Anticancer Drug Des, 1993 Dec, 8(6), 417 - 28 Inhibitory effects of cholera toxin on in vitro growth of human lung cancer cell lines; Kiura K et al.; Cholera toxin (CT) inhibited the growth of three out of 10 small cell lung cancer (SCLC) cell lines and two out of seven non-small cell lung cancer (NSCLC) cell lines when tested by 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) assay . These CT-sensitive as well as CT-resistant cell lines bound well to the non-toxic CT-B subunit-fluorescein isothiocyanate conjugate (FITC-CTB) when assayed by flow cytometry . Using the reaction of horseradish peroxidase-conjugated CT-B (HRP-CTB) on thin-layer chromatography (TLC), we analyzed gangliosides extracted from SCLC cell lines, CT-resistant SBC-1, minimally CT-sensitive SBC-3 and CT-sensitive SBC-5 . HRP-CTB was found to react not only with GM1, but also with Fuc-GM1, GD1b and other gangliosides on TLC . Although CT-resistant SBC-1 cells bound well to FITC-CTB, the binding of gangliosides extracted from SBC-1 cells to HRP-CTB was markedly decreased when compared to those from CT-sensitive SBC-5 cells . The CT resistance of the minimally CT-sensitive SBC-3 cell lines, which binds weakly FITC-CTB and HRP-CTB, was partially reversed by exogenous GM1 pretreatment . These observations suggest that the amount of gangliosides, such as GM1, Fuc-GM1 and GD1b, on the cells, rather than the CT-binding ability to the cells, plays a major role in cytotoxicity by CT. Am J Physiol, 1993 Dec, 265(6 Pt 1), G1050 - 6 Neural mediation of cholera toxin-induced mucin secretion in the rat small intestine; Moore BA et al.; We examined the role of enteric nerves in cholera toxin (CT)-induced mucin secretion in proximal and distal regions of rat small intestine . Stimulation of intestinal loops with 120 micrograms (1.5 mumol) CT using an in vitro open-loop model resulted in an approximately four-fold increase in luminal mucin content over unstimulated controls in both regions of the gut . Prior treatment of loops with tetrodotoxin had no effect on the amount of mucin released in response to CT . However, permanent destruction of primary sensory afferent nerves by neonatal treatment of rats with capsaicin reduced the mucin response to CT to baseline levels in both regions . In normal animals, atropine resulted in approximately 40% inhibition of mucin secretion in both the proximal and distal small intestine . The atropine-sensitive secretory response appears to be a component of the capsaicin-sensitive response . These results suggest that choleraic mucin secretion is mediated primarily by a capsaicin-sensitive neurogenic pathway involving local activation of sensory nerves, which may then elicit mucin secretion through interaction with cholinergic nerves. Biochem J, 1993 Dec 1, 296 ( Pt 2), 335 - 40 Correlation between prolactin secretion and Gs protein expression during sustained cholera-toxin stimulation; Lin JH et al.; We have studied the chronic effect of cholera toxin (CTX) on prolactin synthesis and secretion in GH3 pituitary-tumour cells . Time-course analysis showed that prolactin secretion increased with time of CTX exposure, reached a peak at 3 h, and decreased thereafter . Prolactin synthesis was also shown to be stimulated by CTX . The basic and forskolin-stimulated cyclic AMP levels of the CTX-treated cells followed a biphasic time response similar to that of prolactin secretion . Exposure of cells to CTX for more than 3 h abolished the subsequent CTX-catalysed ADP-ribosylation in vitro . Moreover, a significant decrease in the pertussis-toxin-catalysed ADP-ribosylation was found after cells were exposed to CTX for longer than 6 h . Western-blot analysis indicated that the amount of Gs alpha (alpha-subunit of Gs) protein increased within 3 h, followed by a gradual decrease to 50% of the control level at 24 h . The accumulation of Gs alpha mRNA increased within 6 h of CTX exposure, and decreased thereafter to 40% of the basal level at 48 h . Our findings that prolonged treatment of CTX induced similar patterns of time responses in Gs alpha protein expression, cyclic AMP production and prolactin secretion indicate that CTX-induced changes in Gs alpha protein levels may be responsible for the cellular response leading to prolactin secretion. J Clin Invest, 1993 Dec, 92(6), 2941 - 51 Entry of cholera toxin into polarized human intestinal epithelial cells . Identification of an early brefeldin A sensitive event required for A1-peptide generation; Lencer WI et al.; The effect of brefeldin-A (BFA), a reversible inhibitor of vesicular transport, on cholera toxin (CT)-induced Cl- secretion (Isc) was examined in the polarized human intestinal cell line, T84 . Pretreatment of T84 monolayers with 5 microM BFA reversibly inhibited Isc in response to apical or basolateral addition of 120 nM CT (2.4 +/- 0.5 vs . 68 +/- 3 microA/cm2, n = 5) . In contrast, BFA did not inhibit Isc responses to the cAMP agonist VIP (63 +/- 7 microA/cm2) . BFA had no effect on cell surface binding and endocytosis of a functional fluorescent CT analog or on the dose dependency of CT induced 32P-NAD ribosylation of Gs alpha in vitro . In contrast, BFA completely inhibited (> 95%) the ability of T84 cells to reduce CT to the enzymatically active A1-peptide . BFA had to be added within the first 10 min of CT exposure to inhibit CT-elicited Isc . The early BFA-sensitive step occurred before a temperature-sensitive step essential for apical CT action . These studies show that sequential steps are required for a biological response to apical CT: (a) binding to cell surfaces and rapid endocytosis; (b) early, BFA-sensitive vesicular transport essential for reduction of the A1-peptide; and (c) subsequent temperature-sensitive translocation of a signal (the A1-peptide or possibly ADP-ribose-Gs alpha) to the basolateral domain. Endocrinology, 1993 Dec, 133(6), 2756 - 60 Cholera toxin and dibutyryl cyclic adenosine 3',5'-monophosphate sensitize gonadotropin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: evidence for cross-talk between a cholera toxin-sensitive G-protein and PKC; Barnes SJ et al.; The present study assesses the relationship between G-proteins and protein kinase-C (PKC) in the gonadotrope . Cells were depleted of PKC with 1 microM phorbol 12-myristate 13-acetate for 12 h, followed by medium 199-BSA for 6 h before treatment with vehicle, pertussis toxin (PTX), cholera toxin (CTX), or (Bu)2cAMP (dBcAMP) for 18 h . PTX (10 ng/ml) significantly decreased GnRH-stimulated inositol phosphate (IP) production over a range of 10(-8)-10(-6) M GnRH . The degree of this inhibition was the same in control cells and PKC-depleted cells . Pretreatment with CTX (0.5 microgram/ml) significantly decreased GnRH-stimulated IP production over a range of 10(-9)-10(-6) M GnRH in PKC-depleted cells . This effect was mimicked by pretreatment with 3 mM dBcAMP . Although CTX and dBcAMP both decreased GnRH-stimulated IP production in control cells, this effect was enhanced in PKC-depleted cells . CTX (0.1 microgram/ml) and dBcAMP (3 mM) both enhanced GnRH-stimulated LH release, whereas PTX (100 ng/ml) had no effect . This was observed in control as well as PKC-depleted cells . Both PKA and PKC are capable of regulating IP turnover by phosphorylating phospholipase-C at distinct sites . CTX activates a G-protein that increases cAMP . cAMP can then activate PKA . In PKC-depleted cells, CTX inhibits GnRH-stimulated IP production . This effect is mimicked by dBcAMP, which suggests a role for PKA in the gonadotrope . The results of this study provide evidence for cross-talk between a CTX-sensitive G-protein and PKC. J Diarrhoeal Dis Res, 1993 Dec, 11(4), 227 - 34 Molecular mediators formed in the small intestine in response to cholera toxin; Peterson JW et al.; The molecular mechanism of experimental cholera, leading to increased water and electrolyte secretion, was evaluated with the rabbit intestinal loop model . The levels of cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), and 5-hydroxytryptamine (5-HT) were measured in mucosal tissue or luminal fluids from intestinal segments exposed to cholera toxin (CT) . Each mediator increased in a dose-dependent manner coinciding with the amount of fluid accumulating in the CT-treated loops within the 16 h observation period . Interestingly, a substantial amount of the cAMP in the CT loops was released into the intestinal lumen, along with the fluid . Fluid accumulation was evoked by instillation of PGE2 and CT into the intestinal segments, but not by 5-HT . Large doses of dibutyryl cAMP at 50 mg/ml, but not at 25 mg/ml, also evoked fluid accumulation when injected into the intestine . Further, CT evoked the release of 5-HT from the mucosa, although the high doses of cAMP caused a massive release of 5-HT . PGE2 injection was without effect on 5-HT release . Although CT increased the amount of PGE2 into the luminal fluid, no effect on PGE2 levels was observed by injecting any dose of dibutyryl cAMP or 5-HT into the intestinal lumen . Our interpretation of these data is that CT stimulates the independent synthesis and release of both cAMP and PGE2 . The cAMP appears to cause the release of 5-HT from the enterochromaffin cells . Since dibutyryl cAMP did not evoke a PGE2 response, it was concluded that cAMP could elicit secretion of fluid without the participation of PGE2; however, the cAMP doses were not physiological.(ABSTRACT TRUNCATED AT 250 WORDS) Histochemistry, 1993 Dec, 100(6), 457 - 64 Subunit b of cholera toxin labels interstitial cells of Cajal in the gut of rat and mouse; Anderson CR et al.; Cholera toxin subunit b was found in vivo and in vitro to label interstitial cells of Cajal in the intestine of rat and mouse . Cholera toxin-labelled interstitial cells were present in the subserosa, the myenteric plexus and the deep muscular plexus of mouse small intestine, and the deep muscular plexus only of the rat small intestine . In the large intestine of the mouse, interstitial cells were present in the subserosa and in a plexus associated with the inner surface of the circular muscle, while in the rat they were only present in the latter location . Macrophages, which were present in many of the same locations as interstitial cells, were also labelled by cholera toxin but could be distinguished from interstitial cells by their ability to take-up fluorescein isothiocyanate-labelled dextran . Labelling with subunit b of cholera toxin is a simple way of labelling interstitial cells of Cajal and which is compatible with a range of physiological and histological procedures. Mol Cell Endocrinol, 1993 Dec, 98(1), 33 - 42 Functional effects of transgenic expression of cholera toxin in pancreatic beta-cells; Wogensen L et al.; Investigation of intracellular pathways of stimulus-secretion signaling in vivo is possible by transgenic expression of agents known to influence specific biochemical interactions in the cells . The objective of the present study was to establish an experimental model for analyzing signal transduction mechanisms in pancreatic beta-cells in vivo, by expressing the cholera toxin A1 subunit under control of the insulin promoter, intending a constant activation of the Gs-protein, and thereby constant generation of cAMP . Surprisingly, the transgenic mice demonstrated mild hyperglycemia and hypoinsulinemia in vivo, and diminished glucose-induced insulin release from the in vitro perfused pancreas, whereas the pancreatic insulin content was normal . These observations suggest a deficiency in either the insulin release mechanisms or glucose recognition . Although the translated cholera toxin A1 subunit was biologically active, there was no increase in the islet content of cAMP . We conclude that the observed phenotype in the cholera toxin transgenic mice may be caused