gave rise to stable proteins which reacted with CTB-specific monoclonal antibodies (mAb) and bound to GM1 gangliosides (GM1), indicating that insertions between these positions do not drastically alter the conformation or the receptor-binding properties of native CTB . Plasmids were also constructed encoding CTB hybrid proteins which had either an 11-aa peptide from hepatitis B virus (HBV) pre-S(2) or one of two peptides related to the heat-stable toxin (STa) of enterotoxigenic Escherichia coli inserted between aa 55 and 64 of CTB . This resulted in the production of HBV::CTB or ST::CTB hybrid proteins and illustrated that the internal permissive site can be used for insertion of peptides of varying aa composition . The reactivity of the inserted epitopes with epitope-specific mAb in GM1-ELISA and immunoblots varied greatly between hybrid proteins and depended on the position in CTB and the aa composition of the inserted peptides . Despite these differences, all the HIV::CTB, ST::CTB and HBV::CTB hybrid proteins could induce low, but significant, levels of serum Ab in mice against gp120, STa or pre-S(2), in addition to strong serum Ab responses against CTB . The Ab response against the internally inserted gp120 peptide was similar to that against the same peptide fused to the N-terminus of CTB, indicating that internally placed peptides had similar immunogenicity to the same peptides added terminally.

J Immunol, 1995 Nov 15, 155(10), 4621 - 9
Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4; Marinaro M et al.; Despite pathophysiologic effects including diarrhea, cholera toxin (CT) is a potent mucosal immunogen and adjuvant . We investigated the influence of CT on T helper (Th)-type 1 (Th1) and Th2 cell-regulated Ag-specific B cell isotype and IgG subclass Ab responses elicited when the toxin was co-administered orally with different protein Ags . When mice were orally immunized with tetanus toxoid (TT) and CT as adjuvant, this regimen induced TT-specific secretory IgA responses in the gastrointestinal tract as well as serum IgG, including IgG1 and IgG2b subclasses, and IgA responses . This oral regimen also induced TT- and CT-B-specific IgE responses . In addition, CT also elicited adjuvant effects for Ag-specific IgG1, IgE, and IgA responses when two other protein Ags, OVA and hen egg white lysozyme, were given by the oral route . Quantitative reverse transcriptase-PCR was performed to assess levels of mRNA for Th1 (IFN-gamma) and Th2 (IL-4) cytokine expression in TT-stimulated CD4+ T cell cultures . Both Peyer's patches and splenic CD4+ T cells expressed markedly increased levels of IL-4-specific message, but did not result in changes in IFN-gamma mRNA expression . To determine whether the route of immunization influenced IgE responses, mice were immunized s.c . with TT and CT as adjuvant . Significant increases in total and TT-specific IgE Abs were induced when CT was co-administered . Taken together, these results show that CT acts as a mucosal adjuvant to enhance Th2-type responses and in particular, the IL-4 produced results in a characteristic Ab isotype pattern associated with this cytokine.

J Mol Recognit, 1995 Nov-Dec, 8(6), 358 - 62
Solution dynamics of the oligosaccharide moiety of ganglioside GM1: comparison of solution conformations with the bound state conformation in association with cholera toxin B-pentamer; Richardson JM et al.; The solution dynamics of the oligosaccharide moiety of ganglioside GM1 have been determined by use of a combination of 1H rotating frame Overhauser effect measurements and restrained molecular dynamics simulations . It is found that the Galbeta1-3 and NeuNAc moieties which are primarily recognized by cholera toxin both exhibit considerable torsional flexibility about their respective glycosidic linkages . A comparison with the bound state conformation of the ganglioside in association with cholera toxin B-pentamer, shows that a low energy conformation of the oligosaccharide, which closely approximates the global minimum, is selected upon binding.

Gen Pharmacol, 1995 Nov, 26(7), 1597 - 602
Multiplicative interaction between intrathecally and intracerebroventricularly administered morphine for antinociception in the mouse: effects of spinally and supraspinally injected 3-isobutyl-1-methylxanthine, cholera toxin, and pertussis toxin; Suh HW et al.; 1 . Either intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration of morphine alone at the dose of 0.2 microgram slightly increased inhibition of the tail-flick response . However, combined i.t . and i.c.v . injections of morphine at the same dose increased the inhibition of the tail-flick response in a synergistic manner . 2 . Cholera toxin (CTX, 0.05 to 0.5 microgram) pretreated i.t . or i.c.v . for 24 hr or pertussis toxin (PTX, 0.05 to 0.5 microgram) for 6 days dose-dependently attenuated inhibition of the tail-flick response induced by combined i.t . and i.c.v . injection of morphine . 3 . 3-Isobutyl-1-methylxanthine (IBMX, 0.001 to 0.1 ng) pretreated i.t . for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by combined i.t . and i.c.v . injections of morphine . However, IBMX pretreated i.c.v . for 10 min was not effective in attenuating the inhibition of the tail-flick response induced by combined i.t . and i.c.v . injections of morphine . 4 . It is concluded that both spinal and supraspinal CTX- and PTX-sensitive G-proteins are involved in the antinociception produced by morphine-induced multiplicative interaction between spinal and supraspinal sites . However, only spinal but not supraspinal cAMP phosphodiesterase is involved in mediating antinociception induced by morphine-induced multiplicative interaction.

Biol Pharm Bull, 1995 Nov, 18(11), 1509 - 12
Cholera toxin accentuates the antagonism by acetylcholine of higenamine-induced positive chronotropy is isolated right atria of mice; Kimura I et al.; +/- -Higenamine (demethylcoclaurine), a cardiotonic principle from aconite root, chronotropic and inotropic actions mediated through beta 1-adrenergic receptors . We have investigated the influence of cholera toxin (CTX), a Gs-protein activator, and pertussis toxin (PTX), a Gi-protein inhibitor on the chronotropic interaction between higenamine and a muscarinic agonist, acetylcholine (ACh) in the isolated right atria of mice . CTX (100nm, 1h) pretreatment accentuated the inhibitory responses to cumulative applications of ACh (30nM--30 microns for the positive chronotropic effects induced by higenamine (100nM), isoproterenol (3 and 10 nM) or dobutamine (100nM) . In normal atria (CTX-untreated), ACh physiologically antagonized the positive chronotropic effects of these beta-adrenergic agonists . Pretreatment with PTX (150 microgram/kg, i.p., 3d) abolished the CTX (100nm, 1 h)-induced accentuation in the inhibitory effect of ACh against higenamine . PTX pretreatment also attenuated the physiological antagonism by ACh against higenamine in normal atria . The negative chronotropic effect of ACh was not affected by a submaximal concentration of forskolin (1 micron) . The These results suggest an accentuated antagonism between higenamine and ACH in CTX-treated, but not in untreated, isolated right atria of mice, which may occur through a functional interaction between the beta1-adrenergic-Gs and muscarinic-Gi systems.

Am J Physiol, 1995 Nov, 269(5 Pt 1), C1271 - 9
Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body; Cachero TG et al.; The regulation of the chemoreceptor cell function by G proteins has been studied by measuring the release of 3H-labeled catecholamines ({3H}CA) in carotid bodies (CBs) treated with fluoride, cholera toxin (CTX), and pertussis toxin (PTX) . Fluoride augmented the basal release of {3H}CA in a dose- (5-20 mM) and Ca(2+)-dependent manner . Nisoldipine (1 microM) and ethylisopropyl amiloride (EIPA; 10 microM) inhibited this effect by approximately 60%, and both drugs combined inhibited it in full . BAY K 8644 (1 microM) doubled the effect of fluoride . The effects of fluoride on the stimulus-evoked release of {3H}CA varied with the type of stimulus and the duration of the treatment . Simultaneous application of fluoride with the stimulus increased by five times the release evoked by hypoxia and by two times that by K+ and dinitrophenol (DNP) . Preincubation with fluoride for 1 h caused an inhibition (approximately 70%) of the release evoked by high K+ and veratridine, whereas that evoked by DNP and low PO2 was still augmented (approximately 2 times) . Preincubation (4 h) of the CBs with CTX (3 micrograms/ml) reduced by 54% the release of {3H}CA evoked by 35 mM K+ but did not affect that evoked by low PO2 or DNP . A similar treatment with PTX (1 microgram/ml) affected only the release of {3H}CA evoked by DNP, reducing it by 65% . The data show that fluoride, CTX, and PTX have different effects on the release of {3H}CA evoked by high external K+, DNP, and low PO2, indicating that the stimulus-secretion coupling process for each stimulus is differently regulated by G proteins.

Proc Natl Acad Sci U S A, 1995 Oct 24, 92(22), 10094 - 8
Transcytosis of cholera toxin subunits across model human intestinal epithelia; Lencer WI et al.; Cholera toxin (CT) elicits a massive secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and ultimately activating basolateral effectors (adenylate cyclase) . The mechanism of signal transduction from apical to basolateral membrane, however, remains undefined . We have previously shown that CT action on the polarized human intestinal epithelial cell line T84 requires endocytosis and processing in multiple intracellular compartments . Our aim in the present study was to test the hypothesis that CT may actually move to its site of action on the basolateral membrane by vesicular traffic . After binding apical receptors, CT entered basolaterally directed transcytotic vesicles . Both CT B subunits and to a lesser extent CT A subunits were delivered intact to the serosal surface of the basolateral membrane . The toxin did not traverse the monolayer by diffusion through intercellular junctions . Transcytosis of CT B subunits displayed nearly identical time course and temperature dependency with that of CT-induced Cl- secretion--suggesting the two may be related . These data identify a mechanism that may explain the link between the toxin's apical receptor and basolateral effector.

Am J Clin Nutr, 1995 Oct, 62(4), 813 - 9
Herman Award Lecture, 1995: infection-induced malnutrition--from cholera to cytokines; Beisel WR; Infection-induced malnutrition, the most common form of cytokine-induced malnutrition, results from the actions of proinflammatory cytokines, ie, tumor necrosis factor (TNF) and interleukins 1,6, and 8 (IL-1, IL-6, and IL-8) . During acute generalized infections, these cytokines initiate the acute-phase reaction . This reaction is quite stereotyped, and includes fever, malaise, myalgia, headaches, cellular hypermetabolism, and multiple endocrine and enzyme responses . In addition, there is heightened catabolism of muscle proteins and many amino acids; flux of free amino acids into the liver; hepatic synthesis of acute-phase plasma proteins; sequestration of iron and zinc; gluconeo-genesis; insulin resistance; impaired cellular uptake of fatty acids from plasma triglycerides; sizable losses of body nitrogen, potassium, magnesium, phosphate, and zinc; retention of body salt and water; heightened metabolic degradation and/or loss of vitamins; and an activation of the immune system . The pathogenesis of cytokine-induced malnutrition is thus vastly different from the malnutrition caused by uncomplicated starvation . Cytokine-induced malnutrition can have a devastating effect on the immune system and its functions . Although proinflammatory cytokines are found in mucosal fluids, where they contribute to the pathogenesis of inflammatory bowel diseases, it is not known whether cytokines play a role in toxigenic, secretory diarrheas such as cholera, which cause huge losses of body water, electrolytes, and bicarbonate while exhibiting no systemic manifestations of an acute-phase reaction.

Am J Physiol, 1995 Oct, 269(4 Pt 1), G548 - 57
Signal transduction by cholera toxin: processing in vesicular compartments does not require acidification; Lencer WI et al.; In the polarized human intestinal epithelial cell line T84, signal transduction by cholera toxin (CT) follows a complex series of events in which CT enters the apical endosome and moves through multiple vesicular compartments before it activates adenylate cyclase . As with processing of many other surface ligands, it has been suggested that CT must enter acidic vesicles to exert its downstream effects . To determine if intravesicular pH may regulate signal transduction by CT, we examined the cAMP-dependent Cl- secretory response {short-circuit current (Isc)} to CT in T84 cell monolayers treated with chloroquine (500 microM), methylamine (50 mM), NH4Cl (10 mM), nigericin (4 microM), or bafilomycin A1 (1 microM) . Each of these reagents collapsed intravesicular pH gradients as confirmed by accumulation of acridine orange within subcellular compartments of living T84 cells imaged by confocal epifluorescence microscopy . Both acidotropic amines and nigericin inhibited the cAMP-dependent Cl secretory response in T84 cells . However, none of these reagents specifically affected adenylate cyclase itself or coupling of adenylate cyclase with the heterotrimeric guanosinetriphosphatase Gs as judged by the secretory response to the adenosine 3',5'-cyclic monophosphate (cAMP) agonists vasoactive intestinal peptide (VIP), forskolin, or 8-bromo-cAMP . In vitro enzyme-linked immunosorbent assay showed that CT binding to ganglioside GM1 was not dependent on pH between 5.0 and 10 . Maximal Isc elicited by apical CT relative to maximal Isc elicited by VIP was not affected by pretreatment with chloroquine, methylamine, NH4Cl, or bafilomycin AI . Nigericin was the only reagent to inhibit CT-induced Isc (5 +/- 2% maximal response to VIP) . The data indicate that low intravesicular pH will have little or no effect on CT association with its membrane receptor GM1, or on subsequent processing/signal transduction events.

J Immunol, 1995 Sep 15, 155(6), 2877 - 87
Paradoxical IgA immunity in CD4-deficient mice . Lack of cholera toxin-specific protective immunity despite normal gut mucosal IgA differentiation; Hornquist CE et al.; Using normal and CD4 gene-targeted (CD4-/-) mice, we asked whether mucosal immune responses and IgA B cell differentiation require the presence of CD4+ T helper cells . We found that CD4-/- mice had numerous B cell germinal centers in Peyer's patches and other gut-associated lymphoid tissues . Membrane IgA+ B cells were found to co-localize to germinal center areas and CD4-CD8- double negative CD3+ T cells had replaced CD4+ T cells in the follicular areas of the Peyer's patches . CD4-/- mice had normal levels of IgA-producing cells in gut-associated lymphoid tissues, and gut lavage contained unaltered levels of total IgA . However, despite T cell help for IgA B cell differentiation, CD4-/- mice did not respond with Ag-specific intestinal IgA following oral immunization with the powerful mucosal immunogen cholera toxin (CT) . By contrast, these mice demonstrated serum alpha-CT IgG following oral immunization, suggesting that double negative CD3+ T cells provided some help for systemic immune responses after oral immunization . Perorally immunized CD4-/- mice were completely unprotected against CT-induced diarrhea while both normal and CD8-/- mice were well protected and also demonstrated high levels of gut mucosal alpha-CT IgA . After reconstitution of the CD4-/- mice by adoptive transfer of naive mesenteric lymph node CD4+ T cells, the mice acquired the ability to respond with specific mucosal immune responses following oral immunization and also developed resistance against CT-induced diarrhea . Thus, paradoxically, although IgA B cell differentiation appears to proceed normally in CD4-/- mice, specific gut mucosal immune responses are grossly impaired in the absence of CD4+ T cells.

J Immunol Methods, 1995 Sep 11, 185(1), 31 - 42
Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens; Harokopakis E et al.; Several immunoadjuvant systems have been proposed to enhance mucosal immune responses of orally administered purified antigens . Cholera toxin (CT) or its B subunit (CTB) have been found to promote immune responses to antigens when they are co-administered via mucosal routes . Oral administration of antigens incorporated into liposomes has also been shown to result in enhanced mucosal immune responses . Here, we describe the covalent coupling of CT and CTB to small unilamellar liposomes for targeting these vesicles to Peyer's patch M cells, following their oral administration . Conjugation was done by means of a thioether bond using succinimidyl(4-N-maleimidomethyl)cyclohexane-1-carboxylate to modify the dipalmitoylphosphatidyl-ethanolamine constituent of liposomes and N-succinimidyl-3-(2-pyridyldithio)propionate to thiolate CT or CTB . The biological activity of CT or CTB bound to liposomes was confirmed by a hemagglutination assay using GM1-enriched human erythrocytes . Furthermore, oral administration of CT-conjugated liposomes to rats resulted in the induction of serum IgG and salivary IgA anti-CT responses . CT-conjugated liposomes may prove to be a useful system for targeted delivery and immunoenhancement of weakly immunogenic antigens.

J Neurosci Methods, 1995 Sep-Oct, 61(1-2), 127 - 38
A retrograde double-labeling technique for light microscopy . A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate; Ruigrok TJ et al.; A light microscopical, non-fluorescent, retrograde double-labeling technique is described . Cholera toxin B-subunit (CTb) and a conjugate of wheatgerm agglutinin and bovine serum albumin coupled to 10 nm gold particles (gold-lectin) are both excellent retrograde tracers and, when visualized by means of immunohistochemistry and silver intensification, respectively, may be readily identified within the same cell . This light microscopical retrograde double-labeling technique is illustrated in rat with experiments designed to investigate the collateralisation (1) of vestibular neurons to the spinal cord and oculomotor complex, (2) of spinal neurons to the left and right lateral reticular nucleus, and (3) of inferior olivary neurons to the uvula of the cerebellum . Advantages over fluorescent double-labeling experiments are found in the fact that the diaminobenzidine reaction product as well as the silver/gold deposits do not fade and can be examined in counterstained sections . Moreover, the injection sites can be kept quite small and may be guided by electrophysiological recording through the injection pipette.

Gut, 1995 Sep, 37(3), 340 - 5
Role of 5-hydroxytryptamine type 3 receptors in rat intestinal fluid and electrolyte secretion induced by cholera and Escherichia coli enterotoxins; Mourad FH et al.; Cholera toxin and Escherichia coli heat labile toxin (LT) induced intestinal secretion has in the past been attributed exclusively to an increase in intracellular cAMP whereas E coli heat stable toxin (ST) induced secretion is mediated through cGMP . Evidence is accumulating on the importance of 5-hydroxytryptamine (5-HT) in cholera toxin induced secretion, but its role in LT and ST is not well established . This study therefore investigated in vivo the effect of 5-HT3 receptor antagonist, granisetron, on intestinal fluid and electrolyte secretion induced by cholera toxin, LT, and ST . Granisetron (30, 75, 150, or 300 micrograms/kg) was given subcutaneously to adult male Wistar rats 90 minutes before instillation of 75 micrograms cholera toxin or 50 micrograms LT in isolated whole small intestine . In situ small intestinal perfusion was performed with an iso-osmotic plasma electrolyte solution (PES) to assess fluid movement . In a second group of animals, granisetron (300 micrograms/kg) was given subcutaneously and two hours later small intestinal perfusion with PES containing 200 micrograms/l ST was performed . Cholera toxin induced net fluid secretion (median -50.1 microliters/min/g (interquartile range -59.5 to -29.8)) was found to be dose dependently decreased or abolished by granisetron (plateau effect at 75 micrograms/kg: 18 (-7.8 to 28), p < 0.01) . Granisetron in high dose (300 micrograms/kg), however, failed to prevent LT or ST induced secretion (-52 (-121 to -71) v -31 (-44 to -18), and (-39 (-49 to 17) v (-22 (-39 to -3)), respectively) . Sodium and chloride movement paralleled that of fluid . In conclusion, these data show that 5-HT and 5-HT3 receptors play an important part in cholera toxin induced secretion but are not involved in E coli heat stable or heat labile toxin induced secretion.

Cell Growth Differ, 1995 Aug, 6(8), 955 - 64
Serum response element and flanking sequences mediate the synergistic transcriptional activation of c-fos by 12-O-tetradecanoylphorbol-13-acetate and cholera toxin in AKR-2B cells; Harvat BL et al.; 12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin have been shown previously to act synergistically to stimulate traverse of G0-G1 and entry into S phase in quiescent mouse fibroblasts . These agents also have a synergistic effect on the induction of the endogenous c-fos gene, as well as a transfected reporter construct containing the mouse fos promoter/enhancer region from -397 to +1 cloned upstream of luciferase . A detailed mutational analysis of the c-fos-regulatory region revealed that the synergy between TPA and cholera toxin requires multiple discrete elements, including the binding sites for the serum response factor (-308 to -299), p62/Elk-1 (-316 to -309), on the 5' side of the serum response element, and a CCAAT or E box-binding protein(s) on the 3'-flanking side of the serum response element (-303 to -295 or -297 to -292, respectively) . The putative cyclic AMP response element (-65 to -58), shown to be activated in a number of cell types after increases in cyclic AMP levels, mediated an induction by TPA but not by cholera toxin in AKR-2B cells, and was not required for the synergistic transactivation induced by the combination of TPA and cholera toxin.

Gastroenterology, 1995 Aug, 109(2), 422 - 30
Ultrastructural changes in the upper small intestinal mucosa in patients with cholera; Mathan MM et al.; BACKGROUND & AIMS: Small intestinal mucosal ultrastructural abnormalities were reported in a limited number of patients with cholera in the 1970s . This study extends these observations by examining distal duodenal biopsy samples from 19 patients with cholera and 10 controls . METHODS: Endoscopic biopsy samples obtained, usually during the first 24 hours of illness, were processed for electron microscopy . RESULTS: Widening of intercellular spaces and alteration of apical junctional complexes were prominent in the villus epithelium, whereas blebbing of microvillus border and mitochondrial changes were more prominent in the crypt epithelium . The apical junctional and intercellular space changes were not altered by oral rehydration . Degranulation of argentaffin cells, mucosal mast cells, and eosinophils; increase in neutrophil polymorphs; and changes in the enteric nerve fibers and microvasculature were also present . The extent of the changes correlated with clinical severity . CONCLUSIONS: The differential involvement of the villus and crypt suggests that factors responsible for secretion may act differentially on surface and crypt epithelium and that both regions may contribute to secretion . The contribution of the enteric nervous system, vasculature, argentaffin cells, mucosal mast cells, eosinophils, and neutrophils in the secretory process and in determining the severity of the clinical illness must be determined by further clinical studies.

