Cholera

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 activation by cholera toxin B subunit is not mediated through the phosphatidylinositol second messenger system; Woogen SD et al.; Although cholera toxin B subunit is a potent mucosal immunogen in vivo, its predominant effect in vitro is inhibition of T cell and B cell activation . We reported earlier that this inhibition was not mediated through activation of adenylate cyclase and increases in intracellular cAMP . There is increasing evidence that T cell activation is initiated through the phosphatidyl inositol second messenger system in which phosphatidyl inositol bisphosphate is hydrolyzed by phospholipase C, producing inositol trisphosphate (IP3) and diacylglycerol . IP3 increases cytosolic calcium and diacylglycerol binds, translocates, and activates protein kinase C (PKC) . These signals lead to a complex series of events eventuating in activation of a number of genes important in cell proliferation . In this study, we asked whether the mechanism of T cell inhibition by B subunit of cholera toxin (CT-B) was due to interference with the phosphatidyl inositol second messenger system . We found that substitution of ionomycin and PMA for IP3 and diacylglycerol, respectively, in culture induced T cell proliferation but only if both were present simultaneously . Such proliferation was inhibited by CT-B even if added hours after the start of culture . An assay for cytosolic PKC activity demonstrated that PMA translocation of PKC from cytosol to membrane was not inhibited by CT-B, indicating that CT-B does not inhibit activation of PKC . There was no inhibition of Con A-stimulated T cell phosphoinositol turnover . Moreover, Con A added to Fura-2 AM-loaded cells caused a rapid rise in cytosolic calcium, which CT-B preincubation did not alter . These results indicate that CT-B did not inhibit IP3 generation or action . We next looked at expression of genes involved in T cell proliferation . CT-B inhibited the production of IL-2 by mitogen-activated T cells; Northern analysis showed that this inhibition was associated with decreased levels of IL-2 mRNA . Expression of IL-2R and of transferrin receptors was only modestly reduced . Despite the presence of IL-2R on the T cells exposed to CT-B, the addition of exogenous IL-2 to the cultures did not reverse the CT-B-induced T cell inhibition . We conclude that the T cell inhibition by CT-B is not mediated by interference with the activation of the phosphatidylinositol second messenger system but occurs at a later stage of T cell activation.

J Mol Biol, 1993 Apr 5, 230(3), 890 - 918
Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin; Sixma TK et al.; Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 multimer structure, in which the B pentamer has a membrane binding function and the A subunit is needed for enzymatic activity . The LT crystal structure has been solved using a combination of multiple isomorphous replacement, fivefold averaging and molecular dynamics refinement . Phase combination using all these sources of phase information was of crucial importance for the chain tracing . The structure has now been refined to 1.95 A resolution, resulting in a model containing 6035 protein atoms and 293 solvent molecules with a crystallographic R-factor of 18.2% and good stereochemistry . The B subunits are arranged as a highly stable pentamer with a donut shape . Each subunit takes part in approximately 30 inter-subunit hydrogen bonds and six salt bridges with its two neighbors, whilst burying a large surface area . The A subunit has higher temperature factors and less well-defined secondary structure than the B subunits . It interacts with the B pentamer mainly via the C-terminal A2 fragment, which runs through the highly charged central pore of the B subunits . The pore contains at least 66 water molecules, which fill the space left by the A2 fragment . A detailed analysis of the contacts between A and B subunits showed that most specific contacts occur at the entrance of the central pore of the B pentamer, while the contacts within the pore are mainly hydrophobic and water mediated, with the exception of two salt bridges . Only a few contacts exist between the A1 fragment and the B pentamer, showing that the A2 fragment functions as a "linker" of the A and B parts of the protein . Interacting with the A subunit by the B subunits does not cause large deviations from a common B subunit structure, and the 5-fold symmetry is well maintained . A potential NAD(+)-binding site is located in an elongated crevice at the interface of two small sheets in the A1 fragment . At the back of this crevice the functionally important Arg7 makes a hydrogen bond connecting two strands, which seems to be conserved across the ADP-ribosylating toxin family . The putative catalytic residue (A1:Glu112) is located nearby, close to a very hydrophobic region, which packs two loops together . This hydrophobic region may be important for catalysis and membrane translocation.

J Pharmacol Exp Ther, 1993 Apr, 265(1), 16 - 23
Differential properties of pre- and postsynaptic 5-hydroxytryptamine1A receptors in the dorsal raphe and hippocampus: II . Effect of pertussis and cholera toxins; Blier P et al.; The injection of 1 microgram of pertussis toxin, which inactivates Gi/o proteins, in the rat dorsal raphe nearly abolished the responsiveness of serotonin (5-HT) neurons to microiontophoretic applications of 5-HT and selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) without altering their responsiveness to of gamma-aminobutyric acid (GABA) . In contrast, the injection of 1 microgram of cholera toxin, which causes an activation of Gs proteins, did not alter the responsiveness of 5-HT neurons to 5-HT, 8-OH-DPAT or GABA . Such in situ injection of either toxin in the dorsal hippocampus decreased by about 90% the responsiveness of CA3 pyramidal neurons to microiontophoretic applications onto their cell body of 5-HT and 8-OH-DPAT, but not of GABA . The effectiveness of the stimulation of the ascending 5-HT pathway in suppressing the firing activity of the same neurons, which results from the release of 5-HT at the level of their dendritic tree, was also markedly decreased in the cholera toxin-treated rats, but intriguingly not in the pertussis toxin-treated rats . These results indicate that, on 5-HT neurons, the somato-dendritic 5-HT1A autoreceptor is coupled to Gi/o, but insensitive to the persistent activation of Gs proteins . In the CA3 region of the hippocampus, there would be two subsets of postsynaptic 5-HT1A receptors on the pyramidal neurons: those apposed to 5-HT terminals on their dendritic tree (denoted intrasynaptic) and those located on their cell body (denoted extrasynaptic) . The former are cholera toxin sensitive, whereas the latter are sensitive to both pertussis and cholera toxins.

Scand J Immunol, 1993 Apr, 37(4), 452 - 8
Cholera toxin enhances alloantigen presentation by cultured intestinal epithelial cells; Bromander AK et al.; In the present study we show that cholera toxin (CT) strongly potentiates antigen presentation by intestinal epithelial cells, probably by enhancing co-stimulation . This was demonstrated in an allogeneic system using cells from the IEC-17 rat epithelial cell line as antigen presenting cells (APC) . These cells were induced by optimal concentrations of IFN-gamma to express good amounts of Ia antigen and cultured for 24-48 h in the presence or absence of CT . Thereafter the cells were thoroughly washed and added to cultures containing MHC-incompatible spleen cells as responder cells . Epithelial cells exposed to CT demonstrated greatly enhanced ability to trigger allogen-specific T-cell proliferation as compared with IEC-17 cells treated with IFN-gamma alone . The mechanism for the enhanced APC function was investigated by analysing CT-treated IEC-17 cells for increased class II MHC antigen expression or enhanced production of cytokines with known co-stimulatory function . We found no significant increase in class II MHC antigen expression . By contrast, CT strongly promoted, in a dose-dependent fashion, the production of both IL-1 and IL-6 cytokines by IEC-17 cells as compared with untreated epithelial cells . This effect of CT was specific and not due to contaminating endotoxin because excess amounts of soluble toxin receptor, ganglioside GM1, added to the IEC-17 cultures completely abrogated the cytokine response to CT . These results together with our previous findings of enhanced antigen presentation by macrophages stimulated by CT suggest that the potent adjuvant function of CT for induction of mucosal immune responses might be attributed to an enhanced co-stimulating ability of several putative APC in the mucosal immune system: macrophages, B cells and epithelial cells.

J Neurochem, 1993 Apr, 60(4), 1540 - 7
The effect of the B subunit of cholera toxin on the action of nerve growth factor on PC12 cells; Mutoh T et al.; Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF) . We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity . CTB treatment (1 micrograms/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation . CTB plus NGF also caused a greater inhibition of {3H}thymidine incorporation into DNA than did NGF alone . These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca2+ in the medium . 125I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells . These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.

Eur J Biochem, 1993 Apr 1, 213(1), 295 - 303
Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages . Differential receptor/G-protein interaction assessed by pertussis and cholera toxins; Levistre R et al.; The involvement of guanine-nucleotide-binding regulatory proteins (G proteins) in the regulation of arachidonic-acid release induced by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or platelet-activating factor (PAF) was examined in guinea-pig alveolar macrophages . We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet-Leu-Phe or PAF) and of GTP (or GTP{S}) . Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic-acid release and of the labeling of a 40-kDa protein in membranes incubated with {32P}NAD and pertussis toxin . fMet-Leu-Phe, but not PAF, allows the ADP-ribosylation of a 40-KDa protein by cholera toxin in the presence of Mg2+ . This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin . The 40-kDa protein ADP-ribosylated seems to be alpha i1 and/or alpha i2 . Stimulation of GTPase activity by fMet-Leu-Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin . Prior treatment of alveolar macrophages with cholera toxin, which ADP-ribosylates Gs, inhibits PAF-stimulated and fMet-Leu-Phe-stimulated arachidonic-acid release to the same extent, via a cAMP-protein-kinase-A cascade . The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet-Leu-Phe and PAF is associated with a strong increase of in-vitro {32P}NAD labeling of Gi proteins either by pertussis or by cholera toxin . This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein-phosphatase inhibitor, suggesting the involvement of protein-kinase A in this process . In conclusion, our results demonstrate that fMet-Leu-Phe and PAF receptors interact differently with Gi1/2 proteins in guinea-pig alveolar macrophages . Gi1/2 proteins are a possible target of the cross-regulation of arachidonic-acid release by a Gs-mediated pathway.

J Immunol, 1993 Apr 1, 150(7), 2599 - 606
Cholera toxin inhibits T cell receptor signaling by covalent modification of the CD3-zeta subunit; Haack BM et al.; In the Jurkat T cell line, triggering of the TCR leads to activation of phospholipase C, resulting in an increase in inositol trisphosphate (IP3) release followed by a rise in intracellular Ca2+ . This signaling pathway is interrupted by cholera toxin (CTX) treatment . To possibly explain this inhibition, we demonstrate that CTX can affect the TCR/CD3 complex itself by causing a covalent modification of the CD3-zeta subunit . After exposure of Jurkat cells to CTX, CD3-zeta increases its apparent m.w . and becomes more acidic in isoelectric focusing . The time course of the modification correlates well with the reduction in IP3 generation and Ca2+ release after CTX treatment, suggesting that the modification of zeta might be the cause of the impaired TCR/CD3 signaling . As is true for the CTX-mediated decrease in TCR signaling, the change in CD3-zeta was cAMP-independent and cannot be evoked by the enzymatically inactive CTX-B subunit alone.

Enfoque, 1993 Apr, 20(1), 12 - 4
{Cholera . Its nursing care}; Araujo MS et al.; Nursing care to cholera patients . Authors have described procedures used on treatment of cholera patients at Hospital Joao de Barros Barreto Belem-Para . They have shown how to send samples to lab tests, and treatment of contaminated hospital materials . They have also described how to make up a cholera treatment unit.

J Vet Med Sci, 1993 Apr, 55(2), 233 - 6
Reverse interference method for measurement of hog cholera virus (HCV) and anti-HCV antibody; Tsuchiya Y et al.; A new procedure was developed for the assay of the hog cholera virus (HCV) and anti-HCV antibody . Initially, the suppression effect of HCV on interferon (IFN) by HCV production was confirmed . Swine kidney cell cultures preinfected with HCV produced no IFN, even following the addition of IFN inducers . However the sensitivity of the cell to IFN was not influenced by the infection with this virus . Based on these results, a new method, named reverse interference method, was established . In this method, infective titer of HCV was determined by the appearance of cell pathogenic effects (CPE) induced by vesicular stomatitis virus (VSV), which is caused by the suppression effect on the heterologous interference of GPE- strain of HCV against VSV infection in swine kidney cell cultures . This method showed nearly the same sensitivity as the END method . There was no difference in the infective titer of HCV and antibody titer against HCV as estimated by this method and the END method . The reverse interference method had advantages in rapidity and objectivity compared with the END method.

Tidsskr Nor Laegeforen, 1993 Mar 30, 113(9), 1082 - 5
{Michael Sars and cholera in Manger 1849}; Sandvik H; In December 1848, cholera struck the city of Bergen, Norway . During the following months the distant parish of Manger was also affected . The local doctor was among the first victims, and although he survived, he was unable to take part in the fight against the epidemic . Instead, the local priest, Michael Sars (1805-69), took command of the situation . He organized new graveyards, materials for coffins, medicines, a cholera doctor from Bergen, and a local cholera hospital . Some of the peasants who were unwilling to comply with the regulations for burial of cholera victims were reported to the police . Michael Sars left the priesthood in 1854, and was appointed professor of zoology . He became one of Norway's most famous marine biologists.

Tidsskr Nor Laegeforen, 1993 Mar 30, 113(9), 1078 - 82
{The Board of Health and its activities during the cholera epidemic in Bergen in 1848-49}; Oeding P; In Norway temporary regulations were issued for control of cholera . An important provision was that Municipal Boards of Health should be appointed . The author describes the duties of the Board of Health, and how it functioned during the cholera epidemic in Bergen in 1848-49 . The cholera broke out on 10 December 1848, and a Board of Health was appointed exactly one week later . The Board held frequent meetings and the minutes provide good information on the development of the epidemic and the activities of the Board . The fact that the Chief Medical Officer was not a member of the Board was in violation of the regulations . However, he usually attended the meetings and strongly influenced the decisions . It may have been considered an advantage that the Chief Medical Officer acted relatively freely, even if the overall responsibility lay with the Board . This led, however, to some doubt about responsibility, and concerning what were the official channels.

Brain Res, 1993 Mar 12, 605(2), 214 - 20
mu and delta opioid agonists at low concentrations decrease voltage-dependent K+ currents in F11 neuroblastoma x DRG neuron hybrid cells via cholera toxin-sensitive receptors; Fan SF et al.; In a previous study, we showed that microM concentrations of mu or delta opioid agonists increase voltage-dependent outward K+ currents in neuroblastoma x DRG neuron hybrid F11 cells via pertussis toxin-sensitive receptors . The present study demonstrates that much lower concentrations (fM to nM) of these opioids (DAGO and DPDPE) decreased voltage-dependent outward K+ currents during step depolarization . The opioid antagonist, naloxone (3 nM) prevented these decreases in K+ current as did the cholera toxin subunits A or B (ca . 1 nM) . Furthermore, the specific mu opioid receptor antagonist, beta-funaltrexamine (5 nM) blocked the decrease by DAGO and the specific delta antagonist, naltrindole (1 nM) blocked that by DPDPE . Acute GM1 ganglioside (1 microM) treatment markedly enhanced the efficacy of opioid-induced decrease in K+ current . After treating the cells with pertussis toxin (1 microgram/ml) for 2 days or more, these opioids decreased the K+ current even when tested at concentrations as high as 1 microM . These results indicate that the decrease in K+ current elicited in F11 cells by low concentrations of mu and delta opioid agonists resembles the opioid-induced prolongation of the action potential duration and decrease in voltage-dependent K+ conductance that occur in DRG neurons in primary cultures . The F11 cell line provides therefore a valuable model system for correlative pharmacologic, electrophysiologic and biochemical analyses of Gs-coupled, GM1 ganglioside-regulated excitatory opioid receptor functions, in addition to G(i)/G(o)-coupled inhibitory receptor functions, in sensory neurons.

Immunology, 1993 Mar, 78(3), 498 - 504
An interspecies idiotope associated with the anti-cholera toxin response detected by a monoclonal auto-anti-idiotypic antibody; Lucas GP et al.; We have produced an auto-anti-idiotypic (auto-anti-id) monoclonal antibody (mAb) which reacted with syngenic mouse polyclonal anti-cholera toxin (anti-CT) IgG antibody (Ab) and six/eight different anti-CT IgG mAb, but not with normal mouse BALB/c IgG . The binding of this auto-anti-id mAb on one anti-CT mAb was significantly inhibited by polyclonal anti-CT sera of rats, rabbits and mice . Thus the idiotope on anti-CT Ab recognized by this auto-anti-id mAb was public and expressed in different species . Because of the absence of competition between CT and this auto-anti-id mAb for the binding to the six anti-CT mAb, the anti-id mAb was classified as an Ab2 alpha . The efficiency of this auto-anti-id mAb to induce anti-CT Ab3 was tested with success in rabbits and rats . Auto-anti-id mAb-immunized rats were significantly protected against an intraintestinal CT challenge.

Hawaii Med J, 1993 Mar, 52(3), 62 - 4
A rare case of cholera in Hawaii; Yamada GM; Cholera is the most fatal of the infectious diarrheas but only rarely encountered in Hawaii . Two cases previously have been documented in the Islands . We describe an elderly patient, without obvious risk factors, who contracted cholera . Early consideration of cholera as a diagnostic possibility is recommended in patients with unexplained, profuse diarrhea . The unique features of this case are discussed in this report.

Vet Microbiol, 1993 Mar, 34(3), 233 - 48
Detection of hog cholera virus antigens in experimentally-infected pigs using an antigen-capture ELISA; Shannon AD et al.; An antigen-capture ELISA was used to detect hog cholera virus (HCV) antigens in blood and tissues taken from pigs infected with 2 different strains of virus . Specific antigens were demonstrated in peripheral blood leucocytes (PBLs) and a wide range of tissue samples 4-6 days after infection of pigs with a moderate-high virulent HCV strain (Weybridge virus) . Strong signal to noise (S/N) ratios were obtained in the ELISA for PBLs and lymphoid tissues such as spleen, tonsil and mesenteric lymph nodes at 5-7 days after infection with the Weybridge virus, S/N ratios varying between 8.1-19.7 for blood samples and 4.3-19.1 for spleen samples . High positive ELISA results were also obtained for duodenum and ileum samples (S/N ratios 10.3-18.6) taken from these pigs, reflecting severe pathological changes observed in the gut at post mortem . In contrast, the antigen-capture ELISA gave strong positive results for PBLs and spleen samples only at 7-9 days after infection of pigs with a low virulent strain of HCV (New South Wales virus) . The ELISA S/N ratios averaged 9.5 for PBLs and 8.9 for spleen samples in these animals . Although virus isolation detected infection earlier in the infected pigs, the ELISA returned positive results on PBLs and spleen samples around the time all of the animals first showed typical signs of classical swine fever . The technique does not require tissue culture and takes less than 36 h to return a definitive result.

