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