BMJ . 1995 Jul 8;311(6997):77.
Cholera epidemic threatens Sierra Leone; Dyer O; PIP: Sierra Leone faces the threat of a major epidemic of cholera with the onset of the rainy season, according to the World Health Organization (WHO) . The situation is particularly grave for the two million people displaced by the country's civil war . Already 1709 cases of cholera have been registered in Freetown, with 57 deaths . Freetown's population has doubled since the start of the war in 1991 with 750,000 refugees camping out in the town . The insurgent Revolutionary United Front is now within 32 km of the capital . Provinces are cut off from the capital, medical supplies are scarce . Doctors and aid workers are forced to rely on a private helicopter service for personal transport . As many as 10,000 people were affected by the disease last year . WHO experts predict that pneumonia is likely to claim the lives of many children, and a highly drug resistant strain of Plasmodium falciparum malaria is also looming . The greatest problems are the lack of safe drinking water and the attendant risks of cholera and dysentery . At one site in Freetown the 6000 refugees have to fetch water from a well and have no latrines . As a result there have been 277 cases of cholera and 2 deaths already among that group . The health department has set up five centers to treat cholera in Freetown and is organizing mobile clinics . WHO's Sierra Leone office is assisting the government mobile health teams, which provide free primary care to displaced people . Medicines and vaccines, however, are lacking . Many of the staff of the 13 district health authorities have been displaced to Freetown . Aid agencies such as Medecins Sans Frontieres and Oxfam have stepped into the role in many districts . Ironically, one of the Revolutionary United Front's main demands is for a free national health service .

Indian Pediatr, 1995 Jul, 32(7), 755 - 61
Clinical profile of cholera in young children--a hospital based report; Amin V et al.; Clinical profile of cholera was studied in children attending Diarrhea Training and Treatment Unit from January-December 1993 . Out of a total 8714 cases of acute watery diarrhea, 64 children (0.7%) were suspected to have cholera on the basis of acute onset loose water/rice watery stools, high purge rate with or without excessive vomiting and/or severe dehydration . Stool culture was positive for cholera in 33 cases (51.6%) . All the isolates were V . cholerae 01 biotype El Tor serotype Ogawa . Sixty four per cent of stool culture positive cases were below 5 years of age . The results assume importance because out of 28 children < 2 years with clinical suspicion of cholera, 11 cases (39.3%) were culture positive for V . cholerae, youngest child being 3 months old . Comparison of various parameters revealed that presence of vomiting > 4 episodes/ day (p < 0.005), frequency of stools >12/24 hours (p <0.002), rice watery stools (p < 0.01) and presence of severe dehydration (p < 0.01) were significant parameters associated with positive stool culture . Beside examination of stool sample by hanging drop method was an excellent diagnostic tool (p < 0.001) with a sensitivity of 51.5%, specificity 100% and positive predictive value of 100% . The isolates of V . cholerae were susceptible to furazolidone, cephelexin, nalidixic acid, norfloxacin and gentamicin . Our observations indicate that cholera is not uncommon in infants and young children . Like children in the older age group, acute onset diarrhea with watery/rice watery stools and high purge rate with or without excessive vomiting and/or rapid development of severe dehydration should arouse suspicion of cholera in younger children also . They should be investigated for cholera even in non-endemic areas and in the absence of cholera outbreaks.

Vaccine, 1995 Jul, 13(10), 933 - 7
Gene fusion of cholera toxin B subunit and HBV PreS2 epitope and the antigenicity of fusion protein; Shi CH et al.; A unique EcoRI site was introduced at the 3' end of cholera toxin B subunit (CTB) gene by site-directed mutagenesis, polynucleotides encoding 120-145aa epitope of HBV PreS2 were chemically synthesized and fused to the 3' end of cholera toxin B subunit gene . The fused gene was over-expressed (about 30 micrograms ml-1) in E . coli, and more than 95% of the fusion protein was secreted into the medium . The fusion protein expressed was purified by affinity chromatography . The chimera protein obtained bound to ganglioside GM1, and had the antigenicity of both cholera toxin B subunit and HBV PreS2 as confirmed by ELISA . After mice were immunized intramuscularly with the fusion protein, anti-CTB antibody and anti-PreS2 antibody were produced . These results indicated that the fusion protein retained not only the biological function of CTB but also the antigenicity and the immunogenicity of cholera toxin B subunit and HBV PreS2 epitope . This work provided a sound basis for further studies on the construction of engineered peptide vaccine.

Vopr Virusol, 1995 Jul-Aug, 40(4), 182 - 6
{Natural killers and cytotoxic lymphocytes in classical hog cholera}; Shubina NG et al.; The formation of immune mechanisms directed at elimination of infected cells and including the activity of natural killers and cytotoxic lymphocytes was assessed in pigs infected with hog cholera virus . In acute disease natural killer activity in the blood is reduced, while in vaccinal process it is increased . Vaccination in parallel with cyclophosphamide immunodepression lead to inhibition of natural killer activity . Leukocytes and lymphocytes of immunized pigs can cause cytolysis of autologous targets infected with hog cholera virus.

Structure, 1995 Jun 15, 3(6), 561 - 70
Surprising leads for a cholera toxin receptor-binding antagonist: crystallographic studies of CTB mutants; Merritt EA et al.; BACKGROUND: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance . The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability . The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin . The molecular basis for these effects was not readily apparent from analysis of an earlier crystal structure of the wild-type toxin B pentamer in a complex with the receptor pentasaccharide . RESULTS: We now report at a resolution of 2.0 A the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution . The observed conformation of the Asp33 side chain suggests that the loss in binding affinity is due to a steric clash with atoms C9 and O9 of the sialic acid moiety of the receptor, ganglioside GM1 . The crystal structure also reveals an unexpected mode of pentamer-pentamer interaction in which pairs of toxin pentamers are joined by reciprocal insertion of the imidazole ring of His13 from one subunit of each pentamer into one of the receptor-binding sites on the other . The surface of interaction at each pentamer-pentamer interface is on the order of 500 A2, and primarily involves contact of residues 10-14 with the receptor-binding site on the associated pentamer . This same pentamer-pentamer interaction is also present in the crystal structure of a second recombinant CTB containing an Arg-->Asp substitution at residue 35, which we have determined at 2.1 A resolution . CONCLUSIONS: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-His-Asn, corresponding to residues 10-14 of CTB, may constitute lead compounds for the design of binding-site inhibitors.

Biochim Biophys Acta, 1995 Jun 6, 1256(3), 275 - 83
The role of calcium influx in cellular proliferation induced by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin; Buckley NE et al.; The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts . It was previously shown that the B subunit had no effect on cAMP, protein kinase C or phosphoinositide turnover, but did cause an increase in the influx of calcium from extracellular sources (Spiegel, S . and Panagiotopoulos, C . (1988) Exp . Cell Res . 177, 414-427) . In contrast to the action of known growth factors, the B subunit induced significant DNA synthesis after only a 1-3 h treatment . We utilized this unique property to determine whether the increase in calcium influx plays a role in B subunit-induced mitogenicity . Cells were briefly treated with the B subunit in the presence of calcium channel blockers, followed by removal of the blockers and further incubation in B subunit-free medium for the remaining time required to measure DNA synthesis . When 1 mM cobalt was only present during the first 3 h incubation . DNA synthesis induced by either the B subunit or fetal bovine serum was completely abolished . However, both nickel (1 mM) adn the L-type voltage-gated calcium channel inhibitor nicardipin (10 microM) inhibited B subunit-induced cell proliferation without abrogating the response to fetal bovine serum . Using a gel retardation assay, we found that the B subunit markedly stimulated specific DNA-binding activity of the transcription factor, activator protein-1 (AP-1), which functions as a major convergence point coupling early events induced by a variety of mitogens to long term growth responses . Presence of c-Fos protein in the AP-1 complex was demonstrated as a supershift band in the gel mobility assay using c-Fos polyclonal antibody . Cobalt, which markedly inhibited B subunit-induced DNA synthesis, also completely abolished AP-1 DNA-binding activity stimulated by the B subunit . In sharp contrast, cobalt had no effect on DNA-binding activity of AP-1 induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate . Our results suggest that calcium influx is a key element for both DNA-binding activity of AP-1 and cell proliferation induced by binding of the B subunit of cholera toxin to cell surface ganglioside GM1.

J Gen Virol, 1995 Jun, 76 ( Pt 6), 1371 - 80
Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus; Muller CP et al.; An efficient mucosal vaccination has a number of obvious advantages over invasive routes of immunization . The immune response to measles virus (MV) was investigated after intranasal and intragastric co-immunization of mice with cholera toxin B (CTB) as an adjuvant . High titres of virus-specific IgG antibodies and a transient IgA response were detected in the sera after intranasal but not after intragastric immunization when CTB was used . In the presence of CTB, higher titres were reached with less antigen and fewer intranasal boosts . Neutralizing antibodies were found in all animals only after co-immunization with MV and CTB . In the nasal wash and the saliva, IgG and IgA titres were significant only in the MV plus CTB groups; IgG levels were comparable to those found after intraperitoneal (i.p.) immunization with complete Freund's adjuvant . Specific IgA was detected in the mucosal fluids only after intranasal immunization with MV plus CTB but not after i.p . or intragastric immunization . The antibody response consisted of 99% IgG1 after MV immunization . In the CTB groups 10% IgG2b and 1% IgG2a were detected in addition to the predominant IgG1 antibodies.

J Diarrhoeal Dis Res, 1995 Jun, 13(2), 95 - 8
Epidemic of cholera among the aborigines of northern Argentina; Mazzafero VE et al.; In February 1992, an epidemic of cholera began in Argentina . The first known case appeared in an aborigine population living by the Pilcomayo river on the Bolivian border . By the end of that year, 551 cases and 15 deaths were reported to the health authorities . Epidemiological analysis helped identify the pattern of transmission of the disease in this region of South America . Polluted watercourses and little pools, formed after the rainy season, were identified as the single source of infection for subsequent cases . Then, the epidemic adopted a person-to-person pattern of transmission and was propagated over a long course with weekly occurrence . Anthropological, cultural, and other factors that contributed to the origin and spread of cholera in northern Argentina are described in this study.

J Indian Med Assoc, 1995 Jun, 93(6), 237 - 8
Cholera outbreaks: role of oral rehydration therapy; Bhattacharya SK; PIP: Acute diarrheal diseases are an important cause of morbidity and mortality especially in children in developing countries . Cholera is endemic in many Asian and African countries . Indeed, since the beginning of recorded history, cholera has been endemic in the Ganges and Brahmaputra deltas of eastern India and Bangladesh . In endemic areas, the disease may account for 5-10% of hospitalized patients with diarrhea in all age groups, but it affects mostly children aged 2-9 years; severe cases are frequently encountered . In newly invaded areas, young adults are most often the initial victims . The discovery that glucose enhances the absorption of sodium and water in the gut and that no structural changes occur in the gut epithelium in cholera led to the development of oral rehydration therapy (ORT) . This discovery is considered one of the most important medical advances of the century . ORT is a simple, cost-effective weapon to fight against diarrheal dehydration, which is the most important cause of death from cholera and other secretory diarrheas . Numerous clinical studies have shown the effectiveness of ORT in all age groups and in all etiologies of diarrhea . While a strong CDD program is the best preparation for a cholera epidemic, the best control measures during an outbreak are health education and the early detection and treatment of people with disease .

Vaccine, 1995 Jun, 13(9), 817 - 20
A method to screen T lymphocyte epitopes after oral immunisation of humans: application to cholera toxin B subunit; Castello-Branco LR et al.; The response to oral immunisation of humans with classical biotype cholera toxin B subunit was studied to identify immunodominant T lymphocyte determinants . The in vitro proliferative response to pools of 12-mer peptides and larger peptides used individually was analysed by a novel statistical approach, and identified an immunodominant region in residues 70-79 in immunised subjects, when either pools or individual peptides were employed . In contrast, a patient infected with El Tor biotype had a dominant response to residues 40-60 . The statistical software employed in this study may enable efficient screening of antigens for immunodominant T lymphocyte determinants when blood precursor frequencies are low following immunisation, and may therefore be of special relevance to mucosal vaccines.

Vaccine, 1995 Jun, 13(9), 803 - 10
Immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB); Nashar TO et al.; The immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB) is reported . Oral delivery of rEtxB to congenic mice of several different H-2 haplotypes resulted in H-2 dependent serum IgG responses (H-2d > H-2b = H-2q > H-2a > H-2k) and a similar spectrum of intestinal IgA responses in those strains tested . Responses to rEtxB and rCtxB were found to be differentially modulated by the H-2 locus, with significant differential effects in H-2b and H-2d congenic strains (H-2d > H-2b for rEtxB; H-2b > H-2d for rCtxB) . Additionally, it was found that when rEtxB was fed to mice previously primed (orally) with either rEtxB or rCtxB only those mice primed with rEtxB exhibited a booster response . A second booster immunisation with rEtxB in rCtxB-primed mice produced an H-2 dependent spectrum of responses characteristic of those elicited by rEtxB, with the antibodies predominantly directed against rEtxB and not rCtxB . These results indicate that the differential response to rEtxB and rCtxB is set at the T- and B-cell level . Also, immunoregulation of antibody responses to rEtxB by intra-H-2 I-E in mice transgenic for the entire IEka gene was investigated . No significant difference between responses in transgene-positive and -negative mice was found, suggesting that antigen presentation does not involve I-E, but occurs in the context of I-A.(ABSTRACT TRUNCATED AT 250 WORDS)

MMWR Morb Mortal Wkly Rep, 1995 May 26, 44(20), 385 - 6
Cholera associated with food transported from El Salvador--Indiana, 1994; Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP; Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, IsraelCholera toxin (CT) consists of a pentameric B subunit which binds with high affinity to ganglioside GM1, and an A subunit which stimulates adenylate cyclase, resulting in the elevation of cAMP . We now examine the effect of cationic amphiphilic drugs (CADs) on the internalization of rhodamine (Rh)-CT in cultured hippocampal neurons . CADs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and on endosomes (Wang, L.-H., Rothberg, K . G., and Anderson, R . G . W . (1993) J . Cell Biol . 123, 1107-1117) . Rh-CT was internalized by an energy- and temperature-dependent (presumably vesicular) mechanism to the Golgi apparatus . Internalization to the Golgi apparatus was completely but reversibly blocked by CADs, and the ability of CT to stimulate the elevation of cAMP was significantly reduced . In control cells, cAMP levels were elevated 2.3-fold after 20 min of incubation with CT, but in CAD-treated cells cAMP levels were only elevated 1.3-fold . The effect of CADs on CT internalization was not due to a direct effect of CADs on the Golgi apparatus . Our data demonstrate that CADs inhibit vesicular transport of CT to the Golgi apparatus and imply that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both pathways are inhibited by CADs.

J Immunol, 1995 May 15, 154(10), 4956 - 64
cAMP-independent effects of cholera toxin on B cell activation . III . Cholera toxin A subunit-mediated ADP-ribosylation acts synergistically with ionomycin or IL-4 to induce B cell proliferation; Francis ML et al.; To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin . Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding . Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect . Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells . However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway . Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig . IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells . Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.

J Mol Biol, 1995 May 5, 248(3), 507 - 12
Atomic force microscopy of cholera toxin B-oligomers bound to bilayers of biologically relevant lipids; Mou J et al.; Cholera toxin B-oligomer was imaged by atomic force microscopy (AFM) on biologically relevant model membranes, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and egg yolk phosphatidylcholine at room temperature in solution at a resolution in the range of 1 to 2 nm . In addition, two-dimensional arrays were grown directly on these model membranes without any special treatment, and were also imaged by AFM . These results demonstrate the ability of AFM for imaging membrane proteins at high resolution without the need of chemical cross-linking, either within the membrane or to the substratum.

Dig Dis, 1995 May-Jun, 13(3), 190 - 8
Cholera in the United States; Hellinger WC et al.; Cholera remains a threat to human health in many parts of the world, including the United States . The epidemiology of cholera is reviewed to prepare for identification and prevention of the disease in appropriate clinical settings . The clinical manifestations of cholera and the pathophysiology of the toxin-induced diarrhea are reviewed to introduce and to clarify appropriate therapeutic and preventive interventions.

Clin Infect Dis, 1995 May, 20(5), 1409 - 19
Cholera: calamitous past, ominous future; Lacey SW; From the pandemics of the 19th century to the recent disaster in Goma, Zaire, cholera has left an indelible mark on human and medical history . Cholera pandemics in the 19th and 20th centuries drove the development of epidemiology as a serious science . Cholera has continued to press advances in the concepts of disease ecology, basic membrane biology, and transmembrane signaling and in the application of scientific information to treatment design . Furthermore, the lessons learned from the study of pandemic cholera are likely to provide insights into the best means of stopping other pandemics . In spite of tremendous scientific and clinical progress, however, the seventh pandemic has lasted 33 years, and the eighth pandemic appears to have started.

J Neuroendocrinol, 1995 May, 7(5), 361 - 9
Melatonin receptors couple through a cholera toxin-sensitive mechanism to inhibit cyclic AMP in the ovine pituitary; Morgan PJ et al.; The nature of melatonin receptor-G-protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins . ADP-ribosylation of ovine PT membrane proteins using 32P-NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins . Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX . Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for 125I-MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%) . A CTX-sensitive component also mediates the inhibition of forskolin-stimulated cyclic AMP accumulation as pre-treatment of PT cells with CTX (5 micrograms/ml) for 16 h blocked this response . Gs alpha is a major substrate for ADP-ribosylation by CTX, and 16 h pre-treatment of PT cells with CTX (5 micrograms/ml) caused a down-regulation of Gs alpha . Northern analysis showed only one major transcript of Gs alpha of about 2 kb, which would encompass all of the known splice variants of the Gs gene . Screening of a cDNA library from ovine PT for Gs-related genes and sequencing of clones, combined with RT-PCR of PT mRNA, revealed no novel products . On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the Gs class of G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sci, 1995 May, 4(5), 841 - 8
Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding; Shoham M et al.; Cholera is a widespread disease for which there is no efficient vaccine . A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine . Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5 . Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells . This loop exhibits remarkable conformational plasticity induced by environmental constraints . The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3 . In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix . Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins . Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies . Thus, saccharide binding confers rigidity upon the loop . The conformation of CTP3 in complex with TE33 is quite different . The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR-derived solution structure . Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively . Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity . This suggests a different interaction of TE33 with intact CT.

Infect Immun, 1995 May, 63(5), 2021 - 5
Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates; Bergquist C et al.; This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate . After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations . Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB . Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations . Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested . Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung . Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations . Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations . This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate . The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization.

Ugeskr Laeger, 1995 Apr 17, 157(16), 2325 - 6
{Cholera in Denmark--an imported case of illness}; Lange KH et al.; A case of cholera imported to Denmark from the Pacific is presented . The patient was successfully treated with rehydration and antibiotics . A survey of the ongoing seventh pandemic of cholera is given and the possible emergence of a new eighth pandemic is discussed . Guidelines for prophylactic and therapeutic measures are discussed . Although V . cholerae colonies can be recognized on routine cultivation media, low numbers require selective media, and this is not included in routine investigations of stools for pathogens.

J Immunol, 1995 Apr 1, 154(7), 3611 - 7
Prevention of acute graft-versus-host disease by treatment with a novel immunosuppressant . Cholera toxin B subunit; Yankelevich B et al.; Transplantation of allogeneic bone marrow (BM) is often complicated by the development of acute graft-vs-host disease (GVHD) caused by contaminating T cells in BM inocula because of activation of the host reactive T effector cells . Using a murine model for acute GVHD caused by injection of parental C57Bl/6 splenocytes into unirradiated (C57Bl/6 x DBA/2) F1 hybrids, we demonstrated that pretreatment of the inocula with a novel immunosuppressant, B subunit of cholera toxin (CT-B) impaired the ability of C57Bl/6 T cells to induce acute GVHD in F1 recipients . F1 mice injected with CT-B-treated C57Bl/6 splenocytes did not develop significant splenomegaly, and no antihost CTLs were found in their spleens . Moreover, these mice did not suffer from aplastic anemia, nor from general immunosuppression . Immunofluorescence studies suggest that treatment of the inducing inocula with CT-B selectively prevents accumulation of the host-reactive CD8+ T cells in F1 mice . Furthermore, our experiments demonstrated that CT-B treatment does not impair the ability of BM progenitors to form colonies in semisolid culture or in lethally irradiated hosts . Thus, taken together, our data suggest that ex vivo CT-B treatment can be used in allogeneic BM transplantation to prevent acute GVHD.

Infect Immun, 1995 Apr, 63(4), 1452 - 61
Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli; Peterson JW et al.; The mechanisms which enable cholera toxin (CT) and the Escherichia coli heat-stable enterotoxins (STa and STb) to stimulate intestinal secretion of water and electrolytes are only partially understood . CT evokes the synthesis of 3',5'-cyclic AMP (cAMP), and STa is known to elevate intestinal levels of 3',5'-cyclic GMP (cGMP) . Neither of these recognized second messengers appears to mediate E . coli STb responses . We compared the secretory effects of CT, STa, and STb using the pig intestinal loop model and also measured the effects of toxin challenge on the synthesis of cAMP, cGMP, and prostaglandins (e.g., prostaglandin E2 {PGE2}), as well as on the release of 5-hydroxytryptamine (5-HT) from intestinal enterochromaffin cells . All three enterotoxins elicited fluid accumulation within a 2-h observation period . A combination of maximal doses of STa with STb yielded additive effects on fluid accumulation, which suggested different mechanisms of action for these toxins . Similarly, challenge of pig intestinal loops with a combination of CT and STb resulted in additive effects on fluid accumulation and luminal release of 5-HT . Unlike its effect on intestinal tissues from other animals, CT did not appear to elicit a dose-dependent cAMP response measurable in mucosal extracts from pig small intestine . In contrast, luminal fluid from CT-challenged pig intestinal loops contained dose-related amounts of cAMP and PGE2 that had been secreted from the mucosa . cAMP responses to STa or STb could not be demonstrated in either mucosal tissue or luminal fluid . In contrast, cGMP levels were increased in the intestinal fluid of loops challenged with STa but not in those challenged with STb . While the mechanisms of action of CT and STa are thought to involve impulse transmission via the enteric nervous system, we demonstrated significant stimulation of PGE2 synthesis and 5-HT release for CT and STb but very little for STa . We conclude from these data that the mechanisms of action of STa, STb, and CT are distinct, although the mode of action of STb may have some similarity to that of CT . Since STb stimulated the release of both PGE2 and 5-HT from the intestinal mucosa, the data suggested the potential for an effect of STb on the enteric nervous system.