Exp Parasitol, 1993 Mar, 76(2), 182 - 91
Trichinella spiralis: the effect of oral immunization and the adjuvancy of cholera toxin on the mucosal and systemic immune response of mice; deVos T et al.; The effect of oral immunization and the adjuvancy of cholera toxin (CT) were examined in mice infected with Trichinella spiralis . The mean of total muscle larvae was reduced by 36% in mice infected with Trichinella larvae in combination with CT . In mice fed soluble, particulate, or soluble/particulate antigens in combination with CT on Days 0, 14, and 21, and challenged with Trichinella larvae on Day 28, there was a significant reduction in adult worm fecundity (50%), worm size (20-30%), and the mean of total muscle larvae (75%) but no apparent effect on the rate of expulsion on Day 6 postchallenge . Following antigen feeding, but prior to challenge with Trichinella (Day 28), the immunoglobulin response was positive in only a small proportion of mice . On Day 6 following challenge with Trichinella larvae, the biliary immunoglobulin response was enhanced approximately 10-fold (P < 0.05) in all antigen-feeding treatments which included CT, compared with nonimmunized controls or antigen-feeding treatments which did not include CT . Similarly, the serum IgG response was enhanced following challenge with Trichinella larvae in treatments fed soluble, particulate, or soluble/particulate antigens with CT . The response was also enhanced in the particulate, but not in soluble or soluble/particulate antigen treatments without CT.

J Clin Microbiol, 1993 Mar, 31(3), 565 - 8
Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach; Katz JB et al.; Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) are closely related pestiviruses . BVDV and BDV are found worldwide but seldom cause disease in swine . In contrast, HCV has been successfully eradicated from swine in several nations but poses a potentially devastating threat to them because of its great virulence . Rapid differential diagnosis of HCV from BVDV and BDV infections in swine is vital for detection of the possible reintroduction of HCV into national herds from which it has been eradicated . Nested polymerase chain reactions (PCRs) for each of two pestiviral genomic segments are described . Amplification of the relatively conserved 5' genomic terminus identified 59 of 61 HCV, BVDV, and BDV isolates generically as pestiviruses . Nested amplification of the second region was designed to differentiate HCV from BVDV and BDV by exploiting relatively conserved differences in the nucleotide sequences that encode the major envelope glycoprotein . This second PCR correctly identified 36 of 36 diverse HCV isolates while failing to recognize any of 25 BVDV and BDV isolates . Multiple restriction fragment length analyses confirmed the identities of both external and nested PCR products . The two sets of PCRs may help confirm the generic identity of most pestiviruses and may permit presumptive differential diagnosis of HCV from BVDV and BDV.

Peptides, 1993 Mar-Apr, 14(2), 297 - 301
In vivo inhibitory effect of lanreotide (BIM 23014), a new somatostatin analog, on prostaglandin- and cholera toxin-stimulated intestinal fluid in the rat; Botella A et al.; The antisecretory action of subcutaneously (SC) administered somatostatin(1-14), octreotide, and lanreotide on jejunal net flux of water under basal, prostaglandin E1 (PGE1)- and cholera toxin (CT)-stimulated secretory conditions was determined in vivo on isolated intestinal loop in anesthetized rats . Both PGE1 and CT induced intestinal hypersecretion in the rats . This secretory effect was not affected by SC administration of saline . Lanreotide (1, 10, and 100 micrograms/kg) reduced the maximal PGE1-induced secretion, while 200 micrograms/kg had no effect . Similarly, octreotide (1 and 10 micrograms/kg) and somatostatin (1-14) (0.1 and 1 microgram/kg) reduced the increase of net water flux induced by PGE1 . However, higher doses of octreotide (100 and 200 micrograms/kg) and somatostatin(1-14) (10 and 100 micrograms/kg) had no effect on PGE1-induced secretion . Lanreotide, octreotide, and somatostatin(1-14) (1 and 10 micrograms/kg) abolished the maximal secretion induced by cholera toxin . However, 100 micrograms/kg of lanreotide, octreotide, and somatostatin(1-14) had no effect on cholera toxin-induced secretion . The present study shows that lanreotide, octreotide, and somatostatin(1-14) reduce the secretion induced by PGE1 and abolish that induced by CT . These effects were obtained with doses of less than 100 micrograms/kg of the products, higher doses being ineffective . The higher efficacy against CT-induced hypersecretion as compared to PGE1-induced hypersecretion suggests a direct antisecretory effect at the enterocyte level and indicates the usefulness of these products as antidiarrheal agents in nonhormonally mediated diarrhea.

Endocr Res, 1993 Mar, 19(1), 33 - 46
The effect of isobutylmethylxanthine, forskolin, and cholera toxin on growth hormone release from pituitary cell cultures of perinatal and mature rats; Cuttler L et al.; The factors that regulate growth hormone (GH) release during the perinatal period are not well understood . Circulating GH levels are markedly elevated in mammalian fetuses and newborns compared with mature animals, and the immature pituitary is highly responsive to the GH-stimulatory effect of GH-releasing factor (GHRF) . The etiology of these developmental changes in GH secretion is not known . In order to investigate the mechanisms underlying GH release from immature pituitaries, we tested the effects of agents that increase intracellular cyclic adenosine 3',5' monophosphate (cAMP) production independent of the GHRF receptor on GH release from pituitaries of developing and mature rats . Pituitary cell cultures from fetal (day 20 of gestation), newborn (postnatal day 2), juvenile (postnatal day 12-15), adult male (3-4 months), and adult female (3-4 months) rats were tested with isobutylmethylxanthine (IBMX; 0.001-1.0 mM), forskolin (0.01-10 microM), and cholera toxin (0.025-25 ng/ml) . IBMX, forskolin, and cholera toxin stimulated GH release in a dose-dependent manner from pituitary cultures of all age groups . However, the magnitude of the GH responses to these agents was highly age-dependent . Perinatal pituitaries exhibited markedly greater GH responses to IBMX, forskolin, and cholera toxin than did those of mature animals (P < 0.001 for age effect with each agent) . GH release in response to the highest dose of IBMX (1 mM) was 301 +/- 8, 389 +/- 37, 296 +/- 33, 198 +/- 14, and 187 +/- 19% of control values from pituitary cell cultures of fetal, newborn, juvenile, adult male, and adult female rats, respectively (P < 0.001) . In response to the highest dose of forskolin (10 microM) GH release was 537 +/- 46, 601 +/- 75, 274 +/- 22, 270 +/- 37, and 248 +/- 35% of control values in the same respective age groups (P < 0.001) . Similarly, the highest dose of cholera toxin (25 ng/ml) stimulated GH release to 407 +/- 55, 365 +/- 43, 249 +/- 26, 186 +/- 11, and 186 +/- 1% of controls in these respective age groups (P < 0.003) . The marked stimulation of GH release from perinatal pituitaries by IBMX, forskolin, and cholera toxin is consistent with the concept that cAMP is a potent mediator of GH release from immature as well as mature somatotrophs . The developmental changes in the GH secretory response to these agents further suggest that signal transduction pathways mediating GH release may undergo maturation, at least in part, at intrasomatotroph loci distal to the GHRF receptor.

MMWR Morb Mortal Wkly Rep, 1993 Feb 12, 42(5), 89 - 91
Update: cholera--Western Hemisphere, 1992; Synergistic effect of cholera toxin with cyclosporine and azathioprine on survival of rat renal allografts; Department of Urology, National Defense Medical College, Saitama, JapanA combination of 10 mg/kg/d of AZA and 0.1 mg/kg/d of CT significantly prolonged graft survival (P < .05) over 10 mg/kg/d of AZA alone . A combination of 2.5 mg/kg/d of CyA and 0.1 mg/kg of CT given only once significantly (P < .05) prolonged graft survival over 2.5 mg/kg/d of CyA alone.

Infect Immun, 1993 Feb, 61(2), 384 - 90
Reduction in oral immunogenicity of cholera toxin B subunit by N-terminal peptide addition; Dertzbaugh MT et al.; The mucosal adjuvanticity of cholera toxin and the potential of the B subunit of cholera toxin (CtxB) to serve as an oral vaccine carrier have prompted interest in the coupling of immunogenic peptides to this protein . The purpose of this study was to determine how such fusions affect the function of CtxB . Oligonucleotides were genetically fused to the 5' terminus of the ctxB gene to encode additional amino acids of 8, 12, and 24 residues in length . None of these additions affected the ability of CtxB to oligomerize, as determined by nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Circular dichroism revealed no difference in conformation between the modified B subunits, regardless of the length of the addition . However, when compared with native CtxB, additions to the N terminus induced a consistent change in the net conformation of the protein . By using a competitive enzyme immunoassay, the affinity of the modified B subunits for GM1 ganglioside was shown to gradually decrease with increasing length of the N-terminal addition . A similar pattern was observed for the ability of the chimeras to inhibit proliferation of concanavalin A-stimulated spleen cells in vitro, which is a previously described functional property of CtxB that is dependent on its binding to cells . Lastly, the oral immunogenicity of these chimeras was found to be less than that of native CtxB . These results indicate that large fusions to the N terminus of CtxB can significantly affect its biological properties and could reduce its value as a mechanism for effective mucosal immunization.

Zentralbl Veterinarmed B, 1993 Feb, 40(1), 46 - 54
Clinical, virological and serological findings after intranasal inoculation of pigs with bovine viral diarrhoea virus and subsequent intranasal challenge with hog cholera virus; Dahle J et al.; Five groups of weaner pigs were intranasally inoculated with constant doses of bovine viral diarrhoea virus (BVDV) strain OSLOSS/2482 . Four weeks post primary inoculation (p.p.i.) the animals were intranasally challenged with decreasing doses of hog cholera virus (HCV) strain Alfort/187 . Clinical signs were not observed apart from a short febrile period (2 days, > 40 degrees C) in one animal . Another animal died intercurrently without showing any pathological signs . Virus isolation from leucocyte samples taken regularly during one week post challenge detected HC viraemia in most animals that had received HCV doses > 100 TCID50 per animal . Using monoclonal antibody (mab) analysis all isolates obtained were proven to be HCV . Serological investigations using the virus neutralization test (VNT) yielded HC neutralizing antibodies in all groups with higher titres in those animals having received HCV doses > 100 TCID50 . However, HCV specific neutralizing antibodies never exceeded the BVDV antibody titre . A complex trapping blocking (CTB) ELISA applying a HCV specific mab detected HCV specific antibodies in animals that had gone through HC viraemia while discriminating BVDV specific antibodies.

Br J Cancer, 1993 Feb, 67(2), 279 - 83
Inhibition of human pancreatic cancer cell (MIA PaCa-2) growth by cholera toxin and 8-chloro-cAMP in vitro; Ohmura E et al.; The effects of cholera toxin (CT) and 8-chloro-cAMP (8-Cl-cAMP) on cell growth were investigated using two human pancreatic carcinoma cell lines (MIA PaCa-2, Panc-1) . CT, which catalyses the ADP ribosylation of Gs, suppresses the proliferation of MIA PaCa-2(PC) cells . CT at the low dose of 0.1 pg ml-1 was inhibitory of PC cell growth, and the maximum suppression (70%) was achieved at a CT concentration of 100 pg ml-1 . This phenomenon was reversible . The production of cAMP by CT (100 pg ml-1) in PC cells was enhanced 320-fold compared with the control . In addition, cAMP analogues (8-Cl-cAMP, 8-Br-cAMP) and forskolin decreased the growth rate of PC cells in a dose-dependent manner . These results support the view that CT suppresses PC cell growth by stimulating cAMP production . Conversely, Panc-1 cells were far less sensitive to CT in cell growth and cAMP production . 8-Cl-cAMP was also less effective on Panc-1 cell growth . The binding of an insulin-like growth factor (IGF)-I and transforming growth factor (TGF)-alpha, which has been shown to stimulate PC cell growth in an autocrine manner, to PC cells was not modified in cells treated with CT or 8-Cl-cAMP . The results suggest that the inhibitory actions of these substances do not occur at the level of the receptor for IGF-I or EGF/TGF-alpha . We have previously shown that phorbol esters, which decrease the binding of TGF-alpha to PC cells, has an anti-proliferative activity on these tumour cells . Inhibited cell growth by maximum suppressive dose of CT or 8-Cl-cAMP was further inhibited by TPA . In addition, an oncogene product of K-ras which is commonly activated in pancreatic cancer, was increased by CT and 8-Cl-cAMP . It is concluded that CT and 8-Cl-cAMP inhibit PC cell growth, presumably in a similar manner, and their mechanism(s) of action may be different from that of TPA . The anti-proliferative effect of CT or 8-Cl-cAMP was enhanced by TPA, implying that the combination of these substances results in increased inhibition of the PC cell growth.

J Mol Biol, 1993 Jan 20, 229(2), 286 - 90
New approach for atomic force microscopy of membrane proteins . The imaging of cholera toxin; Yang J et al.; We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer . A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol % ganglioside (GM1) was transferred onto the surface of mica from a Langmuir trough . Cholera toxin, both the B-subunit and the complete molecular randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm . The pentameric structure of the B-subunit oligomers was well resolved . This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Biochem J, 1993 Jan 15, 289 ( Pt 2), 469 - 73
Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors; Schepers TM et al.; N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes . Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation . The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested . The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits . Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5'-{beta gamma-imido}triphosphate and GDP in a concentration-dependent manner . Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide . In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect . Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5'-{gamma-thio}triphosphate and in the absence of guanine nucleotides . Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP . These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.

Eur J Pharmacol, 1993 Jan 5, 230(1), 95 - 7
Cholera toxin but not pertussis toxin inhibits angiotensin II-enhanced contractions in the rat portal vein; Zhang J et al.; Angiotensin II (Ang II)-enhanced phasic contractions in the rat portal vein were concentration dependently inhibited by cholera toxin (0.1-10 micrograms/ml) and dibutyryl cyclic AMP (0.1-1 mM), but not by pertussis toxin (1 micrograms/ml), which suggests that Gi is not involved in the Ang II signal transduction pathway . It also seems likely that the effect of cholera toxin is due to its ability to increase cyclic AMP production through Gs.

Nippon Jibiinkoka Gakkai Kaiho, 1993 Jan, 96(1), 66 - 76
{Autonomic neurons sending fibers into the canine laryngeal nerves--using a retrograde tracer technique with cholera toxin}; Uno T; The distribution of the autonomic neurons sending fibers into the canine superior and inferior laryngeal nerves was investigated by an immunohistochemical technique with cholera toxin B subunit as a retrograde tracer . Cholera toxin was applied to the right internal branch of the superior laryngeal nerve (SLNI), external branch of the superior laryngeal nerve (SLNE) or inferior laryngeal nerve (ILN) in 2-month-old dogs . In every case of cholera toxin application, labeled neurons were seen mainly in the caudal portion of the ipsilateral superior cervical ganglion, whereas none were seen in the stellate ganglion . The number of labeled neurons in the superior cervical ganglion after application of cholera toxin to the SLNI was over 20 times that observed after application to the SLNE or ILN . When cholera toxin was applied to the SLNI or SLNE, labeled neurons were found mainly in the ipsilateral dorsal motor nucleus of the vagus (DMNV), and a few labeled neurons were found in the ipsilateral reticular formation . The neurons in the DMNV were localized, on the average, between 2.7 mm and 5.3 mm rostral to the obex . The number of neurons in the DMNV labeled retrogradely from the SLNI was much larger than that from the SLNE . In contrast, no labeled neurons were detected in the DMNV after application to the ILN . These results demonstrate the following: 1) The sympathetic neurons innervating the canine larynx are distributed mainly in the caudal portion of the superior cervical ganglion and they send their fibers mainly into the SLNI . 2) The parasympathetic neurons innervating the canine larynx have a limited distribution in the dorsal motor nucleus of the vagus and their main pathway is also the SLNI.

Acta Otolaryngol, 1993 Jan, 113(1), 43 - 7
Cholera toxin B-HRP and wheat germ agglutinin-HRP tracing of tensor tympani muscle motoneurons and processes in rabbits; Counter SA et al.; The brain stem position, organization and number of motoneurons innervating the rabbit tensor tympani muscle (TTM) were determined by retrograde axonal transport of cholera toxin B/horseradish peroxidase conjugate (CTB-HRP) and wheat germ agglutinin HRP conjugate (WGA-HRP) tracers . The synaptic input to the TTM motoneurons was examined with WGA-HRP . The results show the motoneurons of the TTM to be localized in a cluster ventro-lateral to the outer margin of the ipsilateral trigeminal motor nucleus (VMN) and dorso-lateral to the superior olive . The number of labeled cells was greater in the combined CTB-HRP/WGA-HRP injected cases . The TTM motoneurons were triangular and elongated in shape and smaller than those of the VMN . An extensive network of dendritic branches was present ventro-laterally in the vicinity of the superior olive . Similar, but less extensive collections of dendritic processes were observed to course dorso-medially, rostrally and caudally . Axons were observed to project first dorsally or laterally, towards the trigeminal motor root, then after a sharp turn coursed ventrally within the trigeminal motor root (VMR) . Transneuronal transport of the WGA-HRP was not accomplished in any preparation, suggesting among other things, system or species differences in the effectiveness of the WGA-HRP conjugate as a transynaptic tracer . It is concluded that the TTM acoustic reflex in rabbits and other mammals, its threshold, prolonged contraction capacity, and its influence on middle ear sound transmission may be related to its demonstrated extensive synaptic field in the reflex chain, particularly in the area of the superior olive, while its many other physiological functions may be made possible by the number, location, and multi-dimensional orientation of its motoneurons and dendrites.

Vaccine, 1993, 11(2), 119 - 21
Circulating cellular immune response to oral immunization of humans with cholera toxin B-subunit; Lewis DJ et al.; Peripheral blood mononuclear cells were taken from subjects before and after oral immunization with cholera toxin B-subunit . Cells obtained from naive volunteers before immunization did not proliferate in vitro to B-subunit . Oral immunization induced a proliferative response in all volunteers with a peak stimulation index of 20, and was detected up to 1 year later . The proliferative response kinetics suggest the appearance in the blood of primed T cells from the gut coinciding with the disappearance of primed plasmablasts from the circulation, supporting the concept of a common mucosal immune system in man for T and B cells.