by a deleterious effect of the transgene itself on beta-cell function, or that counter regulatory mechanisms may compensate for the transgene-induced changes in intracellular enzymatic pathways. J Biol Chem, 1993 Nov 15, 268(32), 23769 - 72 Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin; Degtyarev MY et al.; The alpha subunit of the heterotrimeric Gs protein that couples the beta-adrenergic receptor to adenylyl cyclase undergoes post-translational palmitoylation . We examined the dynamics of this modification of alpha s by metabolic labeling of COS and S49 lymphoma cells under different conditions . The endogenous alpha s proteins were immunoprecipitated with a peptide-specific antibody, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by fluorography and densitometry . A pulse-chase study of COS cells incubated with {3H}palmitate or {35S}methionine showed that for alpha s the palmitate turnover (t1/2 approximately 50 min) was significantly faster than the protein degradation . Treatment of cells with 10 microM isoproterenol, a beta-adrenergic receptor agonist, in the presence of {3H}palmitate led to a rapid 4-10-fold increase in the palmitoylation of alpha s . This increase in palmitoylation was concentration-dependent (EC50 approximately 0.9 microM) and blocked by the antagonist propranolol . The mutant alpha s proteins in the unc and H21a S49 cell lines did not show an increase in {3H}palmitate incorporation with isoproterenol treatment . Cholera toxin treatment of COS cells increased the {3H}palmitate incorporation into the alpha s subunits . These data indicate that palmitoylation of the alpha s subunit is dynamic and regulated by activation of the alpha s subunit. Gene, 1993 Nov 15, 133(2), 227 - 32 Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit; Gonzalez RA et al.; A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB) . Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3' end of the DNA that codes for the leader peptide, and the 5' end of the gene encoding mature CTB . The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside . The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide . The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity . Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed. Infect Immun, 1993 Nov, 61(11), 4919 - 24 Effect of cholera toxin on vaccine-induced immunity and infection in murine schistosomiasis mansoni; Akhiani AA et al.; Intradermal vaccination of mice with soluble adult worm antigen (SWAP) in combination with Mycobacterium bovis BCG (Swedish strain) induced significant protection against subsequent infection with Schistosoma mansoni cercariae . When cholera toxin (CT) was used as an adjuvant in combination with SWAP or fraction A, no significant protection was observed . However, intradermal vaccination in combination with CT triggered a strong anti-SWAP antibody response and induced a strong delayed-type hypersensitivity response to schistosome antigens (SWAP or fraction A), one significantly higher than that in the SWAP-BCG group . In addition, vaccinating mice intranasally with SWAP or cercarial antigen together with CT as adjuvant failed to induce any significant protection . Surprisingly, mice given CT alone intranasally revealed a significantly enhanced worm burden . These findings suggest that mucosal application of CT may modulate the host-parasite relationship in favor of parasite survival. Infect Immun, 1993 Nov, 61(11), 4637 - 44 Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect of inflammatory cytokines; McGee DW et al.; Oral administration of cholera toxin (CT) induces a strong mucosal immune response to CT as well as having a potent adjuvant effect . Since one of the first cell types to encounter CT during cholera infection or after oral administration is the epithelial cell, we studied the effect of CT on interleukin-6 (IL-6) secretion by the rat intestinal epithelial cell line IEC-6 . CT was found to rapidly enhance IL-6 secretion and IL-6 gene expression by these cells . The addition of dibutyryl cyclic AMP (cAMP) to cultures of IEC-6 cells had little effect on IL-6 secretion, yet mRNA levels were elevated, suggesting that the response may have been regulated by cAMP . Purified B subunit of CT did not significantly enhance IL-6 secretion or mRNA expression . CT and transforming growth factor beta 1 synergistically enhanced IL-6 secretion in IEC-6 cells . The addition of CT with either IL-1 beta or tumor necrosis factor alpha gave even greater synergistic enhancement of IL-6 secretion, and dibutyryl cAMP could mimic CT's synergy with IL-1 beta . These results indicate that the intestinal epithelial cell is capable of secreting high levels of IL-6 after encountering CT, especially in the presence of inflammatory cytokines . This high level of IL-6 secretion could be a very important component of the mucosal immune response to CT and may also account for a portion of the adjuvant effect of CT. Hear Res, 1993 Nov, 70(2), 173 - 86 Input from the inferior colliculus to medial olivocochlear neurons in the rat: a double label study with PHA-L and cholera toxin; Vetter DE et al.; The inferior colliculus provides a strong descending influence capable of modulating the excitability levels of olivocochlear neurons (Rajan, 1990) . In an attempt to anatomically demonstrate this pathway in rats, an experimental paradigm was designed by which anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), which delineates axonal arbors, and retrogradely transported cholera toxin B subunit alone (CT-B) or conjugated to horseradish peroxidase (CT-HRP), which delineate dendritic arbors, are visualized in the same brainstem sections . PHA-L was injected unilaterally into the central nucleus of the inferior colliculus of adult rats 5-9 days prior to injection of CT-B or CT-HRP into either the contralateral or the ipsilateral cochlea . Descending collicular axons labeled with PHA-L densely innervate the ventral nucleus of the trapezoid body (VNTB), which contains neurons of the medial olivocochlear system (MOCS), but do not enter the lateral superior olive, where the neurons of the lateral olivocochlear system (LOCS) are found . The collicular projection to VNTB is largely ipsilateral and supplies mostly the ventral half of the nucleus . Within VNTB, the collicular fibers intermingle with dendrites and, to a lesser extent, cell bodies of MOCS . Collicular boutons, predominantly of the en passant type, are often observed in close apposition to dendrites and, less frequently, cell bodies of both crossed and uncrossed MOCS . These light microscopic results suggest the existence of direct, synaptic contacts between descending collicular axons and ipsilateral crossed and uncrossed MOCS . Numerous collicular boutons were also seen at a distance from MOCS, suggesting that they establish synapses with other neuron types of the VNTB that do not send their axons to the cochlea. J Emerg Med, 1993 Nov-Dec, 11(6), 717 - 21 Imported cholera in a 31-year-old Peruvian female; Kyriacou DN et al.; We present the case of a 31-year-old Peruvian female with severe dehydration due to diarrhea and vomiting . The patient was one of a number of travelers arriving in Los Angeles on an international flight from Argentina and Peru . Because of the travel history and clinical presentation, cholera was suspected and ultimately confirmed by stool culture . The patient's clinical course is outlined, and discussion of the relevant epidemiology and clinical management of cholera is provided . Physicians should suspect cholera when treating patients with severe gastroenteritis . The short incubation period, rapid onset of dehydration and shock, and high case fatality rate of untreated cholera require a consideration of cholera in patients with diarrhea and recent travel to areas where cholera is prevalent. J Int Med Res, 1993 Nov-Dec, 21(6), 323 - 33 Effects of tropisetron, a 5-hydroxytryptamine type 3 receptor blocker, on intestinal secretion induced by cholera toxin or deoxycholic acid in rabbits in vivo; Bardhan PK et al.; It has been suggested that 5-hydroxytryptamine is involved in the pathogenesis of various intestinal hypersecretory states including cholera . In this study, the effect of tropisetron (ICS 205-930), a specific 5-hydroxytryptamine type-3 receptor blocker, on jejunal and colonic fluid secretion induced respectively by cholera toxin and deoxycholic acid was investigated in rabbits using isolated loops of intestine in vivo . Marked fluid accumulation in both the jejunal and colonic loops was observed after exposure to cholera toxin and deoxycholic acid respectively . Elevation of jejunal and colonic mucosal cyclic adenosine monophosphate concentrations was also noted . Intraperitoneal administration of tropisetron dose-dependent inhibited jejunal secretion induced by cholera toxin . In contrast, no significant anti-secretory effect of tropisetron was observed against colonic secretion induced by deoxycholic acid . Tropisetron did not affect elevated mucosal cyclic adenosine monophosphate concentrations . The inhibitory effect of tropisetron on intestinal secretion induced by cholera toxin, which was independent of cyclic adenosine monophosphate formation, suggests that 5-hydroxytryptamine plays an important role in this type of secretion. Pharmacol Biochem Behav, 1993 Nov, 46(3), 623 - 9 Inhibition of the antianalgesic action of dynorphin A in mice by cholera toxin; Arts KS et al.; Dynorphin A-(1-17) (Dyn A) administered intrathecally (IT) or released spinally in the mouse produces an antianalgesic action . The present experiments indicate that IT administration of cholera toxin inhibited the antianalgesic action of Dyn A . When clonidine was administered intracerebroventricularly (ICV) to release spinal Dyn A, IT cholera toxin inhibited the antianalgesic action of Dyn A so that the analgesic component of action of clonidine became evident . Dyn A given IT inhibited the analgesic action of morphine given ICV . Cholera toxin given IT eliminated the antagonistic action of Dyn A . These results, in addition to others, indicate that IT cholera toxin antagonized the action of Dyn A . When the antianalgesic action of Dyn A was attenuated by pretreatment with dynorphin antiserum or by pretreatment that produced desensitization to Dyn A, cholera toxin had no effect . These results suggested that the antianalgesic action of Dyn A is mediated by activation of opioid receptors that are positively coupled to adenylate cyclase through a Gs regulatory protein. Gastroenterology, 1993 Nov, 105(5), 1286 - 93 Involvement of the myenteric plexus in the cholera toxin-induced net fluid secretion in the rat small intestine; Jodal M et al.; BACKGROUND: The enteric nervous system is responsible in vivo for most of the change in fluid transport induced by cholera toxin . The aim of the present study was to investigate the importance of the myenteric plexus in the Intramural reflex responsible for this secretion . METHODS: Long-term ablation of the myenteric plexus was achieved by serosal application of benzalkonium chloride on jejunal segments in rats . RESULTS: The treated segments without functioning myenteric plexus showed a normal net fluid absorption . Cholera toxin in this segment only induced a reduction of fluid absorption, whereas in a nontreated ileal segment it concomitantly induced a conspicuous net fluid secretion . Intravenous hexamethonium did not change the cholera toxin response in the treated jejunal segments, whereas vasoactive intestinal polypeptide elicited a marked secretion . CONCLUSIONS: Benzalkonium chloride treatment eliminated the ability of cholera toxin to induce intestinal secretion . Thus, all afferent fibers in the intramural secretory reflex activated by cholera toxin are probably conveyed via the myenteric plexus, which functions as the integrating center in the enteric nervous system . The Ussing chamber technique using stripped intestinal preparations cannot be used when studying effects of luminal secretagogues. J Biol Chem, 1993 Oct 15, 268(29), 21626 - 31 Regulation of the human serotonin transporter . Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding; Ramamoorthy S et al.; Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4-fold) serotonin transport activity in these cells . Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation . Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA . Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected . Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- {125I}iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity . Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels. Eur J Pharmacol, 1993 Oct 12, 243(1), 65 - 71 Cholera toxin antagonizes morphine-induced catalepsy through a cyclic AMP-independent mechanism; Massi P et al.; We studied the effect of intracerebroventricular pretreatment with pertussis toxin and cholera toxin on morphine catalepsy in rats . Pertussis toxin (1 micrograms/rat, two, three and six days before) did not affect catalepsy evoked by central morphine . Cholera toxin (1 micrograms/rat) did not affect morphine catalepsy after 24 h and 48 h, but significantly reduced it (about 60%) after three and five days . Ten days later the morphine response had totally recovered . This effect was selective, since morphine analgesia was not modified . The reduction of catalepsy appeared unrelated to the ability of cholera toxin to raise cAMP levels, as demonstrated by the different time course of changes in striatal cholera toxin-stimulated adenylate cyclase activity . The effect required an intact cholera toxin molecule and did not occur with a similar dose of cholera toxin-B subunit . These findings demonstrate that catalepsy is an opioid effect not linked to pertussis toxin-sensitive G proteins and suggest that the Gs protein might be involved. J Exp Med, 1993 Oct 1, 178(4), 1309 - 20 Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues; Xu-Amano J et al.; Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses . We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses . Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum . PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells . Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted . Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures . However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum . Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP . Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract. Epilepsy Res, 1993 Oct, 16(2), 137 - 46 Epileptic focus induced by intrahippocampal cholera toxin in rat: time course and properties in vivo and in vitro; Williams SF et al.; A small dose (0.5-1.0 micrograms) of cholera toxin injected into rat hippocampus induced an epileptic focus which discharged intermittently for 7-10 days . Epileptic discharges lasting from 70 ms to 2 min were recorded in vivo through implanted electrodes . The longer bursts could generalize to the neocortex, and occasionally caused motor seizures . The epileptic bursts reached a maximum 3-4 days after injection, and then declined to occasional brief interictal discharges by 9 days . Postmortem histology revealed no evidence of gross pathology or neuronal loss . Hippocampal slices prepared from rats < 8 days after injection of cholera toxin, and maintained in vitro, generated brief spontaneous and evoked epileptic bursts, usually lasting < 1 s . Spontaneous bursts always started in subregion CA3c, and propagated through the pyramidal layer at a mean of 0.18 m/s . Intracellular recordings from CA3 pyramidal layer cells always revealed simultaneous paroxysmal depolarization shifts during epileptic bursts . Epileptic activity, both in vivo and in vitro, required the whole toxin molecule . Injections of either the B subunit or the vehicle solution were not epileptogenic . Therefore binding of the toxin to neuronal membranes, which is mediated by the B subunit, was not sufficient for the epileptogenic effects of cholera toxin . This suggested that the activation of Gs which requires the whole molecule, was necessary . Gs activation is known to stimulate cyclic AMP production, but forskolin, which directly stimulates adenyl cyclase, failed to produce epileptic activity, even though it depressed action potential accommodation and afterhyperpolarizations (AHPs) . While further work is required to resolve the basic mechanisms of cholera toxin induced epileptic foci, we propose that they require the activation of Gs, which can enhance Ca2+ currents and modify excitatory synaptic transmission directly . Cyclic AMP induced changes in these properties cannot be excluded . However, cyclic AMP induced reductions in action potential accommodation and AHPs, which are found in cholera toxin foci, may contribute to, but are not sufficient for, epileptogenesis . Cholera toxin differs from the commonly used epileptic agents in that its main action is on G proteins and second messenger systems, rather than on synaptic transmission directly . Furthermore it has a prolonged time course, and does not cause gross pathology . These features combine to make it a distinctive model for epilepsy and neuronal synchronization. Can J Physiol Pharmacol, 1993 Oct-Nov, 71(10-11), 791 - 9 Effects of pertussis and cholera toxins on alpha-adrenoceptor function in rat tail artery: differences in hypertension; Li XF et al.; The alpha 1- and alpha 2-adrenoceptor-stimulated contractile responses of rat tail artery rings were compared in Sprague-Dawley (SD), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) rats that were untreated, treated with pertussis toxin, or treated with cholera toxin . The maximal responses, expressed as milligrams of tension, induced by clonidine (an alpha 2-adrenoceptor agonist) and cirazoline (a selective alpha 1-adrenoceptor agonist) were significantly greater in SHR than in SD or WKY, and the tissues were more sensitive to the agonists in SHR or SD than in WKY . Yohimbine (0.1 microM), a selective alpha 2-adrenoceptor antagonist, shifted the dose-response curves for clonidine to the right . The effects of yohimbine were greater in SD than in WKY or SHR, but not different between WKY and SHR . Prazosin (0.05 microM), a selective alpha 1-adrenoceptor antagonist, shifted the dose-response curves of cirazoline to the right, but the effects of prazosin were not different among these three strains of rats . Nifedipine (0.05 microM) completely blocked the response to clonidine in SD and WKY; however, in SHR, approximately one-third of the response to clonidine was resistant to nifedipine . Nifedipine, at 0.05 microM, only partially inhibited responses to cirazoline in SD, SHR, and WKY, and no differences were noted between the strains . Pertussis toxin pretreatment (50 micrograms/kg, 3 days before experiment) almost completely blocked the responses to clonidine, but only partially inhibited those to cirazoline . After pertussis toxin pretreatment, the responses (maximal effects and EC50s) to clonidine and cirazoline were not significantly different in arteries from the three strains of rats.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Virol, 1993 Oct, 74 ( Pt 10), 2053 - 60 Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia; van Rijn PA et al.; Four antigenic domains (A, B, C and D) on envelope glycoprotein E1 (gp51-54) of hog cholera virus strain Brescia have been specified by using 13 monoclonal antibodies (MAbs) that recognize non-conserved and conserved epitopes . It was shown that the non-conserved epitopes map to the N-terminal half of E1 by analysis of chimeric E1 proteins of strains Brescia and C . Conserved epitopes, however, could not be mapped using this approach . Here we describe mapping of both conserved and non-conserved epitopes on E1 by the use of an extensive set of single and double deletion mutants of E1 of strain Brescia . Deletion mutants were transiently expressed in COS1 cells and analysed by immunostaining with the 13 MAbs directed against strain Brescia and four MAbs directed against strain C . All MAbs bound to the N-terminal half of E1, i.e . amino acids 690 to 866 encoded by the sequence of strain Brescia . Domain B and one epitope in domain C are located between residues 690 and 773 . Other epitopes in domain C are located on an extended region, i.e . between residues 690 and 800 . Conserved epitopes of domain A are mapped between residues 766 and 866, whereas the only non-conserved epitope in this domain is located between residues 766 and 813 . Domain D, represented by one MAb, is located in the same region as this non-conserved epitope of domain A, i.e . between residues 766 and 800 . The results suggest the presence of two distinct antigenic units on E1, one consisting of domains B and C and the other consisting of domain A. Immunology, 1993 Oct, 80(2), 197 - 203 Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant; Vajdy M et al.; In the present study we investigated immunological memory at the cellular level following oral immunization using cholera toxin (CT) as the mucosal adjuvant . We found that memory cells, isolated from mice orally primed with keyhole limpet haemocyanin (KLH) admixed with CT adjuvant 8 months earlier, responded by increased proliferation to antigen-challenge in vitro . In contrast, unstimulated memory cells or KLH-stimulated cells from naive mice did not respond . Memory cells were isolated from different lymphoid tissues; spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) as well as the intestinal lamina propria (LP) . Thus, oral immunization using CT adjuvant promoted the generation of memory cells that were present in both systemic and local intestinal lymphoid tissues . The demonstration of lymphokine production in the KLH-responsive cultures indicated the presence of antigen-specific memory T cells . Lymphokine production early in culture was dominated by interleukin-2 (IL-2), which peaked on day 2-3, followed by IL-5 and, in particular, interferon-gamma (IFN-gamma) which increased over time . Lamina propria memory cells were found to proliferate poorly to recall antigen in vitro compared to lymphocytes from SP or MLN . In contrast, very significant production of IL-5 and, in particular, IFN-gamma was demonstrable in LP cell cultures . The use of CT adjuvant also stimulated the generation of antigen-specific memory B cells following oral immunization . This was evidenced by KLH-specific antibody production in antigen-challenged memory lymphocyte cultures . The memory B cells produced IgM anti-KLH, while no detectable antigen-specific IgG or IgA was found . Unstimulated memory cells or naive cells failed to produce anti-KLH antibodies . These in vitro findings provide evidence that oral immunization using CT adjuvant stimulates both antigen-specific memory T and B cells . Furthermore, our data suggest the existence of memory B cells following oral CT adjuvant immunization which have retained the ability to produce IgM and which therefore probably have not undergone terminal isotype switch differentiation to other isotypes and thus have not deleted the mu constant heavy-chain gene . Finally, our data also suggest that memory T and B cells, either sessile in the various lymphoid tissues or recirculating, can be activated by antigen in situ in, for example, lymph nodes and spleen and, more importantly, in the intestinal LP itself. Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1153 - 8 Interaction of pyrene-labeled monosialoganglioside GM1 micelles with cholera toxin; Picking WD; Monosialoganglioside GM1 labeled with 1-pyrene-dodecanoic acid was used to monitor interactions between cholera toxin and its receptor . Binding of cholera toxin to labeled ganglioside caused a decrease in pyrene fluorescence intensity at 480 nm concomitant with an increase in fluorescence intensity at 380 and 398 nm . The observed fluorescence changes are similar to those observed when fluorescent ganglioside is moved from an aqueous solvent to an organic solvent . The data are consistent with cholera toxin-bound ganglioside undergoing a process resembling solvent-dependent molecular dispersion. Eur J Immunol, 1993 Sep, 23(9), 2136 - 43 Cholera toxin adjuvant greatly promotes antigen priming of T cells; Hornquist E et al.; Cholera toxin (CT) given perorally is a powerful mucosal immunogen and adjuvant . Information that explains the adjuvant effect of CT may be used for the development of more effective oral vaccines and might also contribute to our understanding of the mechanisms involved in regulating mucosal immunity . The present study was undertaken to investigate if CT administered together with keyhole limpet hemocyanin (KLH) would act to promote or inhibit priming of KLH-specific T cells and whether the adjuvant effect of CT is restricted to mucosal immune responses or is a generalized phenomenon due to direct immunomodulating effects of CT . We found that CT adjuvant greatly augmented the effectiveness of a single oral priming immunization with KLH: re-challenge with KLH in vitro 1 week following immunization gave several-fold stronger proliferation in KLH-specific spleen, mesenteric lymph node, Peyer's patch and gut lamina propria T cells from KLH + CT adjuvant as opposed to KLH only-treated mice . Moreover, several-fold stronger cytokine production, i.e . interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and interferon-gamma accompanied the enhanced proliferative response of T cells from CT adjuvant-treated mice . The adjuvant effect of CT was not restricted to mucosal immune responses but was evident also following a single parenteral immunization with KLH + CT . Limiting dilution analysis revealed that CT adjuvant promoted a 20- to 40-fold increase in the frequency of primed KLH-specific T cells . Phenotypic and functional analyses clearly demonstrated that CT adjuvant primarily enhanced priming of CD4+ rather than CD8+ T cells and the pattern of lymphokine secretion disclosed that CT most probably promoted antigen priming of both Th1 and Th2 type of CD4+ T precursor cells. Infect Immun, 1993 Sep, 61(9), 3952 - 7 Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine; Wiedermann U et al.; To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet . The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine . The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency . The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats . Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group . Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum . Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals . In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine. Proc Soc Exp Biol Med, 1993 Sep, 203(4), 424 - 7 Effects of cholera and pertussis toxins on prolactin stimulation of lactose synthesis and ornithine decarboxylase activity in mouse mammary gland explants; Koduri PB et al.; Studies indicate that G proteins are likely involved in the signal transduction pathway for prolactin's stimulation of mitogenesis in Nb2 cells . In the mammary gland, little is known about the possible role of G proteins in the prolactin (PRL) stimulation of milk product synthesis . Therefore, the effects of cholera and pertussis toxin, enzymes that modify G protein activity, were tested on several actions of prolactin on mouse mammary tissue in culture . At concentration of 0.1-0.5 micrograms/ml, cholera toxin stimulated ornithine decarboxylase activity in a dose-response fashion; when tested in concert, cholera toxin and prolactin caused an additive response . Cholera toxin by itself did not affect the rate of lactose synthesis, but at concentrations above 0.5 micrograms/ml, it attenuated the magnitude of the prolactin stimulation of lactose synthesis . Pertussis toxin (0-0.5 micrograms/ml), both by itself and in concert with PRL, had no effect on ornithine decarboxylase activity . At concentrations of 25 ng/ml and above, pertussis toxin inhibited the PRL stimulation of lactose synthesis, whereas at 0.2 and 0.5 micrograms/ml, pertussis toxin abolished the PRL response . These observations suggest that a G protein, but not Gs, may be involved in prolactin's mechanism of signal transduction in the mouse mammary gland. J Virol, 1993 Sep, 67(9), 5435 - 42 Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera; Hulst MM et al.; The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector . Recombinant virus BacE1{+} expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV . The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H) . This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell . In contrast, recombinant virus BacE1{-}, which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells . The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive . The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa) . Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells . This amount was about 3-fold higher than that of cell-associated E1 in BacE1{-} and 10-fold higher than that of cell-associated E1 in BacE1{+}-infected Sf21 cells . Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms) . The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine. Eur J Epidemiol, 1993 Sep, 9(5), 563 - 5 The early stage of the recurrent cholera epidemic in Luanda, Angola; Colombo MM et al.; The recurrent cholera epidemic in Angola has been occurring in the rainy hot season since 1987 . About 350 cases were registered in the Luanda province in the first quarter of 1992 . Out of 110 analysed cases, 13 were positive for V . parahaemolyticus and 57 were positive for multiresistant V . cholerae O1 . Such strains were also isolated in the Bengo river, which feeds the Luanda water supply. Vaccine, 1993 Sep, 11(12), 1179 - 84 Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems; Holmgren J et al.; Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects which alone or in combination might explain their strong adjuvant action in stimulating mucosal IgA and other immune responses to admixed unrelated antigens after oral immunization . These effects include, depending on animal species and experimental systems, enhanced antigen presentation by a variety of cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and complex stimulatory as well as inhibitory effects on T-cell proliferation and lymphokine production . This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT and LT associated with enhanced cyclic AMP formation in the affected cells, and thus it may prove difficult to eliminate the enterotoxic activity without loss of adjuvanticity . However, through a separate mechanism, as an antigen-carrier system providing specific binding to epithelium including the M cells of intestinal Peyer's patches, both CT and its non-toxic binding subunit moiety (CTB) have been shown to markedly enhance the mucosal immune response to various foreign antigens or epitopes covalently linked to these molecules . This gives promise for the future use of CTB or related non-toxic binding derivatives as vehicles to facilitate induction of mucosal immune responses to a broad range of antigens for human vaccination purposes. Rev Sci Tech, 1993 Sep, 12(3), 879 - 85 Application of a blocking enzyme-linked immunosorbent assay for serological monitoring of hog cholera (classical swine fever) in Poland; Pejsak Z et al.; Between 1990 and 1992, serum samples from 55,478 domestic swine were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of hog cholera virus (HCV) antibodies . The amount of antibody in the sera was expressed as the mean percentage inhibition (PI) . For diagnosis, the tested sera were diluted 1:2 and considered positive if the PI was less than 25% . Sera giving PI values in the range of 25-50% were retested against HCV and bovine virus diarrhoea virus (BVDV), by neutralising peroxidase-linked assay . Comparison of the serum titres obtained was used for serological diagnosis of hog cholera; the tested sera were considered negative for hog cholera if the titre for BVDV was higher than that obtained for HCV . All sera with a PI higher than 50% were considered negative for HCV and BVDV . All sera were found to be free of antibodies to HCV . BVDV antibodies were demonstrated in 0.40% of the sera tested in 1990, in 1.80% in 1991 and 1.06% in 1992. Vaccine, 1993 Sep, 11(12), 1205 - 13 Induction of CD8+ cytotoxic T cells by immunization with killed influenza virus and effect of cholera toxin B subunit; Mbawuike IN et al.; The MHC class I cytotoxic T-lymphocyte (CTL) response in mice given formalin-inactivated influenza whole-virus vaccine (WVV) with or without cholera toxin B subunit (CTB) was studied . Intraperitoneal injection of Balb/c (H-2d) mice with high doses of A/Taiwan/1/86 (H1N1) WVV stimulated influenza A virus-specific CTL response in a dose-dependent manner . A dose of 4.4 or 44 micrograms induced CTL response equal to or greater than live influenza virus infection . Coadministration of vaccine with 5 or 25 micrograms of CTB resulted in a higher level of CTL than with vaccine alone . CTL lysed A/Taiwan and A/Shanghai (H3N2) virus-infected class I-expressing P815 (H-2d) but not virus-infected EL-4 (H-2b) target cells nor B/Yamagata virus-infected target cells . Virus-infected MHC class II- and class I-expressing A20 (H-2d) targets were also lysed . Depletion of Lyt-2+ (CD8+) T cells with monoclonal antibody completely abrogated lysis of P815 target cells and resulted only in a slight reduction of lysis of A20 target cells . Depletion of L3T4+ (CD4+) T cells or NK cells had minimal effect on lysis of either P815 or A20 target cells . Using limiting dilution analysis, the precursor CTL (pCTL) frequency paralleled CTL activity . Significant CTL activity was detected 7 months after immunization . These results demonstrate that adequate doses of influenza WVV with or without CTB can induce long-lasting influenza A cross-reactive MHC class I-restricted CD8+ CTL response in mice . Thus, coadministration of influenza WVV with CTB may lead to an effective vaccine that stimulates both CTL and antibody responses. Electrophoresis, 1993 Sep, 14(9), 899 - 901 Checkerboard immunoblotting recognizes twenty epitopes among the B subunit proteins of the cholera enterotoxin family; Qu ZH et al.; Checkerboard immunoblotting (CBIB) was used to analyze the reactions of a series of monoclonal antibodies with proteins of the cholera enterotoxin (CT) family, including heat labile enterotoxins (LTs) produced by diarrheagenic strains of Escherichia coli and genetically engineered chimeric proteins in which single amino acids of the CT-B subunit protein or human (H) LT-B subunit protein were substituted for corresponding residues in porcine (P) LT-B . The result indicated that there were at least twenty different patterns of reactivity suggesting that there are at least twenty recognizable epitopes among the proteins studied . An epitope which includes Ala46 appears to be particularly important . This epitope is common to CT and H-LT but not P-LT, and the epitope is not blocked by the Gm1 ganglioside . Human convalescent sera react with this epitope . CBIB is a versatile technique for epitope analysis. Gastroenterol Clin North Am, 1993 Sep, 22(3), 639 - 60 Epidemiology of cholera in the Americas; Blake PA; A persisting reservoir of the Gulf Coast strain of toxigenic V . cholerae O1 in Louisiana and Texas marshes and the shipment of seafood from