Infect Immun, 1995 Apr, 63(4), 1349 - 55
Oral immunization with the dodecapeptide repeat of the serine-rich Entamoeba histolytica protein (SREHP) fused to the cholera toxin B subunit induces a mucosal and systemic anti-SREHP antibody response; Zhang T et al.; The intestinal protozoan parasite Entamoeba histolytica causes amebic dysentery, a major cause of morbidity worldwide . The induction of a mucosal antibody response capable of blocking amebic adhesion to intestinal cells could represent an approach to preventing E . histolytica infection and disease . Here we describe the expression of a chimeric protein containing an immunogenic dodecapeptide derived from the serine-rich E . histolytica protein (SREHP), fused to the cholera toxin B subunit (CtxB) . The CtxB-SREHP-12 chimeric protein was purified from Escherichia coli lysates and retained the critical GM1 ganglioside-binding activity of the CtxB moiety . Mice fed the CtxB-SREHP-12 fusion protein along with a subclinical dose of cholera toxin developed mucosal immunoglobulin A and immunoglobulin G and systemic antibody responses that recognized recombinant and native SREHP . Our study confirms the feasibility of inducing mucosal immune responses to immunogenic peptides by their genetic fusion to the CtxB subunit and identifies the CtxB-SREHP-12 chimeric protein as a candidate oral vaccine to prevent E . histolytica infection.

Mol Immunol, 1995 Apr, 32(6), 425 - 31
The sequence 130-137 of human interferon-alpha 2 is involved in the competition of interferon, prothymosin alpha and cholera toxin B subunit for common receptors on human fibroblasts; Zav'Yalov VP et al.; 125I-labelled recombinant human interferon alpha 2 (rHuIFN-alpha 2) capable of high-affinity binding (Kd = 2.46 +/- 0.18 x 10(-10) M) with receptors expressed on mouse thymocytes was obtained . Prothymosin alpha (proTM-alpha) but not cholera toxin was found to compete with radiolabelled IFN-alpha 2 for binding to the same receptor (Ki = 3.68 +/- 0.21 x 10(-11) M) . The synthetic peptide covering the sequence 130-137 of IFN-alpha 2 (authors' definition: alpha-peptoferon) was shown to have the capacity to displace the labelled IFN-alpha 2 from the IFN-alpha 2/receptor complex (Ki = 7.19 +/- 0.12 x 10(-11) M) . It was shown that receptors of this type are localized in plasmatic membrane fraction . Using {125I}-alpha-peptoferon, specific and saturable binding was detected on human fibroblasts and the data fitted a single binding site . Scatchard analysis yielded a Kd of 9.63 +/- 0.17 x 10(-8) M . The binding was competitively inhibited by IFN-alpha 2 (the Ki value in competition assays was 1.37 +/- 0.12 x 10(-8) M), proTM-alpha(Ki = 2.2 +/- 0.2 x 10(-7) M) and cholera toxin B subunit (Ki = 5.5 +/- 0.2 x 10(-7)) . The present study has demonstrated that the sequence 130-137 of HuIFN-alpha 2 is involved in the competition of HuIFN-alpha 2, proTM-alpha and cholera toxin B subunit for common receptors on human fibroblasts.

Epidemiol Infect, 1995 Apr, 114(2), 249 - 55
Epidemic cholera in rural El Salvador: risk factors in a region covered by a cholera prevention campaign; Quick RE et al.; In response to the Latin American cholera epidemic, El Salvador began a prevention programme in April 1991 . The first case was confirmed in August, and 700 cases were reported within 3 months . A matched case-control study was conducted in rural La Libertad Department in November 1991 . Illness was associated with eating cold cooked or raw seafood (odds ratio {OR} = 7.0; 95% confidence limits {CL} = 1.4, 35.0) and with drinking water outside the home (OR = 8.8; 95% CL = 1.7, 44.6) . Assertion of knowledge about how to prevent cholera (OR = 0.2; 95% CL = 0.1, 0.8) and eating rice (OR = 0.2; 95% CL = 0.1, 0.8) were protective . More controls than patients regularly used soap (OR = 0.3; 95% CL = 0.1, 1.0) . This study demonstrated three important points for cholera prevention: (1) seafood should be eaten cooked and hot; (2) populations at risk should be taught to treat household drinking water and to avoid drinking water outside the home unless it is known to be treated; and (3) education about hygiene can be an important tool in preventing cholera.

Eur J Pharmacol, 1995 Mar 16, 292(3-4), 293 - 9
Pertussis and cholera toxins modulate kappa-opioid receptor agonists-induced hypothermia and gut inhibition; Shukla VK et al.; In mice pretreated intracerebroventricularly (i.c.v.) with either saline (10 microliters/mouse), pertussis (1 microgram/mouse) or cholera (2.5 micrograms/mouse) toxins, effect of kappa-opioid receptor agonists on the colonic temperature and charcoal meal transit time were assessed . The kappa-opioid receptor agonist, trans-(+)-3,4-dichloro-N-methyl-{2-(1- pyrrolidinyl)cyclohexyl}-benzeneacetamide methane sulfonate hydrate (U-50488H, 50, 100 and 200 micrograms/mouse, i.c.v.) produced dose dependent hypothermia . Pertussis toxin pretreatment (72 and/or 144 h before) antagonized (P < 0.05) the hypothermic effect of U-50488H (100 micrograms/mouse) and (+)-trans-N-methyl-N-{2-(1- pyrrolidinyl)cyclohexyl{benz{b}-thio-phene-4-acetamide (PD 117302, 30 micrograms/mouse) . In contrast, cholera toxin pretreatment (48 and/or 96 h before) did not antagonize the hypothermic effect of the kappa-opioid receptor agonists . Moreover, both i.c.v . and intrathecal (i.t.) administration of kappa-opioid receptor agonists, U-50488H, }{5R-(5 alpha,7 alpha,8 beta)}-(+/-)-N-methyl-N-{7-(1- pyrrolidinyl)-1-oxaspiro{4,5}dec-8-yl}-benzeneacetamide inverted question mark (U-69593) and PD 117302, produced dose dependent inhibition of the charcoal meal transit . Cholera toxin pretreatment (48 and 96 h before) augmented (P < 0.05) the antitransit effect of i.c.v . administered U-50488H (100 micrograms/mouse), U-69593 (100 micrograms/mouse) and PD 117302 (50 micrograms/mouse) . However, pertussis toxin pretreatment did not affect the gastrointestinal inhibitory effect of the kappa-opioid receptor agonists . The present results extend our previous results on the effect of kappa-selective agonists on gastrointestinal motility and indicate, like the prototype opiate agonist morphine, kappa-opioid receptor agonists are effective in inhibiting the gastrointestinal motility when administered either by intrathecal or intracerebroventricular routes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1995 Mar 15, 306 ( Pt 3), 765 - 9
The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A; Levistre R et al.; Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine . The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA) . (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation . By contrast, the Gi alpha concentration was unchanged . (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5'-{gamma-thio}triphosphate (GTP{gamma S}) . (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-{methyl-amino}ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor . (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA . In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation . Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

Brain Res, 1995 Mar 13, 674(1), 107 - 11
Evidence that cholera toxin B subunit (CTb) can be avidly taken up and transported by fibers of passage; Chen S et al.; It has been reported that cholera toxin B subunit (CTb) is a sensitive neuronal tracer with unique features . However, the possible uptake of CTb by non-terminal fibers passing through the injection site has not been examined thoroughly . In the present study, small iontophoretic injections (current = +2 microA) of CTb were made in the olivocerebellar pathway in the rat ventrolateral medulla . A large number of retrogradely labeled neurons were seen in the contralateral inferior olive . In addition, prominent anterogradely labeled climbing fibers/terminals were found in the cerebellum ipsilateral to the injection site . This study, in contrast to previous report(s), indicates that CTb can be taken up avidly by fibers of passage and transported both anterogradely and retrogradely.

Gac Med Mex, 1995 Mar-Apr, 131(2), 213 - 7
{A novel treatment of cholera by a Mexican physician in the 19th century}; Rodriguez-de-Romo AC; Doctor Felipe Castillo, head of the Hospital de San Pablo during the cholera epidemic of 1850, used "Salty water" as treatment for the patients who attended the hospital . The etiology and pathogenesis of this sickness were unknown in those days, so Castillo's conduct was surprising . This study is based on an unpublished report, classified as anonymous, that Castillo gave to the Governor of Mexico City during the cholera epidemic.

Vaccine, 1995 Mar, 13(4), 339 - 41
Mechanism of enhancement of the immune responses to influenza vaccine with cholera toxin B subunit and a trace amount of holotoxin; Tamura S et al.; Cholera toxin B subunit (CTB) (1 microgram) and a trace amount of cholera toxin (CT) (0.1-10 ng), when inoculated intranasally into Balb/c mice together with influenza vaccine, induced synergistically a greater delayed-type hypersensitivity (DTH) response to the vaccine than did a trace amount of CT alone . In parallel with the in vivo response, normal peritoneal macrophages that were incubated in vitro with the vaccine and the CT-containing CTB, induced a higher adenylate cyclase activity and a greater ability to transfer DTH response into naive recipient mice than did the macrophages incubated with the vaccine and CT . The treatment of macrophages with the vaccine and CTB failed to induce either adenylate cyclase or DTH response . From these results, the mechanism by which CTB and a trace amount of CT enhance immune responses synergistically could be explained by the enhancement of the CT action on macrophages or by the efficient binding of a trace amount of CT to antigen-presenting cells in the presence of a relatively large amount of CTB, resulting in enhanced cyclic AMP formation followed by enhanced antigen presentation.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Mar-Apr, Suppl 2, 9 - 22
{The epidemiological characteristics of cholera in the Republic of Dagestan . An assessment of the epidemic-control measures}; Onishchenko GG et al.; Retrospective analysis of epidemic cholera manifestations was made in Daghestan using the data of operative epidemic analysis of the break in 1994 . Unexpected prolongation of epidemic process of cholera for Daghestan, which was imported by pilgrims from Southern-Western Asia, has been shown using climate-geographical social-demographical and sanitary-hygienic peculiarities . Common laws of development of epidemic complications were demonstrated, as well as the main ways of infection transmission of great number of Daghestan settlements in epidemic process . The importance of antiepidemic means and significant role of created specialized antiepidemic groups have been emphasized in rapid carrying out of means in infection focus, including massive investigation of people in settlements.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Mar-Apr, Suppl 2, 52 - 6
{The cholera outbreak in one of the central regions of Dagestan}; Savel'ev VN et al.; The data on the epidemiological analysis of cholera cases in the epicenter of this infection in the Daghestan, viz . in the village of Gerga, Kaiakent District, are presented . The outbreak of cholera was due to the import of this infection by pilgrims from their hajj to Saudi Arabia . The causative agent of the outbreak was the epidemic variant of V . cholerae eltor Ogawa . Everyday contacts were the main route of the transmission and spread of this infection, the water factor playing an insignificant role . Two epidemic waves of cholera were registered in Gerga, each of them provoked the penetration of the infection to other regions of the Republic of Daghestan.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Mar-Apr, Suppl 2, 36 - 9
{The characteristics of cholera prevalence and of the performance of epidemic-control measures in small settlements of Dagestan}; Terent'ev AN et al.; The present work deals with summarizing the experience obtained by the specialized antiepidemic brigade of the Rostov-on-Don Research Institute for Plague Control in the work on the liquidation of cholera in some regions of Daghestan with a view to discussing the problems of improvement of anticholera measures . The characteristic features of the epidemic process were its explosive character, sparseness of the foci of infection, the prevalence of its transmission through everyday contacts (family contacts and intensive tribal contacts) and essential delays in taking anticholera measures due to sudden appearance of outbreaks, remoteness of small settlements and the lack of manpower and means for carrying out anticholera measures at a given place and time, as well as delays in epidemiological analysis carried out by local health service bodies . Delays in carrying out such measures led to the spread of infection both within settlements and in the whole region and further in the republic . The epidemic process was complicated by the antibiotic resistance of V.cholerae strains circulating on this territory . All these factors formed specific epidemic situation which introduced amendments into the organization of anticholera measures.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Mar-Apr, Suppl 2, 27 - 30
{The mechanisms of the intrafocal and territorial spread of cholera in the Republic of Dagestan}; Grizhebovskii GM et al.; The description of the epidemic manifestations of cholera in the Republic of Daghestan at the period of the seventh pandemic, linked with the action of such common transmission factors as water, food and everyday contacts, is presented and their role in the territorial spread of this infection is evaluated . The analysis of family foci in the Derbent and Kaiakent regions in 1994 is given; the conclusion is made that a low sanitary level of human dwellings leads to a wide spread of cholera among close relations due to the action of water, alimentary and contact factors of transmission.

Scand J Gastroenterol, 1995 Mar, 30(3), 242 - 5
The presence of bicarbonate in oral rehydration solution does not influence fluid absorption in cholera; Sarker SA et al.; BACKGROUND: On the basis of human perfusion studies it has been speculated that bicarbonate ions in oral rehydration salt solutions (ORS) to treat diarrhoea are more efficiently absorbed from the small bowel . We evaluated the role of bicarbonate in ORS by using a reduced purging rate in cholera as a proxy indicator for absorption efficiency in cholera-like severe diarrhoea . METHODS: In a double-blind randomized trial 60 patients received standard ORS containing bicarbonate or an identical solution except that sodium bicarbonate was replaced by an equimolar amount of sodium chloride (sodium, 90 mmol/l; potassium, 20 mmol/l; chloride, 80 mmol/l; bicarbonate, 30 mmol/l; glucose, 111 mmol/l; and osmolality, 331 mmol/l) after initial intravenous rehydration to correct initial dehydration and shock and until diarrhoea ceased . RESULTS: Five patients receiving standard ORS and eight receiving bicarbonate-free ORS required unscheduled intravenous therapy for recurrence of severe dehydration in spite of receiving ORS solution . ORS intake and purging rate, in ml/kg body weight/day, both including and excluding stool output during unscheduled intravenous therapy are closely similar in the two treatment groups . CONCLUSION: The results indicate that bicarbonate-containing ORS solution does not have any clinically significant effect on the absorption efficiency of ORS, either beneficial or adverse, and its use is relevant only for correction of metabolic acidosis of diarrhoeal dehydration.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 417 - 9
Inhibition of cholera toxin by human milk fractions and sialyllactose; Idota T et al.; The effects of human milk fractions on clolera toxin B subunit binding to monosialoganglioside 1 (GM1) were investigated . Human milk, human defatted milk, whey, and a low-molecular-weight fraction of human milk inhibited the binding, but casein did not inhibit it . The inhibitory activity of whey from bovine-milk-based infant formula was less than that of whey from human milk . Differences in composition between human and bovine whey seemed to influence the extent of the inhibitory activity . Sialylated oligosaccharides were considered to be the possible components that inhibited cholera toxin . The effects of sialyllactose, a predominant sialylated component of human milk, on cholera toxin-induced diarrhea were investigated by the rabbit intestinal loop method . Sialyllactose inhibited the cholera toxin inducing fluid accumulation, although neither sialic acid nor lactose had an effect on it . The results suggest that sialyllactose is responsible for the inhibitory activity of milk on cholera toxin.

Neuroscience, 1995 Mar, 65(1), 119 - 60
Afferent projections to the rat locus coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris leucoagglutinin; Luppi PH et al.; The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin . To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties . After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described . We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kolliker-Fuse nucleus, (4) mesencephalic reticular formation . Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus . No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus . Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus . These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus . These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents . These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin . Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kolliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunology, 1995 Mar, 84(3), 446 - 52
Effects of prostaglandin E2, cholera toxin and 8-bromo-cyclic AMP on lipopolysaccharide-induced gene expression of cytokines in human macrophages; Zhong WW et al.; Prostaglandin E2 (PGE2) appears to regulate macrophage cytokine production through the stimulatory GTP-binding protein (Gs protein)-mediated cyclic AMP (cAMP)-dependent transmembrane signal transduction pathway . In this study, we used PGE2, cholera toxin (CT; a direct G alpha s protein stimulator) and 8-bromo-cAMP (a membrane permeable cAMP analogue) to stimulate this pathway, and investigated their influence on cytokine gene expression in lipopolysaccharide (LPS)-activated human macrophages . The mRNA expression for interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-8 were determined employing reverse transcription polymerase chain reaction (RT-PCR) using specific primers . We demonstrated that PGE2, CT and 8-bromo-cAMP inhibited the LPS-induced gene activation of TNF-alpha and IL-1 alpha, and had no effect on the gene activation of IL-1 beta and IL-8 . Further, our data indicate that PGE2 suppressed the gene activation of IL-6 following LPS stimulation, but neither CT nor 8-bromo-cAMP had an effect . These data suggest that PGE2 alters LPS-stimulated gene activation of only some of the early macrophage cytokines, and does so either by a Gs transmembrane cAMP-dependent or an independent system.

Horm Metab Res, 1995 Mar, 27(3), 137 - 40
Effect of cholera toxin and pertussis toxin on the growth of A431 cells: kinetics of cyclic AMP and inositol trisphosphate in toxin-treated cells; Kamiya Y et al.; Cholera toxin and pertussis toxin were inhibitory to the incorporation of thymidine into A431 cells in serum-free culture . Cholera toxin enhanced the growth inhibitory effect of epidermal growth factor (EGF) on A431 cells, whereas pertussis toxin attenuated the effect . Cholera toxin increased the concentration of intracellular cyclic AMP (cAMP) to three-times the initial concentration at 120 minutes and it increased the concentration of intracellular inositol trisphosphate (IP3) rapidly but transiently . Pertussis toxin reduced the concentration of IP3 both in EGF treated and untreated A431 cells at 10 minutes . cAMP was not involved in pertussis toxin-mediated effects . In conclusion, the intracellular cAMP and IP3 concentrations in CT-treated A431 cells are compatible with previous reports regarding the growth inhibitory effects on A431 cells . The inhibitory effect of PTX on the EGF-induced increase of intracellular IP3 is thought to be compatible with the finding that PTX attenuated the EGF-induced growth inhibition.

Biochim Biophys Acta, 1995 Feb 22, 1247(1), 65 - 73
Fluorescence analysis of the interaction between ganglioside GM1-containing phospholipid vesicles and the B subunit of cholera toxin; Picking WL et al.; Binding of cholera toxin B protomer (CT-B) to a pyrene-labeled analogue of its ganglioside GM1 receptor (pyrene-GM1) in the absence and presence of phosphatidylcholine vesicles was monitored using steady-state fluorescence spectroscopy . CT-B association with pyrene-GM1 micelles induces changes in the fluorescence properties of this ganglioside analogue that are consistent with its conversion from an excimer to a monomer form . Incubation of pyrene-GM1 with preformed vesicles of phosphatidylcholine (PC) results in complete conversion of pyrene-GM1 to its monomer form, however, unlike with CT-B binding, incorporation of pyrene-GM1 into PC vesicles occurs with a concomitant loss of fluorescence quenching by the small polar quenching agent acrylamide . Subsequent binding of CT-B to the PC-GM1 composite vesicles causes no further change in the pyrene fluorescence emission spectrum but does appear to increase acrylamide accessibility . These data lead to the conclusion that cholera toxin binding to a cell membrane alters membrane packing at the site of attachment . Furthermore, this phenomenon appears to be influenced by environmental conditions such as pH . A pH of about 4.0 or less causes acrylamide quenching to decrease to approximately the levels observed in the absence of CT-B . These results may be useful in describing the dynamics of the interaction between cholera toxin and target cell membranes . Moreover, these data could provide clues to the mechanism by which the toxic portion of CT is able to enter the cytoplasm of target cells.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 529 - 35
The rat liver ecto-ATPase/C-CAM cDNA detects induction of carcinoembryonic antigen but not the mercurial-insensitive ecto-ATPase in human hepatoma Li-7A cells treated by epidermal growth factor and cholera toxin; Knowles AF; The rat liver ectoATPase has reportedly been cloned . The cDNA, a member of the carcinoembryonic antigen (CEA) gene family, was shown to increase aggregation of transfected cells, but ATPase activity was not evaluated . Using this cDNA as a probe to clone the mercurial-insensitive ectoATPase (MI-ectoATPase) of human hepatoma Li-7A cells, the cDNA obtained was that of CEA which has no ATPase activity . The probe also did not detect increased transcription when MI-ectoATPase activity was induced in Li-7A cells . It is concluded that the "rat liver ectoATPase cDNA" codes for a cell adhesion molecule but does not code for an ectoATPase . It was also discovered that expression of four CEA transcripts in Li-7A cells was markedly stimulated by a single growth modulator, EGF, and was further stimulated by a cAMP elevating agent, cholera toxin.

Brain Res, 1995 Feb 6, 671(1), 27 - 37
Serotonergic and non-serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study; Datiche F et al.; Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat . After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD) . A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups . Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups . In the RD, 30% of CTb stained cells were immunoreactive to 5-HT . After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70% . These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment . To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed . Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero-posterior differences.

J Immunol, 1995 Feb 1, 154(3), 1032 - 40
Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit; Elson CO et al.; Despite the mucosal immunogenicity and adjuvanticity in vivo of cholera toxin (CT), both CT and CT B subunit are strong inhibitors of T cell activation in vitro . This study asked whether such T cell inhibition is relevant to the mucosal effects of CT in vivo . The activation of T cells pulsed in vitro for only 15 to 120 min with CT or CT B subunit, respectively, was inhibited, consistent with the expected short exposure times in vivo . Although both CD8+ and CD4+ T cells were inhibited in vitro, CD8+ T cells bound more toxin and were inhibited to a greater degree than were CD4+ T cells . Intestinal gavage of mice with 10 micrograms CT did not alter the overall composition of Peyer's Patch, mesenteric lymph node, or spleen but did cause a marked depletion of intraepithelial lymphocytes, mainly CD8+ T cells, and of lymphocytes in the dome epithelium over Peyer's Patch . To determine whether such inhibition of T cells was functionally relevant in vivo, T cells from mice fed keyhole limpet hemocyanin (KLH) were adoptively transferred into naive recipients, who were then parenterally immunized . T cells from mice fed KLH alone inhibited both the systemic IgG and secretory IgA anti-KLH response, but T cells from mice fed KLH plus CT did not, indicating that mucosally applied CT was able to abrogate the induction of this suppressor T cell . We conclude that one of the mechanisms of CT's mucosal effects in vivo is the inhibition of certain mucosal T cell functions and alteration of the regulatory T cell environment in gut-associated lymphoid tissue.