Hear Res, 1993 Jan, 64(2), 151 - 65
Localization of the GM1 ganglioside in the vestibular system using cholera toxin; Mancini P et al.; Cholera toxin is an ubiquitous activator of intracellular adenylate cyclase and is divided in two major components: A and B . The B-component consists of several subunits that specifically bind to the external cell membrane . The receptor for the toxin, the GM1 ganglioside, is concentrated in nervous tissues . The B subunit of the cholera toxin, conjugated to different molecules (i.e., choleragenoid) is therefore a sensitive anatomical tracer and has been used to detect the presence of GM1 in mammalian tissues . Using choleragenoid, unlabeled and labeled with FITC, we have determined the distribution of the GM1 ganglioside in the vestibular system of the chinchilla . Vestibular tissues were fixed in 4% paraformaldehyde in phosphate buffer, decalcified in 10% EDTA and prepared as either whole-mount, surface-preparations, or for radial cryosections . Positive control tissue consisted of binding to normal brain tissues . Negative controls consisted of several treatments: masking of the GM1 receptors with unlabeled choleragenoid, tissue extraction of GM1 using ethanol, and preabsorbing the choleragenoid with bovine GM1 . In addition, to exclude staining of glycoproteins that may have a carbohydrate structure similar to GM1, tissues were digested with trypsin prior to choleragenoid exposure . In the vestibular system, a strongly positive reaction was observed in: the sensory stereocilia and supporting cells of the maculae and cristae, epithelial cells of the planum semilunatum, and polygonal cells of the semicircular canal . Positive but less strong reactivity was observed in the sensory cell body of maculae and cristae, nerve fibers, epithelial cells of utricle and ampulla walls and flattened epithelial cells of the semicircular canals . No reactivity was present in the supporting connective tissue cells and fibrils, blood vessels, gelatinous cupula of the cristae ampullaris and statoconial membranes . Brain tissue showed strong choleragenoid reactivity . The negative controls showed no or greatly reduced reactivity to choleragenoid . Trypsin digestion did not decrease reactivity to choleragenoid.

Am J Physiol, 1993 Jan, 264(1 Pt 1), G86 - 94
Cholera toxin-sensitive neurons in guinea pig submucosal plexus; Jiang MM et al.; Cholera toxin (CT) increases intestinal secretions by direct stimulation of mucosal enterocytes; enteric neurons also may play a role . We tested the latter possibility by retrograde labeling of mucosal terminals in guinea pig small intestine with the B subunit of CT (B-CT) and by intracellular recordings from submucosal neurons during superfusion with CT . All vasoactive intestinal peptide (VIP)-positive neurons, and only VIP-positive neurons, were labeled with B-CT . Fluorogold (FG) was used to retrogradely label nerve terminals in submucosal arterioles in preparations in which B-CT labeled mucosal terminals; colocalization of B-CT with FG was observed in neurons up to 3 mm from the site of FG application . CT selectively depolarized neurons known to contain VIP . We conclude that all VIP-containing neurons, and only VIP neurons, in guinea pig submucosal plexus possess B-CT binding sites and can be activated by CT . Some of these neurons provide a dual innervation to both arterioles and mucosa . We suggest that one functional consequence of CT may be to activate vasodilator nerves, thus increasing vascular perfusion of the mucosa to further stimulate intestinal secretions.

Arch Virol, 1993, 132(3-4), 429 - 35
Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus; Muyldermans G et al.; After amplification by PCR, the 5' region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced . The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses . By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.

Vaccine, 1993, 11(2), 113 - 8
Adjuvant action of cholera toxin and pertussis toxin in the induction of IgA antibody response to orally administered antigen; Wilson AD et al.; The ability of cholera toxin B-subunit to bind to intestinal epithelium and in particular the dome epithelium of the Peyer's patch can account for its potency as an immunogen . However, pure B-subunit is a less effective immunogen than whole cholera toxin . The immunogenicity of B-subunit may be restored by the addition of either traces of whole toxin or by pertussis toxin . Cholera toxin and pertussis toxin were both able to stimulate a response when fed in conjunction with keyhole limpet haemocyanin, whereas recombinant B- subunit of heat-labile toxin from Escherichia coli had no effect, demonstrating that the adjuvant action is a property of the enzymically active A-subunits . The adjuvant activity of both pertussis toxin and cholera toxin may be due to their ability to cause an increase in the activity of adenylate cyclase via their action on GTP-binding regulatory proteins . However, feeding of forskolin, a direct activator of adenylate cyclase, had no effect on the mucosal immune response, indicating a role for cholera and pertussis toxin which is independent of enhancement of adenylate cyclase activity in the regulation of the immune response . Antibody to pertussis toxin was not detected, which was attributed to inadequate absorption of pertussis toxin.

Microbiol Immunol, 1993, 37(4), 317 - 23
Cholera toxin inhibits lethal hit stage of natural killer cell-mediated cytotoxicity; Watanabe M et al.; Cytolytic process which was affected by cholera toxin (CT) resulting in the loss of natural killer (NK) cell activity was analyzed . Conjugate formation assay, membrane phospholipid methylation assay and serine esterase (granzyme A) release assay were used to determine the stage of the CT-induced inhibition of NK cell-mediated cytotoxicity . A human NK cell line YT cell-mediated cytotoxicity was completely abolished by CT pretreatment or addition of CT to the assay system . The conjugate formation assay revealed that the binding between YT cells and target cells was not affected by CT . The defined triggering stage which is coupled with membrane phospholipid methylation was not affected by CT treatment, either . On the other hand, the lethal hit stage which is represented by serine esterase (SE) release was completely inhibited by CT treatment of YT cells . Therefore, CT inhibits the stage after binding and triggering--i.e., lethal hit stage of NK cell-mediated cytotoxicity . The results also suggest that there exists a CT-sensitive negative cytotoxic signal transduction pathway as well as usual positive signal transduction pathway and these pathways might cross talk each other in the NK cell cytotoxic process.

Reg Immunol, 1993 Jan-Feb, 5(1), 60 - 7
Regional dichotomy of interleukin-4 and -5 regulation of senescent B cell responses specific for cholera toxin in Peyer's patches, lamina propria, and mesenteric lymph nodes; Haq JA et al.; The capacity of exogenous interleukin-4 (IL-4) and IL-5 to augment the antigen-specific senescent Peyer's patch (PP), lamina propria (LP), and mesenteric lymph nodes (MLN) B cells was investigated in the present studies . CTx-primed lymphocytes in the PP, LP, and MLN were obtained from 4-and 24-month old C57BL/6J male mice, 14 days after oral immunization with cholera toxin (CTx) . Cells were cultured optimally for 4 days either alone, with lipopolysaccharide (LPS), with LPS plus recombinant IL-4 or IL-5, or with LPS plus IL-4 or IL-5 plus monoclonal antibodies specific for the respective interleukin . Culture supernantants were tested for IgA and IgG anti-CTx antibodies by an ELISA assay . The data indicated impaired antibody responses of aged PP and MLN B cells by the lack of LPS plus IL-4 induced enhancement of anti-CTx IgG antibody production as compared with the young group . For anti-CTx IgA production, aged LP and MLN B cells did not respond equally as well to LPS plus IL-5 stimulation as the young group . In contrast, aged PP B cells responded equally as well by anti-CTx IgA production to LPS plus IL-5 stimulation as the young group . Both anti-IL-4 and anti-IL-5 Mabs blocked the respective IL-4 and IL-5-induced enhancement of antigen-specific IgG or IgA antibody production . The findings suggest a regional dichotomy of IL-5 induced enhancement of aged IgA-producing B cells specific for CTx in the Peyer's patches, mesenteric lymph nodes, and lamina propria.

Arch Virol, 1993, 131(3-4), 475 - 81
The skin, tongue, and brain as favorable organs for hog cholera diagnosis by immunofluorescence; Pan IC et al.; Hog cholera virus antigens were found densely distributed in skin and tongue of pigs experimentally infected with hog cholera virus . The finding described here warrants the usage of ear biopsies for hog cholera diagnosis on a herd basis.

Bull Pan Am Health Organ, 1993, 27(4), 331 - 6
Private sector response against the cholera threat in Trinidad and Tobago; Hospedales J et al.; During the first half of 1992 the threat of cholera to Trinidad and Tobago prompted a strong health education effort by public authorities and the private sector . To help assess the private sector effort, the cost of cholera-related advertisements and private announcements placed in the country's two leading newspapers during January-June 1992 were reviewed . The review indicated that an estimated TT$ 540,660 was spent on these ads and announcements, that they contributed strongly to keeping cholera prevention continuously in the public eye, and that most of the messages published were accurate, specific, and safe . The strength and success of the private contribution to cholera prevention in this case suggests that similar approaches could be applied to other health problems and to the cholera problem outside Trinidad and Tobago . Overall, the lesson appears to be that if one can find congruence between private sector motives and public health interests, then the potential prospects for a successful partnership are great.

Bull Pan Am Health Organ, 1993, 27(4), 313 - 30
Waterborne cholera in Riohacha, Colombia, 1992; Cardenas V et al.; Between 1 January and 31 July 1992 a cholera epidemic caused 548 reported cases (an incidence of about 8 cases per 1,000 inhabitants) in Riohacha, Colombia . Following an initial review of hospital and laboratory data, a cross-sectional household survey and case-control study were conducted to investigate this epidemic . The cross-sectional survey found an increased risk of cholera between November 1991 and September 1992 among subjects who usually drank unchlorinated piped water from the municipal water system (prevalence odds ratio, POR = 5.7; 95% confidence interval, CI = 1.2-41.1), as well as an increased risk of acute diarrheal disease in the 2 weeks preceding the survey interview among these same subjects (POR = 3.3; 95% CI = 1.1-11.2) . The case-control study revealed an association between cholera and drinking unboiled tap water (OR = 7.2; 95% CI = 1.6-32.2), and also between cholera and limited availability of water (< 1,400 liters per week) within the household (OR = 3.6; 95% CI = 0.8-16.4) . These findings strongly suggest that most of the Riohacha cholera cases were transmitted by contaminated municipal water, a conclusion supported by descriptive evidence of problems affecting Riohacha's municipal water and sewerage systems.

Vaccine, 1993, 11(9), 929 - 34
Intestinal antibody response after oral immunization with a prototype cholera B subunit-colonization factor antigen enterotoxigenic Escherichia coli vaccine; Ahren C et al.; A prototype oral enterotoxigenic Escherichia coli (ETEC) vaccine containing formalin-inactivated whole bacteria expressing colonization factor antigens CFA/I and CFA/II and cholera B subunit (CTB) has been tested for safety and immunogenicity in 20 adult Swedish volunteers . When given in three doses with 2-week intervals the vaccine was found to be safe and to give rise to specific IgA antibody responses in intestinal lavage fluid in most of the volunteers (CFA/I 82%, CFA/II 82% and CTB 91%) . The frequencies and magnitudes of these responses, which were already maximal after two doses, were comparable with those previously found in patients convalescing from severe ETEC diarrhoea . All the vaccinated volunteers also responded with antitoxin IgA as well as IgG antibodies in serum, whereas the serum antibody responses against the CFAs were weaker and mainly of the IgA isotype.

Trop Geogr Med, 1993, 45(6), 269 - 73
Cholera: developments in prevention and cure; van Loon FP; The current pandemic of cholera coincides with a flurry of research activities in cholera prevention and cure . This article highlights important developments in cholera research, including key findings of a large-scale oral cholera vaccine trial, new insights in cholera epidemiology and risk factors, and recent directions in rehydration therapy and medical treatment of cholera.

Jpn J Physiol, 1993, 43(4), 553 - 60
Modification of Ca(2+)-sensitivity of Ca(2+)-activated Cl- channel by vasopressin and cholera toxin; Marunaka Y; The apical membrane of renal epithelium (A6) had a Ca(2+)-activated, outwardly rectified chloride channel with the single channel conductance of 3 pS for outward current and 0.5 pS for inward current . The outward rectification was dependent on the cytosolic Ca2+ concentration . Vasopressin (arginine vasopressin, AVP) and cholera toxin (CTX) increased the single channel conductance for inward current through an increase in the Ca(2+)-sensitivity about 100-fold.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1993, 64(6), 345 - 50
Effects of pertussis and cholera toxin on the interferon-gamma stimulated immunocytochemical staining of ICAM-1 and inositol phosphate formation in a human renal carcinoma cell line; Hansen AB et al.; We have recently shown that interferon-gamma (IFN-gamma) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1 . In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-gamma signalling . Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-gamma induced ICAM-1 staining . Preincubation with PT or CT for 24 h also inhibited IFN-gamma induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation . Our findings suggest that IFN-gamma induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway . This may reflect a role for G-proteins in the coupling of IFN-gamma receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.

Arch Virol, 1993, 131(3-4), 405 - 17
Characterization of structural and non-structural proteins of hog cholera virus by means of monoclonal antibodies; Muyldermans G et al.; A panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays . Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized . One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV . The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance . On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions . The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.

Arch Dermatol Res, 1993, 285(1-2), 27 - 31
Immunohistochemical study of cell proliferation and differentiation in epidermis of mice after administration of cholera toxin; Rahman SA et al.; Cholera toxin causes reversible epidermal hyperplasia . We observed maximal thickness of the epidermis on the fourth day after treatment and a return to pretreatment values by day 7 . The increase in thickness occurred in the basal and intermediate layers, with these layers becoming two to three times thicker than those of normal epidermis . The time sequence of epidermal proliferation was studied using bromodeoxyuridine (BrdU) labelling . We observed a maximum number of labelled basal cells within the first 24 h . Only a few cells were labelled 7 days after toxin injection . Griffonia simplicifolia-IB4 (GSA-IB4), Ulex europaeus-I (UEA-I) and Griffonia simplicifolia-II (GSA-II) lectins were used for the analysis of epidermal cell differentiation in the tissue sections . To study keratinocyte differentiation, further immunological staining was performed using two anticytokeratin antibodies, PKK2 and PKK3 mouse monoclonal antibodies . From the immunocytochemical results, we conclude that synchronous differentiation of the epidermis occurs after cholera toxin administration.

Vaccine, 1993, 11(2), 235 - 40
Current progress in the development of the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as carriers for the oral delivery of heterologous antigens and epitopes; Nashar TO et al.; The development of non-living carrier systems for delivery of protective antigens or epitopes to the immune system represents both a fundamental and an applied aspect of vaccinology . A wide range of carrier systems, ranging from inert supports to proteins that exert direct immunomodulating effects on the immune response, are being studied . In this overview we describe the current progress in the development of the B-subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as potential protein carriers for the oral delivery of chemically and genetically attached antigens and epitopes.

J Toxicol Environ Health, 1993 Jan, 38(1), 1 - 18
Acute-phase plasma protein response to cholera intoxication in healthy and diabetic rats; Fouad FM et al.; The aim of the present study is twofold: to establish the response of hepatic machinery of plasma protein biosynthesis to cholera intoxication, and to examine the same response of alloxan-diabetic hepatocytes with minimal capacity of synthesis of plasma proteins . Direct lesion of hepatic plasma membranes via ip administration of cholera toxin to male rats resulted in a typical acute-phase response (APR) of plasma proteins, which had regressed to levels similar to those of healthy controls approximately at 240 h postintoxication . The d 2 response to a single 0.16 mg/kg body weight dose was typified by a 23% reduction in the level of albumin, but a 6- and 24-fold increase in the levels of fibrinogen and alpha-1-acid glycoproteins, respectively . This response was similar (in direction but not in magnitude) to the acute-phase reaction to a simple subcutaneous administration of carrageenan . The intoxication was accompanied by a massive leakage, into the peritoneal cavity, of plasma fluid, which embraced the complete profile of acute-phase reactants . A three-step mechanism is proposed to account for the observations as follows: (1) There is a rapid formation of a stable complex between subunit B of the toxin and ganglioside GM1 of hepatic plasma membrane . An APR is induced in response to the alteration(s) of hepatic plasma membranes . (2) The release, from the choleragen-membrane complex, of polypeptide A1 and its subsequent penetration of the hepatic membrane result in both activation of adenylate cyclase and increased vascular permeability of hepatic membranes . This leads, in turn, to exudation of components of plasma fluid in the peritoneal cavity of intoxicated rats . An alternate rationale for this exudation is the slow leakage of plasma proteins out of the blood vascular system (possibly through microvesicles) into the peritoneal cavity of cholera intoxicated rats . The spectrum of acute-phase hepatic secretory components was mirrored in the corresponding peritoneal exudate . (3) The increased hepatic membrane flow provides the continued renewal of plasma membrane proteins required for its eventual repair by either endocytosis or sloughing off the toxin-bound membrane segments into the circulatory system, thus producing regression of APR . Livers of diabetic rats, an already established model in terms of APR, responded to ip administration of cholera toxin by increased biosynthesis of the identified plasma proteins and a marked reduction in total free-glucose in serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Immunol, 1992 Dec, 22(12), 3179 - 82
Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit; Pierre P et al.; Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally . Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline . Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses . Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).

Kansenshogaku Zasshi, 1992 Dec, 66(12), 1645 - 50
{The first cholera case diagnosed early in the clinical laboratory by DNA probe method}; Miyagi K et al.; An alkaline-phosphatase-labelled synthetic oligonucleotide probe was applied to detect the structural gene of cholera enterotoxin (ctx) . This DNA probe has a complementary base sequence to 30 base of the CT-A subunit . This method was, for the first time, applied to diagnosis of a diarrheal patient . The probe detected ctx rapidly and simply as compared with reversed passive latex agglutination (RPLA) and Beads-ELISA . The cfu minimal dose for detection with the probe was about 10(6-7)/ml . This method can be easily performed in any clinical laboratory because the procedure is safe, simple and rapid (it can be completed within about 3 hours).