these areas throughout the United States means that sporadic cases and outbreaks of cholera may occur anywhere in the country for the foreseeable future . Such cases are most likely to occur during warm months, especially between July and October . Physicians should think of cholera when consulted for severe watery diarrhea, even when the patient has no history of travel, and alert the laboratory . Experience has shown that US food and water sanitation is good enough to make either secondary transmission or large outbreaks unlikely; however, as long as we have foodborne and waterborne outbreaks of bacterial enteric diseases, the Gulf Coast strain may appear in a situation in which it can multiply and be ingested in large numbers by many people . The Latin American cholera epidemic has caused more cases of cholera in the United States in 2 years than the total of Gulf Coast strain cases identified during the past 20 years . The epidemic's future is uncertain . Despite knowing a great deal about cholera epidemiology, we cannot fully explain the ebb and flow of cholera epidemics . We do not know why cholera was apparently eliminated from the Western Hemisphere by 1900, nor can we predict which areas will be affected next or whether cholera will remain in a given area transiently or become endemic . The fact that cholera disappeared from the Western Hemisphere in the last century does not necessarily mean that it will disappear again . The situation is different now in several ways . The current pandemic is caused by the El Tor biotype, which persists better in the environment than does the classical biotype . Travel is now more frequent and more rapid . Finally, the population of the Western Hemisphere is about 14 times larger now than it was in 1850 and produces about 80,000 metric tons of human feces each day, of which only a fraction is treated . Thus, cholera will probably become endemic in Latin America and persist indefinitely. Vaccine, 1993 Sep, 11(12), 1233 - 9 Immune response related to the molecular structure of a peptide from the cholera toxin B subunit; Halimi H et al.; Three forms of a peptide P50-75 from the cholera toxin B subunit in the absence of carrier or adjuvant were administered orally or intraperitoneally to C57Bl/6J mice . Mice were given P50-75 as the free monomer, as an octamer synthesized on the seven polylysine core proposed by Tam (S), or as an octamer synthesized on the epsilon-amino groups of a chain of eight Lys-Gly-Gly units (C) . P50-75, presented to the immune system, as monomer or polymers, generated similar serum titres of anti-cholera toxin (CT) antibodies . However, mice immunized orally with the polymers S and C were better protected against the intestinal effects of the toxin than mice immunized with the free monomer P50-75 . S and C are more effective than P50-75 or the B subunit in increasing the amounts of total IgA secreted into the intestine and, moreover, the anti-CT IgA neutralized toxin activity . The amounts of anti-CT subclasses (IgG1, IgG2a, IgG2b, and IgG3 plus IgM) produced by the antigens depended on how the peptide P50-75 was presented for priming to the mice boosted thereafter with the B subunit. J Biol Chem, 1993 Aug 15, 268(23), 17038 - 44 Orientation of cholera toxin bound to target cells; Orlandi PA et al.; Cholera toxin (CT) consists of a pentameric B subunit that binds to specific cell surface receptors identified as ganglioside GM1 and an A subunit that activates adenylylcyclase . The A subunit consists of A1 and A2 peptides linked by a disulfide bond; A2 acts to connect A to B, whereas A1 is an ADP-ribosyltransferase that modifies the alpha subunit of the stimulatory G protein (Gs) . How the toxin is oriented when it binds to the cell surface and the related issue of the mechanism by which A1 gains access to Gs alpha are not known . In the present study, we used subunit-specific antibodies and their corresponding Fab fragments to assess their affects on holotoxin binding to target cells and their immunoreactivity to cell-bound toxin . Our results suggest that CT binds with A1 facing away from the membrane . Our hypothesis is further supported by the ability to assemble active CT on the cell surface of cultured human intestinal and neurotumor cells by the sequential addition of purified B and A subunits . We also observed that when cells containing bound CT were incubated at 37 degrees C, both subunits rapidly became inaccessible to their respective antibodies . We propose that the holotoxin binds with its A subunit facing away from the membrane and must enter the cell in order for A1 to be released, gain access to Gs alpha, and activate adenylylcyclase. J Biol Chem, 1993 Aug 15, 268(23), 17026 - 9 ADP-ribosylation of Gs by cholera toxin is potentiated by agonist activation of beta-adrenergic receptors in the absence of GTP; Bornancin F et al.; Purified Gs is a substrate for ADP-ribosylation catalyzed by cholera toxin (CTx) . In S49 cyc- membranes complemented with in vitro translated Gs alpha, the beta-adrenergic agonist isoproterenol enhanced the ADP-ribosylation rate . This effect was maximal if all guanyl nucleotides were suppressed but was blocked by the beta-adrenergic antagonist alprenolol . Enhancement was partially diminished if addition of GDP followed that of isoproterenol . When added in the absence of agonist, the GTP analogues guanosine 5'-O-(gamma-thiotriphosphate) and guanosine 5'-(beta, gamma-imido)triphosphate potentiated CTx-catalyzed ADP-ribosylation of Gs alpha consistent with their activating ADP-ribosylation factors . However, this effect was lessened when the same nucleotides were tested in the presence of agonist . Taken altogether, these results indicate that like Gt and Gi, Gs is an optimal substrate for CTx when coupled to an agonist-activated receptor and depleted of nucleotide . Therefore, coupling to the receptor and subsequent departure of the GDP turn out to be the common features underlying the sensitivity of all GTP-binding proteins to CTx-catalyzed ADP-ribosylation. J Steroid Biochem Mol Biol, 1993 Aug, 46(2), 203 - 8 Cholera toxin directly stimulates pregnenolone generation with increasing Ca2+ efflux in bovine adrenocortical mitochondria; Nishikawa T et al.; The present experiments demonstrated that the holotoxin as well as the A- and B-subunits of cholera toxin were able to directly enhance pregnenolone synthesis when isolated intact mitochondria, prepared from bovine adrenocortical tissue, were incubated; they were not, however, able to enhance pregnenolone synthesis when the inner mitochondrial fraction was similarly incubated, suggesting that the conformational structure of mitochondria is very important for activation of cholesterol side-chain cleavage by cholera toxin . Data are also presented demonstrating that cholera toxin can enhance Ca2+ release from isolated mitochondria, while pertussis toxin could activate neither pregnenolone generation nor increase Ca2+ efflux from mitochondria . Thus it is suggested that cholera toxin may activate pregnenolone synthesis by regulating Ca2+ movement in mitochondria. Dtsch Tierarztl Wochenschr, 1993 Aug, 100(8), 330 - 3 Antibody prevalence of hog cholera, bovine viral diarrhoea and Aujeszky's disease virus in wild boars in northern Germany; Dahle J et al.; During the hunting season 1990/1991 a total of 841 blood samples was collected from shot wild boar corresponding to about 2.11% of the total hunting bag in Lower Saxony . All the sera were screened for neutralizing antibodies (nAb) to hog cholera virus (HCV) and bovine viral diarrhoea virus (BVDV) by direct neutralizing peroxidase linked antibody (NPLA) assay . For the detection of antibodies (Ab) against HCV a Complex Trapping Blocking (CTB) ELISA was used . Cytotoxic sera were retested using an indirect immunoperoxidase technique . Additionally all sera were tested for antibodies to Aujeszky's disease virus (ADV) in a commercial indirect ELISA and 722 sera for ADV nAb in a virus neutralization test (VNT) with and without the addition of guinea pig complement . Screening of 841 sera yielded six sera neutralizing HCV strain ALFORT/187 and two sera with a positive CTB-ELISA reaction . A total of seven sera not identical with the HCV seropositive sera yielded BVDV neutralizing antibodies . The majority of HCV nAb positive samples were found in a region close to the former border to the German Democratic Republic (GDR) west of the State of Sachsen-Anhalt . Screening for ADV detected five sera positive in ELISA amongst 12 sera yielding nAb against ADV . Positive samples were obtained in the regions of Brunswick, Luneburg and Hannover. Infect Immun, 1993 Aug, 61(8), 3282 - 6 Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A; Donta ST et al.; Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined . Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin . It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. Scand J Immunol, 1993 Aug, 38(2), 201 - 8 B-cell activation in duodenal mucosa after oral cholera vaccination in IgA deficient subjects with or without IgG subclass deficiency; Nilssen DE et al.; Alterations in duodenal Ig-producing cells induced by two oral cholera vaccinations were studied by two-colour immunofluorescence in mucosal tissue sections from adults with selective IgA deficiency (IgAD), either with (n = 7) or without (n = 9) frequent infections, infection-prone patients with combined IgAD and IgG subclass deficiency (IgGSD) (n = 7), and normal control subjects (n = 11) . The proportion of IgG-producing cells prior to immunization tended to be lower in the symptomatic IgAD subjects than in the clinically healthy ones . In the first subgroup the absolute number of IgG cells per intestinal length unit was significantly increased after immunization (P < 0.04), and this tendency was also observed in the healthy IgAD subjects (6/9) and in those with combined deficiency (5/7) . Very few IgAD subjects responded with an increase of IgM-producing cells . The normal controls responded variably in all major immunocyte classes, in the order IgA > IgG > IgM . Compared with these controls, the patients with combined IgAD and IgGSD showed significantly increased IgG1 (P < 0.01) and reduced IgG2 (P < 0.006) proportions, which was in accordance with their serum subclass levels . Our study showed that oral cholera vaccination preferentially activates intestinal IgG-producing cells in IgAD subjects . This result agreed with data recently obtained by ELISPOT in the same patients with regard to antibody-forming cells specific for cholera toxin . Both methods suggested that IgG rather than IgM antibodies are elicited as compensation for a lacking IgA response . However, our overall results showed that intestinal B-cell activation is quite variable after oral cholera vaccination . Although such vaccination might be of importance for enhancing mucosal immunity also in IgAD patients, a concurrent gut disease could possibly be aggravated by IgG-mediated mucosal immunopathology in the absence of anti-inflammatory IgA antibodies. Acta Physiol Scand, 1993 Aug, 148(4), 393 - 401 Transcellular fluid secretion induced by cholera toxin and vasoactive intestinal polypeptide in the small intestine of the rat; Sjoqvist A et al.; The permeation of intravenously administered 51Cr-EDTA and {14C}mannitol to the perfused intestinal lumen was measured in anaesthetized rats together with the net intestinal fluid . Net fluid secretion was induced by cholera toxin or by intravenous infusion of vasoactive intestinal polypeptide (VIP) . The plasma clearance of Cr-EDTA and mannitol was 0.9 +/- 0.1 and 1.4 +/- 0.2 microliters min-1 g-1 intestine during the control period prior to the secretion and the net fluid absorption was about 7 +/- 5 microliters min-1 g-1 . Cholera toxin induced a net fluid secretion of about 30 +/- 7 microliters min-1 g-1 but the clearance did not rise but decreased significantly . The findings for VIP-induced secretion were similar . No indication of solvent drag was seen . Thus it is concluded that the fluid was secreted in channels which were smaller than the probes and we propose that the secreted fluid entered the intestinal lumen through the epithelial cells and not by the paracellular route . The decreased permeation of Cr-EDTA and mannitol from plasma to lumen during volume secretion suggest that there was a decreased mucosal permeability during the secretion . The decrease in permeability was consistent with a decrease in pore size . One explanation of the data is that the pore radius contracted from about 35 to 15 A during cholera if we assume a homogenous pore population . However, the data indicated that there was not a uniform size of the pore.