J Biol Chem, 1995 Jan 6, 270(1), 21 - 4
Different ARF domains are required for the activation of cholera toxin and phospholipase D; Zhang GF et al.; ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform . ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD . A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method . To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1 . The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive . The two chimeric proteins had quite opposite effects on PLD activity . rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly . It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

Biopolymers, 1995, 37(6), 383 - 9
Two-dimensional NMR studies of the interactions between a peptide of cholera toxin and monoclonal antibodies; Anglister J et al.; To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens . For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation . To extract the information about antibody-peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab-peptide complex (Fab: antibody fragment made of Fv--the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen--the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed . Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied . The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site . The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies' combining sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Chin J Biotechnol, 1995, 11(2), 79 - 86
A secretion expression system using promoter and signal peptide of cholera toxin B subunit gene; Cao C et al.; A secretion expression plasmid vector pMC05S was constructed taking advantage of the promoter, signal peptide, and transcriptional terminator of cholera toxin B subunit gene and beta-galactosidase was overexpressed in E . coli and most of the expressed enzyme was secreted into periplasma when the lacZ gene was inserted downstream of the signal peptide sequence of pMC05S . The yield of beta-galactosidase by engineered E . coli reached 30 mg/L and most of the beta-galactosidase retained the activity of the enzyme . The appropriate host strain and medium were also investigated . This system provided a new approach for the expression of proteins that easily form inclusion bodies.

Virus Genes, 1995, 10(2), 185 - 7
Characterization of the hog cholera virus 5' terminus; Katayama K et al.; Hog cholera virus (HoCV) 5' terminus of the ALD and GPE(-) strains were analyzed by using rapid amplification of cDNA end method (5'RACE) . An additional nine nucleotides were found at the 5' termini of genomic RNA in the ALD and GPE(-) strains of HoCV . These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5' terminus by computer-assisted analysis . It seems possible that the secondary structure and/or the 5' terminus sequence has a significant role in the HoCV virus genome.

Ophthalmic Res, 1995, 27(3), 153 - 7
Inhibition of platelet-activating factor-induced retinal impairments by cholera and pertussis toxins; Cluzel J et al.; Platelet-activating factor (PAF) has been shown to alter the trans-retinal potential recorded from light-stimulated isolated retina . In the present study, we investigated the effect of cholera and pertussis toxins on PAF-induced impairment of the electroretinogram (ERG) . Administered alone, 2 x 10(-7) M PAF induced a very marked and rapid drop in the b-wave amplitude . When 75 micrograms/l of cholera toxin was coadministered with PAF in the perfusion solution, no b-wave drop was observed, suggesting that the effect of PAF on retinal function was mediated by GTP-binding protein (G protein) . Similarly, a low dose of pertussis toxin (5 micrograms/l) was sufficient to antagonize the action of PAF on the ERG . Our results suggest that the irreversible and deleterious effect of PAF on ERG is mediated by a G protein mechanism, located in the neural retina.

Gastroenterology, 1995 Jan, 108(1), 34 - 9
Cholera toxin-induced small intestinal secretion has a secretory effect on the colon of the rat; Nocerino A et al.; BACKGROUND/AIMS: Little information is available on the role of colon during small intestinal secretion . The aim of this study was to examine the effects of secretory changes in the small intestine on the colonic transport of electrolytes and water in vivo . METHODS: The jejunum and colon of the rat were perfused in vivo simultaneously but separately, and jejunal secretion was induced by exposing the jejunum to cholera toxin, 8-bromo-cyclic guanosine monophosphate, or hyperosmolarity . RESULTS: Jejunal perfusion with a hyperosmolar mannitol solution (600 mOsm/L) or with 8-bromo-cyclic guanosine monophosphate (0.5 mmol/L) resulted in net secretion of water in the jejunum but did not affect the baseline rate of water transport in the colon . On the contrary, addition of cholera toxin (1 microgram/loop) to the jejunal segment not only induced a significant local secretory change but also resulted in a similar change in the colon, which was not exposed to cholera toxin . The intestine was transected immediately below the jejunum, thus interrupting the anatomical continuity of the enteric nervous system . This procedure eliminated the distant secretory effect of cholera toxin, thus allowing the conclusion that the enteric nervous system is involved in the distant propagation of the local secretion induced by cholera toxin . CONCLUSIONS: Cholera toxin, but not other secretagogues, triggers a secretory response that is not only local but also extends to distal segments via the enteric nervous system.

Berl Munch Tierarztl Wochenschr, 1995 Jan, 108(1), 20 - 5
Assessment of safety and protective value of a cell culture modified strain "C" vaccine of hog cholera/classical swine fever virus; Dahle J et al.; The protective value of a commercial strain "C" vaccine of classical swine fever (CSF) was tested in weaner pigs . Vaccinated animals were challenged intranasally with the virulent hog cholera virus (HCV) strain ALFORT/187 in groups of four pigs each at one to four weeks post vaccination, respectively . Non-vaccinated control animals were challenged in the same manner . Some vaccinated pigs seroconverted as early as one week post vaccination with all pigs yielding neutralizing antibodies (nAb) against the vaccine virus at two weeks post vaccination . After challenge no clinical signs were observed in any of the vaccinated animals whereas non-vaccinated control animals developed fever starting in general on the fourth day post challenge . In vaccinated pigs no challenge virus could be isolated from leucocyte samples taken on days 3 to 7 post challenge while HCV was isolated from buffy coat leucocytes of all non-vaccinated animals . Six out of eight control animals were sacrificed and viral antigen was detected in tonsils, mandibular lymph node and spleen in four animals, exclusively in tonsils in one animal and none in another animal . Two non-vaccinated animals that survived the experiment seroconverted after challenge and developed nAb against the HCV strain ALFORT/187.

Arch Virol, 1995, 140(8), 1385 - 91
Comparison of the entire nucleotide and deduced amino acid sequences of the attenuated hog cholera vaccine strain GPE- and the wild-type parental strain ALD; Ishikawa K et al.; We have determined the complete nucleotide sequences of a live attenuated hog cholera virus (HCV) and its progenitor strain . The viral RNA of each strain consisted of 12,298 nucleotides including untranslated regions of 373 and 228 bases at the 5' and 3' end, respectively . There was a single large open reading frame spanning 11,697 nucleotides which could encode a large protein of 3,899 amino acids with a calculated molecular weight of 438-kDa . We have found 225 nucleotide difference between the two strains, of which six were located in the untranslated region . Four-sixths of these differences resulted in amino acid substitutions.

Vet Res, 1995, 26(4), 300 - 9
Partial sequencing of hog cholera virus Alfort strain genome and its comparison with other pestivirus strains; Bakkali Kassimi L et al.; After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined . This sequence was compared with homologous parts of previously published pestivirus genomes . The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67% . Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively) . Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC . These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage . Genomic relationships among the pestiviruses are discussed.

Adv Exp Med Biol, 1995, 371B, 1507 - 12
Cholera toxin increases T lymphocyte responses to unrelated antigens; Hornquist E et al.; We have found that CT adjuvant greatly augmented oral priming immunizations . Limiting dilution analysis revealed strong increases in the frequency of primed antigen specific T lymphocytes subsequent to the use of CT adjuvant . Moreover, several fold stronger lymphokine production accompanied the strong proliferative responses seen with CT-treated and KLH-primed T cells . The lymphokine pattern secreted by these T cells suggested that TH1 as well as TH2 types of cells were effectively primed by KLH, and that CT adjuvant did not exert a selective effect on any of these T-cell subsets . These effects of CT were truly due to immunomodulation and not simply the result of a more efficient uptake of gut luminal antigens in the presence of CT, because CT enhanced equally well T cell priming by peroral as well as parenteral immunization with KLH . Moreover, the CT-induced enhanced responsiveness to antigen was also observed with CD4(+)-enriched T cells suggesting that CT primarily affected T cell priming rather than the balance between CD4+ and CD8+ T cell subsets.

Tidsskr Nor Laegeforen, 1994 Dec 10, 114(30), 3612 - 5
{Hospital records from the cholera epidemic in Bergen 1848-1849}; Oeding P; During the great cholera epidemic in Bergen in 1848-49, three cholera hospitals were established . Records from the three hospitals have been found and studied . Of the 1,024 cholera patients in the city, 707 were hospitalized, and of these 430 (60.8 per cent) died . Overall, slightly more women than men were admitted to hospital . The women were older than the men but the mortality was the same . The Christi Krybbe hospital had the oldest patients but the lowest mortality . The staff probably acquired more experience at Christi Krybbe, which was in action during the whole epidemic, and the nursing may have been better there . The mortality in the hospitals reached a peak during the first weeks of the epidemic and later decreased considerably.

Tidsskr Nor Laegeforen, 1994 Dec 10, 114(30), 3608 - 11
{Cholera hospitals in Bergen}; Oeding P; As early as 1831 premises for a cholera hospital were discussed in Bergen, but no decision was taken . On the same day that Bergen was declared to be cholera-infected, i.e . December 18th, 1848, a Board of Health was appointed . The Board immediately established the city's first cholera hospital, the Christi Krybbe hospital . Two additional hospitals were later established in other parts of Bergen . The hospitals had a total of 200 beds for a population of 24,000 . Bergen was therefore relatively well equipped with hospital beds for cholera patients . A number of other plans for cholera hospitals were discussed . The Board wanted the county to establish a cholera hospital outside Bergen, to avoid new infection being introduced into the city . However, no hospital other than the three mentioned above was taken into use.

Surgery, 1994 Dec, 116(6), 1048 - 52; discussion 1052-3
An in vitro model of thyroid neoplasia: permanently transfected FRTL-5 cells with thyroglobulin promoter-cholera toxin A1 subunit minigene; Saji M et al.; BACKGROUND . Activating mutations of protein Gs alpha that stimulates adenylyl cyclase are present in a subset of thyroid adenomas . Cholera toxin A1 subunit (CT) mimics these mutations via adenosine diphosphate-ribosylation of Gs alpha . METHODS . To test the role of activated Gs alpha in thyroid neoplasia we developed an in vitro model . With molecular genetic techniques a transgene (TG-CT) in which the thyroglobulin gene (TG) promoter directs expression of CT was made . This transgene was transfected into rat thyroid follicular cells (FRTL-5) . Integration of TG-CT transgene and its expression were examined . RESULTS . Polymerase chain reaction of DNA from transfected FRTL-5 cells identified the TG-CT transgene in six cell lines . The TG-CT transfected clones exhibited up to a 65-fold (1.29 +/- 0.37 pmols cyclic adenosine monophosphate (cAMP)/micrograms DNA) increase in cAMP over nontransfected FRTL-5 cells (0.02 +/- 0.001 pmols cAMP/micrograms DNA) . Insulin, a known stimulator of the TG promoter, further induced CT gene expression and led to a 209-fold (10.43 +/- 0.10 pmols cAMP/micrograms DNA) increase in cAMP over nontransfected cells (0.05 +/- 0.00 pmols cAMP/microgram DNA) . CONCLUSIONS . By mimicking a known mutation associated with thyroid neoplasms, these permanently transfected FRTL-5 cell lines will serve as a model to examine the long-term potential neoplastic effects of activated Gs alpha on thyroid follicular cells.

J Bone Miner Res, 1994 Dec, 9(12), 1927 - 34
Cholera toxin-stimulated bone resorption in cultured mouse calvarial bones not inhibited by calcitonin: a possible interaction at the stimulatory G protein; Ransjo M et al.; We examined the effect of calcitonin in cultured mouse calvarial bones after prestimulation with different activators of adenylyl cyclase . Calcitonin (100 ng/ml), added after 48 h of culture, inhibited bone resorption (assessed as release of 45Ca from prelabeled bones cultured for 96-144 h) stimulated with parathyroid hormone (PTH, 10 nM; 0-144 h) or the adenylyl cyclase stimulator forskolin (2 microM; 0-144 h) . However, no effect of calcitonin was demonstrated when bone resorption was prestimulated with the adenylyl cyclase stimulator cholera toxin, at and above 1 ng/ml, at any time point studied . In contrast, two other types of inhibitors of bone resorption in vitro, the carbonic anhydrase inhibitor acetazolamide (10 microM) and the aminobisphosphonate AHPrBP (10 microM), significantly inhibited cholera toxin-stimulated bone resorption . No cyclic AMP response to calcitonin was seen after preculture for 48 h with cholera toxin (0.1-100 ng/ml), although bones precultured in basic medium, in the absence or presence of forskolin, were still able to respond to calcitonin with elevation of cyclic AMP . Binding studies with {125I}calcitonin demonstrated that the preculture with cholera toxin did not affect the binding of calcitonin to the receptor . In summary, our data show that cholera toxin pretreatment makes calvarial bones insensitive to calcitonin-induced inhibition of bone resorption as a result of an interaction with cholera toxin at the level of calcitonin receptor-linked signal transduction . We suggest that the interaction, distal to the calcitonin receptor, is caused by the irreversible activation of Gs produced by cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Promot Educ, 1994 Dec, 1(4), 15 - 7, 47
{Fighting cholera in shanty-town . Successful experience of a Quebec project adapted to Peru}; Frechette L et al.; Originally designed in Quebec, the MOI project was a collaboration between two professors of social work from Quebec and two members of the Peruvian NGO called SUR in Villa de Salvador, one of poorest slum areas on the outskirts of Lima . The approach is founded on the notion that the body is the primary instrument through which a person can interact with the world around him or her, and that the physical and mental health of an individual exists within the context of healthy conditions of life that must include at least a basic social and health infrastructure as well as healthy hygiene practices on the part of individuals, families and the local community . Preschool children (ages 4-6), study a different part of the body and its proper care each week through classroom observation games . Parents' help is requested to modify unhealthy conditions, at the same time to enrich the children's experience and to mobilize the community to improve health conditions . During the 1991 cholera epidemic, not a single case was counted in the experimental district, despite its clearly socio-economically impoverished status, and despite the fact that the Ministry of Health recorded 86,650 cases in the Lima-Callao district, accounting for about 40% of the total number of cases in the Peru . The prior work made it easier to explain how cholera is spread and what special new measures needed to be taken in addition to the hygiene habits already taught.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1994 Dec, 116(6), 1269 - 74
On the role of the carboxyl group of sialic acid in binding of cholera toxin to the receptor glycosphingolipid, GM1; Lanne B et al.; The carboxyl group of the natural cholera toxin receptor, the ganglioside GM1, Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-Cer, has been converted to a number of C(1)-amides of NeuAc . The binding of cholera toxin B-subunit to these derivatives was monitored by exposing the modified glycolipids, on solid phases, to radiolabeled toxin . Binding was obtained, although substantially reduced, with the amide and to a lesser extent with the benzylamide and also the C(1)-alcohol . In the assay system used, the methyl-, ethyl-, or propylamides did not bind . It was concluded that the hydrogen bonding capacity of a carboxyl or amide group is needed for strong binding . This is in agreement with the recently published crystal structure of the B-subunit in complex with the GM1 pentasaccharide.

Glycoconj J, 1994 Dec, 11(6), 533 - 40
Comparison of the glycolipid-binding specificities of cholera toxin and porcine Escherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine; Teneberg S et al.; The binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxin were investigated by binding of 125I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells . The binding of cholera toxin was restricted to the GM1 ganglioside . The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide . The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin . Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences . By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose . Interactions which may explain the relatively high toxin affinity for this receptor were found.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 10795 - 9
Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance; Sun JB et al.; Oral administration of antigens, including allergens and autoantigens, may be an efficient way to prevent diseases associated with untoward immune responses to self- and non-self-antigens . However, this approach has met with limitations because it usually requires repeated administrations of large doses of antigen and is less efficient in an already immune host, and the effect is of short duration . We report that a single oral administration of minute amounts of particulate or soluble antigen coupled to the B subunit of cholera toxin (CTB) can markedly suppress systemic immune responses in naive and in systemically immune animals . Both early (2-4 hr) and late (24-48 hr) delayed type-hypersensitivity reactivities were strongly suppressed after feeding a single dose of CTB-conjugated antigen . Serum antibody responses were also decreased, although moderately, after oral administration of CTB-conjugated antigen . This strategy of tolerance induction, based on oral administration of small amounts of antigens conjugated to a mucosa-binding molecule, may find broad applications for preventing or abrogating untoward immune responses.

Tijdschr Diergeneeskd, 1994 Nov 1, 119(21), 629 - 33
{Postmortem findings in swine: non-selected submissions from hog cholera protection areas of 1992 versus selected submissions of 1991-1992}; Peperkamp NH et al.; This article presents a survey of death-causes of all spontaneously died pigs, n = 851, from a restricted area in the province of South Holland during a 2 1/2 months lasting hog-cholera epizootic in 1992 . 23 pigs from 5 submissions showed a positive IFT against hog-cholera virus . Those animals and pigs from sero-positive farms were excluded from this survey . The results of the post-mortems were compared with the post-mortem findings of the normally submitted, selected, animals in 1991 and 1992, n = 904 + 745, from the western parts of the Netherlands, in which the above mentioned province is situated . By means of classification of the animals in age-classes and of the post-mortem findings to disease or diseased organsystem, insight is gained in the prevalence of the various causes of death per age-class . No distinct differences were found between the findings in the groups of animals from 1991 and 1992 . In the group of the hog-cholera-period comparatively more animals belonged to the neonatal and suckling period . In contrast to 1991-1992 the number of weaned and fattening pigs in the hog-cholera-group was lower . In both groups 40% of the death-causes was due to diseases of the digestive tract and 30% was a result of respiratory-tract infections . The third main cause of death in both groups was septicaemia and related diseases as endocarditis, pleuritis, peritonitis, polyserositis and polyarthritis . Comparison of the prevalence of infectious diseases per organsystem in successive age-classes demonstrated a similar tendency in the hog-cholera-group as in the year-groups 1991 and 1992.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1994 Nov, 62(11), 4948 - 54
Deficient induction of the immune response to oral immunization with cholera toxin in malnourished rats during suckling; Flo J et al.; Malnourished rats during suckling were orally immunized with cholera toxin (CT) after different periods of refeeding . Intestinal fluids, sera, and supernatant fluids from cultured mesenteric lymph node (MLN) cells were obtained after rats were given three doses of CT and analyzed by enzyme-linked immunosorbent assay (ELISA) to evaluate the specific antibody response . Serum-specific immunoglobulin G (IgG), IgA, and IgM were severely diminished in malnourished rats immunized with three doses of CT after 1 week of refeeding when compared with those of controls . Also, a decreased IgA ELISA titer of the intestinal fluids and abrogation of the capacity to neutralize the CT in the intestinal ligated loop test were found . When a booster was given at 113 days of age, the immune response continued to be affected in the serum and the intestinal fluid . The results from the analysis of the supernatant fluids from cultured MLN cells were coincident with those mentioned above . When one dose of CT was administered into Peyer's patches (PP) after 1 week of refeeding, an impaired immune response was found in the intestinal fluid of malnourished rats during suckling compared with that of controls . This result together with the analysis of supernatant from MLN and PP cell cultures suggests that antigen triggering in the PP was affected . When the refeeding period was extended to 30 days and then the first dose of CT was administered, the antibody immune responses in intestinal fluid serum and supernatant fluid approached control values . These observations reinforce the fact that the gut-associated lymphoid tissue immaturity of the rats when they received the first CT dose (at 28 days old) was the main reason for the decreased immune response observed in the experimental group.

Histochem J, 1994 Nov, 26(11), 856 - 62
Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods; Dederen PJ et al.; In this report a comparison is made of three different visualization methods of rat cervical motoneurons retrogradely labelled with cholera toxin B subunit (CTb) . CTb conjugates such as CTb-HRP and CTb-FITC or CTb-TRITC, which can be visualized after histochemical detection and by fluorescence microscopy, respectively . The following results were obtained . (1) Immunochemical detection of CTb with peroxidase and DAB-Ni incubation provides the best labelling of the cell bodies and their processes, whereas immunochemical detection with FITC produces less effective labelling of the dendrites . (2) Histochemical visualization of CTb-HRP conjugate gives results similar to those of CTb immunochemistry but produces a much more granular appearance of the label, which may affect the identification of distal dendrites . In addition, direct electron-microscopic analysis of labelled structures can be achieved . (3) CTb-FITC and CTb-TRITC visualization permit double-labelling experiments but the labelled cells exhibit fluorescence only in their somata and proximal dendrites . (4) Factors other than labelling intensity, e.g . double-labelling, preservation of the label, compatibility with other techniques and even economic reasons must be taken into consideration when a selection of visualization methods is to be made.

Science, 1994 Oct 7, 266(5182), 107 - 9
Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model; Gabriel SE et al.; The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model . CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT . Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

Endocr J, 1994 Oct, 41(5), 593 - 7
Cholera toxin can ADP-ribosylate Gs as well as Gi in ACTH-unresponsive human adrenocortical cancer; Nishikawa T et al.; It is well known that cholera toxin (CT) stimulates ADP-ribosylation of Gs and also pertussis toxin (PT) does Gi . Each GTP-binding protein has its own action in the regulation of adenylate cyclase . A human non-functioning adrenocortical cancer tissue showed an unresponsiveness in adenylate cyclase to ACTH although ACTH and CT activated adenylate cyclase in a non-functioning adrenal adenoma tissue . CT ADP-ribosylated 43 kDa protein of the plasma membrane of the cancer tissue while CT and PT could ADP-ribosylate 43 kDa and 38 kDa protein in the adenoma tissue, respectively . Immunoblotting analysis of the cancer tissue demonstrated that 40 kDa protein was detected by anti-Gs antibody as well as by anti-Gi antibody . The present experiments demonstrated that CT could ADP-ribosylate Gs which has stimulatory action on adenylate cyclase and also Gi which inhibits adenylate cyclase . Thus it is suggested that CT can activate the ADP-ribosylation of Gs and also Gi in a human adrenocortical cancer tissue, partly resulting in abnormal regulation of adenylate cyclase which may be crossly related to ACTH-unresponsiveness.