Eur J Biochem, 1992 Nov 15, 210(1), 33 - 44
Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation; Bornancin F et al.; Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively . These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes . Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-{gamma-thio}triphosphate (GTP{gamma S}), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP . ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha . Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP{gamma S} in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP{gamma S} and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive . Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)

J Neurosci Res, 1992 Nov, 33(3), 466 - 75
Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates intracellular free calcium in sensory neurons; Milani D et al.; The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types . To explore a role for such changes in calcium in the growth regulatory function of cell-associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit . To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 microM) and then exposed to B subunit . Incubation of naive cultures with 1 microgram/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura-2 fluorescence imaging . Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure . These increases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers . Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1 . However, the morphological changes did not result in altered neurofilament protein expression . Immunogold electron microscopy using anti-choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1 . The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types.

Arch Surg, 1992 Nov, 127(11), 1330 - 4
Cholera toxin pretreatment protects against tumor necrosis factor lethality without compromising tumor response to therapy; Block MI et al.; Antitumor therapy with tumor necrosis factor is limited by systemic toxic effects . We studied whether cholera toxin, a bacterial exotoxin that adenosine diphosphate-ribosylates the alpha-subunit of Gs proteins, could separate the lethal from the antitumor effects of tumor necrosis factor . A single dose of intravenous cholera toxin protected non-tumor-bearing mice from a lethal dose of Escherichia coli endotoxin administered 6 or 24 hours later . On the basis of these results, tumor-bearing mice were randomized to receive either cholera toxin or saline, followed 6 hours later by either human tumor necrosis factor (400 micrograms/kg) or saline . Tumor-bearing mice pretreated with cholera toxin had (1) reduced treatment-related mortality (0/11 vs 5/11 for saline controls) and (2) tumor regression similar to that of controls . In a separate experiment in tumor-bearing mice, intravenous human tumor necrosis factor treatment induced an increase in serum levels of murine tumor necrosis factor to a peak of 500 pg/mL at 1 hour in saline-pretreated controls, while a similar increase could not be detected in those mice pretreated with cholera toxin . These results suggest that pretreatment with cholera toxin can reduce the endogenous tumor necrosis factor response to administered tumor necrosis factor and separate the lethal from the antitumor effects . Cholera toxin may prove to be a useful tool for investigating the mechanisms underlying the varied effects of tumor necrosis factor.

Fundam Appl Toxicol, 1992 Nov, 19(4), 575 - 83
Suppression of the murine gut mucosal IgA response to cholera toxin with oral cyclosporine; Kawabata TT et al.; The gut mucosal immune system may be a primary target for many ingested chemicals . Methods have been developed to examine the effects of chemicals on the systemic humoral immune response; however, studies to evaluate various methods of assessing the local gut mucosal immune response in a toxicology assay have been limited . The objectives of this study were to examine the effects of the known immunosuppressive compound, cyclosporine (CYS), on the generation of a cholera toxin (CT)-specific gut mucosal IgA response and evaluate the methods used to measure the gut IgA response . Groups of female B6C3F1 mice were left untreated or were treated daily, p.o., with corn oil (vehicle) or CYS at doses of 10 and 50 mg/kg for 20 days . On Days 3 and 13, mice were sensitized p.o . with CT . On Day 21, mice were terminated, gut washings were collected, and lamina propria lymphocytes were extracted from gut tissue with collagenase treatment . Cholera toxin-specific IgA in the gut washings was measured by an ELISA . The numbers of CT-specific IgA (CT-IgA) and total IgA antibody-forming cells (spot-forming cells, SFC) obtained from the lamina propria were determined by the ELISPOT method . A dose of 50 mg/kg CYS produced a significant decrease in the amount of CT-IgA in gut washings . This dose also decreased the number of cells recovered from the lamina propria by at least 50% . The amount of CT-specific SFC/million lamina propria cells decreased with a dose of 10 mg/kg CYS, whereas 50 mg/kg CYS did not alter the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Signal, 1992 Nov, 4(6), 619 - 25
Inhibition of oxytocin-stimulated phosphoinositide turnover in rat myometrium by pertussis and cholera toxins may involve protein kinase A activation; Singh SP et al.; Both pertussis and cholera toxins inhibit oxytocin-stimulated phosphoinositide turnover in rat myometrium . The actions of pertussis and cholera toxins as well as those of CPTcAMP are reversed by H-8, an inhibitor of protein kinase A . H-8 does not have a major effect on cAMP elevation by the toxins in the presence of oxytocin . The results suggest that the stimulation by oxytocin of phosphoinositide turnover does not involve direct obligatory coupling to a pertussis toxin-sensitive GTP-binding protein . Rather, indirect effects on protein kinase A activation may contribute to the inhibitory effects of both cholera and pertussis toxins . This study suggests that caution must be exercised in interpreting inhibition of phosphoinositide turnover by pertussis toxin in whole cell experiments as indicative of direct involvement of a toxin-sensitive GTP-binding protein.

Pharmacol Toxicol, 1992 Nov, 71(5), 391 - 4
Forskolin but not cholera toxin bypasses flufenamate-induced inhibition of cAMP production in anterior pituitaries; Bourne GA; The pharmacological activators of adenylyl cyclase (cholera toxin and forskolin) were utilized in the present study to determine whether they could bypass the inhibitory effects of flufenamate on cAMP production in rat hemipituitaries . During 2 hr incubations, 10 microM flufenamate inhibited gonadotropin-releasing hormone (GnRH)-stimulated (25 nM) cAMP production . Flufenamate did not affect GnRH-receptor interactions as evidenced by its inability to significantly affect either the binding affinity or the binding capacity for GnRH . Additionally, flufenamate inhibited the cholera toxin-stimulated cAMP production, but was ineffective against forskolin-induced activation of adenylyl cyclase . These results indicate that forskolin can be used to restore cAMP production in the presence of flufenamate . Since GnRH and cholera toxin stimulate cAMP production via the GnRH receptor and the Gs protein respectively, and forskolin exerts its stimulatory effects via the catalytic component, the data are consistent with the possibility that flufenamate exerts its inhibitory effect at the level of the Gs protein.

Mol Endocrinol, 1992 Nov, 6(11), 1815 - 24
Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A; Liu YF et al.; The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1) . In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation . In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells . However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ({Ca++}i) by mobilization of intracellular calcium rather than by calcium influx . This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells . Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs . The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g . Gs) may mediate these actions . The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal {Ca++}i . Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal {Ca++}i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and {Ca++}i . From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.

Biochim Biophys Acta, 1992 Oct 27, 1137(2), 237 - 41
Regulation of lipoprotein lipase by dibutyryl cAMP, cholera toxin, Hepes and heparin in F1 heart-cell cultures; Friedman G et al.; Regulation of lipoprotein lipase was studied in mesenchymal rat heart-cell cultures . Treatment of the cultures with dibutyryl cyclic AMP or with cholera toxin resulted in an increase in LPL activity and a comparable increase in LPL mRNA . When the cells were exposed to 100 mM Hepes for 24 h, total enzyme activity rose 2-fold and LPL mRNA increased 2.4-fold . After 72 h, there was a 3-fold increase in LPL mRNA and a 4-fold rise in cellular LPL activity, while medium activity increased 20-fold . Exposure of the cultures to heparin for 24 h resulted in a 3.2-fold increase in total activity and a 36-fold increase in medium activity . This increase was not accompanied by any rise in LPL mRNA . Addition of actinomycin D to control dishes for 24 h resulted in a 33% reduction in LPL mRNA and a 43% reduction in enzyme activity . These values were 71% and 56%, respectively, in Hepes-treated cells, indicating that no stabilization of LPL mRNA occurred under these conditions . It can be concluded that in mesenchymal rat heart-cells in culture cAMP and cholera toxin upregulate lipoprotein lipase at the level of transcription . The increase in LPL activity after 24 h exposure to Hepes could be compatible with transcriptional regulation, while exposure to heparin is not accompanied by a change in LPL mRNA.

Chem Phys Lipids, 1992 Oct, 62(3), 269 - 80
Detection of protein mediated glycosphingolipid clustering by the use of resonance energy transfer between fluorescent labelled lipids . A method established by applying the system ganglioside GM1 and cholera toxin B subunit; Antes P et al.; Glycosphingolipids labelled in the ceramide moiety with 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH) or 6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)aminohexanoic acid (NBD) were incorporated into small unilamellar lecithin liposomes . They were used in resonance energy transfer (RET) experiments between the donor fluorophore DPH and the acceptor NBD to study glycosphingolipid distribution . In pure lecithin liposomes the fluorescent derivatives of GM1, GA1, galactosylceramide and sulfatide behaved almost identically and Ca2+ ions (5 microM or 150 mM) did not influence their transfer efficiencies . But cholera toxin B subunit (CTB) specifically clustered GM1 and enhanced the transfer efficiency . This RET-based method facilitated determination of binding specificity, complex stoichiometry (CTB/GM1 = 1:5), halftime of complex formation (5 s), cooperativity in binding and had a maximal sensitivity at a liposome dotation rate of just 0.25 mol% . In contrast to this, anisotrophy of the fluorophores and the excimer to monomer ratio of pyrene-GM1 were not affected by CTB . This demonstrates the advantage of the presented technique in detection of protein mediated glycosphingolipid clustering.

Biochem Int, 1992 Oct, 28(1), 161 - 8
Cholera toxin diminishes tyrosine kinase activity of the insulin receptor; Muller-Wieland D et al.; We have examined the effect of cholera toxin (CT) on the insulin receptor tyrosine kinase . Incubation of intact rat hepatoma cells FaO with CT (1 microgram/ml/2h) inhibited insulin-induced receptor autophosphorylation by 30% in vivo . This effect persisted after receptor purification in vitro . CT did not alter hormone binding of the insulin receptor, indicating that the toxin affects signal transduction of insulin at the level of the receptor kinase . Experiments using chinese hamster ovary (CHO) cells transfected either with the human insulin receptor (HIR) or a mutant lacking the last 43 amino acids of the receptor beta-subunit (HIR delta CT) showed, that the carboxy-terminal tail of the insulin receptor does not play a role in the suppressive effect of the toxin on the insulin receptor kinase.

Aust Vet J, 1992 Oct, 69(10), 245 - 8
A comparison of the pathogenicity of two strains of hog cholera virus . 2 . Virological studies; Kamolsiriprichaiporn S et al.; Quantitative and qualitative differences were demonstrated in the amount of virus in a range of tissues from pigs infected with either the Weybridge or New South Wales (NSW) strains of hog cholera (HC) virus . The titre of the Weybridge strain in samples, as assessed by either virus titration in cell culture or by the density of specific fluorescing cells in tissue sections, was higher than that for the NSW strain . This correlated with the greater severity of the clinico-pathological syndrome induced by the Weybridge strain . The implications of the differences in the virus content of tissues in the diagnosis of HC is discussed as is the use of monoclonal antibodies to differentiate HC and bovine virus diarrhoea viruses.

Aust Vet J, 1992 Oct, 69(10), 240 - 4
A comparison of the pathogenicity of two strains of hog cholera virus . 1 . Clinical and pathological studies; Kamolsiriprichaiporn S et al.; The virulence of a strain of hog cholera virus isolated during an outbreak of mild disease in pigs in New South Wales in 1960/61 (the NSW strain) was compared over 11 days with that of a virulent strain by inoculating 8 pigs with each virus and comparing the ensuing clinical signs and pathology . Both viruses caused persistent pyrexia and leukopenia, the NSW strain 4 to 5 days and the virulent strain 3 days, after inoculation . Few other clinical signs were observed in the pigs inoculated with the NSW strain . In contrast, all pigs inoculated with the virulent strain became progressively depressed and incoordinated, and were killed between days 6 and 9 . Bronchopneumonia and swollen, reddened lymph nodes were observed in pigs inoculated with both viruses . Few other gross lesions were observed with the NSW strain, but some pigs receiving the virulent strain had pustules in the tonsil and the anterior oesophagus, petechial haemorrhages in the kidney, and small infarcts at the margins of the spleen . There were marked differences in the histopathology, both in the variety of organs affected and the severity of lesions in individual organs . Suppurative bronchopneumonia occurred in both groups . Other changes in the pigs affected with the NSW strain were colitis, mild cerebral vasculitis, necrosis, haemorrhage and neutrophil infiltration in some lymph nodes and spleens . In pigs infected with virulent virus the cerebral vasculitis was so severe that there was necrosis of cells within the vessel walls.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biochem, 1992 Oct, 50(2), 210 - 8
Cholera toxin potentiates TPA-induced mitogenesis and c-fos expression in BALB/c-3T3-derived proadipocytes; Smyth MJ et al.; Treatment of quiescent density-arrested A31T6 proadipocytes with medium supplemented with either 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or cholera toxin alone did not stimulate G0/G1 traverse and initiation of DNA synthesis . Combinations of either TPA and cholera toxin or insulin and cholera toxin caused a small stimulation of proliferation . Addition of medium supplemented with TPA and insulin caused a marked stimulation of cell cycle traverse which was significantly potentiated by the coaddition of cholera toxin . The actions of cholera toxin were mimicked by forskolin . Expression of c-fos was regulated in a manner that reflected the results of the mitogenic experiments . TPA caused a marked induction of expression, while only a small increase in transcript levels was seen after treatment with cholera toxin . Addition of a combination of cholera toxin and TPA caused a synergistic induction of c-fos expression . The model system described in this paper allows a detailed analysis of the regulation, by independent second messenger systems, of the transcription of a gene in a mitogenically relevant manner.

Aliment Pharmacol Ther, 1992 Oct, 6(5), 619 - 27
A new model of human secretory diarrhoea using cholera toxin; Hunt JB et al.; Secretory diarrhoea is a major cause of morbidity and mortality worldwide . However, there is no biologically relevant test system in man for assessing new anti-diarrhoeal therapies prior to clinical trial . We have used highly purified cholera toxin in combination with the triple lumen jejunal perfusion technique to establish a subclinical model of cholera in man . Cholera toxin was administered either by mouth with sodium bicarbonate or directly into a 30 cm 'open' or 'closed' (isolated between two inflated balloons) jejunal segment in healthy adult volunteers . Both oral dosing and direct delivery into an 'open' jejunal segment failed to produce consistent secretion of water and electrolytes . In contrast 15 micrograms or 25 micrograms of cholera toxin elicited secretion of water and sodium 3 h after instillation into the balloon occluded 'closed' jejunal segment (P less than 0.05 vs . controls) . The rate of secretion was constant over the maximal period studied (4.5 h) and was similar to that reported in human cholera . None of the subjects experienced troublesome diarrhoea . We believe this model offers a relevant test system for assessing anti-diarrhoeal therapy in man.

Pathology, 1992 Oct, 24(4), 296 - 301
Effects of cholera toxin on human colon carcinoma cell lines; Barkla DH et al.; This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT) . Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment . However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment . At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions . The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material . The second population was membrane bound . Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells . At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology . By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance . The response to CT was dose-dependent . Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells . This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurosci Methods, 1992 Oct-Nov, 45(1-2), 23 - 33
Notes on a light and electron microscopic double-labeling method combining anterograde tracing with Phaseolus vulgaris leucoagglutinin and retrograde tracing with cholera toxin subunit B; Bruce K et al.; Investigations of monosynaptic connections in the central nervous system have been hindered by the lack of compatible markers that can be used at both light and electron microscopic levels . In attempts to determine synaptic contacts between fibers originating in the substantia nigra and neurons projecting to the spinal cord, we have developed a double immunolabeling technique using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde transport of unconjugated cholera toxin subunit B (CTB) . In this report, we describe technical modifications which consistently produced superior labeling together with adequate ultrastructural preservation of the tissue and discuss the advantages of the two tracers.

MMWR Morb Mortal Wkly Rep, 1992 Sep 11, 41(36), 664 - 7
Cholera associated with international travel, 1992; Survival of hog cholera virus (HCV) in sausage meat products (Italian salami); Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, ItalySurvival of hog cholera virus (HCV) was determined in several sausage meat products (Italian salami) prepared with meats from experimentally infected hogs slaughtered at the peak of disease . Meats were processed following the technology applied by the main factories of the typical Italian production . The survival of HCV was assessed through inoculation in both PK 15 cell monolayers and fully susceptible piglets . In all types of sausages examined HCV was detected up to 75 days of curing by piglet inoculation . This technique was much more sensitive than use of cell culture.

J Diarrhoeal Dis Res, 1992 Sep, 10(3), 133 - 8
Perfusion studies in cholera: methods and procedures; van Loon FP et al.; This paper reviews the characteristics of perfusion techniques in the study of intestinal functions by specifically examining the methods and procedures of perfusion in patients with diarrhoea due to infection with V . cholerae 01 . Because of abundant jejunal secretion of water and electrolytes in cholera, perfusion studies require special approaches with regard to patient preparation, use of tubing material, selection of markers, and rate of perfusion . A discussion on specific problems involved in marker perfusion techniques in cholera and on the interpretation of the results is followed by practical recommendations.

Gut, 1992 Sep, 33(9), 1174 - 8
Effect of cholera toxin on the human jejunum; Petritsch W et al.; In order to develop a model for secretory diarrhoea and to confirm the in vitro effects of cholera toxin in man in vivo the effect of intrajejunally administered cholera toxin was investigated in healthy volunteers . An intestinal perfusion technique with an occluding balloon proximal to the infusion site was used . The jejunum was perfused under steady state conditions with a plasma like electrolyte solution containing polyethylene glycol as a non-absorbable volume marker . After two control periods of one hour each, during which water was absorbed at a rate of 104 (14) (mean (SEM), n = 15) and 94 (15) ml/30 cm/h, respectively, three different doses of cholera toxin (6.25 micrograms, 12.5 micrograms, 25 micrograms) were administered by bolus into the lumen of the jejunum . Cholera toxin reduced absorption of water and electrolytes progressively over four hours and induced secretion in a dose dependent fashion . In the fourth hour net secretion amounted to 22 (23), 36 (24), and 88 (40) ml/30 cm/h (each n = five) with doses of 6.25, 12.5, and 25 micrograms cholera toxin, respectively . The movement of sodium, chloride, and bicarbonate paralleled water movement . Our results suggest that cholera toxin may serve as a secretory model in the human jejunum which might allow testing of new antisecretory agents.

J Clin Microbiol, 1992 Sep, 30(9), 2518 - 20
Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin; Yam WC et al.; The effectiveness of a latex agglutination assay kit for the detection of Escherichia coli heat-labile toxin and cholera toxin was determined for the identification of natural isolates of the corresponding enteric pathogens in Southeast Asia . By selection of the appropriate culture media, the sensitivity of the assay was improved from 90.6% (for the detection of heat-labile toxin) and 75% (for the detection of cholera toxin) to 100%, and the results were confirmed with bioassays and DNA hybridization assays for both clinical and environmental isolates.