(ABSTRACT TRUNCATED AT 250 WORDS) J Smooth Muscle Res, 1993 Aug, 29(4), 101 - 9 Relatively immediate relaxant effects of cholera toxin on isolated rabbit blood vessels; Tsuru H et al.; A study was made on the relatively immediate relaxant effect of cholera toxin (CTX) on the isolated ear artery, thoracic aorta and saphenous vein of the rabbit . Both preparations of CTX, containing sodium azide (NaN3) and azide-free, showed no effect on the non-precontracted artery, but CTX containing NaN3 relaxed the moderately precontracted blood vessels with methoxamine promptly, i.e., with a time course of min order . However, the immediate relaxation produced by CTX containing NaN3 was attributed mainly to NaN3 . Azide-free CTX, on the other hand, at 1-10 micrograms/ml gradually produced concentration-dependent relaxation of the precontracted vessels . The relaxant effects of CTX on the vessels were slow and long-lasting, i.e., with a time course of 10 min order . The relaxation induced by CTX was not influenced by the removal of endothelium nor by pretreatment with 10 microM indomethacin, 3 microM atropine or 3 microM propranolol . Activation of protein kinase C by a phorbol ester inhibited the relaxant effect of CTX . These results indicate that CTX relaxes the blood vessels by directly acting on the smooth muscles, without mediation by known endogenous relaxing factor, such as endothelium-derived relaxing factor (EDRF = NO) or prostaglandin I2 (prostacyclin) and by muscarinic receptor or beta-adrenoceptor. Acta Physiol Scand, 1993 Aug, 148(4), 387 - 92 On the involvement of tachykinin neurons in the secretory nervous reflex elicited by cholera toxin in the small intestine; Sjoqvist A et al.; The possible involvement of tachykinins in the nervous reflex activated by exposing the intestinal mucosa to cholera toxin was investigated in cats and rats . Three types of experiments were performed . In cats the release of tachykinins into blood was followed after placing cholera toxin in the intestinal lumen . In rat experiments a tachykinin receptor antagonist (Spantide II) was given close i.a . and its effect on cholera toxin-evoked fluid secretion was studied . Finally, in rats the effect of cholera toxin on the SP contents in the intestinal mucosa was studied . No release of tachykinins could be demonstrated . Spantide II did not change the rate of cholera toxin induced secretion . The SP content in the intestinal mucosa was not influenced by placing the toxin in the intestinal lumen . Hence, no experimental evidence was obtained for the involvement of a tachykinin neuron in the intestinal secretory nervous reflex activated by cholera toxin . Based on observations reported in the literature the involvement of an acetylcholine/tachykinin neuron in the reflex is tentatively discussed. Cell Signal, 1993 Jul, 5(4), 485 - 93 An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins; Milligan G et al.; Cholera toxin-catalysed {32P}ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the alpha 2-C10 adrenergic receptor . As well as incorporation of radioactivity into 45,000 and 42,000 M(r) polypeptides which represent forms of Gs alpha, there was weak labelling of a 40,000 M(r) polypeptide(s) . Addition of the alpha 2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M(r) polypeptide(s) but did not alter labelling of the forms of Gs . We have previously demonstrated that the 40,000 M(r) polypeptide(s) labelled in such a manner represents a combination of the alpha subunits of Gi2 and Gi3 {Milligan et al . (1991) J . biol . Chem . 266, 6447-6455} . Mercuric acetate treatment of membranes prelabelled with {32P}ADP-ribose by exposure to pertussis toxin and {32P}NAD removed totally the radiolabel from both Gi2 and Gi3 . However, cholera toxin-catalysed {32P}ADP-ribosylation of either the alpha subunits of the Gi-subtypes or of forms of Gs was unaffected by such treatment . By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the Gi-subtypes and from Gs but did not remove {32P}ADP-ribose incorporated by pertussis toxin from the Gi-subtypes . It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue . It is suggested that this residue may be Arg 179 in Gi2 alpha and Arg 178 in Gi3 alpha. Rev Alerg, 1993 Jul-Aug, 40(4), 91 - 4 {Cholera immunology and the molecular biology of cholera toxin . Recent progress and future prospects}; Carrada Bravo T; Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects that might explain their adjuvant action in stimulating secretory mucosal IgA after oral immunization . In mice experimental model, these effects include: enhanced antigen presentation by macrophages and other cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and other important effects on T cell proliferation and lymphokine production . The adjuvant activity is linked to the ADP-ribosylating action of CT with increased cyclic AMP formation in the affected cell, and thus it may be difficult to eliminate the enterotoxic activity without loss of adjuvanticity . However, both CT and its non-toxic B subunit moiety (CTB) have been shown to enhance the mucosal immune response to various epitopes or antigens covalently linked to these molecules . This now give promise that those antigens could become a useful vehicle to facilitate the induction of specific secretory IgA response to a broad range of antigens for human vaccination against cholera and other enteric infections. Neuroscience, 1993 Jul, 55(1), 45 - 56 Epileptic focus induced in rat by intrahippocampal cholera toxin: neuronal properties in vitro; Watts AE et al.; Injecting 0.5-1.0 microgram of cholera toxin into rat hippocampus induces a chronic epileptic focus which generates interictal discharges and brief epileptic seizures intermittently over the following seven to 10 days . Here we examined the electrophysiological properties of hippocampal slices prepared from these rats three to four days after injection, at the height of the epileptic syndrome . These slices generated epileptic discharges in response to electrical stimulation of afferent pathways . In many cases epileptic discharges occurred spontaneously in the CA3 subregion; these usually lasted < 200 ms, but they could last < 0.6 s . Intracellular recordings from pyramidal layer cells revealed depolarization shifts synchronous with the epileptic field potentials . These depolarization shifts had slow onsets compared with those induced by blocking inhibition with bicuculline (depolarizations started a mean of 57 ms before, and reached 5.2 mV by, the onset of the cholera toxin epileptic field potential, compared with 12 ms and 3.6 mV respectively for 70 microM bicuculline methiodide) . Extracellular unit recordings showed that the slow predepolarization seen in the cholera toxin focus was associated with an acceleration of the firing of other pyramidal layer neurons . The epileptic activity in this model cannot be attributed to the loss of synaptic inhibition, because inhibitory postsynaptic potentials could be evoked when the synchronous bursts were blocked by increasing {Ca2+}o from 2 to 8 mM . Observations of monosynaptic inhibitory postsynaptic currents isolated by application of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione, 50 microM DL-2-amino-5-phosphonovaleric acid and 100-200 microM 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid showed a small effect of the toxin only on the time course of the inhibitory postsynaptic current . On the other hand, there were significant changes in the intrinsic properties of individual neurons . The membrane potentials of cells in the cholera toxin focus did not differ from those in slices from rats injected with vehicle solution, but their input resistances were significantly increased . Unlike the other cellular changes in this model, the increase in input resistance was not seen in slices exposed acutely to 1 micrograms/ml cholera toxin for 30 min, suggesting there may be morphological changes in the chronic focus . Action potential accommodation and the slow afterhyperpolarization were depressed in both acute and chronic epileptic tissue, indicating impairments of Ca(2+)- and/or voltage-dependent K+ currents, and we conclude that these provide the most likely basis for cholera toxin epileptogenesis. Comp Biochem Physiol C, 1993 Jul, 105(3), 397 - 400 Cholera toxin: radio-iodination and uptake by the intestine of sucking rats; Aye-Kyaw et al.; 1 . The chloramine-T procedure was employed to radio-iodinate cholera toxin using Na125I . The procedure was found to be efficient and reproducible . 2 . Intragastric injections of both the labelled and the unlabelled toxin produced (a) significant increases in intestinal fluid accumulation as measured by the fluid accumulation ratio; (b) significant increases in cAMP levels; and (c) significant decreases in cAMP-phosphodiesterase activities when compared with the controls suggesting that radio-iodination did not impair the biological activity of the toxin . 3 . In vivo uptake studies of the labelled toxin by different parts of the intestine indicated that the uptake by the duodenum and jejunum was high and rapid when compared with the ileum implying that there are more binding sites (or receptor proteins) for cholera toxin in the duodenum and jejunum than in the ileum. J Biol Chem, 1993 Jun 5, 268(16), 12010 - 6 Brefeldin A blocks the response of cultured cells to cholera toxin . Implications for intracellular trafficking in toxin action; Orlandi PA et al.; Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase . The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase . There is a distinct lag phase between toxin binding and activation of adenylylcyclase . Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane . We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT . Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner . BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml . BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells . Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells . BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP . In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay . Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells . BFA treatment did not prevent the internalization of CT but did inhibit its degradation . BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum . BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae . We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells . The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action. MMWR Morb Mortal Wkly Rep, 1993 Jun 4, 42(21), 407 - 9; 415-6 Epidemic cholera--Burundi and Zimbabwe, 1992-1993; Hyperactivity and sensitization to psychostimulants following cholera toxin infusion into the nucleus accumbens; Department of Psychology, Northeastern University, Boston, Massachusetts 02115Although manipulation of second messenger systems is widespread in cell biology, there are few experiments examining the consequences of such manipulation on behavior . In three separate experiments, we extended earlier work by Miller and Kelly (1975) that examined the behavioral effects of microinfusion of cholera toxin (CTX) into the nucleus accumbens (N . Acc.) in rats . CTX is a bacterial toxin that ADP-ribosylates the Gs transducer protein and stimulates production of cAMP . For Experiment I, three groups of rats received either saline or CTX (50 or 500 ng/microliter) into the N . Acc . Locomotor activity was measured for 4 hr following a single CTX infusion and subsequently for 4 hr on 6 consecutive days . No acute effects on motor activity were observed . However, the 500 ng dose of CTX induced long-lasting hyperactivity that was apparent 24 hr later and that lasted 4 d . A smaller but significant hypermotility occurred on days 4 and 5 following infusion of the 50 ng dose . Site specificity of this effect was investigated in Experiment II by infusion of CTX (250 ng/microliter) into either the N . Acc . or the posterior dorsal striatum (PDS) . CTX treatment of the PDS had no behavioral effects while the long-lasting hyperactivity following treatment of the N . Acc . was replicated . In Experiment III the effect of intra-accumbens pretreatment with saline or CTX (10 ng/microliter) on d-amphetamine (0.5 mg/kg, i.p.)- and cocaine (7 mg/kg, i.p.)-induced motor activity was investigated.(ABSTRACT TRUNCATED AT 250 WORDS) J Diarrhoeal Dis Res, 1993 Jun, 11(2), 97 - 100 Comparative effects of nicotinic acid and nicotinamide on cholera toxin-induced secretion in rabbit ileum; Briend A et al.; Nicotinic acid reduces the cholera-toxin induced fluid secretion in experimental animals but its toxicity at high doses prevent its therapeutic use in patients suffering from cholera . This study aimed to determine whether nicotinamide, the non toxic amide derivative of nicotinic acid, is as effective as nicotinic acid in inhibiting cholera toxin induced intestinal secretion in vivo . Four intestinal loops, with their blood supply intact, were isolated in 30 rabbits and injected with either (i) 30 mM mannitol, (ii) 30 mM mannitol + 10 micrograms cholera toxin, (iii) 30 mM glucose, or (iv) 30 mM glucose + 10 micrograms cholera toxin . These rabbits were then randomly assigned to three groups receiving intraluminally either 100 mg/kg of nicotinic acid, 100 mg/kg of nicotinamide, or 10 ml/kg of Ringer solution . Measurement of intestinal fluid accumulation showed that nicotinic acid, but not nicotinamide, significantly reduced cholera toxin induced intestinal secretion. J Diarrhoeal Dis Res, 1993 Jun, 11(2), 67 - 74 In vitro uptake of amino acids in the jejunal mucosa of patients with cholera; Khin Maung U; In vitro uptake of 14C-labelled amino acids was studied in jejunal mucosa biopsy specimens from 64 adults admitted for treatment of cholera (proven by stool culture) within 48 hours of onset of watery diarrhoea to determine the state of amino acid carriers in the jejunal mucosa during actively purging disease . Continued absorption of amino acids by the NBB carrier (for neutral amino acids), the Y+ system (for dibasic amino acids), and the PHE carrier were operative even during the actively purging stage of watery diarrhoea due to cholera . The IMINO carrier for absorption of N-substituted amino acids was found to be inoperative during cholera but the imino acids could be absorbed by the PHE carrier . This study demonstrates continued intestinal absorption of amino acids during cholera, provides scientific basis for use of amino acids in "improved" oral rehydration solutions utilising amino acid transport systems which are linked to the absorption of sodium (and water) so that reduction in diarrhoeal stools can be achieved, and emphasises the importance of maintaining feeding during acute diarrhoea to prevent the development of malnutrition. Biochem J, 1993 Jun 1, 292 ( Pt 2), 401 - 8 Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1; Banno Y et al.; Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies . Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein . Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment . Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin . Cholera toxin also caused an increase in BK receptor number . Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number . The specific binding of {3H}BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg{Hyp3,Thi5,8,D-Phe7}BK, but not by the B1 BK receptor agonist des-Arg9-BK . These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP. J Immunol, 1993 Jun 1, 150(11), 4810 - 21 Cholera toxin promotes B cell isotype switching by two different mechanisms . cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GM1-receptor binding enhances later events in differentiation; Lycke NY; In a recent study we provided evidence that isotype switching in LPS-stimulated murine B cells was greatly enhanced by cholera toxin (CT) . We found that CT acted synergistically with IL-4 to promote IgG1 differentiation at the gene transcriptional level by strongly enhancing the expression of germ-line gamma 1-RNA transcripts . In this study we ask which mechanisms are responsible for the isotype-switching effect of CT on B cells and which second messenger systems are involved in this process . We found that at least two different mechanisms are involved: 1) increased intracellular cAMP levels stimulated by the A subunit potentiates isotype switching early in differentiation by augmenting the formation of sterile germ-line gamma 1-RNA transcripts and 2) the binding of the nontoxic B subunit to the membrane GM1-ganglioside receptor promotes later stages of differentiation . Although the whole toxin gave up to a ninefold increase in IgG1 differentiation the cAMP-independent effect of rCTB gave at most a fivefold increase in IgG1 differentiation as compared to that seen with IL-4 alone . However, on a molar basis whole CT was at least a 1000-fold more efficient at promoting B cell switch-differentiation as compared to rCTB . Moreover, IL-4 did not stimulate cAMP in murine B cells and its effect on LPS-stimulated B cell differentiation was not decreased by inhibitors of cAMP-dependent protein kinases . However, CT's effect on B cell switch differentiation was blocked by inhibitors of protein kinases and could be partially mimicked by dibutyryl cAMP . In contrast to CT, the enhancing effect of rCTB on IgG1-differentiation was not affected by blocking of the protein kinases and the combination of rCTB and dBcAMP was as potent as the intact CT in promoting IL-4-stimulated IgG1 differentiation . Finally, the IL-4 pathway, but not the CT pathway, was sensitive to phorbol esters: In IL-4 plus LPS-stimulated B cell cultures IgG1 production was almost completely blocked by PMA . This inhibition was not associated with a decreased B cell proliferation or expression of germ-line gamma 1-RNA transcripts . The addition of CT, and to a significantly lesser extent rCTB, to these cultures enhanced IgG1-differentiation and expression of germ-line gamma 1-RNA transcripts to the same extent as in cultures without PMA . The existence of dual mechanisms operating together on B cell differentiation help to explain the strong adjuvant function by CT on IgG and IgA antibody responses after oral and parenteral immunizations. J R Army Med Corps, 1993 Jun, 139(2), 76 - 8 The blue epidemic cholera--some aspects of treatment in the mid 19th century; Bonnici W; Cholera was first recorded in the Indian sub-continent in 1817 from where pandemics reached Europe via trade and pilgrim routes . Notwithstanding the numerous therapeutic methods prescribed, the virulence of cholera remained unabated . This article describes some of the proposed treatment regimes in relation to the assumed pathophysiology. Appl Environ Microbiol, 1993 May, 59(5), 1264 - 8 Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin; Abouzied MM et al.; The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production . Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA) . When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization . The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice . Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde . Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol . No reactivity was observed with 3- or 15-acetyldeoxynivalenol . The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein . DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis. Am J Trop Med Hyg, 1993 May, 48(5), 597 - 602 Epidemic cholera in the Amazon: the challenge of preventing death; Quick RE et al.; Epidemic cholera struck Peru in January 1991, and spread rapidly . The national cholera case-fatality rate (CFR) was less than 1% in the first six months of the epidemic, but in some rural areas, the CFR exceeded 10% . We investigated cholera mortality in the rural Amazon region, an area with a CFR of 6.3% . We conducted a case-control study, comparing 29 decedents with 61 survivors of recent cholera-like diarrheal illness in 12 villages with a combined CFR of 13.5% . Of 29 decedents, 28 (96%) died in the village or en route to a health facility . Death occurred within 36 hours of illness onset for 83% of the decedents . In 11 (92%) villages, the first or second recognized case was fatal . Death was associated with receiving treatment only at home (odds ratio indeterminate; 95% confidence interval 3.5, indeterminate) . Treatment with oral rehydration salts (ORS) was not protective against death for patients who received treatment only at home . Treatment with homemade sugar-salt solution (SSS) was also not protective; fewer than one-third of respondents knew the correct SSS recipe . Most decedents experienced multiple barriers to health care . Cholera victims died rapidly and early in village outbreaks, and few patients had access to health care . Provision of threatened villages with ORS supplies and education in their use before cholera strikes is essential to reducing cholera mortality in this region. Infect Immun, 1993 May, 61(5), 2162 - 71 Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit; Menge AC et al.; The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes . CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate . While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response . Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate . Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups . IgM antibody titers were generally negligible in the different secretions . An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues . A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats . In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate. Infect Immun, 1993 May, 61(5), 2082 - 8 Oral immunization with Toxoplasma gondii antigens in association with cholera toxin induces enhanced protective and cell-mediated immunity in C57BL/6 mice; Bourguin I et al.; Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo) in association with either cholera toxin (CT) or CT B subunit, the T . gondii-specific in vitro proliferation of splenic T lymphocytes was determined . Cytokines produced by these T cells were then characterized . After oral challenge with T . gondii 76K cysts, the percentage of cumulative survival was assessed, as was the number of brain cysts in the mice which survived . The TSo-specific proliferation of splenic T lymphocytes was greatly enhanced by the use of CT, whereas CT B subunit alone did not lead to amplification of splenic T-cell proliferation . The use of CT was associated with an increase of interleukin-2 (IL-2) and gamma interferon synthesis by TSo-stimulated splenic T cells, whereas no enhancement of IL-5 and IL-6 production was observed . IL-4 was not detected . A significant protection of mice immunized orally with TSo plus CT was observed in comparison with those immunized with TSo alone . This protection was associated with a large decrease in the number of brain cysts compared with the number found in naive mice infected orally with a sublethal dose of T . gondii 76K cysts . Further studies, using well-defined T . gondii proteins which are known to induce both mucosal and systemic immune responses, are needed to confirm the value of CT in the enhancement of protection against oral toxoplasmosis. J Virol, 1993 May, 67(5), 2922 - 7 Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen; Kimman TG et al.; T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect . Therefore, little is known about cellular immunity to HCV . In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs . We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals . However, such cells proliferated in vitro upon stimulation with viable HCV antigen . The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV . Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response . The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain . A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains . This immunological phenomenon may mask a specific T-cell response to the virus. J Clin Microbiol, 1993 May, 31(5), 1148 - 54 Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction; Wirz B et al.; Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses . For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses . Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested . In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains . By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates . By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung . Such extracts were used directly for RT-PCR without prior RNA isolation . We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction . This allowed the differentiation of HCV from BVDV and BDV in one step . To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining . The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses. Mol Immunol, 1993 May, 30(7), 627 - 35 Agonistic and antagonistic effects of cholera toxin on human B lymphocyte proliferation; Garrone P et al.; In our attempts to elucidate the mechanisms regulating the IL2- and IL4-induced proliferation of human B lymphocytes, we studied the effects of cholera toxin (CT) and other agents increasing adenosine 3', 5'-cyclic monophosphate (cAMP) levels on tonsil B cells activated through their antigen receptors . CT enhanced proliferation of anti-IgM-costimulated B cells in a dose-dependent fashion (1 ng/ml to 10 micrograms/ml), a property shared in part by other agents inducing cAMP, such as forskolin, prostaglandin E2 and dibutyryl-cAMP, but not by the purified B subunit of CT . However, when cytokine-dependent proliferation was studied, CT and cAMP-increasing agents inhibited IL2-induced DNA synthesis of anti-IgM-activated B cells . This blockade was not due to a modification of the kinetics of proliferation, but was rather a consequence of partial inhibition of IL2 receptor expression . In contrast CT and cAMP-elevating agents enhanced the latest phases of the IL4-induced DNA synthesis of anti-IgM-activated B cells . These results indicate that CT displays agonistic and antagonistic effects on human B cell proliferation, most of these effects being reproduced by cAMP-elevating agents . Thus limited activation of the cAMP pathway in B cells may facilitate the development of TH2-type immune responses. Endocrinology, 1993 May, 132(5), 2131 - 5 A cholera toxin-sensitive guanyl nucleotide binding protein mediates the movement of pituitary luteinizing hormone into a releasable pool: loss of this event is associated with the onset of homologous desensitization to gonadotropin-releasing hormone; Janovick JA et al.; Cholera toxin (CTX; 5 micrograms/ml), but not pertussis toxin (100 ng/ml), when preincubated with pituitary cells for 18 h, enhances the percentage of cellular LH released in response to continuous or pulsatile administration of 5 x 10(-9) M GnRH . This effect occurs without increasing total (intracellular plus extracellular) LH, indicating that it is best explained by redistribution of LH from a nonreleasable to a releasable pool . This site of action is consistent with the observation that CTX-pretreated cells are also sensitized to stimulation of LH release by the Ca2+ ionophore A23187 . The observations that CTX stimulates the production of cAMP in these cells and that the sensitizing action of CTX is mimicked by (Bu)2cAMP (1 mM) are consistent with the view that a CTX-stimulated guanyl nucleotide binding protein, capable of activating adenylyl cyclase, is mediating this sensitization . We used a perifused cell system to show that the movement of LH into a releasable pool is lost with the onset of homologous desensitization due to high pulse frequency or constant administration of GnRH (5 x 10(-9) M, continuous or a pulse each 15 min) . Sensitization to CTX is restored by stimulation with a high concentration of GnRH (10(-6) M) or by resetting the pulse frequency to the rate measured in vivo (a pulse each 90 min) . Both of these treatments also circumvent the desensitized state, restoring LH release . These results identify a novel lesion associated with the development of desensitization in the gonadotrope and support the role of a CTX-sensitive guanyl nucleotide binding protein in regulation of pituitary responsiveness to GnRH. Endocrinology, 1993 May, 132(5), 2124 - 30 Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action; Hawes BE et al.; A growing body of evidence suggests a role for guanyl nucleotide binding proteins (G proteins) in GnRH action . G protein activation provokes LH release, inositol phosphate (IP) production, decreased gonadotrope responsiveness to GnRH, increased gonadotrope responsiveness to the calcium ionophore A23187, and decreased GnRH receptor binding . The specific G proteins involved in these actions, however, are not known . This study uses pertussis toxin (PTX) and cholera toxin (CTX), which affect the activity of a number of G proteins by ADP ribosylation of a Cys or an Arg residue, respectively, of the alpha-subunit . Although not an effective LH secretagogue in itself, CTX enhanced GnRH-, NaF-, and A23187-stimulated LH release after an 18-h pretreatment period . CTX pretreatment did not affect GnRH- or NaF-stimulated IP production . Conversely, 18 h pretreatment with PTX reduced GnRH- and NaF-provoked IP production compared to control values, but did not affect LH release . In addition, pretreatment with either CTX, PTX, or Bt2cAMP provoked a decrease in GnRH receptor binding compared to control . The results of this study suggest that: 1) GnRH stimulates IP production, but not LH release, through a PTX-sensitive G protein; 2) A distinct CTX-sensitive G protein appears to provoke gonadotrope sensitization by stimulating an increase in intracellular cAMP levels; and 3) there appears to be a distinct G protein, insensitive to PTX and CTX, capable of mediating LH release independent of IP production and cAMP. Salud Publica Mex, 1993 May-Jun, 35(3), 268 - 82 {Preparing ourselves for cholera: a rapid evaluation of the quality of oral rehydration activities in Guatemala}; Hermida J; The directorate of the North Health Area of Guatemala along with the National Nutrition Institute of Centroamerica and Panama in 1991 carried out a rapid evaluation of the quality of care provided to the population for oral rehydration, acute diarrhea and cholera . The purpose was to collect data to facilitate the implementation of efficacious activities to improve quality and optimize the use of resources . The current article contains the results of the evaluation of twenty health centers of the North Health Area of Guatemala, and the consequent activities to improve the process of care . The main failures in performance where: deficient distribution of inputs; errors in the performance of physical exams of the children in the determination of the severity of dehydration; poor use of antibiotics and in the treatment of those with severe dehydration; and specially the failure to educate the mother about the proper feeding for a sick child . The delivery of inputs improved as a outcome of the actions product of the evaluation . Another activity was a workshop, combining theory and practice, of the treatment of cholera . Currently, local authorities prepare and carry out longer term interventions taking into account the results of the evaluation. Virus Res, 1993 May, 28(2), 203 - 8 Cloning and nucleotide sequence determination of the major envelope glycoprotein (gp55) gene of hog cholera virus (Weybridge); Yu M et al.; A 1.7 kb cDNA fragment corresponding to the coding region of the major envelope glycoprotein (gp55) of pestivirus hog cholera (Weybridge) was obtained using the polymerase chain reaction (PCR), and then cloned into pUC 8 . The deduced amino acid sequence of gp55 showed a strong homology to that of HCV strains Brescia (94%) and Alfort (90%), and to a lesser extent to the closely related gp53 of bovine viral diarrhoea virus strain, NADL (65%) . Eighteen cysteine residues were identified in the sequenced region, all of which were conserved between the gp55/gp53 sequences . This suggests that although the homology at the protein level may vary, there are strong conformational motifs which are conserved among the pestivirus envelope proteins. Morfologiia, 1993 May-Jun, 104(5-6), 39 - 47 {Changes in the neuronal ultrastructure of the plexuses of the small intestine in suckling rabbits with experimental cholera}; Bardakhch'ian EA et al.; Ultrastructural changes in the small intestine nerve cells have been studied in 34 suckling rabbits infected intragastrically with V . cholerae 01 . A correlation between neuron reactions and vascularization of the intramural ganglia have been revealed . Initial alterations (1.5 hr) in nerve cells were characterized by adoptive reorganization of the cytoplasmic organelle . During the period of V . cholerae adhesion (4 hr) ultrastructural alterations were accompanied by cytoplasmic chromatolysis . 1-2 days later, i.e . during the period of clinical manifestations of experimental cholera, changes in the ultrastructure of neurons became stable and most of the injuries were irreversible . The involvement of small intestine nerve elements in this process may be one of pathogenetic links of cholera infection. Lancet, 1993 Apr 24, 341(8852), 1049 - 51 Bioimpedance monitoring of rehydration in cholera; McDonald JJ et al.; Measurement of bioimpedance (BI) is a simple non-invasive technique that relies on the different conductivity of tissues to define body composition and can be easily adapted to automated monitoring . We assessed the accuracy of BI in monitoring rehydration and acute fluid fluxes in 35 Peruvian cholera patients . Patients were monitored throughout the acute phase of diarrhoea and followed up at 3 and 10 days . BI was compared with other objective measures of dehydration including packed cell volume, serum protein, and calculated fluid balance . BI rapidly detected inadequate treatment and acute fluid flux, correlating highly with intravascular hydration as measured by serum protein and packed cell volume . BI values during dehydration were significantly raised compared with 10-day convalescent values and age-matched controls (p < 0.05) . We also encountered an unexpected difference in the bioelectrical response to dehydration and rehydration between sexes . We conclude that BI has uses in monitoring dehydrated patients, in oral rehydration trials, and in physiological studies. J Immunol, 1993 Apr 15, 150(8 Pt 1), 3274 - 83 Inhibition of murine T cell activ