Indian J Med Res, 1994 Oct, 100, 184 - 9
Octreotide (SMS 201-995) as an antisecretory agent in cholera toxin & bile acid induced intestinal secretion in an in vivo animal study; Bardhan PK et al.; The effect of Octreotide (SMS 201-995), synthetic somatostatin analogue on small intestinal and colonic fluid secretion induced respectively by cholera toxin (CT) and deoxycholic acid (DCA) was investigated in rabbits using in vivo isolated loops . After exposure to CT and DCA, marked fluid accumulation was observed in the small intestinal and colonic loops, along with elevation of jejunal and colonic mucosal cyclic AMP concentrations . Octreotide inhibited CT and DCA induced small intestinal and colonic secretion, dose-dependently . This anti-secretory effect was observed after both intramuscular and oral administration of octreotide . In contrast, octreotide did not affect the elevated mucosal cyclic AMP concentrations . These results suggest that octreotide inhibits CT and DCA induced intestinal secretion, and this anti-secretory effect is produced by affecting processes beyond cyclic AMP formation.

Eur J Clin Invest, 1994 Oct, 24(10), 664 - 8
Effect of 5-hydroxytryptamine antagonists on cholera toxin-induced secretion in the human jejunum; Eherer AJ et al.; In rats, the combined administration of the 5-HT2 antagonist ketanserin and the 5-HT3 antagonist tropisetron inhibits cholera toxin-induced intestinal secretion . We investigated whether these agents and the 5-HT3 antagonist ondansetron can inhibit cholera toxin-induced secretion in the human jejunum using a segmental perfusion technique . In a first control period the subjects' jejunums were perfused continuously with a plasma-like electrolyte solution . In a second control period they either received a combination of tropisetron plus ketanserin, or tropisetron or ondansetron alone . Cholera toxin 6.25 micrograms was then administered intrajejunally and the experiments were continued for 4 h . Net water movements during the 4th hour after CT administration minus net water movement during the first control period was used for further calculation and was referred to as net luminal gain . In perfusion studies with tropisetron plus ketanserin resp . ondansetron the net luminal gain of water (+ 161 +/- 26 resp . 189 +/- 28 ml 30 cm-1 h-1, mean +/- SEM) was significantly higher compared to perfusion studies with cholera toxin alone (+ 94 +/- 30) . Treatment with tropisetron did not change the CT-induced net luminal gain of water (+ 108 +/- 41) . Movements of sodium, chloride, bicarbonate and potassium paralleled the movement of water . In agreement with these observations we found a deterioration of clinical parameters after the end of the perfusion studies in four of five subjects treated with CT 25 micrograms plus ketanserin and tropisetron.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Neurosci, 1994 Oct 1, 6(10), 1633 - 40
Cholera toxin beta subunit induces the differentiation of human medulloblastoma cell line DEV in a neuronal pathway; Dufay N et al.; Medulloblastomas are primitive neuroectodermal tumours that are thought to arise from multipotent precursor cells in the cerebellum . Medulloblastoma cells may be undifferentiated or exhibit glial, neuronal or ependymal characteristics, suggesting that they may conserve their ability to differentiate in appropriate circumstances . Medulloblastoma cell lines may thus provide models to study the commitment and differentiation of multipotent CNS progenitor cells . A human medulloblastoma cell line, DEV, has previously been shown to differentiate in an astrocytic pathway after infection by the retrovirus HTLV-1 . In this study immunofluorescence flow cytometry shows that cholera toxin beta subunit (CT beta), which binds to the ganglioside GM1, induces a twofold increase in the number of DEV cells differentiating towards a neuronal pathway, as shown by the increased proportion and labelling intensity of cells stained by an anti-neurofilament antibody . Immunocytochemistry shows that after 3 days in culture with CT beta, DEV cells develop processes which stain positive for neurofilaments and MAP-1 . This suggests that CT beta induces DEV cells to express a more neuronal phenotype.

Vaccine, 1994 Oct, 12(13), 1238 - 40
Effects of cholera toxin adjuvant on IgE antibody response to orally or nasally administered ovalbumin; Tamura S et al.; Effects of cholera toxin B subunits supplemented with 0.1% cholera toxin (CTB*) on systemic IgE antibody responses to ovalbumin (OVA) were examined in BDFI (H-2b/d), Balb/c (H-2d) and C3H (H-2k) mice given OVA intragastrically or intranasally . Two successive doses of OVA intragastrically administered to Balb/c and C3H mice induced little IgE response and resulted in almost complete unresponsiveness to subsequent intraperitoneal challenge with OVA in Al(OH)3, while the intragastric administration to BDF1 mice induced high IgE response and resulted in abrogation of the unresponsiveness to the subsequent challenge . The intranasal administration of OVA induced little IgE response and suppressed response to the subsequent challenge in any strain of mice . On the other hand, two successive doses of intragastric or intranasal OVA together with CTB* enhanced IgE response in all three strains and the subsequent challenge with OVA in Al(OH)3 induced higher IgE responses . These results suggest that CTB* augments IgE response to OVA and abrogates the unresponsiveness when administered orally or intranasally into mice together with OVA, regardless of the H-2 haplotype of the mice.

Scand J Gastroenterol, 1994 Oct, 29(10), 908 - 15
Ketanserin and granisetron reduce cholera toxin-induced hypersecretion in pig jejunum; Hansen MB et al.; BACKGROUND: Serotonin antagonists have been proven antisecretory in cholera toxin (CT)-induced hypersecretion in the small intestine of rodents . The pig small intestine is a good model for the human small intestine with regard to physiologic and pharmacologic processes . METHODS: The antisecretory effect of intraluminally administered methysergide, renzapride, ketanserin, granisetron, and tropisetron on CT-induced hypersecretion was tested in isolated pig jejunal loops in vivo . RESULTS: Methysergide, ketanserin, and granisetron reduced the hypersecretory effect of CT maximally by 25%, 80%, and 50%, respectively . Tropisetron enhanced whereas renzapride did not alter the CT response . Combination of ketanserin and granisetron gave a maximal inhibitory effect of about 85% . Surprisingly, renzapride, granisetron, and tropisetron each induced hypersecretion . Taking into account the hypersecretory effect of the antagonists, they all reduced this CT-elicited hypersecretion . CONCLUSIONS: Results suggest involvement of the 5-hydroxytryptamine-2 and 5-hydroxytryptamine-3 receptor subtypes as mediators in CT-induced hypersecretion in pig jejunum, and antidiarrheal therapeutic potentials of ketanserin and granisetron.

J Clin Microbiol, 1994 Oct, 32(10), 2600 - 2
Reverse transcriptase-PCR assay for detection of hog cholera virus; Harding M et al.; A reverse transcriptase-PCR strategy was developed for the detection of hog cholera virus . Hog cholera virus template was amplified from tissue culture fluids and from tissues and blood of infected pigs, but not from samples containing other pestiviruses . Restriction endonuclease analysis identified samples as historic or recent isolates.

Rev Saude Publica, 1994 Oct, 28(5), 332 - 6
{Cholera epidemiology in Mozambique: 1973-1992}; Aragon M et al.; The results of an epidemiological analysis of cholera in Mozambique from 1973 to 1992 are described . The project sought to assess the influence of socio-economic and ecological factors the spread of cholera in a country at war . Information about the incidence of cholera and the fatality rate were related to the rainfall and the annual average growth rate of the population in the main cities . Water supply, sanitation and food hygiene were also studied . The high annual average growth rate of the population was found to have a direct linear correlation to the incidence of cholera . The drought of 1991-1992 also played an important role in the increased number of cases of the disease . Cholera has presented an endemic-epidemic pattern determined by: a) the uncontrolled growth of urban population, b) the deterioration of sanitation in urban centers, c) the unhygienic commercialization of food and d) the drought.

Int Arch Allergy Immunol, 1994 Oct, 105(2), 195 - 7
Effects of B subunit of cholera toxin on histamine release from rat peritoneal mast cells; Sugimoto K et al.; We studied the effects of B subunit of cholera toxin (CTB) on secretagogue-induced histamine release from rat peritoneal mast cells . CTB inhibited the release of histamine in response to compound 48/80, but not in response to mastoparan, substance P, concanavalin A or ionophore A23187 . The results suggest that CTB can be used as a tool for studying the cellular events involved in histamine release from mast cells.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9655 - 8
Role of platelet activating factor in the intestinal epithelial secretory and Chinese hamster ovary cell cytoskeletal responses to cholera toxin; Guerrant RL et al.; With the recent heightened concern about cholera around the world come new questions about the mechanism by which cholera toxin causes diarrhea . Peterson and Ochoa have suggested that prostaglandin synthesis is key to both the intestinal epithelial secretory and the CHO cell responses to cholera toxin {Peterson, J . W . and Ochoa, G . (1989) Science 245, 857-859} . Because platelet activating factor (PAF) can be a potent stimulus for prostaglandin synthesis, we examined its role in the intestinal and tissue culture effects of cholera toxin . We report that the specific PAF receptor antagonists BN 52021 and SR 27417 inhibit the effects of cholera toxin on intestinal secretion in rabbit ileal loops in vivo and on the cytoskeleton of Chinese hamster ovary cells in vitro . We also show that PAF itself can cause net fluid secretion in the rabbit model and that PAF potentiates the effects of cholera toxin on intestinal secretion . Finally, we demonstrate that cholera toxin stimulates significant PAF production (2.6-fold) in isolated T-84 intestinal epithelial cells . We conclude that cholera toxin stimulates PAF production and that PAF is involved in both the secretory and cytoskeletal responses to cholera toxin . These findings further support the involvement of additional mediators of cholera toxin effects other than mucosal cell cyclic AMP and help explain the effects of cholera toxin on prostaglandin synthesis.

Biochim Biophys Acta, 1994 Sep 8, 1223(2), 296 - 305
Interaction of a cholera toxin derivative containing a reduced number of receptor binding sites with intact cells in culture; De Wolf MJ et al.; Hybrid CTB (hCTB), having only one or two functional binding sites, has been constructed from two chemically inactivated derivatives of CTB . One inactive derivative consisted of CTB formylated in the lone Trp-88 of each beta-chain (fCTB), whereas the other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site (sssCTB) . hCTB, fCTB and sssCTB were able to reassociate with CTA and form the corresponding holotoxins hCT, fCT and sssCT as measured by gel filtration chromatography . In contrast to fCT and sssCT, hCT could increase the cAMP content of intact Vero cells in a time- and dose-dependent way: concentrations as low as a few nanograms of hCT per milliliter caused a significant increase in the intracellular cAMP level . The maximal cAMP level induced by hCT (1 microgram/ml) was, however, more than 2-fold lower than that elicited by its native counterpart . At saturating ligand concentrations and at 37 degrees C, the lag periods and rates of CT and hCT induced cAMP accumulation were essentially the same . Treatment of Vero and HeLa cells with GM1 did not affect their difference in response to CT and hCT . When Vero cells treated with hCT were incubated for longer periods of time, a further slow accumulation of cAMP occurred until after about 20 h cAMP levels of cells exposed to CT or hCT were essentially the same . In contrast to Vero and HeLa cells, human skin fibroblasts exhibited an almost identical response to CT as well as to hCT . Acidotropic agents such as chloroquine and monensin affected the CT and hCT induced increase in cAMP content of Vero cells, fibroblasts and GM1 treated Hela cells in a similar way . The results are consistent with the view that CT receptor recognition domains are shared between adjacent beta-chains, that pentavalent binding appears not to be essential for cytotoxicity and that in the cell types studied intracellular processing of CT, hCT is involved.

Biochim Biophys Acta, 1994 Sep 8, 1223(2), 285 - 95
Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives; De Wolf MJ et al.; In order to test the hypothesis that binding sites of cholera toxin for its receptor, the monosialoganglioside GM1, are shared between adjacent beta-polypeptide chains, two inactive chemical derivatives of the B subunit of cholera toxin (CTB) were prepared and were subsequently used for the construction of hybrid CTB pentamers . One inactive derivative consisted of CTB specifically modified in the single essential Trp-88 residue of each beta-chain . This residue was modified by formylation, a treatment preserving the structural integrity of CTB . The other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site . Using {1,4-14C}succinic anhydride for the site-specific succinylation and analysis of radiolabeled tryptic fragments of S-carboxymethylated {14C}sssCTB revealed that the amino groups specifically modified were the alpha-amino group of Thr-1 and the epsilon-amino groups of respectively Lys-34 and Lys-91 . Upon submitting equal amounts of formylated CTB and site-specific succinylated CTB to a denaturation-renaturation cycle, hybrid pentamers were formed which in contrast to the parental compounds were able to bind GM1 . The affinity of hybrid CTB for GM1, as estimated by a competitive solid-phase radiobinding assay was unexpectedly high and only 2.5-fold lower than that of its native counterpart . The number of active binding sites on hybrid CTB was determined from: (i) titration with the oligosaccharide moiety of GM1 (oligo-GM1) and monitoring the reversal of the Trp fluorescence quenching by iodide ions and (ii) rapid gel filtration over a superdex HR column of a mixture of hybrid CTB and an excess of 3H-labeled oligo-GM1 . The data are in agreement with the formation of one active binding per four reconstituted binding sites in hybrid CTB, which is consistent with a random association of CTB monomers during the denaturation-renaturation cycle.

J Am Vet Med Assoc, 1994 Sep 1, 205(5), 742 - 5
Detection of a cell line contaminated with hog cholera virus; Bolin SR et al.; Cell lines from the repository of the American Type Culture Collection were examined for possible contamination with bovine viral diarrhea virus . During testing, hog cholera virus (HCV) was detected in the IB-RS-2 D10 porcine kidney cell line . This variant of HCV was avirulent for pigs and seldom induced detectable concentrations of antibody against reference viruses (HCV-Ames or bovine viral diarrhea virus-NY1) in serum of inoculated pigs . Additionally, this variant of HCV did not confer protection to pigs against virulent HCV . The contaminated cell line had been distributed to > 20 laboratories in the United States . The cell line was not used in field studies and has been destroyed.

Neuron, 1994 Sep, 13(3), 657 - 69
VIP inhibits N-type Ca2+ channels of sympathetic neurons via a pertussis toxin-insensitive but cholera toxin-sensitive pathway; Zhu Y et al.; The best characterized Ca2+ channel modulation in mammalian sympathetic neurons is an inhibition of N-type channels via a pertussis toxin (PTX)-sensitive heterotrimeric G protein . Here, we show that vasoactive intestinal polypeptide (VIP), an abundant neuropeptide in the PNS and CNS, inhibited N-type Ca2+ channels in rat sympathetic neurons in a voltage-dependent, membrane-delimited manner . The effect of VIP was insensitive to PTX but was attenuated by cholera toxin or anti-Gs alpha antibodies . VIP-mediated inhibition was independent of cAMP-dependent protein kinase A (PKA) . The results provide evidence for a new signal transduction pathway in which N-type Ca2+ channel modulation requires activation of Gs alpha but is independent of PKA-mediated phosphorylation.

Mol Cell Biochem, 1994 Sep, 138(1-2), 157 - 66
ADP-ribosylation factors: a family of approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin; Welsh CF et al.; ADP-ribosylation factors (ARFs) comprise a family of approximately 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of Gs alpha, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin . ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis . Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure . ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species . They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.

Microvasc Res, 1994 Sep, 48(2), 212 - 35
Mechanisms of cholera toxin prevention of thrombin- and PMA-induced endothelial cell barrier dysfunction; Patterson CE et al.; Thrombin-induced endothelial cell (EC) activation leads to compromise of monolayer barrier function due to cellular retraction/contraction and intercellular gap formation . Cyclic AMP induces relaxation in other contractile cells and promotes barrier function in EC . To investigate mechanisms involved in cAMP protection in thrombin-induced permeability, we pretreated bovine pulmonary arterial EC monolayers with 1 microgram/ml cholera holotoxin which catalyzed ADP ribosylation of Gs and increased synthesis of cAMP . The holotoxin, but not the binding subunit, reduced basal permeability and prevented gap formation and permeability following challenge with 1 microM thrombin, 100 microM thrombin receptor-activating peptide, or 1 microM phorbol myristate acetate (PMA) . Furthermore, thrombin-induced gap formation and permeability were reversed by cholera toxin post-treatment . Pretreatment with 5 microM forskolin or 1 mM dibutyryl cAMP, with or without 1 mM isobutyl methylxanthine, but not cGMP analogs, protected against thrombin-induced EC permeability, mimicking the cholera toxin effect . Although downregulation of protein kinase C attenuated both thrombin- and PMA-induced permeability, cholera toxin did not alter either PMA-induced protein kinase C activation or thrombin-induced Ca2+ mobilization . In contrast, cholera toxin attenuated thrombin-induced myosin light chain phosphorylation and largely prevented actin redistribution . These studies suggest that cholera toxin: (1) protects endothelial barrier function and reverses established dysfunction via increased cAMP (2) does not alter thrombin receptor interaction or early signal events such as Ca2+ mobilization and PKC activation, (3) attenuates myosin light chain kinase activation and actomyosin contractile interaction subsequent to thrombin activation, and (4) abrogates contractile processes subsequent to PKC activation, which is also an important mechanism in thrombin-induced permeability but is independent of myosin light chain kinase activation.

Infect Immun, 1994 Aug, 62(8), 3594 - 7
Successful immunization against gastric infection with Helicobacter species: use of a cholera toxin B-subunit-whole-cell vaccine; Lee A et al.; In previous studies we found that immunizing mice with a sonicate of Helicobacter felis and adjuvant cholera toxin (CT; 10 micrograms) protected the animals against challenge with viable H . felis . The aim of this study was to determine whether a low dose of CT or its nontoxic B subunit (CTB) was effective as an adjuvant in Helicobacter oral vaccines . Significant protection against viable H . felis challenge was achieved in the animals immunized with H . felis antigen plus the combination of 0.5 microgram of CT and 10 micrograms of CTB (96%), with H . felis antigen plus 0.5 microgram of CT (95%), and with H . felis antigen plus 10 micrograms of CTB (83%) . No protective effect was found in the mice immunized with either H . felis antigen alone or adjuvant CTB and CT alone . Twenty-six percent of mice immunized with Helicobacter pylori antigen plus CT (10 micrograms) were protected against H . felis challenge, confirming the value of the model in predicting effects of immunization in humans . The observation that immunity can be induced with a nontoxic adjuvant CTB opens the way for human studies with H . pylori vaccines and is a further step along the road to effective strategies of prevention of gastroduodenal diseases of major world significance.

Mol Microbiol, 1994 Aug, 13(4), 745 - 53
Galactose-binding site in Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT); Merritt EA et al.; The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coli is an attractive target for the rational design of potential anti-cholera drugs . In this paper we analyse the molecular structure of this binding site as seen in several crystal structures, including that of an LT:galactose complex which we report here at 2.2 A resolution . The binding surface on the free toxin contains several tightly associated water molecules and a relatively flexible loop consisting of residues 51-60 of the B subunit . During receptor binding this loop becomes tightly ordered by forming hydrogen bonds jointly to the GM1 pentasaccharide and to a set of water molecules which stabilize the toxin:receptor complex.

J Immunol, 1994 Jul 15, 153(2), 647 - 57
Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin; Snider DP et al.; Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags . We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX . The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA . However, when given a subsequent i.p . challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death . Within 10 min of i.p . challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues . These mice had detectable serum IgE Ab . Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice . Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis . Ag given s.c . without adjuvant could also sensitize for systemic and intestinal anaphylaxis . Sensitization with HEL given s.c . was dose dependent and correlated with a critical amount of HEL in the circulation . HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL . Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis . These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.

Ginecol Obstet Mex, 1994 Jul, 62, 178 - 81
{Complications and treatment of cholera during pregnancy}; Figueroa Damian R et al.; Since 1961 cholera has spread in many countries reaching a pandemic form . Since 1991 Mexico has been involved in this pandemia . Near 20% of all cases of cholera in our country happen in fertile women, so the possibility of the association between cholera and pregnancy is high . We present the case of a pregnant woman, who during her third trimester presented a episode of cholera, developing premature labor . Furthermore is revised the medical literature about the general principles of the management of cholera, and the association between pregnancy and the intestinal infection.

J Med Microbiol, 1994 Jul, 41(1), 3 - 9
Interactions of intestinal mediators in the mode of action of cholera toxin; Peterson JW et al.; Cholera toxin (CT) and prostaglandin E2 (PGE2) increased the synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) in rabbit intestinal mucosa, which appeared to be responsible for inducing the release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells into the intestinal lumen . With isolated intestinal cells, CT induced the synthesis of PGE2 more efficiently from epithelial cells than from lamina propria cells; however, the basal amount of this eicosanoid produced by lamina propria cells was approximately six-fold more than that formed by the epithelial cells . The CT-induced stimulation of arachidonate metabolism appeared to be generalised in nature, as PGF2 alpha and leukotrienes were synthesised in addition to PGE2 . Injection of dibutyryl cAMP into the intestinal lumen in vivo markedly reduced both basal levels of PGE2, as well as CT-induced levels of PGE2, released into the luminal fluid . Similarly, when biopsy samples of tissue from rabbit intestinal loops, challenged in vivo with dibutyryl cAMP, were washed and incubated in vitro, the amount of PGE2 synthesis remained below basal levels . In contrast, when biopsy samples of normal small intestinal tissue were exposed in vitro to dibutyryl cAMP, PGE2 synthesis increased . Thus, cAMP appeared to down-regulate the levels of intestinal eicosanoids in vivo, despite its innate capacity to evoke PGE2 synthesis from mucosal tissue in vitro . Thus, the data indicate that CT-induced mediators exhibit interactive effects that alter their cellular concentrations, that in turn could affect the biological responses.