Epidemiol Bull, 1992 Sep, 13(3), 9 - 11
The economic impact of the cholera epidemic, Peru, 1991; Vaccination by cholera toxin conjugated to a herpes simplex virus type 2 glycoprotein D peptide; Department of Pathology, McMaster University, Hamilton, Ontario, CanadaImmunization of BALB/cJ mice with a peptide corresponding to residues 1 to 23 of glycoprotein D {gD(1-23)} from herpes simplex virus type 2 (HSV-2) elicits antibody responses which correlate with protection against lethal HSV-2 infection . In the present study, we examined the ability of cholera toxin (CTX) to act as an immunogenic carrier for gD(1-23) . The number of gD(1-23) residues conjugated to CTX affected its binding to GM1 ganglioside and physiological toxicity, both of which are factors affecting oral immunogenicity . The antibody response elicited after intraperitoneal (i.p.) immunization with the CTX-gD(1-23) conjugate was protective against a lethal i.p . challenge with HSV-2 . In other experiments, mice were immunized i.p . on day 0 and subsequent immunizations conducted on days 14 and 28 were administered either intragastrically or intravaginally (i.vag.) . Intraperitoneal priming followed by either i.p or intragastric boosting resulted in anti-HSV-2 antibodies in vaginal washings and in protection against a lethal i.vag . challenge with HSV-2 . Intraperitoneal priming followed by i.vag . boosting did not elicit anti-HSV-2 antibodies in vaginal washings and did not protect mice against a lethal i.vag . challenge with HSV-2 . These results suggest that CTX can act as a systemic and an oral delivery molecule for the covalently linked gD(1-23) peptide and that such conjugates can elicit protective immune responses against systemic and genital HSV-2 infection.

Clin Exp Immunol, 1992 Sep, 89(3), 378 - 83
Characterization of an anti-idiotypic MoAb bearing an internal image of the receptor-binding epitope of cholera toxin; Lucas GP et al.; A mouse anti-cholera toxin (CT) MoAb, mAb1, specific for the GM1-binding epitope of CT, was used to raise a syngenic anti-idiotypic MoAb, mAb2 . Purified mAb2 was specific for mAb1 as shown by latex particle counting immunoassay and ELISA . Several experiments of competition between mAb2 and CT for binding to mAb1 demonstrated that mAb2 bore an internal image of the GM1-binding epitope of CT . Binding of mAb2 to GM1 unambiguously corroborated the mAb1-paratopic specificity of mAb2 . Furthermore, mAb2 acted as a CT-surrogate antigen: rabbits injected with mAb2 produced some anti-CT antibodies, Ab3, which resembled mAb1 in specificity as expected . The potential use of this mAb2 as vaccine or as prophylactic agent to prevent CT from binding to its cellular receptor is discussed.

J Clin Invest, 1992 Sep, 90(3), 759 - 65
Platelet-activating factor-mediated transmembrane signaling in human B lymphocytes is regulated through a pertussis- and cholera toxin-sensitive pathway; Mazer BD et al.; Platelet-activating factor (PAF) stimulates human B cells, resulting in elevation of intracellular calcium and the release of inositol phosphates . This signaling pathway is inhibited in the presence of pertussis (PT) or cholera toxin (CT) . Preincubation of human B cells with either toxin, but not their inactive subunits, for 3 h blocked these PAF-induced responses in two B-lymphoblastoid cell lines . This effect was time dependent, with some inhibition noted at 30 min, but only after preincubation for 2-3 h was maximum inhibition achieved . This inhibitory activity was also dose dependent . The toxins blocked both PAF-induced transmembrane uptake of Ca2+ as well as release of Ca2+ from internal stores, and were selective in that activation events after cross-linking of surface IgM were not affected . Further, the toxins did not appear to act through elevation of intracellular levels of cAMP . These data, coupled with previous observations on the absence of heterologous desensitization between PAF and sIgM receptors, may delineate distinct signaling pathways in human B cells . This may reflect different roles for GTP-binding proteins in the activation of human B cells.

Regul Pept, 1992 Aug 13, 40(3), 339 - 49
Difference between the antisecretory mechanisms of opioids and the somatostatin analogue octreotide in cholera toxin-induced small intestinal secretion in the rat; Sjoqvist A; The antisecretory effect of morphine and the somatostatin analogue octreotide was studied on cholera toxin-induced secretion in anaesthetized rats . Small intestinal secretion was induced with cholera toxin . Morphine (6 mg/kg b.wt.) and the somatostatin analogue octreotide (3 micrograms/kg b.wt.) reduced the cholera secretion in rats whose intestines had been subjected to sympathetic denervation . This was in contrast to the secretion elicited by helodermin which was unaffected by octreotide and morphine in the presence of nicotinic ganglionic blockade . The alpha-adrenergic receptor blocker phentolamine (1-2 mg/kg b.wt . i.v.) and the inhibitor of sympathetic transmitter release guanethidine (5 mg/kg b.wt . i.v.) abolished the antisecretory effect of morphine on the cholera secretion in contrast to the antisecretory effect of somatostatin which was unaffected by the alpha-blockade . It is proposed that the antisecretory effect of morphine and octreotide on cholera toxin-induced secretion was conducted at a step prior to the activation of the secretory epithelium and that the antisecretory effect of morphine was mediated indirectly by interaction with sympathetic nerve terminals in the intestine . The findings are consistent with a model where octreotide and morphine inhibit the nervous secreto-motor reflex activated by the cholera toxin.

Biochim Biophys Acta, 1992 Aug 12, 1136(2), 219 - 22
Amylin inhibits insulin-stimulated glucose uptake in C2C12 muscle cell line through a cholera-toxin-sensitive mechanism; Sheriff S et al.; Rat amylin inhibits insulin-stimulated glucose uptake with an IC50 of 12.1 +/- 4.1 pM in C2C12 myotubes . The maximal inhibition is 64 +/- 5.4% observed at a 100-pM dose of the peptide . Consistently, presence of 100 pM amylin shifted the dose-response curve of insulin to the right, increasing the ED50 from 0.71 to 16 nM . No effect of amylin is observed on basal glucose uptake in these cells . Cholera-toxin treatment of the cells did not affect the insulin-stimulated glucose uptake, while the inhibitory effect is completely lost in toxin-treated cells . These findings strongly suggest that rat amylin is active at a physiological concentration and the amylin inhibition of glucose uptake is mediated through a cholera-toxin-sensitive mechanism.

J Immunol, 1992 Aug 1, 149(3), 981 - 8
Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit; Tamura S et al.; Cross-protection against influenza virus infection was examined in mice, immunized intranasally with a nasal site-restricted volume of inactivated vaccines together with cholera toxin B subunit (CTB) as an adjuvant . The mice were challenged with either a small or a large volume of mouse-adapted virus suspension, each of which gave virgin mice either a predominant upper or lower respiratory tract infection . A single dose of a monovalent influenza A H3N2 virus vaccine with CTB provided complete cross-protection against the small-volume challenge with a drift virus within the same subtype, but a slight cross-protection against the large-volume challenge . A second dose of another drift virus vaccine increased the efficacy of cross-protection against the large-volume challenge . Similar cross-protection against H1N1, H3N2, or B type drift virus challenge was provided in the mice having received a primary dose of a mixture of H1N1, H3N2, and B virus vaccines with CTB and a second dose of another trivalent vaccine . The degree of cross-protection against the small- and the large-volume infection paralleled mainly the amount of cross-reacting IgA antibodies to challenge virus hemagglutinin in the nasal wash and that of cross-reacting IgG antibodies in the bronchoalveolar wash, respectively . On the other hand, in mice immunized subcutaneously with the trivalent vaccines having no cross-reacting IgA antibodies, the efficacy of cross-protection was not so high as that of nasal vaccination . These results suggest that the nasal inoculation of trivalent vaccines with CTB provides cross-protection against a broader range of viruses than does the current parenteral vaccination.

Anal Biochem, 1992 Aug 1, 204(2), 244 - 9
Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces; Uwiera RE et al.; A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface . The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1 . Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin . Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added . This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface . The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine . SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay . The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface . We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.

Curr Opin Immunol, 1992 Aug, 4(4), 387 - 91
Cholera as a model for research on mucosal immunity and development of oral vaccines; Holmgren J et al.; During the past year, the extensive investigational use of a recently developed oral vaccine against cholera has led to significant advances in our understanding of both immunity to cholera and related diarrhoeal diseases, and the mucosal immune response in general after oral immunization . The oral cholera vaccine has been shown to protect, through its cholera toxin B subunit component, against travellers' diarrhoea caused by enterotoxigenic Escherichia coli . The elaboration of sensitive new techniques has allowed detailed clonal analyses of the activation of specific B and T cells and immunologic memory in intestinal mucosa in humans after oral cholera vaccination . These techniques have also been used to demonstrate a transient appearance after immunization of specific gut-derived IgA antibody-producing cells in the circulation and also, a few days later, in a distant mucosal tissue such as the salivary glands.

Jpn J Med Sci Biol, 1992 Aug, 45(4), 199 - 202
Decreased phosphodiesterase activity in cholera toxin-induced hypersecretion in suckling rats; Aye-Kyaw et al.; During cholera toxin (CT)-induced hypersecretion in suckling rats, the rise in the intestinal cAMP concentration was found to be accompanied by a decrease in the cAMP-phosphodiesterase activity . The results suggest the involvement of phosphodiesterase (PDE) as one of the factors governing the rise of cAMP.

J Mol Biol, 1992 Jul 5, 226(1), 23 - 8
A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography; Mosser G et al.; Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1 . Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A . A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data . Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees . Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density . The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively . The apparent projected density of the outer ring is larger than that of the inner ring . We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.

Infect Immun, 1992 Jul, 60(7), 2874 - 9
Cholera toxin as a mucosal adjuvant: effects of H-2 major histocompatibility complex and lps genes; Elson CO; In previous studies we found that cholera toxin (CT) can act as a mucosal adjuvant; i.e., it can stimulate an intestinal secretory immunoglobulin A (S-IgA) response to an unrelated protein antigen when both are fed together to mice . The purpose of this study was to determine whether the mucosal adjuvanticity of CT is restricted by either H-2 major histocompatibility complex or lps genes by using congenic inbred strains that differ at only a single genetic locus . Groups of five mice each were fed saline, CT (10 micrograms), keyhole limpet hemocyanin (KLH) (5 mg), or both CT and KLH on four different days, and samples of intestinal secretions and plasma were obtained 1 week after the last feeding . In the mice fed both CT and KLH, the intestinal S-IgA anti-KLH response was higher in H-2b congenic strains than in H-2k congenic strains, and in addition there was a highly significant positive correlation between the intestinal S-IgA anti-KLH and S-IgA anti-CT responses in the intestinal secretions of individual mice . Similarly, in the lps congenic strains, mice of the endotoxin-responsive strain that were fed both CT and KLH had substantially higher S-IgA and plasma IgG responses to KLH than did mice of the endotoxin-unresponsive strain . The effect of CT on the induction of oral tolerance to KLH in the H-2 congenic strains was also examined . In contrast to the results above, the abrogation of oral tolerance to KLH by CT occurred in all strains regardless of H-2 haplotype . Similarly, the adjuvant effect of CT on plasma IgG anti-KLH responses after both were given together intraperitoneally was not restricted by H-2 . I conclude that the mucosal adjuvanticity of CT is influenced by both the H-2 and lps genetic loci and that it appears to depend on a vigorous mucosal immune response to CT itself.

J Infect Dis, 1992 Jul, 166(1), 2 - 14
The treatment of cholera: clinical science at the bedside; Carpenter CC; In 1959-1961, two major international centers for the study of cholera were established in Calcutta and in Dacca, Bangladesh . As the result of collaborative work in these centers, a simple effective oral therapy for cholera, using ingredients available in virtually every part of the world, was defined . Through the well-coordinated efforts of the World Health Organization (WHO), knowledge of how to prepare and administer oral rehydration therapy has now been disseminated throughout most of the world . With this background, when Peru was attacked in 1991 by a massive and totally unanticipated outbreak of cholera, a remarkably well-organized national response to the epidemic achieved a survival rate greater than 99% in greater than 300,000 cholera patients during the first year of the epidemic . Thus the results of clinical research on the Indian subcontinent, widely disseminated through educational programs by the WHO, have resulted in unparalleled success in the treatment of the largest epidemic outbreak of cholera in the 20th century.

Comp Immunol Microbiol Infect Dis, 1992 Jul, 15(3), 221 - 8
A tissue culture vaccine with lapinized chinese (LC) strain of hog cholera virus (HCV); Ferrari M; The lapinized chinese (LC) strain of hog cholera virus (HCV), was adapted to grow in a cell line from minipig kidney (MPK) where it reached a titer, as determined by immunofluorescence, significantly higher than in rabbits . Inasmuch as the immune serum to HCV neutralized the culture-adapted virus, it was concluded that its antigenicity did not undergo any change after adaptation to MPK cells . The MPK-LC adapted virus (MPK-LC-HCV) showed also a higher immunogenic activity in rabbits, in comparison with the original LC virus . The MPK-LC-HCV protected pigs against challenge infection with virulent HCV . Thus, the vaccinated pigs did not show any clinical signs of disease, nor have they been responsible of virus shedding after they were exposed to the challenge infection 1 month or 6 and 11 months later . All vaccinated pigs seroconverted after vaccination and the antibody titers were on the same range of those reported in pigs vaccinated with the traditional vaccine prepared in rabbits . In the same pigs the antibody concentration underwent a booster effect following challenge infection . It was suggested the MPK-LC-HCV vaccine as an alternative product that might be used to prevent HCV infection . prevent HCV infection.

Acta Physiol Scand, 1992 Jul, 145(3), 229 - 37
Actions of serotonin antagonists on cholera-toxin-induced intestinal fluid secretion; Sjoqvist A et al.; The effects of several 5-hydroxytryptamine (5-HT) receptor antagonists were tested in rats in vivo on the intestinal fluid secretion evoked by cholera toxin . Five receptor antagonists were used, namely 2-bromolysergic acid diethylamine (2-bromo-LSD), granisetron, ketanserin, methysergide and ondansetron . The drugs were used in doses that inhibited the arterial hypertension and/or bradycardia evoked by 5-HT given i.v . Granisetron and ondansetron markedly diminished cholera-toxin-evoked secretion, whereas ketanserin was without any effect . Methysergide also diminished cholera-toxin-induced fluid secretion particularly when the drug was given as an i.v . infusion . The results are considered in relation to the pathophysiology of cholera secretion and to the current views of receptor subtypes for 5-HT . It is proposed that the receptor involved is a 5-HT3 receptor, possibly also a receptor of the 5-HT1 type . Results from experiments in which 5-HT (20 mM) was placed in the intestinal lumen to evoke an intestinal secretion suggest that the 5-HT3 receptor is located in the villus tissue . It was also demonstrated that zimeldine, an inhibitor of presynaptic 5-HT reuptake, diminished choleraic secretion, an effect that may be ascribed to a 5-HT tachyphylaxis caused by an accumulation of 5-HT in a synaptic cleft.

Comp Immunol Microbiol Infect Dis, 1992 Jul, 15(3), 213 - 9
Hog cholera diagnostic techniques; Pearson JE; Clinical signs and lesions can sometimes provide the basis for a presumptive diagnosis of hog cholera (HC) . However, an accurate diagnosis requires laboratory testing . The usual procedure for the detection of viral antigen is the examination of cryostat sections stained with fluorescein-conjugated HC antiserum . A more definitive technique is isolation of the virus in PK-15 cell cultures and identification of the viral antigen in cells using an HC fluorescent antibody conjugate . As bovine viral diarrhea (BVD) virus will cross-react with HC virus, isolation must be confirmed by the comparison of BVD and HC staining or, preferably, by the use of monoclonal antibodies that can differentiate between HC and BVD viruses . Hog cholera surveillance must rely on serology . The fluorescent antibody virus neutralization (FAVN) test is the classical technique, and HC and BVD antibody can usually be differentiated if HC-positive serum samples are tested against both viruses . Recently the enzyme-linked immunosorbent assay (ELISA) and peroxidase-labeled antibody tests have become the commonly used techniques.

Comp Immunol Microbiol Infect Dis, 1992 Jul, 15(3), 189 - 201
The hog cholera virus; Moennig V; Hog cholera virus (HCV) is a spherical enveloped particle of about 40-60 nm dia . The viral genome is a single strand RNA of about 12,000 bases with positive polarity . One single large open reading frame codes for presumably four structural, i.e . three glycoproteins and a core protein, and about three to five nonstructural proteins . The functional role is not yet fully clear for all viral proteins . HCV belongs to the pestivirus group and it is closely related to bovine viral diarrhoea and border disease viruses . The relationship extends to morphology, antigenicity, host spectrum and molecular properties . Pestiviruses hold generic status in the family Flaviviridae.

J Med Assoc Thai, 1992 Jul, 75(7), 413 - 7
A common source foodborne outbreak of E1 Tor cholera following the consumption of uncooked beef; Swaddiwudhipong W et al.; In July 1988, an outbreak of cholera with 71 culture-confirmed cases of biotype El Tor, serotype Ogawa, occurred in a non-endemic area in Mae Sot district, Tak province . Fifty-two cases had diarrhea and 19 had asymptomatic cholera infection . No cases died . Epidemiological investigation revealed a significant association between cholera infection and the consumption of uncooked beef . Increased risk of cholera infection was observed when the incriminated beef was kept at room temperature and consumed later without cooking . The beef was possibly contaminated with V . cholerae O1 from an infected butcher who may have contracted the disease from his foreign worker who was found to have asymptomatic cholera infection . Early detection of the outbreak and rapid identification of transmission source permitted prompt appropriate control measures which, therefore, prevented the outbreak from spreading to other communities . As outbreaks of foodborne diseases, including cholera, have been reported frequently after the consumption of raw food in many parts of Thailand, preventive educational efforts should be directed towards modifying the traditional behavior patterns of consuming raw food among these Thai people.