Cell Immunol, 1994 Jul, 156(2), 402 - 13
Unique T cell differentiation markers: gangliosides with cholera toxin receptor activity on murine fetal thymocytes; Noguchi M et al.; Cholera toxin B subunit receptors (CTBR; gangliosides GM1a and GM1b-GalNAc-Gal) and GM1b-type gangliosides were examined during T cell development in BALB/c mice by FACS or TLC immunostaining . Sixty-two percent of CD4-8- early fetal thymocytes express CTBR {58% low affinity (GM1a+), 4% high affinity (GM1a2+)} at 13 days gestation (Day 13); GM1a2+ was expressed preferentially on Thy-1+ fetal thymocytes after Day 13 . GM1a2+ Thy-1+ cells increased from 4% on Day 13 to 95% on Day 17 . Surface GM1a2+ decreased beginning on Day 15 while GM1b-GalNAc-Gal (and CD4 and CD8) increased . GM1b-type+ cells increased from 8% on Day 13 to 51% on Day 17 and then decreased to 16% at 4 weeks; few asialo GM1+ cells (< 5%) are seen during gestation . Thus, synthesis of GM1a- and GM1b-type series was active until Days 15 and 17, respectively, and then was suppressed; GM1b-GalNAc-Gal was synthesized from GM1b following Gestational Day 15 . GM1a- and GM1b-type identify immature T cells in mice, while GM1b-GalNAc-Gal identifies mature T cells.

Lijec Vjesn, 1994 Jul-Aug, 116(7-8), 169 - 74
{Cholera threatens again}; Lukas D et al.; The etiology, epidemiology, pathogenesis, clinical manifestations and treatment procedures of cholera with the point to new experience are presented . This work was motivated by the intensifying interest for this old disease which has reappeared in Europe in epidemic form.

Cell Signal, 1994 Jul, 6(5), 487 - 92
Altered guanine nucleoside triphosphate binding to transducin by cholera toxin-catalysed ADP-ribosylation; Wieland C et al.; The influence of cholera toxin (CTX)-catalysed ADP-ribosylation on binding of guanine nucleoside triphosphates to transducin was studied by measuring the binding of the GTP analogue, guanosine 5'-{gamma-thio}triphosphate (GTP{gamma S}), to illuminated bovine rod outer segment (ROS) membranes treated with or without CTX . Besides the well-documented inhibition of the transducin GTPase activity, CTX treatment inhibited binding of GTP{gamma S} to illuminated ROS membranes . This inhibition was due to an approximately two-fold lower apparent affinity for the nucleotide, while the density of binding sites was not altered . CTX decreased the association rate of GTP{gamma S} by a factor of about two . Competition experiments with GTP, guanosine 5'-{beta, gamma}iminotriphosphate or GDP showed that the apparent affinities for both guanine nucleoside triphosphates, but not for GDP, were lowered by about two-fold upon CTX treatment . In contrast to CTX, pertussis toxin treatment of ROS membranes reduced the density of binding sites available to GTP{gamma S}, while the apparent affinity of the remaining sites was unchanged . It is concluded that ADP-ribosylation of transducin by CTX not only inhibits its GTPase activity but also decreases the affinity for guanine nucleoside triphosphates, data which suggest that the arginine moiety modified by CTX is involved in both binding and hydrolysis of GTP.

Int J Immunopharmacol, 1994 Jul, 16(7), 547 - 60
Myeloid cell proliferation stimulated by Steel factor is pertussis toxin sensitive and enhanced by cholera toxin; Hendrie PC et al.; Effects of G-protein toxins on Steel factor (SLF) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulated proliferation of human factor-dependent cell line, M07e, were evaluated . Pertussis toxin pretreatment suppressed GM-CSF- or Steel factor-induced proliferation by 54 +/- 8%; however, proliferation induced by the combination of GM-CSF plus Steel factor was suppressed to a much lesser extent (14 +/- 8%) . Pretreatment of M07e cells with cholera toxin, suppressed GM-CSF- and GM-CSF plus Steel factor-stimulated proliferation by 57 +/- 6% and 79%, respectively, but increased the proliferative response to Steel factor alone by twofold . Similar effects of pertussis toxin and cholera toxin were observed on proliferation of normal myeloid progenitor cells from human umbilical cord blood . Pertussis toxin treatment of M07e cells for 4 h resulted in the ADP-ribosylation of 40-42 kDa protein band but did not significantly increase cyclic AMP levels . Cholera toxin pretreatment was associated with a 10-fold increase in intracellular cyclic AMP levels . These results implicate pertussis toxin sensitive pathways for both GM-CSF and Steel factor, but suggest that these pathways may not be required for synergistic proliferation stimulated by the combination . In addition, proliferation stimulated by GM-CSF, +/- Steel factor, is sensitive to cholera toxin pretreatment; whereas cholera toxin pretreatment enhanced proliferation stimulated by Steel factor, possibly via increased cyclic AMP . This suggests divergent signal transduction pathways for the two cytokines.

J Histochem Cytochem, 1994 Jun, 42(6), 705 - 16
Identification and localization of the GM1 ganglioside in the cochlea using thin-layer chromatography and cholera toxin; Santi PA et al.; Using high-performance thin-layer chromatography, we identified GM1, GM3, GD3, GD1a, GT1b, GQ1b, and other gangliosides in chinchilla cochlea and cerebellum . GM1 was also identified on chromatograms with the B-subunit of cholera toxin (BCT) . BCT was also used to determine the distribution of GM1 in fixed and unfixed tissues from cochlea, cerebellum, and sciatic nerve . Positive control tissues showed expected labeling of GM1 by BCT . Negative controls showed expected suppression of BCT binding to GM1 after GM1 extraction and GM1 absorption . In the cochlea, GM1 appeared abundant in plasma membranes of most epithelial cells lining the endolymphatic surface of the scala media, including the interdental, inner supporting, pillar, Deiters, Hensen, Claudius, Boettcher, spiral prominence, and external sulcus . GM1 appeared less abundant in cells of the stria vascularis, Reissner's membrane, and in nerve fibers . In hair cells, the stereocilia appeared to contain GM1; however, the endolymphatic surface of the cuticular plate and the body of the outer hair cells appeared to contain little GM1 . In addition, the tectorial membrane, connective tissue of the spiral limbus, and amorphous layer of the basilar membrane also appeared to contain little GM1 . Enzymatic degradation of glycoproteins and transformation of polysialogangliosides to GM1 increased the reactivity of BCT to cochlear GM1 . This further supported the presence of GM1 and other gangliosides in the cochlea . Although the functional significance of GM1 and other gangliosides in the cochlea is not yet known, they are likely to play important roles in membrane function.

Vaccine, 1994 Jun, 12(8), 731 - 5
Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit; Oien NL et al.; Local IgA and IgG antibodies against respiratory syncytial virus (RSV) were induced in the respiratory tract of mice following intranasal vaccination with an RSV chimeric FG glycoprotein and cholera toxin B (CTB) as a mucosal adjuvant . Local antibody production was not induced following parenteral immunization with FG administered in alum adjuvant . While both vaccination protocols induced serum antibodies against RSV and protected the lower respiratory tract from RSV infection, only intranasal FG/CTB afforded protection of the upper respiratory tract . These data suggest that vaccination via the mucosal route may be superior to vaccination by a parental route in providing complete protection against RSV.

AIDS, 1994 Jun, 8(6), 779 - 85
Immune response following oral administration of cholera toxin B subunit to HIV-1-infected UK and Kenyan subjects; Lewis DJ et al.; OBJECTIVE: To determine the effect of HIV-1 infection on immunoglobulin (Ig) G and IgA antibody response and circulating antibody forming cell response to oral immunization with the B subunit of cholera toxin . DESIGN: Healthy UK volunteers, and HIV-1-positive UK and Kenyan volunteers at different clinical stages of HIV-1 infection received two oral immunizations . CD4+ T cells, serum beta 2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response . METHODS: Serum antitoxin IgG and IgA measured by enzyme-linked immunosorbent assay and antitoxin IgG, IgA and IgM antibody-forming cells detected by enzyme-linked immunospot assay at different times after two oral immunizations . RESULTS: UK HIV-positive volunteers (mean CD4+ T cell count, 52 x 10(6)/l) responded poorly to primary and booster immunization . HIV-infected Kenyans (752 x 10(6)/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 x 10(6)/l had an insignificant primary, but significant booster response . Two oral immunizations induced antibody responses in HIV-positive Kenyan groups (who may have prior immunity from exposure to environmental bacterial toxins) of similar or greater magnitude to healthy UK volunteers . CONCLUSIONS: Mucosal immunization may recall immune memory and be of benefit in early and moderately advanced clinical HIV disease . The findings have important clinical implications in that mucosally targeted vaccines are potentially useful in this group of patients.

APMIS, 1994 Jun, 102(6), 465 - 73
Antisecretory factor enhances in vivo internalization of cholera toxin and of horseradish peroxidase into rat enterocytes; Lange S et al.; The in vivo effect of antisecretory factor (ASF, derived from pig plasma) on the ability of cholera toxin (CT) and of horseradish peroxidase (HRP) to bind to and penetrate into epithelial cells of the rat small intestine was evaluated in the absence of anesthetics . The potency of intravenously administrated ASF was demonstrated by some 70% inhibition of CT-induced secretion in ligated small intestinal loops . Using immunohistochemical methods for visualization, we found ASF to enhance internalization of both CT and HRP after 30 to 60 min of challenge, without interfering with the initial binding to the enterocyte brush border region . The internalization process started in the upper 2/3 of the villus region . After 5 h, no CT or HRP could be seen bound to the enterocytes . The results suggest that ASF might enhance small intestinal absorption.

Mol Pharmacol, 1994 Jun, 45(6), 1160 - 7
Positive modulation of intracellular Ca2+ levels by adenosine A2b receptors, prostacyclin, and prostaglandin E1 via a cholera toxin-sensitive mechanism in human erythroleukemia cells; Feoktistov I et al.; Human erythroleukemia (HEL) cells express megakaryocyte/platelet membrane markers and thus have been used as a model for studying platelet membrane receptors and their coupling to cell signaling pathways . Our previous studies, however, indicated that platelets and HEL cells possess different subtypes of adenosine A2 receptors . Furthermore, we now report that, whereas adenosine inhibits intracellular Ca2+ increases in platelets, it potentiates the rise in intracellular Ca2+ produced by thrombin, prostaglandin E1, thapsigargin, and the calcium ionophore A23187 in HEL cells . Stable adenosine analogs potentiated intracellular Ca2+ increases with a rank order of potencies of 5'-N-ethylcarboxamidoadenosine (NECA) > (R)-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) >> CGS 21680, suggesting that this effect is mediated by A2b receptors . EC50 values for NECA and R-PIA were 0.8 and 42 microM, respectively . NECA (100 microM) potentiated by 2-3-fold the increase in intracellular Ca2+ produced by 0.3 unit/ml thrombin . This effect was mimicked by cholera toxin and was shared by other Gs-coupled receptors, such as those activated by the prostacyclin analog iloprost and prostaglandin E1, indicating the involvement of Gs proteins . Adenosine analogs also increased intracellular cAMP with the same rank order of potencies . The membrane-permeable analog 8-bromo-cAMP, however, had no effect on intracellular Ca2+ levels, indicating that the potentiation of intracellular Ca2+ increases and the activation of adenylate cyclase are parallel but independent events . The increase in intracellular Ca2+ produced by adenosine is due not to an increase in phosphoinositide hydrolysis but, rather, to an increase in calcium influx, and it is lost if cells are studied in the absence of extracellular Ca2+ . We conclude, therefore, that adenosine A2b receptors in HEL cells are coupled to Gs proteins and their activation leads to stimulation of adenylate cyclase and, independently, to potentiation of the rise in intracellular Ca2+ . We speculate that A2b receptors in HEL cells activate a calcium channel through a cholera toxin-sensitive mechanism that requires an initial increase in intracellular Ca2+.

J Neurosci Methods, 1994 Jun, 52(2), 143 - 8
Differential labeling of converging afferent pathways using biotinylated dextran amine and cholera toxin subunit B; Alisky JM et al.; We report a new technique for 2-tracer anterograde labeling that permits unequivocal identification of the differentially labeled projections in the same section . One pathway is labeled with biotinylated dextran amine and is visualized as a black to dark gray diaminobenzidine (DAB)-cobalt precipitate by an avidin-biotinylated peroxidase reaction . The other pathway is labeled with cholera toxin subunit B and is visualized as a reddish-brown reaction product using DAB without cobalt as the substrate for peroxidase immunohistochemistry . To maintain serial order, sections can be processed mounted on slides without any loss of sensitivity for either tracer.

J Virol, 1994 Jun, 68(6), 3934 - 42
Antigenic structure of envelope glycoprotein E1 of hog cholera virus; van Rijn PA et al.; Envelope glycoprotein E1 (gp51 to gp54) is the most antigenic protein of hog cholera virus or classical swine fever virus (CSFV) . Four antigenic domains, A to D, have been mapped on E1 with a panel of monoclonal antibodies (MAbs) raised against CSFV strain Brescia . The boundaries of these domains have been established by extensive studies on binding of MAbs to transiently expressed deletion mutants of E1 (P . A . van Rijn, E . J . de Meijer, H . G . P . van Gennip, and R . J . M . Moormann, J . Gen . Virol . 74:2053-2060, 1993) . In this study, we used neutralizing MAbs of domains A, B, and C to isolate MAb-resistant mutants (MAR mutants) of CSFV strain Brescia and Chinese vaccine strain ("C") . The E1 genes of MAR mutants were cloned in a eukaryotic expression vector, and the effects of MAR mutations on epitopes were studied with a panel of 19 MAbs by immunostaining of COS1 cells transiently expressing these mutant E1s . Except for the MAR mutation Cys-->Arg at position 792, which abolished binding of all MAbs of domains A and D, amino acid substitutions affected only MAbs belonging to the same domain as the MAb used to select the MAR mutant . However, a MAR mutation in a particular domain did not per se abolish binding of all MAbs recognizing that domain . Furthermore, MAR mutants possessed conservative as well as nonconservative amino acid substitutions . To investigate the significance of a secondary structure for the binding of MAbs, all cysteine residues in the N-terminal antigenic part of E1 were mutated to serine . We found that the cysteines at positions 693 and 737 were essential for binding by MAbs of domains B and C, whereas those at positions 792, 818, 828, and 856 appeared to be essential for the binding of most MAbs of domains A and D . These results fully comply with the previously proposed two-unit structure of the N-terminal half of E1 . One unit consists of antigenic domains B and C, whereas the other unit consists of the highly conserved domain A and domain D . We conclude that the first six cysteines are critical for the correct folding of E1 . A model of the antigenic structure of E1 is presented and discussed.

Arch Biochem Biophys, 1994 May 1, 310(2), 481 - 8
Effect of cholera toxin and pertussis toxin on prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinase production by human monocytes; Corcoran ML et al.; Activation of human monocytes induces the production of matrix metalloproteinases (MMPs) through a prostaglandin E2 (PGE2)-cAMP-dependent pathway . Since G-proteins have been documented to modulate adenylyl cyclase, we examined the effect of G-protein ADP-ribosylating agents, cholera toxin (CT) and pertussis toxin (PT), on the signal transduction pathway that culminates in the production of monocyte MMPs . Although CT elevated cAMP levels in both unstimulated and concanavalin A (Con A)-stimulated monocytes, it enhanced the production of prostaglandin H synthase-2 (PGH synthase-2, PGHS-2) protein, prostaglandins, interstitial collagenase, and 92-kDa type IV collagenase/gelatinase only in Con A-stimulated monocytes . Additionally, the indomethacin-mediated suppression of Con A-induced monocyte interstitial collagenase and 92-kDa type IV collagenase/gelatinase production could be reversed by CT . In contrast to the actions of CT, PT treatment suppressed the levels of cAMP, PGHS-2, PGE2, interstitial and 92-kDa type IV collagenase/gelatinase in Con A-stimulated monocytes . The regulation of MMP production by these toxins appears to be mediated primarily through their effect on adenylyl cyclase since the release of arachidonic acid was relatively unaffected by these agents . These findings provide evidence that G-proteins may be involved in either the enhancement or suppression of the eicosanoid-cAMP-dependent signal transduction pathway that results in the production of monocyte MMPs.

J Neurosci Res, 1994 May 1, 38(1), 81 - 90
Study of the phorbol ester effect on Alzheimer amyloid precursor processing: sequence requirements and involvement of a cholera toxin sensitive protein; Efthimiopoulos S et al.; Phorbol esters (PDBu) stimulate alpha-secretase cleavage and secretion of the Alzheimer amyloid precursor protein (APP) . To determine whether any cytoplasmic residues or sequence motifs mediate the PDBu effect on APP processing, this region of APP was altered by point mutations or deletions . To differentiate the mutated APP from the endogenous APP, the APP751 ectodomain between amino acids 1 and 647 was replaced by a human secreted alkaline phosphatase derivative (SEAP) . The resultant fusion protein (SEAP-APP751) was cleaved by alpha-secretase at the same site as full-length APP, and its secretion was stimulated by PDBu at a level similar to APP751 . However, PDBu-stimulated secretion of the SEAP-APP751 fusion protein reached its maximum level after 30 min of treatment, while secretion of APP751 reached its maximum after 60 min, suggesting that the APP ectodomain affects the kinetics of APP secretion . Mutation of the cytoplasmic serines to alanines had no effect on the PDBu-stimulated secretion of the SEAP-APP, indicating that protein kinase C (PKC) phosphorylation of the cytoplasmic domain of APP is not important for stimulation of APP secretion . Similarly, deletion of the cytoplasmic domain between amino acids 719 and 751 had no effect on the PDBu-stimulated secretion . However, deletion of amino acids 707-751 resulted in a significant increase in the secretory cleavage of the SEAP-APP707 delta C construct, suggesting that the sequence 707-719 is important for the regulated secretion of APP . Cholera toxin, but not pertussis toxin, reduced the PDBu-induced secretion of APP by more than two-fold, suggesting that the PDBu response may be modulated by a cholera toxin sensitive heterotrimeric G-protein.

Vis Neurosci, 1994 May-Jun, 11(3), 441 - 6
Cholera toxin mapping of retinal projections in pigeons (Columbia livia), with emphasis on retinohypothalamic connections; Shimizu T et al.; Anterograde transport of cholera toxin subunit B (CTb) was used to study the retinal projections in birds, with an emphasis on retinohypothalamic connections . Pigeons (Columbia livia) were deeply anesthetized and received unilateral intraocular injections of CTb . In addition to known contralateral retinorecipient regions, CTb-immunoreactive fibers and presumptive terminals were found in several ipsilateral regions, such as the nucleus of the basal optic root, ventral lateral geniculate nucleus, intergeniculate leaflet, nucleus lateralis anterior, area pretectalis, and nucleus pretectalis diffusus . In the hypothalamus, CTb-immunoreactive fibers were observed in at least two contralateral cell groups, a medial hypothalamic retinorecipient nucleus, and a lateral hypothalamic retinorecipient nucleus . To compare retinorecipient hypothalamic nuclei in pigeons with the mammalian suprachiasmatic nucleus, double-label experiments were conducted to study the existence of neurophysin-like immunoreactivity in the retinorecipient avian hypothalamus . The results showed that only cell bodies in the medial hypothalamic nucleus contained neurophysin-like immunoreactivity . The results demonstrate CTb to be a sensitive anterograde tracer and provide further anatomical information on the avian equivalent of the mammalian suprachiasmatic nucleus.

Vaccine, 1994 May, 12(6), 521 - 6
Conversion of orally induced suppression of the mucosal immune response to ovalbumin into stimulation by conjugating ovalbumin to cholera toxin or its B subunit; Stok W et al.; Oral pretreatment of mice with ovalbumin (OVA) not only suppressed a subsequently induced systemic immune response ('oral tolerance') but also suppressed, even more effectively, a subsequently induced intestinal IgA response . In contrast, pretreatment with OVA conjugated to cholera toxin (CT) or its B subunit (CTB) resulted in a stimulative effect . The stimulative effect was enhanced when unconjugated OVA and polymerized OVA were removed from the OVA-CT (B) conjugate mixtures by affinity chromatography . Thus, the effect of oral pretreatment depends on the balance between tolerizing and stimulating components in the conjugate mixture . As OVA-CTB conjugates were at least as effective as OVA-CT conjugates in stimulation of the intestinal immune response, we concluded that the ability of the OVA conjugates to bind to the intestinal mucosa is a prerequisite in inducing the stimulative effect . These observations further demonstrate that conjugation of a protein antigen to an appropriate carrier can convert the nature of the immunization from suppressive into stimulative.

Soc Sci Med, 1994 May, 38(9), 1171 - 91
Cholera diffusion in Russia, 1823-1923; Patterson KD; All six cholera pandemics of the 19th and early 20th centuries struck Russia, causing millions of deaths . Cholera entered Russia from the south, with the Volga river system being a common and efficient route into the heart of the country . Diffusion was predominantly linear, along the navigable rivers and later, along the railroads . In contrast to Pyle's findings for the U.S.A., urban hierarchical diffusion was of only local significance in Russia.

Lik Sprava, 1994 May-Jun, (5-6), 163 - 6
{Disordered water-mineral metabolism in cholera patients}; Nikitin EV et al.; Clinical analysis was carried out of El Tor cholera in 100 patients, plasma concentrations of electrolytes of potassium (K) and sodium (Na) were studied as were those of trace elements of iron (Fe), copper (Cu), magnesium (Mg) and manganese (Mn) in the whole blood before and after rehydration therapy and depending on the extent of dehydration . The results were as follows: the plasma concentration of K and Na was lowered, that of Mg in the whole blood increased in stage III as well as that of Mn in st . I and II of dehydration; the concentration of Fe, Cu was decreased in st . I, II, III, and of Mn in sit . III of dehydration . The activities of transferrin and ceruloplasmin were found to be on the decrease for the duration of the illness . Rehydration promoted the return to normal of water and electrolyte metabolism, haemodynamics, without affecting to any noticeable degree the trace element metabolism.

Dtsch Tierarztl Wochenschr, 1994 May, 101(5), 204 - 6
The influence of protease inhibitors of the organism, especially bovine aprotinin, on the production of virulent hog cholera virus in tissue cultures; Korn G et al.; The influence of biological protease inhibitors, especially aprotinin, on the production of virulent hog cholera virus in cell cultures . Production of number and size of fluorescent plaques after infection PK 15 cells with HC virus depended on properties of fetal calf sera added to the medium . By affinity chromatography on bovine alpha-chymotrypsin bound to CM-cellulose inhibitory proteins against chymotrypsin-like proteases could be eliminated from inhibiting sera . The fraction free from inhibitors did not inhibit plaque formation of HC virus in contrast to the fraction containing the eluted inhibitor(s) . The inhibitory properties of fetal calf sera could be measured by a plaque inhibition test . By this test no inhibitory component could be demonstrated in 30 individual serum samples from pigs which on subsequent infection with HC virus exhibited virus growth to high titres . The serum from one pig, however, was found to inhibit HC virus plaque formation in PK 15 cells . When infected, this animal reacted with an unexpected low virus replication and mild disease symptoms . The production of virulent HC virus could likewise be inhibited by addition to the medium of the bovine protease inhibitor aprotinin; the degree of inhibition being dependent on the concentration . Virulent virus was produced again after elimination of this inhibitor from the medium . A weak inhibitory effect on plaque formation was also achieved by addition of the protease inhibitor chymostatin (3 mg/5 mg) and human alpha 1x plasma inhibitor to the medium of infected cells . The inhibitory effect appears to be very specific.

J Comp Neurol, 1994 Apr 22, 342(4), 603 - 18
Afferents to the nucleus reticularis parvicellularis of the cat medulla oblongata: a tract-tracing study with cholera toxin B subunit; Fort P et al.; The aim of this study was to examine anatomical evidence in cats of whether the nucleus reticularis parvicellularis (Pc) is part of the circuit responsible for the inhibition of brainstem motoneurons during paradoxical sleep . For this purpose, we made iontophoretic injections of the retrograde and anterograde tracer cholera toxin B subunit (CTb) in the Pc . After CTb injections in the Pc, a large number of retrogradely labeled neurons were seen in the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the posterior hypothalamic areas, the mesencephalic reticular formation, the nucleus locus subcoeruleus, the nucleus pontis caudalis, other portions of the Pc, the nucleus reticularis dorsalis, the trigeminal sensory complex, and the nucleus of the solitary tract . We further found that the Pc receives 1) serotoninergic afferents from the raphe dorsalis, magnus, and obscurus nuclei; 2) noradrenergic inputs from the dorsolateral pontine tegmentum; 3) cholinergic afferents from the lateral medullary reticular formation; 4) substance P-like afferents from the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray, and nucleus of the solitary tract; and 5) methionine-enkephalin-like projections from the periaqueductal gray, the nucleus of the solitary tract, the lateral pontine and medullary reticular formation, and the spinal trigeminal nucleus . We further found that the Pc do not receive afferents from brainstem structures responsible for muscle atonia, such as the ventromedial medulla and the dorsomedial pontine tegmentum, and therefore may not be part of the circuit inhibiting the brainstem motoneurons during paradoxical sleep.

J Biol Chem, 1994 Apr 1, 269(13), 9743 - 5
Effect of ADP-ribosylation factor amino-terminal deletions on its GTP-dependent stimulation of cholera toxin activity; Hong JX et al.; It has been proposed that the amino-terminal domain of ADP-ribosylation factor (ARF) is critical for its stimulation of cholera toxin ADP-ribosyltransferase activity . In this study, recombinant ARF1 (rARF1), r delta 13ARF1 (recombinant ARF1 lacking the first 13 amino acids) and rPKA14ARF1 (recombinant ARF1 in which the first 14 amino acids were replaced by the first 7 amino acids of the cAMP-dependent protein kinase catalytic subunit) were used to assess the effect of the amino terminus on the ability of ARF to enhance ADP-ribosylation of agmatine by the cholera toxin A subunit . The GTP-dependent ARF activities of r delta 13ARF1 and rPKA14ARF1 were similar to that of rARF1, whereas the GTP requirement for half-maximal activation of cholera toxin A, was somewhat higher for rARF1 than it was for r delta 13ARF1 and rPKA14ARF1 . These results are consistent with the view that the amino terminus of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin.

Hear Res, 1994 Apr, 74(1-2), 197 - 203
G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins; Ogawa K et al.; In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P2y agonists . Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins) . Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE . Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs) . Ten mM NaF plus 10 microM AlCl3 increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat . Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in saponin-permeabilized tissues . Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-{3-thiotriphosphate} (ATP-gamma-S) was significantly inhibited by 100 microM guanosine 5'-O-{2-thiodiphosphate} (GDP-beta-S) . These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE . ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of Gs- and G(i)-type G-proteins . However, neither CTX nor PTX affected basal or agonist-stimulated release of InsPs . These observations suggest that muscarinic and P2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.

Biophys J, 1994 Apr, 66(4), 935 - 41
Orientation of cholera toxin bound to model membranes; Cabral-Lilly D et al.; The orientation of cholera toxin bound to its cell-surface receptor, ganglioside GM1, in a supporting lipid membrane was determined by electron microscopy of negatively stained toxin-lipid samples . Image analysis of two dimensional crystalline arrays has shown previously that the B-subunits of cholera toxin orient at the membrane surface as a pentameric ring with a central channel (Reed, R . A., J . Mattai, and G.G . Shipley . 1987 . Biochemistry . 26:824-832; Ribi, H . O., D . S . Ludwig, K . L . Mercer, G . K . Schoolnik, and R . D . Kornberg . 1988 . Science (Wash, DC) . 239:1272-1276) . We recorded images of negatively stained cholera toxin and isolated B-pentamers oriented perpendicular to the lipid surface so that the pentamer ring is viewed from the side . The pentamer dimensions, estimated from the average of 100 molecules, are approximately 60 by 30 A . Images of side views of whole cholera toxin clearly show density above the pentamer ring away from the lipid layer . On the basis of difference maps between averages of side views of whole toxin and B-pentamers, this density above the pentamer has been identified as a portion of the A-subunit . The A-subunit may also extend into the pore of the pentamer . In addition, Fab fragments from a monoclonal antibody to the A-subunit were mixed with the toxin prior to binding to GM1 . Density from the Fab was localized to the region of toxin above the pentamer ring confirming the location of the A-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Eye Res, 1994 Apr, 13(4), 311 - 3
Endotoxins in cholera and pertussis toxins interfere with in vivo responses to these agents in the albino rabbit eye; Mittag TW et al.; Intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5-1 microgram/eye) decreased intraocular pressure (IOP) up to 50% in the albino rabbit eye, which lasted up to six days . Both toxins were active on G-proteins as determined by in vitro and in vivo effects on ciliary process adenylyl cyclase activity and by ADP ribosylation of G-protein alpha-subunits with 32P-NAD . However, forty-two hours after toxin injection aqueous humor proteins increased from control levels of 0.8-1.2 mg/ml to 8-25 mg/ml . Both toxins contained 1-3 parts per thousand endotoxin sufficient to cause the IOP and aqueous humor protein responses observed . We conclude that the in vivo responses to intraocular CTX or PTX obtained from commercial sources may not provide unequivocal evidence for the role(s) of G-proteins in aqueous humor dynamics, and must be interpreted with caution.

Vaccine, 1994 Apr, 12(5), 419 - 26
Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine; Tamura S et al.; Cholera toxin B subunit (CTB) and Escherichia coli heat-labile toxin (LTB) (2 micrograms), each supplemented with a trace amount of cholera toxin (CT) (0.02-20 ng), were examined for the adjuvant effect on antibody (Ab) response against influenza inactivated HA (haemagglutinin) vaccine in Balb/c mice . Each mouse received a primary intranasal (i.n.) inoculation of the vaccine (1.5 micrograms) and the CT-containing CTB and in 4 weeks a second i.n . inoculation of the vaccine alone . The primary inoculation of the vaccine with CTB alone did not induce either anti-HA IgA or IgG Ab response, or haemagglutination-inhibition Ab responses in the serum . The vaccine with less than 2 ng of CT also failed to induce Ab response . On the other hand, the vaccine with CT-containing CTB induced a high Ab response, which increased depending on the CT dose . Moreover, the second vaccine induced a response more than ten times higher than the primary one and the response increased depending on the CT dose . Similar enhancement was found in the local anti-HA IgA Ab response in the nasal wash . Such synergistic effects were observed also between LTB and CT . The amount of Ab produced by the synergism was considered to be enough to protect against virus infection . These results suggest that CTB (or LTB) containing a trace amount of CT (about 0.1%) can be used practically as a potent adjuvant for nasal vaccination of humans against influenza.

Infect Immun, 1994 Apr, 62(4), 1460 - 4
Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants; Balanzino LE et al.; The ability of glycoproteins from pig intestinal brush border membranes (BBM) to bind cholera toxin (CT) or heat-labile toxins from strains of Escherichia coli isolated from human (LTh) or pig (LTp) intestines was studied . Glycoproteins capable of binding the toxins are also recognized by antibodies or lectins specific for ABO(H) blood group and related antigens . Pigs expressing A, H, or I antigenic determinants were used for comparison . The toxin-binding capacity of a glycoprotein depends on the toxin type and the blood group epitope borne by the glycoprotein . LTh and LTp preferably bound to several blood group A-active glycoproteins rather than H-active glycoproteins . By contrast, CT practically did not recognize either blood group A- or blood group H-active glycoproteins, while glycoproteins from pigs expressing I antigenic determinants were able to interact with LTh, LTp, and CT . LTh, LTp, or CT glycoprotein binding was selectively inhibited by specific lectins or monosaccharides . Affinity purification of the toxin binding brush border glycoproteins on the basis of their blood group reactivity suggests that such glycoproteins are hydrolytic enzymes . BBM from A+ pigs contain about 27 times more LTh binding sites, in addition to those recognized by CT, than an equivalent membrane preparation from H+ pigs . The present findings may help clarify some previous unclear results on LTh binding to intestinal BBM glycoproteins obtained by use of animals not typed by their ABO(H) blood group phenotype.

Immunology, 1994 Mar, 81(3), 338 - 42
Cholera toxin acts as a potent adjuvant for the induction of cytotoxic T-lymphocyte responses with non-replicating antigens; Bowen JC et al.; Cholera toxin (CT) is a strong systemic and mucosal adjuvant that greatly enhances IgG and IgA immune responses, but its adjuvant effects for cellular immunity, particularly class I-restricted cytotoxic T lymphocyte (CTL) responses, are less well understood . In the present report, CT and the purified non-toxic B component (CTB) were assessed for their ability to facilitate class I-restricted CTL induction to soluble proteins as well as to permit sensitization of target cells for CTL-mediated lysis . Priming for ovalbumin (OVA)-specific CTL occurred following oral exposure to a combination of OVA with CT plus CTB . In addition, CTB mixed with soluble proteins and administered intravenously primed mice for antigen-specific class I-restricted CTL . Target cells could also be sensitized for CTL-mediated killing following their exposure to soluble antigen and CTB in vitro . These results indicate that combinations of CT and CTB not only enhance antibody responses, but also have an immunomodulating effect to allow sensitization and priming for antigen-specific class I-restricted CTL.

Biochem J, 1994 Mar 1, 298 ( Pt 2), 493 - 7
Stimulation of high-affinity GTPase activity and cholera toxin-catalysed {32P}ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and alpha 2C10 adrenoceptor-transfected Rat 1 fibroblasts; Carr C et al.; Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts . This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein . LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor . The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells . LPA was also able to promote cholera toxin-catalysed {32P}ADP-ribosylation of Gi . This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin . LPA-stimulated cholera toxin-catalysed {32P}ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304 . Dose-response curves for LPA in the two assays of G-protein activation were coincident . The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.

Blood, 1994 Mar 1, 83(5), 1299 - 309
Priming effects of granulocyte-macrophage colony-stimulating factor are coupled to cholera toxin-sensitive guanine nucleotide binding protein in human T lymphocytes; al-Aoukaty A et al.; In addition to the mobilization of neutrophils and monocytes, granulocyte-macrophage colony-stimulating factor (GM-CSF) also mobilizes lymphocytes into peripheral blood . We examined the ability of GM-CSF to induce the proliferation of purified human T cells (CD3+ CD4+ CD56- CD16- B1- MO2-) in two major aspects: (1) the mechanisms of GM-CSF interaction with interleukin-2 (IL-2) causing T-cell proliferation, and (2) the intracellular signals transmitted by GM-CSF in T lymphocytes . We observed that concentrations of GM-CSF between 0.01 ng/mL and 10 ng/mL had a synergistic effect with concentrations of IL-2 between 1 U/mL and 10 U/mL in stimulating T-cell proliferation . This effect of GM-CSF was maximal when it was added at the start of the culture . In situ hybridization showed the presence of mRNA for GM-CSF receptors in T cells . Further analysis showed that GM-CSF induced the expression of IL-2 receptor (IL-2R) on the surface of T lymphocytes . These events coincide with the ability of GM-CSF to increase the intracellular levels of both cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) in T cells, to increase the binding of (gamma-35S) GTP to T-cell membranes, and to enhance GTPase activity as determined by increased hydrolysis of 32P-GTP . IL-2 also induced IL-2R expression, cyclic nucleotide secretion, and G-protein activation . However, the presence of IL-2 reduced GM-CSF induction of these activities . Addition of antibodies to the alpha and beta subunits of IL-2R permitted the activation of G protein by GM-CSF even when IL-2 was present . Furthermore, GTP binding and GTPase activity induced by GM-CSF or IL-2 were inhibited by the addition of cholera toxin (CT), but not pertussis toxin (PT) . Cumulatively, these results suggest that in T lymphocytes, receptors for GM-CSF or IL-2 may be coupled to the same CT-sensitive G protein, although other possibilities may exist . The role that G proteins play in mediating the intracellular signaling pathways induced by GM-CSF or IL-2 in human T cells is supported by adenosine diphosphate-ribosylation of a 44-kD or a 39-kD G protein in T-cell membranes by CT and PT, respectively.

Infect Dis Clin North Am, 1994 Mar, 8(1), 183 - 205
Cholera . Lessons from the epidemic in Peru; Gotuzzo E et al.; It is not known why the cholera epidemic, which affected all other continents, has not affected Latin America in the past 30 years . In addition, it is unclear why cholera appeared in Peru in 1991 . Improvements in scientific knowledge and technology have occurred in Peru during the last 30 months . While it is impossible to summarize in only one article all these concepts, this article presents a few of the most important issues and recent developments in the treatment and prevention of cholera.

Brain Res, 1994 Feb 28, 638(1-2), 151 - 6
Cholera toxin-induced Gs alpha down-regulation in neural tissue: studies on the pineal gland; Babila T et al.; Cholera toxin (CT) treatment (50 micrograms/ml) was used to down regulate the alpha subunit of the stimulatory guanine nucleotide binding protein (Gs alpha) in pineal glands in organ culture, as has been seen in non-neural tissue . A 15 h treatment reduces Gs alpha by approximately 75% as measured using semi-quantitative Western blot technology . In contrast, this treatment does not alter the abundance of G beta, Gi alpha or Go alpha . This effect on Gs alpha was still apparent following a 36-h washout period . The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of Gs alpha . This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP (N6,2'-O-dibutyryl adenosine 3',5' monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives . This indicates that a 48-h CT treatment was not generally toxic . In contrast, this treatment blocked subsequent CT stimulation of NAT . The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced alpha- or beta-adrenergic stimulation . alpha 1-Adrenergic activation of the pineal gland elevates {Ca2+}i, which potentiates effects of cyclic AMP; in these studies the response to alpha-adrenergic activation was markedly increased in 48 h CT-treated glands, reflecting Ca2+ potentiation of the effects of elevated levels of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Gut, 1994 Feb, 35(2), 211 - 4
Absorption of a hypotonic oral rehydration solution in a human model of cholera; Hunt JB et al.; The development of oral rehydration solutions (ORSs) has been one of the important therapeutic advances of this century . The optimal formulation, however, of ORSs for both cholera and other infective diarrhoeas is still debated . Part of the problem in developing ORSs has been the lack of adequate test systems for the assessment of new formulations before clinical trial . We have developed a jejunal perfusion, cholera toxin induced, secretory model in humans and have compared net water and solute absorption from a hypotonic ORS (HYPO-ORS: sodium 60 mmol/l, glucose 90 mmol/l, osmolality 240 mOsm/kg) and the British Pharmacopoeia recommended ORS (UK-ORS: sodium 35 mmol/l, glucose 200 mmol/l, osmolality 310 mOsm/kg) in six healthy volunteers . A plasma electrolyte solution (PES) was also perfused in all subjects to confirm a secretory state . Only HYPO-ORS reversed sodium secretion to absorption (p < 0.01) . Both ORSs promoted net water absorption but this was greatest with HYPO-ORS (p < 0.01) . Glucose and potassium absorption rates were similar for both ORSs whereas chloride absorption mirrored sodium absorption and was greatest from HYPO-ORS (p < 0.05) . These results, in a biologically relevant model of secretory diarrhoea, suggest it may be possible to achieve improved rates of rehydration by the use of hypotonic ORS with mid range sodium concentrations.

Clin Exp Immunol, 1994 Feb, 95(2), 222 - 6
Intestinal and circulating antibody-forming cells in IgA-deficient individuals after oral cholera vaccination; Friman V et al.; In search for a possible explanation for the different susceptibility to mucosal infections in IgA-deficient (IgAd) individuals, the frequency of total immunoglobulin-secreting cells (ISC) and vaccine-specific antibody-secreting cells (ASC) in intestinal mucosa and peripheral blood was determined by the enzyme-linked immunospot (ELISPOT) assay before and after peroral vaccination with a B subunit-whole cell cholera vaccine . Two groups of IgAd individuals, frequently infected and non-infected respectively, and normal controls were studied . Before cholera vaccination there were significantly higher frequencies of total IgM and IgG ISC in the gut, but not in the blood, in the IgAd individuals than in the controls . However, there were no significant differences between healthy and infection-prone IgAd individuals in this respect . In response to oral cholera vaccination, intestinal cholera toxin (CT)-specific IgG and IgM ASC were significantly more abundant among the IgAd individuals with a history of frequent infections than among the healthy IgAd individuals and controls . A similar difference in IgG and IgM ASC, although not significant, was also noted in blood . In IgAd individuals with frequent infections the vaccine induced variable anti-CT IgM ASC responses in the gut, ranging from no increase to a few strikingly high responses . In the controls, the CT-specific responses were dominated by IgA ASC . The data show that oral cholera vaccination evoked strong CT-specific IgG ASC responses, and in some cases also strong IgM ASC responses in the intestinal mucosa of IgAd patients with a history of frequent infections . The healthy IgAd individuals unexpectedly responded with lower numbers of CT-specific IgG ASC and did not show any increase of CT-specific IgM ASC in the intestinal mucosa . Thus, inability to mount a mucosal immune response to an oral antigen cannot in itself explain recurrent infections among many IgAd individuals.

Acta Med Okayama, 1994 Feb, 48(1), 17 - 23
Regulatory effect of lymphokine-activated killer cells on epidermal proliferation induced by cholera toxin in mice; Okamoto Y et al.; We investigated the effects of lymphokine-activated killer (LAK) cells on epidermal hyperplasia induced by cholera toxin (CT) . LAK cells showed cytotoxic activity against both tumor cell lines and proliferating normal cells including skin epidermal cells . When 1 x 10(7) LAK cells were injected intradermally together with 1.0 ng of CT, epidermal hyperplasia was markedly suppressed . The LAK effectors inhibiting epidermal hyperplasia showed surface phenotypes of asialo-GM1+, Thy-1+, Lyt-2- and L3T4-, that were different from those of LAK cells killing tumor cells in vitro . Epidermal hyperplasia induced by CT was not suppressed by topical administration of cytokines such as interleukin-2, interferon and tumor necrosis factor . Therefore, the antiproliferative effect of LAK cells might be attributed to their direct action on the epidermal cells.

Mol Chem Neuropathol, 1994 Feb-Apr, 21(2-3), 259 - 71
Inhibition of neurite outgrowth of neuroblastoma Neuro-2a cells by cholera toxin B-subunit and anti-GM1 antibody; Wu G et al.; The role of cell surface GM1 ganglioside in neurite outgrowth of Neuro-2a neuroblastoma cells was investigated by application of anti-GM1 antibody and the B subunit of cholera toxin (cholera B) to cultured cells stimulated to grow neurites in various ways . When the cells were simultaneously treated with stimulatory agent and cholera B, inhibition, as measured by percent of neurite-bearing cells, was observed with most stimuli: neuraminidase; GD1a ganglioside, retinoic acid, and low serum . However, with dibutyryl cyclic AMP the small reduction observed was not statistically significant . The inhibitory effect of cholera B on neurite outgrowth induced by low serum was dose-dependent, reaching a maximum at 200 ng/mL; 48 h after washout of cholera B the cells were released from inhibition and regrew neurites at nearly the previous rate in the presence of low serum . When the cells were exposed to stimulus for 6 h or more the inhibitory effect of subsequent addition of cholera B was reduced or eliminated; inhibition thus occurs during an early stage of neurite initiation . Anti-GM1 antibody at dilutions of 1:100-1:400 had the same inhibitory effect as cholera B with cells stimulated by GD1a or retinoic acid, whereas anti-GM2 antibody had no effect at 1:200 or 1:400; inhibition by the latter antibody at 1:100 dilution was similar to that attained with control ascites fluid . These results point to a pivotal role for cell surface GM1 in Neuro-2a differentiation induced by many (but not all) neuritogenic agents.