Tidsskr Nor Laegeforen, 1992 Jun 30, 112(17), 2214 - 7
{Cholera in Bergen in 1848-1849--what caused the epidemic?}; Oeding P; In December 1848, a big cholera epidemic broke out in Bergen . It lasted until April 1849, by which time 605 victims had died . The cholera spread by contact from person to person . How it came to Bergen, and how it was introduced into the population however, been open to doubt . This question is subject of this article . Primary sources have been studied and compared with contemporary medical reports . There is no doubt that a schooner from the Dutch city of Vlaardingen brought the cholera to Bergen . A passenger died of cholera on the day before the ship arrived, and was buried in Bergen . How the infection was transmitted to the first victim in the city, a watchman's wife who lived on the outskirts, is more difficult to explain . Later information, telling that she had washed the passenger's clothing, has not been confirmed in the primary sources . Most probably, healthy carriers among the crew transmitted the cholera to the population after the quarantine had been suspended . The watchman's wife need not have been the first one to catch the infection.

Cancer Res, 1992 Jun 15, 52(12), 3340 - 6
Growth inhibition by cholera toxin of human lung carcinoma cell lines: correlation with GM1 ganglioside expression; Kaur G et al.; The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented . CT inhibited the growth of nine SCLC cell lines (concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of GM1 ganglioside, the surface receptor for CT . CT-resistant SCLC all had greatly decreased GM1 expression . In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines . Seven of the 11 CT-resistant NSCLC had levels of GM1 comparable to CT-sensitive NSCLC or SCLC . In a limited panel of cell lines, cyclic AMP (cAMP) agonists including forskolin, 8Br{cAMP}, and dibutyryl{cAMP} did not consistently reproduce CT-mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT . Expression of the RI and RII regulatory subunits of cAMP-dependent protein kinase was similar in CT-resistant and CT-sensitive SCLC or NSCLC cell lines . In the presence of isobutylmethylxanthine, intracellular cAMP levels induced by CT in a CT-resistant, GM1(+) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line . We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of GM1, and in the case of SCLC cell lines the presence of GM1 is sufficient . In NSCLC cell lines, expression of GM1 is not sufficient for growth inhibition by CT . These findings imply refractoriness to growth inhibition by cAMP in GM1(+), CT-resistant NSCLC cell lines and the possibility of non-cAMP-related mechanisms for growth inhibition in CT-sensitive cell lines.

Eur J Clin Nutr, 1992 Jun, 46(6), 437 - 43
Lack of therapeutic efficacy of vitamin A for non-cholera, watery diarrhoea in Bangladeshi children; Henning B et al.; Vitamin A deficiency has been postulated to increase childhood mortality, possibly through increasing the severity and case-fatality of infectious diseases like diarrhoea . A clinical trial was conducted to measure the effect of vitamin A therapy on the severity and duration of acute episodes of non-cholera, watery diarrhoea; 83 children with less than 48 h of illness were randomized to receive vitamin A (200,000 IU of retinyl palmitate) orally or placebo during hospitalization at the International Centre for Diarrhoeal Disease Research in Bangladesh . The patients were similar initially with regard to age, nutritional status and severity of diarrhoea prior to admission . No adverse effects of vitamin A were detected . During hospitalization there were no differences between groups in duration of illness or stool output . Thus, vitamin A can be given safely during diarrhoeal illness to augment hepatic reserves and possibly provide a beneficial effect in regard to subsequent episodes of diarrhoea and other infections, but this supplementation should not be expected to have a therapeutic effect on a current episode.

CMAJ, 1992 Jun 1, 146(11), 1947 - 52
Cholera vaccination: a decision analysis; MacPherson DW et al.; OBJECTIVE: To examine the clinical impact and financial cost of a vaccination program for the prevention of cholera in North Americans travelling to endemic and epidemic regions by means of the principles of decision analysis and a decision tree as well as to illustrate the effect of case attack rates on the cost per case prevented by vaccination . DESIGN: Review of the scientific literature to establish the probabilities of each significant outcome as well as a decision analysis and partial economic evaluation . OUTCOME MEASURES: Clinical impact (attack rates for cholera among vaccinated and nonvaccinated travellers), rates of death associated with cholera and vaccine-associated adverse events (VAAEs), and the number of VAAEs and the vaccine cost per case prevented . MAIN RESULTS: On the basis of our assumptions (including a rate of one case of cholera per 500,000 journeys to endemic regions), to prevent one case of cholera a vaccination program would cost $28.67 million and be associated with 105 VAAEs . CONCLUSION: Routine vaccination of travellers to endemic areas cannot be recommended; however, for people travelling to regions with a high transmission rate vaccination should be considered.

J Virol, 1992 Jun, 66(6), 3677 - 82
A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus; Weiland E et al.; Several monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) reacted with the HCV structural glycoprotein gp44/48 and neutralized the virus . The presence of HCV gp44/48 on the viral surface was directly demonstrated by immunogold electron microscopy . Eight anti-HCV gp44/48 MAbs were tested by immunoperoxidase assay against a panel of pestivirus strains . Each MAb showed a distinct pattern of reactivity with HCV strains . It is suggested that the MAbs are well suited for epidemiological investigations of HCV outbreaks.

Voen Med Zh, 1992 Jun, (6), 60 - 4
{The diagnostic and treatment characteristics of El Tor cholera and its combinations with other infectious diseases in an outbreak of multiple intestinal infection}; Ivanov KS et al.; The article summarizes the experience of diagnosis and treatment of El Tor cholera in servicemen during an outbreak of intestinal polyinfection in the conditions of dry hot climate in desert and mountainous terrain of Afghanistan . The authors describe the clinical course of El Tor cholera which is stipulated by the mixed character of dehydration, polymorphism of clinical symptoms and more severe forms of concomitant infections.

J Diarrhoeal Dis Res, 1992 Jun, 10(2), 79 - 86
Cholera epidemics in Bangladesh: 1985-1991; Siddique AK et al.; In 1991, a major epidemic of diarrhoea broke out in Bangladesh . To estimate the extent of cholera during diarrhoea epidemics and to focus on the public health issues related to cholera in Bangladesh, we have used the government figures of the 1991 epidemic and data from our own experience of epidemic interventions in nearly 400 rural upazilas (sub-district) between 1985 and 1989 and in 1991 . Our data showed that V . cholerae 01 was the most frequently (40%) isolated enteropathogen during the epidemics . The disease is widely distributed in the country . Only 24% of the total 1,648 laboratory confirmed cholera patients were below 5 years of age, and children below 2 years of age accounted for only 10% of the total . Access difficulty to medical care and absence of a reliable surveillance were thought to be the constraints to early detection and appropriate intervention, thus, there were more deaths during the epidemics . We have shown that a high proportion (59%) of cholera patients during their illness in the rural areas were not visited by the government surveillance staff and that most (80%) were treated at home . Access to treatment by qualified physicians was limited to 23% of the patients, whereas a large proportion of the patients were treated by the unqualified rural practitioners (68%), and the others (9%) had no access to any health care providers . Our experience also indicated a higher case fatality ratio (14%) prior to intervention by qualified physicians during epidemics and an overall fatality ratio of 4%, despite the significant reduction (less than 1%) achieved by the intervention . Cholera is highly epidemic in Bangladesh.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biol, 1992 Jun, 117(6), 1197 - 1209
Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic; Lencer WI et al.; The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane . Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated . To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined . Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion . The time course of the CT-induced Isc response paralleled the time course of cAMP generation . The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less . At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide . A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C . At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact . Re-warming above 32 degrees C restored CT-induced Isc . Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited . These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells . We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.

Biochemistry, 1992 May 26, 31(20), 4773 - 8
Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins; Pacuszka T et al.; We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced {Pacuszka, T., & Fishman, P . H . (1990) J . Biol . Chem . 265, 7673-7668} . We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP . This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1 . To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf) . The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells . The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation . By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation . Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine . Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin . Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.

J Biol Chem, 1992 May 5, 267(13), 9028 - 34
Characterization of the human gene encoding ADP-ribosylation factor 1, a guanine nucleotide-binding activator of cholera toxin; Lee CM et al.; Mammalian ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin ADP-ribosyltransferase activity, were grouped into three classes based on deduced amino acid sequence . Human ARF 1, a class I ARF, is identical with its bovine counterpart, has a distinctive pattern of tissue and developmental expression, and is encoded by a approximately 1.9-kilobase mRNA . ARF 1 cDNAs were isolated from a human fibroblast cDNA library; one arose via an alternative polyadenylation signal (AA-TACA) 84 nucleotides 5' to the polyadenylation signal (AATAAA) used in the 1815-base pair cDNA . The polyadenylation signals, their respective locations, and the surrounding nucleotide sequences are conserved in human and rat . The human ARF 1 gene, with four introns, spans approximately 16.5 kilobases . Exon 1 (46 base pairs) contains only untranslated sequence . Translation initiates in exon 2, which encodes the sequence GXXXXGK involved in phosphate binding (GTP hydrolysis) . The sequence DVGG is encoded in exon 3, and NKQD, which is involved in the interaction with the guanine ring, is interrupted following the codon for Q by intron 4 . The carboxyl-terminal 53 amino acids and greater than 1110 base pairs of 3'-untranslated region are encoded in exon 5 . Primer extension and mung bean and S1 nuclease mapping indicated multiple transcription initiation sites and were consistent with Northern analyses . The 5'-flanking region has a high GC content but no TATA or CAAT box, as found in housekeeping genes . In addition, the two human class I ARF genes, ARF 1 and ARF 3, have similar exon/intron organizations and use GC-rich promoters.

Gut, 1992 May, 33(5), 643 - 5
Indomethacin decreases jejunal fluid secretion in addition to luminal release of prostaglandin E2 in patients with acute cholera; Van Loon FP et al.; Human cholera is associated with an increased luminal release of prostaglandin E2 (PGE2), but whether inhibition of increased PGE2 synthesis will reduce or control intestinal secretion is uncertain . 'Steady state' perfusions (10 ml/minute) in 12 patients with acute cholera, and repeat perfusions in nine of these patients during the convalescent phase were therefore performed using the triple lumen technique . The proximal jejunum was perfused with isotonic saline containing sodium-sulphobromophthalein as a non-absorbable marker . After intravenous administration of indomethacin (1.0 mg/kg) the jejunal net transfer of fluid and the jejunal flow rate of PGE2 were determined in 30 minute periods for 120 minutes after a 120 minute control period . Indomethacin decreased net fluid secretion (2.1 (0.3-4.2) v 4.5 (2.5-8.4) ml/hour x cm; medians, Q50 ranges, p less than 0.01) and the jejunal flow rate of PGE2 (1.5 (1.2-2.7) v 2.2 (1.4-4.9) ng/minute, p less than 0.05) . The results of similar perfusion studies in 22 patients with acute cholera, used to establish the spontaneous time related change in fluid secretion, showed no significant change in net fluid transfer (3.5 (2.2-6.2) to 3.5 (2.6-11.6) ml/hour x cm, p greater than 0.25) over 240 minutes . These data provide further evidence in favour of the hypothesis that prostaglandins have a role in the cholera toxin induced intestinal fluid secretion in man.

Scand J Immunol, 1992 May, 35(5), 597 - 602
Biliary immune response to orally presented food antigen, ovomucoid, and its potentiation by cholera toxin B subunit; Sugita-Konishi Y et al.; To clarify the biliary immune response against food antigen, we studied biliary antibody response to intravenous and oral primary immunization with ovomucoid (OM) and the effects of cholera toxin B subunit (CTB) on the oral response in mice . Specific antibodies against OM were induced in biliary and serum immunoglobulin (Ig) A, IgG and IgM by intravenous (i.v.) administration of the antigen . However, the antibodies were induced only in biliary Igs, but not in serum Igs, by oral intubation of the antigen . The higher levels of anti-OM in bile than in serum observed in the oral group may favour the assumption that antigen-stimulated lymphoid cells migrate to the liver, gall bladder or bile duct where they produce antibody . Both serum and biliary anti-OM responses to oral immunization were potentiated remarkably by oral administration of CTB with the antigen, the IgM anti-OM response being potentiated to the largest extent . In the CTB-treated mice, the IgA anti-OM level was higher in bile than in serum . Serum level of IgG anti-OM was much lower in the CTB-treated mice than in the i.v.-immunized mice, but the biliary level of IgG anti-OM in the CTB-treated mice was comparable to that in the i.v.-immunized mice . The relationship between serum and biliary IgA and IgG antibodies suggests that CTB potentiates biliary anti-OM responses not solely through increasing systemic levels of the antibodies but through modulating the processes specific to mucosal presentation of antigen.

Biull Eksp Biol Med, 1992 May, 113(5), 549 - 52
{Ultrastructural changes in interneuron connections in intramural ganglia of the small intestine in suckling rabbits with experimental cholera}; Bardakhch'ian EA et al.; Data, received in investigation of the small intestine intramural ganglia of 34 suckling rabbits show, that development of experimental cholera induce in the neurons reactive shifts, changed by dystrophic alterations . Prevaluation of cholinergic influences in autonomic ganglia in small intestine promotes enterocytes hypersecretion and accelerating of motor activity.

Salud Publica Mex, 1992 May-Jun, 34(3), 287 - 91
{Development of oral vaccines based on recombinant proteins derived from cholera toxin}; Sanchez J et al.; In this paper a new approach to create antigens through genetic engineering is discussed . In this particular case the subunits of V . cholerae toxin are used as heterologous epitope carries . In this paper the manipulation of A and B subunits is described . This manipulation allows both the insertion of epitopes to the B subunit and the use of subunit A in the construction of recombinant antigens similar to the ones derived from subunit B.

Reg Immunol, 1992 May-Jun, 4(3), 153 - 61
Adjuvant effects of freely ingested cholera toxin on systemic antibody and DTH responses to protein antigen; Kusnecov AW et al.; Recent evidence suggests that immunological handling of antigen in the gut may be different when animals voluntarily ingest antigen in their drinking fluid . Therefore, we investigated whether the adjuvant effects of CT were evident in mice that voluntarily ingested CT together with keyhole limpet hemocyanin (KLH) in 1.0 ml chocolate milk . Initially, it was found that ingestion of 2 mg KLH for up to four times (one ingestion per week) did not induce serum IgG anti-KLH antibody; however, KLH-specific IgG was detected if KLH was ingested together with 10 micrograms CT . Furthermore, 50 days after only one ingestion of CT+KLH, the serum IgG response to intraperitoneal challenge with 100 micrograms KLH was significantly elevated with respect to the response of mice who previously had ingested either KLH alone, CT alone, or drinking fluid only . This observation was repeated in further experiments showing an enhanced IgG response to 100 micrograms KLH ip given 7 or 14 days after a single ingestion of 2 or 5 mg KLH mixed with different doses (0.5-20 micrograms) CT . The lowest effective CT dose for serum IgG anti-KLH adjuvanticity was found to be 1 microgram; the highest effective dose was 10 micrograms, there being no additive effect with 20 micrograms CT . It was also found that simultaneous ingestion of KLH (2 or 5 mg) with CT (0.5-20 micrograms) followed 7 or 14 days later by 100 micrograms KLH ip, increased the 24 hr footpad DTH response to 50 micrograms KLH administered 43 days after the ip challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1992 Apr 30, 1135(1), 61 - 6
Actions of phorbol esters on levels of cAMP in cholera toxin-treated chief cells from guinea pig stomach; Raufman JP; Agents like carbachol and cholecystokinin (CCK), that activate chief cell phosphoinositidase C, thereby increasing cell calcium concentration, increase cAMP levels in cholera toxin-treated, but not control, gastric chief cells . In the present study, we found that phorbol esters, like PMA, that activate protein kinase C, also cause augmentation of chief cell cAMP levels . The maximal effect with PMA (100 nM) was about 50% of the maximal response with CCK (10 nM) or carbachol (100 microM) . Because protein kinase C is a calcium-dependent enzyme, we examined the effect of modulating cell calcium levels with the ionophore A23187 . The ionophore alone caused a dose-dependent augmentation of cAMP levels . Adding 100 nM PMA caused an additive response, such that a maximal cAMP response, equal to that seen with 100 microM carbachol, was observed with 30 nM A23187 . Carbachol-, A23187-, and PMA-induced augmentation of cAMP levels was progressively reduced by increasing concentrations of calmidazolium, a calmodulin inhibitor . Combination of phorbol esters that activate protein kinase C with ionophores that increase cell calcium mimics the actions of CCK and carbachol on cAMP levels in cholera toxin-treated chief cells.

Am J Epidemiol, 1992 Apr 15, 135(8), 865 - 74
Nonparticipation as a determinant of adverse health outcomes in a field trial of oral cholera vaccines; Clemens JD et al.; The authors estimated the incidence rates of cholera and death between 1985 and 1988 for 32,642 age- and sex-eligible persons who did not participate in a randomized, placebo-controlled field trial of killed oral cholera vaccines in rural Bangladesh . As compared with 20,744 placebo recipients, the relative risk of cholera for all nonparticipants, adjusted for potentially confounding demographic variables, was 1.20 (95% confidence interval (CI) 1.03-1.41); this adjusted relative risk reflected elevated adjusted relative risks in nonparticipants who were medically ineligible (RR = 1.65; 95% CI 1.22-2.22) or refused to participate (RR = 1.19; 95% CI 1.01-1.41), but not in persons absent at the time of vaccination (RR = 1.00; 95% CI 0.78-1.28) . The adjusted relative risk of death was also elevated in nonparticipants as compared with placebo recipients (RR = 1.28; 95% CI 1.10-1.48), with the same pattern of adjusted relative risks for different categories of nonparticipants: for ineligible subjects, 2.64 (95% CI 2.12-3.29); for refusers, 1.20 (95% CI 1.02-1.41); and for absentees, 0.95 (95% CI 0.75-1.22) . The authors concluded that nonparticipation was associated with clinically cogent adverse health outcomes, but that the magnitude of these associations varied according to the reason for nonparticipation . These findings underscore the caution required in assessing vaccine efficacy with controls who are not vaccinated because of choices made by patients or vaccinators.

Brain Res Bull, 1992 Apr, 28(4), 619 - 23
Visualization of efferent retinal projections by immunohistochemical identification of cholera toxin subunit B; Mikkelsen JD; The present study describes the use of cholera toxin subunit B as an anterograde and retrograde neuronal tracer for studying retinal projections of the rat, mouse, gerbil, and hamster . The tracer was pressure injected in the posterior chamber of the eye and the labeled neurons were identified using an avidin-biotin immunoperoxidase technique using diaminobenzidine as chromagen . Doses of 3-8 microliters (30-80 micrograms) cholera toxin subunit B and a survival for 24 h resulted in an optimal transport of the tracer in all rodent species investigated . The cholera toxin subunit B-containing retinal efferents were effectively stained and yielded the presence of axons with delicate boutons on passage and nerve endings . Smooth and thick fibers were also observed, indicating a distinction between passing and terminating axons, respectively . Immunoreactive axons were observed in the hypothalamus, thalamus, ad mesencephalon, and the precise distribution of positive nerves could be identified in counterstained sections, some of them as delicate endings in apposition to neuronal surfaces . Labeled cell bodies were observed in the oculomotor nucleus and the pretectum, indicating that the tracer is transported retrogradely as well . Because the tracer is identified immunohistochemically, the retinofugal and retinopetal pathways can be mapped more precisely, perhaps in combination with immunohistochemical detection of other antigens.

Epidemiol Infect, 1992 Apr, 108(2), 377 - 86
Estimation of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs; Hone J et al.; Infectious diseases establish in a population of wildlife hosts when the number of secondary infections is greater than or equal to one . To estimate whether establishment will occur requires extensive experience or a mathematical model of disease dynamics and estimates of the parameters of the disease model . The latter approach is explored here . Methods for estimating key model parameters, the transmission coefficient (beta) and the basic reproductive rate (RDRS), are described using classical swine fever (hog cholera) in wild pigs as an example . The tentative results indicate that an acute infection of classical swine fever will establish in a small population of wild pigs . Data required for estimation of disease transmission rates are reviewed and sources of bias and alternative methods discussed . A comprehensive evaluation of the biases and efficiencies of the methods is needed.

J Trop Med Hyg . 1992 Apr;95(2):152.
Case report: cholera in a preterm neonate; Ngoma MP et al.; Falling standards of sanitation resulted in the first outbreak of cholera in Lusaka, Zambia, during the rainy season, February 1990 . A total of 2166 cases were handled with 128 (5.9%) deaths . One hundred and eight (108) children, including one preterm neonate, were admitted to the University Teaching Hospital . The neonate went to the Neonatal Intensive Care Unit.

Biull Eksp Biol Med, 1992 Apr, 113(4), 415 - 7
{Ultrastructural and morphometric analysis of the Paneth cell reaction to administration of cholera toxin}; Shakhlamov VA et al.; Experiments were made on 40 immature guinea--pigs which were divided into two groups . 20 animals received intraduodenal injections of cholera toxin and 20 others were injected normal saline (groups I and II, respectively) . Group I animals exhibited a rapid fall in one--cell secretion of XS recorded on hour 6-12 after the injection of cholera toxin . Inhibition of functional activity was associated with defected synthesis of granules in the cells of Paneth.

J Immunol, 1992 Apr 1, 148(7), 1999 - 2005
Cyclic AMP-independent effects of cholera toxin on B cell activation . II . Binding of ganglioside GM1 induces B cell activation; Francis ML et al.; Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth . The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP . CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag . Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event . Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1 . Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane . rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression . Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1 . CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant . These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.

Biochemistry, 1992 Mar 3, 31(8), 2422 - 6
Fuc-GM1 ganglioside mimics the receptor function of GM1 for cholera toxin; Masserini M et al.; The ability of Fuc-GM1 ganglioside to mimic the receptor function of GM1 for cholera toxin (CT) has been investigated . For this purpose, rat glioma C6 cultured cells were enriched with Fuc-GM1 and the responsiveness to CT was compared with that of cells enriched with GM1 ganglioside . Fuc-GM1 was taken up by cells as rapidly and to the same extent as GM1 . When comparable amounts of ganglioside were associated, the cells enriched with Fuc-GM1 bound the same amount of 125I-CT as did cells enriched with GM1 . Under conditions in which GM1- and Fuc-GM1-enriched cells bound comparable amounts of CT, the Fuc-GM1-treated cells accumulated virtually the same amount of cyclic AMP as did GM1-treated cells, and activation of adenylate cyclase was also similar . The lag time preceding the CT-induced cAMP accumulation was the same in Fuc-GM1- and GM1-enriched cells . High-sensitivity isothermal titration calorimetry (ITC) experiments showed that the association constants of CT with Fuc-GM1 or GM1 ganglioside were comparable (4 x 10(7) M-1 and 1.9 x 10(7) M-1, respectively, at 25 degrees C) . Also, the association constants of the B-subunit pentamer with Fuc-GM1 or GM1 ganglioside were comparable (about 3 x 10(7) M-1 and 7 x 10(7) M-1, respectively, at 25 degrees C).

Berl Munch Tierarztl Wochenschr, 1992 Mar 1, 105(3), 73 - 7
{Is feeding of green silage in areas with hog cholera in wild boar a danger for domestic swine herds? Experimental study}; Kaden V et al.; In an experimental study we tested the survival of hog cholera virus (HCV) contained in pieces of muscular tissue and organs from experimentally infected swine after incubation in silage . In big (diameter greater than 20 cm) muscular pieces HCV survived even in excellent mineral acid silage (pH 3.8-4.0) after a storage of 5 months . On the other hand in smaller parts (musculature tissue, organs less than 20 cm diameter) we never found virulent HCV after 3 months of incubation . Independent of the size of the tested organs we did not find any virulent HCV in silage with pH 5.2 after 3 months . The results of our investigations show, that the feeding of green silage in areas with hog cholera among wild boar is a potential risk for the domestic swine population . In conclusion we propose to feed green silage to unvaccinated pigs in such areas only after a storage of 9 month.

Immunology, 1992 Mar, 75(3), 488 - 92
Cholera toxin adjuvant promotes long-term immunological memory in the gut mucosa to unrelated immunogens after oral immunization; Vajdy M et al.; This study was conducted to investigate whether cholera toxin (CT), used as a mucosal adjuvant, would promote the development in mice of immunological memory to unrelated antigens administered by the oral route . We found that oral priming immunizations with keyhole limpet haemocyanin (KLH) in combination with CT adjuvant induced long-term, at least 22 months and perhaps lifelong, immunological memory in the intestinal lamina propria (LP) . In contrast, oral priming immunizations with KLH alone failed to stimulate immunological memory . Moreover, memory responses in the KLH plus CT-immunized mice were elicited by antigen alone, i.e . without CT adjuvant, suggesting that once immunological memory is established in the intestinal mucosa, e.g . by oral vaccination, elicitation of secondary-type responses does not require the presence of CT and thus could result from re-encounter with specific bacterial or viral antigens in the intestine . We also found that a single priming-dose of KLH plus CT adjuvant was sufficient to stimulate long-term, antigen-specific memory in the intestinal mucosa . Finally, the ability of CT to induce immunological memory in the gut mucosa required the whole toxin and could not be achieved by using the toxoid, the cholera toxin B subunit (CTB), which lacks the adenylate cyclase/cAMP-activating property of the whole molecule . The results support the view that mucosal adjuvants, incorporated into oral vaccines, might be an effective means to achieve long-term immunological memory and protection against pathogenic micro-organisms at mucosal surfaces.

Cell Tissue Res, 1992 Mar, 267(3), 419 - 27
Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle, as revealed by the retrograde transport of the cholera toxin B subunit; Hirakawa M et al.; Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle . Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L3-L6 . The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB . Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction . Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus . Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells . The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.

Reg Immunol, 1992 Mar-Apr, 4(2), 79 - 85
Cholera toxin conjugates for intragastric vaccination against herpes simplex virus type 2; Drew MD et al.; In this study, we tested the hypothesis that enteric immunization with cholera toxin (CTX) conjugated to glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) or a peptide corresponding to residues (1-23) of gD (gD(1-23)) would induce relevant antiviral immunity . Intraperitoneal (IP) immunization of mice with CTX-gD(1-23) conjugate induced anti-HSV-2 sera antibody responses which correlated with protection from a lethal IP challenge with HSV-2 . Intragastric (IG) immunization of mice with the same conjugate or a CTX-gD conjugate did not result in measurable anti-HSV-2 responses in sera or vaginal washings and only small numbers of anti-HSV-2 antibody-secreting cells (ASC) were found in intestinal lamina propria cell and splenocyte preparations . In comparison, anti-CTX responses were detected in sera and vaginal washings after IG immunization with CTX and anti-CTX ASC in lamina propria cell preparations accounted for 5-10% of total ASC detected at this site . No significant differences in the survival of mice immunized with the conjugates were noted after a lethal intravaginal (IVAG) challenge with HSV-2 . The poor enteric immunogenicity of gD(1-23) and gD conjugated to CTX was attributable to proteolysis in the gastrointestinal tract . These results indicate that although peptide-CTX conjugates can induce protective immune responses when administered parenterally, it may not be feasible to use peptides as the basis of an oral vaccine unless methods are developed to protect these antigens from degradation in the gastrointestinal tract.

J Med Assoc Thai, 1992 Mar, 75 Suppl 2, 35 - 7
Precaution against nosocomial spread of cholera in Udornthanee Hospital; Kraisriwatana J et al.; From February to April 1988, there was an outbreak of cholera in Udornthanee Province . One hundred and twenty-four culture-documented cases were admitted into Udornthanee Hospital . Prevention of nosocomial spread of V . cholerae was done by the implementation of proper practices, surveillance culture and routine surveillance . There was no nosocomial spread of V . cholerae to patients or medical personnel . It was also shown that these practices were effective in prevention of contamination of the environment . It is concluded that simple measures are effective in the prevention of spread of V . cholerae in health care settings.

Immunology, 1992 Mar, 75(3), 493 - 8
Involvement of antigen-presenting cells in the enhancement of the in vitro antibody responses by cholera toxin B subunit; Hirabayashi Y et al.; The enhancing effect of cholera toxin B subunit (CTB) on primary antibody responses to keyhole limpet haemocyanin (KLH) and the cellular basis of the effect were investigated, using in vitro cultures of mouse spleen cells . CTB (1-100 ng/ml) enhanced anti-KLH IgM, IgG and IgA antibody responses in a dose-dependent manner, when added to the cultures with KLH . This immunoenhancement was antigen specific, but not due to either polyclonal activation of the spleen cells or antigenic cross-reactivity between CTB and KLH . CTB did not affect the kinetics of the anti-KLH antibody responses . Early (Days 0-1) addition of CTB to the cultures enhanced the anti-KLH antibody production, whereas late (Days 5-7) addition of CTB did not . Addition of CTB with KLH to splenic adherent cells (SAC) resulted in a dose-dependent enhancement of the anti-KLH antibody responses, when the SAC were reconstituted with unimmunized non-adherent cells . Moreover, CTB enhanced IL-1 secretion from SAC incubated with KLH . These results suggest that CTB enhances the primary anti-KLH antibody responses in vitro by acting on early events in the responses, and that antigen-presenting cells play a major role in the enhancement.

MMWR CDC Surveill Summ, 1992 Mar, 41(1), 27 - 34
Surveillance for epidemic cholera in the Americas: an assessment; Vugia DJ et al.; In January 1991, epidemic cholera appeared in Peru and quickly spread to many other Latin American countries . Because reporting of cholera cases was often delayed in some areas, the scope of the epidemic was unclear . An assessment of the conduct of surveillance for cholera in several countries identified some recurrent problems involving surveillance case definitions, laboratory surveillance, surveillance methods, national coordination, and data management . A key conclusion is that a simple, well-communicated cholera surveillance system in place during an epidemic will facilitate prevention and treatment efforts . We recommend the following measures: a) simplify case definitions for cholera; b) focus on laboratory surveillance of patients with diarrhea primarily in the initial stage of the epidemic; c) use predominantly the "suspect" case definition when the number of "confirmed" cases rises; d) transmit weekly the numbers of cases, hospitalized patients, and deaths to regional and central levels; e) analyze data frequently and distribute a weekly or biweekly summary; and f) report the number of cholera cases promptly to the World Health Organization.

J Biol Chem, 1992 Feb 15, 267(5), 3230 - 5
GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes; Walker MW et al.; ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP . ARFs have been purified from both membrane and cytosolic fractions . ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity . The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes . A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates . ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate) . GTP, GDP, GMP, and ATP were inactive . Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion . ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation . These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

J Virol, 1992 Feb, 66(2), 1171 - 5
Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus; Susa M et al.; Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus . It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues . Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure . A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.

Int J Immunopharmacol, 1992 Feb, 14(2), 111 - 20
The T-lymphocyte is the primary cellular target for potentiation of the in vitro T-dependent IgM antibody response by the B subunit of cholera toxin; Campbell KS et al.; The B (or binding) subunit of cholera toxin (CTB) was reported previously to potentiate the in vitro T-dependent IgM antibody response by a mechanism independent of the cyclic AMP-generating capacity of the intact toxin . In the present report, experiments were designed to determine the immune cell type mediating potentiation by CTB . Firstly, CTB did not potentiate T-independent antibody responses at concentrations that effectively enhanced T-dependent responses . Secondly, separation/reconstitution studies with splenocytes from CTB- and vehicle-treated mice demonstrated potentiation of T-dependent responses by CTB treatment of either the Sephadex G10 non-adherent population or the T-lymphocyte + macrophage population of cells . Potentiation was not observed by CTB treatment of the plastic adherent population or the B-lymphocyte + macrophage population . The evidence indicates that the T-lymphocyte is the primary cellular target for CTB-induced effects on the T-dependent IgM antibody response . Monosialoganglioside GM1, the putative binding site for CTB, is most likely the site of action for CTB on T-lymphocytes . These studies provide new insight on the mechanism of immunomodulation by cholera toxin, and CTB should provide a useful tool for further understanding the role of gangliosides in cellular immune responses.

Immunology, 1992 Feb, 75(2), 386 - 8
Immune responses of mice to orally administered asialo GM1-specific rabbit IgG in the presence or absence of cholera toxin; Umesaki Y et al.; Cholera toxin (CT) has been shown to be a most potent mucosal immunogen and an adjuvant to orally administered unrelated antigens . We investigated the effect of the oral administration of substances with the ability to bind to intestinal epithelial cells on the immune responses against themselves in the presence or absence of CT . Mice were fed non-specific rabbit IgG (RGG) or rabbit IgG (a-GA1) specific to asialo GM1 glycolipid, a major component of the apical membrane of mouse small intestinal epithelial cells, with or without CT . Oral administration of a-GA1 evoked stronger antibody responses than that of RGG in both the serum and intestinal fluid in the presence of CT . However, when the antigens were administered singly without CT, no significant antibody response was detected . In this case, oral administration of RGG induced severe suppression of the systemic antibody response to a subsequent intraperitoneal injection of RGG . In contrast, a-GA1 could not induce oral tolerance . Together these findings suggest that substances with the ability to bind to intestinal epithelial cells are strong immunogens in the presence of CT and weak tolerogens in the absence of CT.

Biosci Biotechnol Biochem, 1992 Feb, 56(2), 195 - 8
Inhibition by lactoferrin and kappa-casein glycomacropeptide of binding of Cholera toxin to its receptor; Kawasaki Y et al.; Inhibition from binding of Cholera toxin (CT) to Chinese hamster ovary (CHO)-K1 cells and ganglioside GM1 by lactoferrin (Lf) and kappa-casein glycomacropeptide (GMP) from cow's milk was examined . Both Lf and GMP effectively reduced the CT-derived morphological changes in CHO-K1 cells . The competitive binding assay demonstrated that both Lf and GMP inhibited the binding of CT to GM1, although their affinity for CT was lower than that of GM1 . The inhibitory effect of Lf and GMP seemed to be attributed to their terminal sialic acid, although the sugar chain sequence only partially fitted to the CT-receptor.

Proc R Soc Lond B Biol Sci, 1992 Jan 22, 247(1318), 17 - 20
The glutamate-receptor linked cGMP cascade of retinal on-bipolar cells is pertussis and cholera toxin-sensitive; Shiells RA et al.; Whole-cell patch-clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish . Inclusion of the A-subunit of pertussis toxin in the patch-pipette solution resulted in an increase in inward current and membrane conductance, and a block of light-evoked currents of on-bipolar cells . The opposite effect was obtained with the A-subunit of cholera toxin, which blocked light responses, and induced an outward current and a decrease in membrane conductance . These actions were NAD+ dependent . The results show that the G-protein(s) linking glutamate receptors to a cGMP cascade in on-bipolar cells possess sites which are ADP-ribosylated by pertussis and cholera toxins, with no homology to the adenylate cyclase system but possibly with a homology to transducin . Furthermore, inclusion of H-7, a kinase inhibitor in the patch-pipette solution, or of a non-hydrolysable ATP analogue (AMP-PNP) had no effect on light responses, membrane conductance or dark current of on-bipolar cells, suggesting that the components of this cGMP cascade are unlikely to be regulated by protein kinases.

J Biol Chem, 1992 Jan 15, 267(2), 1020 - 6
Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation; Iiri T et al.; The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T . (1989) J . Biol . Chem . 264, 21394-21400) . In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively . The modified Gi-2 displayed unique properties as follows . 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography . 2) When the purified proteins were incubated with {gamma-32P}GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein . 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification . 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification . 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation . 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein . These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated . Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.

Anat Embryol (Berl), 1992, 185(1), 1 - 16
Distribution and dendritic features of three groups of rat olivocochlear neurons . A study with two retrograde cholera toxin tracers; Vetter DE et al.; Cholera toxin B subunit conjugated to horseradish peroxidase, and unconjugated cholera toxin B subunit are useful tools for retrograde tract tracing . Unilateral injection of either cholera toxin preparation into the cochlea results in excellent labeling of olivocochlear neurons, as judged by the Golgi-like filling of cell bodies, dendrites, and even axons . By this approach, we have studied the light microscopic cytology and topographic distribution of olivocochlear neurons and counted their numbers in Sprague-Dawley rats . The olivocochlear system of rats can be divided into three subgroups . The lateral olivocochlear system, composed of small cells located exclusively within the ipsilateral lateral superior olive (relative to the test cochlea), and a medial olivocochlear system, composed of large cells bilaterally dispersed within the ventral nucleus of the trapezoid body, conformed to previous topographic descriptions . A third subgroup of approximately 110 large cells, herein termed shell neurons, was labeled by both tracers, but was not well recognized in previous studies . Shell neurons and their dendrites surround the ipsilateral, and to a much lesser extent the contralateral, lateral superior olive . Lateral olivocochlear neurons do not project their dendrites outside the gray matter of the lateral superior olive, while dendrites belonging to shell neurons penetrate into that nucleus as well as into other auditory brain stem nuclei and the surrounding reticular formation . Medial olivocochlear neurons usually project dendrites ventrally into the trapezoid body and are always excluded from the lateral superior olive.