Eur J Pharmacol, 1994 Jan 15, 266(2), 125 - 9
cAMP analogues and cholera toxin stimulate the accumulation of nitrite in rat peritoneal macrophage cultures; Sowa G et al.; Rat peritoneal macrophages incubated with the two stable analogues of cAMP, dibutyryl-cAMP and 8-bromo-cAMP, as well as with cholera toxin, released nitrite in a dose-dependent manner . Cholera toxin and dibutyryl-cAMP enhanced nitrite release induced by bacterial lipopolysaccharide . The stimulatory effects of all these substances were inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine, dexamethasone and the protein synthesis inhibitor, cycloheximide . Our data indicate that the activation of cAMP-dependent pathway(s) can induce nitric oxide synthesis in rat peritoneal macrophages even in the absence of immunological stimuli, such as exogenous cytokines or lipopolysaccharide, although the exact mechanism of this phenomenon remains to be established.

Vaccine, 1994 Jan, 12(1), 65 - 72
Characterization of the circulating T-cell response after oral immunization of human volunteers with cholera toxin B subunit; Castello-Branco LR et al.; The kinetics and phenotypic characterization of the in vitro cell proliferative response to the B subunit of cholera toxin were studied using peripheral blood mononuclear cells taken from human volunteers at frequent time points after primary and booster oral immunizations . The cells induced to proliferate by oral immunization secreted IL-3, and lipopolysaccharide depletion and depletion of B cells did not affect proliferation . Flow cytometry demonstrated that activated cells were CD3- and CD4-positive . These findings indicate primed T cells proliferating specifically to the B subunit . The kinetics of the response suggested trafficking in the peripheral circulation of primed T cells from the gut, with a peak stimulation index of between 7 and 93 after first immunization, and a precursor frequency of primed cells of between 1 in 25,400 and 1 in 72,390 . There was close correlation between the serum antitoxin IgA antibody levels and observed proliferation.

Exp Cell Res, 1994 Jan, 210(1), 102 - 6
Control of mammary epithelial cell DNA synthesis by epidermal growth factor, cholera toxin, and IGF-1: specific inhibitory effect of prolactin on EGF-stimulated cell growth; Fenton SE et al.; Epidermal growth factor (EGF) stimulates mouse mammary cell proliferation in vivo, but its maximal level in the mammary gland is at midlactation, when little mammary growth takes place . The present studies use a normal murine mammary gland epithelial cell line (NMuMG) to determine the effects of the mammary mitogens EGF, cholera toxin (CT), and insulin-like growth factor-1 (IGF-1) on cell proliferation and how cell growth is altered by addition of the lactogenic hormone prolactin (PRL) . EGF and CT stimulated over a fourfold increase of DNA synthesis in NMuMG cells when compared to basal levels . Only a twofold stimulation of DNA synthesis was observed when cells were treated with IGF-1 . There was a slight increase in the percentage of cells in S-phase when these agents were added in combination with each other . Physiological levels of PRL had no significant effect on CT- or IGF-1-induced DNA synthesis but reduced EGF-stimulated cell proliferation to basal levels . Furthermore, PRL reduced the percentage of cells in S-phase to IGF-1- and CT-induced levels in the presence of EGF . Interestingly, PRL increased cellular levels of EGF mRNA after 2 h of treatment, which is similar to the response of mouse mammary glands cultured in lactogenic hormones . We conclude that even though PRL can increase the amount of EGF mRNA in mammary epithelial cells, it also eliminates EGF-induced mitogenesis.

Am J Trop Med Hyg, 1994, 50(5 Suppl), 42 - 54
Strategies for the induction of immune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier, and adjuvant; Holmgren J et al.; The concept of a common mucosal immune system, through which specific antigen-activated lymphocytes from the gut can disseminate immunity both along the intestinal tract and to various other mucosal and glandular tissues, has generated much current interest in the possibility of developing oral vaccines, not only for enteric infections but also for infections in the respiratory and urogenital tracts . However, to date it has proven difficult in practice to stimulate strong mucosal IgA immune responses by either parenteral or oral-mucosal administration of most antigens, and experience with soluble protein antigens has, on the whole, been disappointing . A notable exception in this regard is cholera toxin (CT) and in humans more than in other species, its nontoxic B subunit pentamer moiety (CTB) . Based on this, CTB has become an important component in recently developed oral vaccines against cholera as well as against diarrhea caused by enterotoxigenic Escherichia coli producing CT-like heat-labile enterotoxin(s) . Since the strong immunogenicity of CT and CTB can, to a large extent, be explained by their ability to bind to receptors on the intestinal mucosal surface, there has recently been much interest in approaches using CTB as an oral delivery carrier system for other vaccine-relevant antigens, and much progress has been made in preparing immunogenic hybrid proteins by coupling various protein or peptide antigens chemically or genetically to CTB . Indeed, in several systems, oral administration of such hybrid antigens has been found to markedly potentiate both intestinal and extraintestinal IgA immune responses against the CTB-coupled antigens and also to elicit substantial circulating antibody responses . Besides the mucosal immunopotentiating effect of either CT or CTB owing to their similar capacity as oral antigen-delivery vehicles, CT, but in most systems tested not CTB, also has strong adjuvant properties for stimulating mucosal IgA immune responses to admixed (not coupled) unrelated antigens after oral immunization . This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT (and specifically of its A subunit) leading to enhanced cyclic AMP formation in the affected cell, and efforts to eliminate the enterotoxic activity without losing adjuvanticity have so far not met with success.

Vaccine, 1994, 12(3), 215 - 22
Comparison of systemic and mucosal priming for mucosal immune responses to a bacterial protein antigen given with or coupled to cholera toxin (CT) B subunit, and effects of pre-existing anti-CT immunity; Wu HY et al.; Intraperitoneal immunization with a bacterial protein antigen conjugated to cholera toxin B subunit (CTB) was generally less effective than intragastric or intranasal immunization in generating mucosal IgA antibodies, and in priming the mucosal immune system to respond to intragastric or intranasal boosting . Previous intragastric or intranasal immunization which generated high levels of mucosal and circulating antibodies to CTB did not suppress mucosal IgA responses induced by intragastric or intranasal immunization with bacterial antigen conjugated to or mixed with CTB, but serum antibody responses were inhibited depending on the route of immunization and whether CTB was conjugated to or mixed with the antigen.

Can J Vet Res, 1994 Jan, 58(1), 71 - 4
Monoclonal antibodies to bovine viral diarrhea virus: cross-reactivities to field isolates and hog cholera virus strains; Deregt D et al.; Monoclonal antibodies to bovine viral diarrhea virus (BVDV) were examined for binding with a large number of North American BVDV isolates and eight strains of the serologically related pestivirus, hog cholera virus (HCV) . No single BVDV monoclonal antibody reacted with all BVDV isolates . The most cross-reactive monoclonal antibody was an anti-p80/p125 antibody which showed a positive reaction with 173 of 180 (96%) North American isolates . From a fewer number of isolates tested, one anti-gp53 monoclonal antibody also showed a high cross-reactivity (94%) . All BVDV isolates showed a positive reaction with at least one of the seven monoclonal antibodies in the panel . Thus, the results indicated that a pool of these monoclonal antibodies may be used in place of polyclonal antisera for the detection of BVDV contamination of cell lines or for virus isolation . For HCV, all three anti-p80/p125 monoclonal antibodies reacted positively with all eight virus strains . In contrast, none of the anti-gp53 monoclonal antibodies were reactive to HCV strains . Thus, the anti-gp53 monoclonal antibodies may be useful for distinguishing between usually innocuous BVDV infections and the highly significant HCV infections in swine for foreign animal disease surveillance.

J Gen Virol, 1994 Jan, 75 ( Pt 1), 117 - 24
Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus; Mulder WA et al.; Pseudorabies virus (PRV) expressing the envelope glycoprotein E1 (E1) of hog cholera virus (HCV) was used as a model to study the potential risks connected with the use of a live herpesvirus vaccine expressing a foreign gene . The gene encoding E1 was inserted into the glycoprotein X (gX) locus of both a virulent PRV strain and a non-virulent PRV strain in which the virulence genes encoding glycoprotein I (gI) and thymidine kinase (TK) had been inactivated . We investigated whether strain M205 (gI-, TK-, gX-, E1+) had a changed cell or host tropism or virulence compared with strain M206 (gI-, TK-, gX-) in pigs, rabbits, hamsters, rats, mice and rhesus monkeys . The insertion of E1 into this non-virulent PRV strain caused no change in cell or host tropism . However, pigs inoculated with M205 shed less virus over a shorter period than pigs inoculated with M206 . Theoretically, virulent PRV strains expressing E1 (gX-, E1+) could arise through transfer of the E1 gene of M205 to a virulent PRV strain . Therefore, we inoculated pigs with strain M12 (gX-, E1+) or the control strain M104 (gX-) and compared the virulence and pathogenesis . M12 and M104 were of approximately equal virulence and the pathogenesis of both strains was similar . We concluded that incorporating E1 of HCV into the gX locus of PRV did not change cell or host tropism, nor did it change the virulence of either non-virulent or virulent PRV.

Microb Pathog, 1994 Jan, 16(1), 71 - 6
Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose; Uesaka Y et al.; A simple method for purification of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and for purification of cholera toxin using immobilized D-galactose column is described . A single run of column chromatography yielded homogeneous toxin as demonstrated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis.

Exp Brain Res, 1994, 98(1), 21 - 30
Thoracolumbar sympathetic preganglionic neurons in the dorsal commissural nucleus of the male rat: an immunohistochemical study using retrograde labeling of cholera toxin subunit B; Hosoya Y et al.; The cell morphology of sympathetic preganglionic neurons (SPNs) in the dorsal commissural nucleus was studied by the retrograde labeling technique using cholera toxin subunit B (CTb) as a tracer . A small amount of an aqueous solution of CTb was injected unilaterally into the major pelvic ganglion of the male rat . Labeled SPNs were detected immunohistochemically using anti-CTb antiserum . Most of the labeled SPNs were observed in L1 to L3, and a very small number in T13 . They were observed bilaterally in the sympathetic nuclei, such as the intermediolateral cell column, intercalated nucleus and the dorsal commissural nucleus . A loose network of longitudinally or transversely oriented SPN dendrites was located within the dorsal commissural nucleus itself . The lateral margin of the dorsal commissural nucleus was roughly demarcated by longitudinally oriented dendrites . Together with the dendrites of the SPNs of the intercalated and intermediolateral cell column, laterally oriented dendrites of the dorsal commissural nucleus converged and formed the transverse dendritic bundles in the intermediate zone that connect the dorsal commissural nucleus and the intermediolateral cell column . The transverse dendritic bundles were arranged periodically . The axons of the SPNs in the dorsal commissural nucleus traveled laterally into the transverse dendritic bundles, then turned ventrally near the intermediolateral cell column, and finally entered the ventral funiculus . After rhizotomy of the ventral roots of the upper lumbar cord, labeled SPNs were found only on the side contralateral to the rhizotomy . The dorsal commissural nucleus appears as a compact single cell column, but our results clearly show that this nucleus actually consists of two adjacent parallel columns of cells.

Biochem Mol Biol Int, 1994 Jan, 32(1), 13 - 20
C5a stimulus-secretion coupling in rat basophilic leukaemia (RBL-2H3) cells transfected with the human C5a receptor is mediated by pertussis and cholera toxin-sensitive G proteins; Monk PN et al.; Rat basophilic leukaemia cells (RBL-2H3) were transfected with either the wild type human C5a receptor or a truncated form lacking the last 23 C-terminal residues . Transfected cells bound human C5a specifically, with affinities in the range 3-20nM, and 12-166,000 receptors per cell, similar values to those obtained on human neutrophils and monocytic cells . The stimulation of secretion by human C5a was completely inhibited by pertussis toxin and partially sensitive to cholera toxin, indicating that both wild-type and mutated receptors are coupled to G proteins . Cells transfected with the mutated receptor were equally sensitive to hC5a, suggesting that this portion of the C terminus is not an absolute requirement for signal transduction.

Dev Biol Stand, 1994, 82, 215 - 27
Mucosal vaccines based on the use of cholera toxin B subunit as immunogen and antigen carrier; Lebens M et al.; Stimulation of strong mucosal IgA immune responses as a basis for vaccine-induced protection against various pathogens has proved difficult . Most soluble protein antigens administered either parenterally or oral-mucosally have given disappointing results . A notable exception in this regard are cholera toxin (CT) and, particularly in humans, its non-toxic B subunit pentamer moiety (CTB) both of which stimulate a strong intestinal IgA antibody response and long-lasting immunological memory . Based on this, CTB has become an important component in recently developed oral vaccines against cholera and diarrhea caused by enterotoxigenic E . coli . The strong immunogenicity of CT and CTB can to a large extent be explained by their ability to bind to receptors on the intestinal mucosal surface . This has promoted much recent interest in the use of CTB as an oral delivery carrier for other vaccine-relevant antigens . Oral administration of antigens coupled to CTB either chemically or genetically has in several systems been found to markedly potentiate both intestinal and extra-intestinal IgA immune responses against the CTB-coupled antigens and to elicit substantial circulating antibody responses . In contrast to CTB, CT also has strong adjuvant properties for stimulating mucosal IgA immune responses to unrelated, non-coupled antigens after oral co-immunization . This adjuvant activity appears to be closely linked to the A subunit-catalyzed ADP-ribosylating action of CT leading to enhanced cyclic AMP formation in the affected cells.

Neurochem Int, 1994 Jan, 24(1), 1 - 12
The phorbol ester TPA potentiates cholera toxin- and isoproterenol-stimulated cyclic AMP-synthesis in primary astrocyte cultures; Gebicke-Haerter PJ et al.; Cellular responses to changes in the extracellular environment are mediated by intracellular signaling systems . One of the most extensively studied systems is adenylate cyclase which generates the second messenger molecule cAMP . Another one is the phosphatidylinositol (PI) second messenger system giving rise to IP3 and diacylglycerol, the latter stimulating protein kinase C . Recently, a third potential signaling system has attracted increased scientific attention: the phospholipase A2 system which generates arachidonic acid . This substance may be used for eicosanoid synthesis or serve as a second messenger molecule . The present report gives more evidence about mechanisms how these signaling pathways interact in cultured astrocytes . Substances commonly used for stimulation of arachidonic acid release and prostaglandin synthesis in these cultures (A23187, TPA) had no influence on intracellular cAMP levels . Pertussis toxin that had previously been shown to inhibit prostaglandin synthesis, had no influence on cAMP levels either . Cholera toxin, however, raised intracellular cAMP significantly, although much less than the beta-adrenoceptor agonist isoproterenol . Cholera toxin also caused a marked change in astroglial morphology even at reduced concentrations (1-10 ng/ml) . A23187 used in combination with Ctx had a moderate stimulatory effect on cAMP synthesis . In contrast, in the presence of Ctx, the PKC-activating phorbol ester TPA synergistically stimulated cAMP production, raising cAMP levels as high as isoproterenol-stimulated levels . The TPA effect was concentration-dependent . It was also dependent on an intact PKC since preincubation of cells with the phorbol ester completely abolished the synergistic effect . The synergistic effect of the phorbol ester was also observed at subthreshold concentrations of isoproterenol . The data reveal that the sole activation of most Gs molecules is a necessary but not sufficient prerequisite to achieve maximal adenylate cyclase activity . The fine-tuning of this activity apparently occurs at the catalytic subunit which is under the (partial) control of phosphorylation by PKC.

Yi Chuan Xue Bao, 1994, 21(6), 479 - 85
{The upstream sequence of cholera toxin B subunit gene: effect on CTB expression}; Cao C et al.; In this work, we have studied the effect of cholera toxin A structure gene on the expression of the distal ctxB gene by the methods of deletion and frame-shift mutation . The results showed that: The expression level of Plasmid pUC19CTB, which was constructed by cloning the XbaI-EcoRI restriction fragment into pUC19 and ctxA gene was out-frame with lacZ' gene, is about 30 micrograms/ml; If a frame shift mutation was introduced at XbaI site of pUC19CTB so that the cholera toxin A gene was inframe with lacZ' and could be translated, the expression level of ctxB was decreased to 12 micrograms/ml; When A further deletion from XbaI to ClaI of cholera toxin A gene (about 550bp) was made and ctxA was outframe with LacZ', ctxB expression was decreased two fold compared to pUC19CTB; If the ctxA was inframe with LacZ' so ctxA gene could be translated, the expression level of CTB is much lower than the plasmid outframe with lacZ' . These observations could not be explained by the current knowledge about genetical regulation of cholera toxin operon . The promoter we found located in the cholera toxin A subunit gene, which is responsible for the expression of cholera toxin B subunit, may answer the question why the 550bp non-coding sequence could enhance the expression of cholera toxin B subunit.

Epithelial Cell Biol, 1994, 3(4), 161 - 7
Stimulatory guanine nucleotide binding protein in pig epidermis: transient increase of the 45KDA cholera toxin substrate (Gs alpha) in the tape stripping-induced hyperproliferative state; Tsutsui M et al.; Cholera toxin catalyzed the transfer of ADP-ribose from {alpha-32P} NAD to 45kDa protein in pig epidermis . Western blot analysis using anti-Gs alpha antibody identified the 45kDa protein to be Gs alpha . In contrast to pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, the cholera toxin-catalyzed ADP-ribosylation was enhanced by the presence of Mg2+ in the reaction mixture . The cholera toxin-catalyzed ADP-ribosylation of the epidermal 45kDa membrane protein was significantly decreased, when samples were prepared from the cholera toxin-pretreated epidermis . The results, coupled with our previous report (Tsutsui and Iizuka 1990), indicate that pig epidermis contains functional G proteins (Gs and Gi), that affect the epidermal adenylate cyclase activity . Tape stripping-induced hyperproliferative epidermis showed an increased cholera toxin-catalyzed ADP-ribosylation of the 45kDa protein (Gs alpha) at 12-24 h following the tape stripping . Immunoblot analysis, however, showed no remarkable change in the level of Gs alpha compared with non-stripping controls . There was no significant difference in the level of the pertussis toxin-induced ADP-ribosylation of 40kDa protein (Gi alpha) in the tape-stripped epidermis . Immunoblot analysis showed no change in Gi content, either . Forskolin-induced cyclic AMP accumulation was markedly increased in the tape stripping-induced hyperproliferative epidermis . Cholera toxin-induced cyclic AMP accumulation was slightly increased, but this was not statistically significant . These results indicate that the alteration of Gs that is documented by cholera toxin-catalyzed ADP-ribosylation, is among the functional derangements of adenylate cyclase of tape stripping-induced hyperproliferative epidermis.

Yale J Biol Med, 1994 Jan-Apr, 67(1-2), 23 - 32
Congruences in Chinese and Western medicine from 1830-1911: smallpox, plague and cholera; Summers WC; A close examination of three examples, smallpox, plague and cholera, suggest that for acute infectious diseases the Chinese viewed the symptomatologies, the causes, and the rational treatments of these illnesses in many ways similar to that of their contemporary Western counterparts . Rather than holding an opposing, clashing or incongruent system of medical thoughts for these common, well-recognized infectious diseases, the Chinese were prepared, by a long tradition of ontological thinking, to be receptive to the adoption, incorporation or modification of Western medical ideas in the late nineteenth century.

Diabetes, 1994 Jan, 43(1), 24 - 32
ADP ribosylation by cholera toxin identifies three G-proteins that are activated by the galanin receptor . Studies with RINm5F cell membranes; Gillison SL et al.; Inhibition of insulin secretion by galanin is pertussis toxin (PTX) sensitive, suggesting the activation of one or more heterotrimeric (alpha, beta, gamma) G-proteins (Gi/Go) . Multiple effectors, including the K+ATP and L-type Ca2+ channels, adenylyl cyclase, and an as yet unidentified system at a site close to exocytosis, are modulated by galanin . Therefore, it is necessary to delineate the particular G-proteins activated by the galanin receptor as a first step to understanding its net cellular response . During specific conditions, cholera toxin (CTX) can ADP-ribosylate the alpha i/alpha o-subunits of the PTX-sensitive substrates but only during receptor/G-protein interaction . Therefore, we used CTX-catalyzed ADP ribosylation to identify galanin receptor-associated G-protein alpha-subunits in RINm5F cells . Galanin enhanced the ADP ribosylation of membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 M(r) . This labeling was blocked in membranes prepared from PTX-treated cells, enhanced by Mg2+, and showed a biphasic dependence on exogenous guanine nucleotides . Identification of the CTX ADP-ribosylated G-proteins by immunoprecipitation with selective antisera indicate activation by the galanin receptor of alpha i1 and alpha i3, which have the same mobility on SDS-PAGE (42,000 M(r)), and alpha i2 (39,000 M(r)) . These studies provide evidence for the activation of multiple G-proteins by receptors for galanin in RINm5F cells.

Endocr Res, 1993 Dec, 19(4), 259 - 71
Epidermal growth factor modulates cholera toxin induced mammary gland development; Sheffield LG et al.; Ovariectomized mice were either sham operated or sialoadenectomized and injected daily for 18 days with saline, estradiol + progesterone, cholera toxin or estradiol + progesterone+cholera toxin . Mammary development score and DNA were increased by estradiol + progesterone, but not by cholera toxin alone . In combination with estradiol + progesterone, cholera toxin increased mammary development score and mammary DNA . Sialoadenectomy reduced the ability of estradiol, progesterone and cholera toxin to induce mammary development . In other experiments, mice were primed with estradiol + progesterone for 10 days, and mammary tissue removed for in vitro culture with various combinations of insulin, aldosterone, cholera toxin and epidermal growth factor . In combination with insulin and aldosterone, cholera toxin increased mammary development in vitro . Sialoadenectomy reduced the ability of cholera toxin to induce mammary development in vitro . The effect of sialoadenectomy on mammary development was alleviated by adding epidermal growth factor to culture medium . Biochemical studies indicated that sialoadenectomy reduced the ability of estrogen and progesterone to induce cyclic AMP dependent protein kinase levels in mammary tissue, and also the ability of cholera toxin to induce accumulation of cyclic AMP in tissues . These effects of sialoadenectomy were reversed by addition of EGF to culture media.

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