Biotechnology, 1992, 20, 53 - 68
Cholera vaccines; Tacket CO et al.; The currently licensed parenteral cholera vaccine has not been a useful public health tool in the control of cholera . Building on the knowledge that primary infection offers significant protection against reinfection and that mucosal immunity mediates this protection, several oral cholera vaccines have been developed . These vaccine candidates or future candidates derived using the techniques of molecular biology will no doubt contribute to the control of cholera.

Appl Environ Microbiol, 1992 Jan, 58(1), 169 - 73
Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant; Azcona-Olivera JI et al.; Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant . Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used . In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective . Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant . A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding . The detection limit for fumonisin B1 in the ELISA was 100 ng/ml . The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid . Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively . These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.

Vaccine, 1992, 10(2), 101 - 6
Stimulation of the transepithelial flux of influenza HA vaccine by cholera toxin B subunit; Gizurarson S et al.; Secretory antibodies in mucosal surfaces are known to play an essential role in protection against various infectious diseases . To enhance the production of such antibodies, influenza HA vaccine was inoculated intranasally into rabbits, together with cholera toxin B subunit (CTB) which is known to augment antibody response to an unrelated antigen . This combination resulted in high levels of serum IgG antibody responses against HA and CTB molecules, 3-4 weeks after inoculation, compared with the inoculation of HA vaccine alone . The adjuvant mechanism for CTB was studied by using Ussing chambers, in which nasal mucosa from rabbits were mounted . CTB was found to enhance the transepithelial flux of HA vaccine, from the mucosal side (lumen) into the serosal side (lamina propria), indicating that the permeability of the membrane was changed by CTB . Moreover, to achieve effective flux of HA vaccines, some interactions between the vaccine and CTB across the membrane were found, which may effect the effectiveness of the vaccine formulation . The results suggest that one of the mechanisms by which CTB enhances the production of mucosal antibody response is to enhance the transepithelial influx of vaccine into the nasal mucosa, where the cells involved in the antibody production are located . CTB may be used as a potent adjuvant to induce antibody response, by nasal vaccination, against pathogens impinging on mucosal surfaces.

World Health Stat Q, 1992, 45(2-3), 208 - 19
{Cholera, 1991--old enemy, new face}; Simeant S; The cholera epidemics of the XIXth century are described and reviewed . The extent, incidence and case-fatality rate for the disease in the seventh pandemic are described . The global epidemiological situation and its trend at the end of 1991 are analysed . A review of cholera epidemiology highlights the factors that might explain the less tragic nature of the disease today . The role of water, food and direct contagion in transmission of cholera over the last 20 years is considered in the light of recent studies and with special reference to the epidemic in Latin America, where the intense emotion aroused by the disease has prompted vigorous action that could produce significant and lasting progress in the health field.

Rev Gastroenterol Peru, 1992, 12(1), 23 - 7
{New epidemic outbreak of cholera in Lima}; Hoyos C et al.; We report a new outbreak of cholera on the beginning of last Summer (Dec . 91 and Jan 92) . Were 281 patients, 63% male and 36% female, treated in our Cholera Unit of treatment; all of them coming from marginal-urban populations . There was a rate of lethality of inpatients of 0.46% and a mortality of 0.25% over the total of patients that we saw in our hospital; on this summer the outbreak is early and greatest than the summer of 1991 . We can conclude that because of epidemic behavior during the whole 1991 and in the time elapsed of 1992, Peru has become an endemic zone of this new disease, while the current epidemiologic settings stay unchanged.

Vaccine, 1992, 10(11), 735 - 6
Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice; Dragunsky EM et al.; Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC) . CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT . Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively . All three killed parenteral vaccines failed to protect against CT . We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.

Rev Epidemiol Sante Publique, 1992, 40(3), 145 - 55
The cholera epidemic in Ecuador: towards an endemic in Latin America; Weil O et al.; We present an epidemiological study of the first wave of cholera in Ecuador in 1991 . One month after the 7th cholera pandemic hit the Pacific coast of Peru, the disease reached the coast of Ecuador and spread to the rest of the country within a few weeks . One year later, 46,320 cases have been notified, giving an incidence of 481 cases per 100,000 inhabitants . The overall mortality rate has been low (697 deaths, i.e . 1.50%), although there have been large differences between the various provinces: in the Andes (Sierra), for example, rates above 8% have been recorded . The first wave peaked about eight weeks after the first case, with 3400 new cases per week throughout the country . This was followed by a gradual decrease towards a baseline of 250-500 new cases per week . A resurgence was observed in most coastal and Andean provinces from November 1991 onwards . The kinetics of the epidemic are compatible with an endemic implantation of the disease in Ecuador, as is probably the case in the entire intertropical Pacific coast region of Latin America . From the data presented, the latin-american episode of the 7th pandemic starting on the Pacific coast, is characterized by a very high attack rate and a low mortality rate as compared to Africa, and the unexpected involvement of populations living on the high Andean plateaux . It is probable from the results collected in Ecuador that cholera will become endemic in Latin America.

Microbiol Immunol, 1992, 36(7), 745 - 56
Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice; Zhou NN et al.; MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse . CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice . Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment . The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice . The latter may reflect increased B cells in relative number in the peripheral lymphoid organs . Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group . Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT . Production of IFN gamma was suppressed by CT treatment in both strains of mice . This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J . Exp . Med . 172: 95-103, 1990) . However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.

Ann Rech Vet, 1992, 23(1), 93 - 100
Characterization and pathogenicity for pigs of a hog cholera virus strain isolated from wild boars; Leforban Y et al.; One hog cholera virus strain isolated from an outbreak of the disease in a wild boar breeding herd in Brittany (France) in 1990 has been characterized with a panel of monoclonal antibodies to hog cholera virus and ruminant pestiviruses: the strain was found to be indistinguishable from that of other domestic pig isolates . The pathogenicity of the strain to domestic pigs was evaluated by infecting intranasally, intramuscularly and by contact 17 specific pathogen-free 6-week- and 12-week-old pigs . Sixteen of the 17 pigs showed symptoms of hog cholera . The virus was detected in the blood of the 16 pigs during all phases of hyperthermia which persisted up to death or the terminal phase, ie between 16 and 29 days post-infection . One animal recovered after presenting a mild form of the disease . This pig was the only one which raised antibodies to the virus . Typical hog cholera lesions were observed in 2 pigs only; the other animal showed very few pathological changes . No relationship between intensity or duration of the disease and pathological changes could be established.

Exp Brain Res, 1992, 89(3), 478 - 83
Morphology of sympathetic preganglionic neurons innervating the superior cervical ganglion in the chicken: an immunohistochemical study using retrograde labeling of cholera toxin subunit B; Hosoya Y et al.; Preganglionic sympathetic neurons (SPNs) in the chicken were demonstrated immunohistochemically using cholera toxin subunit B (CTb) as a retrograde tracer . After injection of CTb-solution into the superior cervical ganglion, labeled SPNs were mainly found in the ipsilateral sympathetic preganglionic column of Terni (the column of Terni), with only a few in the intermediate zone . They were observed from the caudal half of the 15th cervical segment to the rostral tip of the 3rd thoracic segment . Cell somata of SPNs were loosely packed within the column of Terni, where they had an elliptic shape with the long axis oriented rostrocaudally . In the horizontal plane three kinds of dendrites could be discriminated on the basis of their orientation . Longitudinally oriented dendrites emanated from the rostral and the caudal poles of the SPNs . Medially oriented dendrites were observed to cross the midline and enter the contralateral column of Terni, where they further branched to form a loose dendritic plexus; some extended beyond the lateral limit of the contralateral column of Terni to reach the intermediate zone . Laterally oriented dendrites formed periodically arranged dendritic bundles projecting into the intermediate zone . The present findings provide a detailed account of the dendritic organization of SPNs in the chicken, and suggest that avian SPNs share certain structural features in common with mammalian SPNs.

Vaccine, 1992, 10(14), 1015 - 21
Molecular design of cholera vaccines; Manning PA; Cholera is still a serious public health problem in developing countries, particularly those in tropical regions . This has stimulated considerable research into the molecular analysis of pathogenesis resulting in the identification of a number of critical components required for both colonization of the gut mucosa and the disease symptoms . These components are the targets for rational molecular approaches to vaccine development.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1992, 62(3), 189 - 98
Lateral growth and terminal differentiation during repeated epidermal regeneration in vitro . Age dependence and modulation by cholera toxin; Jensen PK et al.; By incubating multilayered primary cultures of human epidermal keratinocytes in a low calcium medium, the suprabasal layers can be stripped off leaving a basal cell monolayer . When this monolayer is refed normal calcium medium a reproducible series of cell kinetic, morphological and biochemical changes take place resulting in the regeneration of a multilayered tissue . The stripping procedure seems to induce the selective proliferation of a cohort of basal cells that is committed to vertical migration and rapid terminal differentiation . In contrast, when the basal cells are allowed to regenerate in the presence of the strong mitogen, cholera toxin, lateral growth and continued proliferation are favoured at the expense of the capacity of the cells to differentiate . Repeated stripping of the same cultures disclosed a considerable heterogeneity in the capacity of the basal cells to regenerate the suprabasal layers . The number of times the basal cells could restore the suprabasal layers after repeated stripping varied from four to nine times . A negative correlation between donor age and regenerative capacity was observed . The experiments with repeated stripping of the same cultures also showed that the capacity to proliferate and to restore the multilayering was fully retained for at least four cycles of stripping-regeneration, whereas the capacity to terminally differentiate was rapidly lost . It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model for the study of epidermal tissue homeostasis and cellular aging.

Braz J Med Biol Res, 1992, 25(9), 913 - 7
A toxoid prepared from cholera toxin by iodination; Heneine IF et al.; A cholera toxoid was prepared by iodinating purified cholera toxin having an activity of 25 Limit of blueing (Lb) doses/1 microgram with 0.8 mumol of iodine monochloride per mg toxin, and the residual lesion capacity was tested in mice . The blueing dose (BD) test was strongly positive for the native toxin, and completely abolished in the iodinated toxoid when tested at up to 25 times on Lb dose . The dermal microscopic lesions with intradermal doses of 1 microgram virulent toxin presented intense leucocyte infiltration, proteinaceous edema and active hyperemia, whereas none of these effects was observed with the same amount of toxoid . To determine antigenicity, two groups of mice received toxin or toxoid, 8.5 micrograms adsorbed to aluminum hydroxide, followed by a booster of 17 micrograms in saline 21 days later . Measurement of antibodies by ELISA at day 28 indicated that the toxoid was 2.5 times more antigenic than the toxin . These data show that iodination converts cholera toxin to an effective toxoid.

Braz J Med Biol Res, 1992, 25(8), 805 - 7
Gangliosides inhibit serological reactions for the detection of cholera and heat-labile enterotoxins of Escherichia coli; Ricci LC et al.; GM1 ganglioside has been identified as the receptor for cholera toxin (CT) and heat-labile (LT) enterotoxin of Escherichia coli in many cell types . Using the radial immune hemolysis (RIH) and indirect hemagglutination (IH) tests described for the detection of these enterotoxins, a study was conducted on the 100% inhibition of these reactions by pre-incubating these enterotoxins with GM1, GD1a and GT1 gangliosides . GM1 was found to be much more efficient than the other two . With respect to the RIH test, GT1 was more efficient than GD1a as an inhibitor of enterotoxin binding . Similar results were obtained with the IH test . These data also showed that sheep red blood cells provide a good model system for the study of receptors for CT, LT and probably other enterotoxins which bind to gangliosides.

Ann Rech Vet, 1992, 23(1), 73 - 82
Protection of piglets born from ruminant pestivirus experimentally infected sows, and their contacts, to the challenge with hog cholera virus; Leforban Y et al.; Two groups, A and B, of two specific pathogen-free pregnant sows were experimentally infected between the 25th and 29th days post-breeding with two strains of ruminant pestivirus: NADL cytopathic bovine viral diarrhoea virus for group A and Aveyron non-cytopathic border disease virus French strain for group B . Two other pregnant sows (group C) were kept uninoculated as control . When 7 weeks old, 8 piglets of group C were put in contact with 4 piglets of group A (group D), and 8 other piglets of group C with 4 piglets of group B (group E) in two separate pens with the purpose of testing the horizontal transmission of the viruses . All animals were kept under observation and serologically controlled at weekly intervals; two pigs of each group were finally submitted to a challenge with hog cholera virus . The two pigs of group E which were put in contact with the offspring of the border disease virus infected sow were protected; all other animals developed typical hog cholera symptoms and died . The relation between neutralizing titres of the sera to ruminant pestiviruses and protection to the challenge with hog cholera virus is discussed . The two protected pigs had high neutralizing antibody titres to border disease virus but no antibody to hog cholera virus at the time of the challenge . Though the two viruses look serologically distant, we surprisingly observed that infection with border disease virus protects against a superinfection with hog cholera virus.

Gen Pharmacol, 1992 Jan, 23(1), 27 - 31
Cholera toxin augments the release of endothelium-derived relaxing factor evoked by bradykinin and the calcium ionophore A23187; Boulanger CM et al.; 1 . Experiments were designed to examine the effect of cholera toxin and forskolin on the release of relaxing factor(s) from superfused cultured endothelial cells under basal conditions and upon stimulation with bradykinin, adenosine diphosphate or the calcium ionophore A23187 . 2 . Exposure of cultured porcine aortic endothelial cells to cholera toxin (30 micrograms/ml, for 3 hr) and forskolin (10(-6) M, for 45 min) significantly increased the intracellular content in cyclic AMP . Cholera toxin but not forskolin stimulated the accumulation of cyclic GMP . 3 . Exposure to cholera toxin did not modify the basal release of endothelium-derived relaxing factor nor that induced by adenosine diphosphate, but significantly increased that evoked by bradykinin and the calcium ionophore A23187 . Forskolin did not significantly affect the basal or the stimulated release of endothelium-derived relaxing factor . 4 . These results suggest that cholera toxin potentiates the release of endothelium-derived relaxing factor (presumably nitric oxide) from endothelial cells by a mechanism other than augmented production of cyclic AMP.

Cell Signal, 1992 Jan, 4(1), 87 - 98
Cholera and pertussis toxins modify regulation of glucose transport activity in rat adipose cells: evidence for mediation of a cAMP-independent process by G-proteins; Honnor RC et al.; Adenylyl cyclase in rat adipose cells is stimulated by ligands for Rs receptors (e.g . isoproterenol) and inhibited by ligands for Ri receptors (e.g . adenosine) . In contrast, Rs receptors mediate inhibition and Ri receptors mediate augmentation of insulin-stimulated glucose transport activity by a process independent of changes in cellular cAMP-dependent protein kinase activity {Kuroda M., Honnor R . C., Cushman S . W., Londos C . and Simpson I . A . (1987) J . biol . Chem . 262, 245-253} . The present study examines the possible role of G-proteins in the regulation of insulin-stimulated glucose transport activity by Rs and Ri receptors . First, conditions were established that permit intoxication of isolated rat adipocytes by cholera and pertussis toxins without compromising cell integrity . Effectiveness of toxin treatment was monitored by examining adenylyl cyclase activity in isolated plasma membranes . Secondly, neither toxin interfered with the ability of a maximal concentration insulin to initiate the glucose transport response . Thirdly, pertussis toxin eliminated the augmenting effects of adenosine on insulin-stimulated glucose transport activity, but enhanced the inhibitory effects of isoproterenol . Findings with ligands for other Ri receptors (nicotinic acid and prostaglandin E2) mirrored those with adenosine . Finally, cholera toxin elicited a modest depression of transport activity, and only in the absence of an Ri ligand (e.g . adenosine) . Furthermore, in contrast to the enhanced stimulation of adenylyl cyclase by isoproterenol and GTP, cholera toxin eliminated the inhibitory effect of isoproterenol on transport activity . The augmentative effects of adenosine on transport activity were unchanged . Measurements of (-/+cAMP) cAMP-dependent protein kinase activity ratios reinforce the notion that modulation of glucose transport activity is independent of changes in cAMP . We conclude that regulation of glucose transport activity by Rs and Ri receptors is mediated by the G-proteins, Gs and Gi (or other toxin substrates), respectively . Inasmuch as such regulation occurs at the plasma membrane and appears to be cAMP-independent, it is suggested that glucose transporters may be direct targets for receptor: G-protein interactions.

Immunology, 1992 Jan, 75(1), 103 - 7
Cholera toxin inhibits interleukin-2-induced, but enhances pertussis toxin-induced T-cell proliferation: regulation by cyclic nucleotides; Maghazachi AA; To understand the signals transmitted by interleukin-2 (IL-2) during T-cell proliferation, the effect of this cytokine was compared to the bacterial product pertussis toxin (PT) . Both IL-2 and PT induced the incorporation of {3H}thymidine into T cells . Cholera toxin (CT) inhibited IL-2-induced, but enhanced PT-induced T-cell proliferation . The effect of CT is mimicked by the cyclic AMP (cAMP) analogue 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dicAMP) or by the phosphodiesterase inhibitors isobutylmethylxanthine and aminophylline . Measurement of the intracellular level of cAMP showed that CT enhanced this level during both IL-2 or PT incubation with T cells . To delineate the differential effects of cAMP on IL-2 versus PT activity, it was observed that the blocker of intracellular calcium (TMB8), or the guanosine triphosphate (GTP) analogue (GTP gamma S) inhibited both PT and IL-2 activities, whereas the protein kinase C (PKC) inhibitor (H7) was without effect for both stimuli . Further experiments showed that both IL-2 and PT stimulate the endogenous level of cGMP and that CT enhanced this level following PT activation, but reduced it following IL-2 activation of T cells . Hence, there is a major difference between IL-2 and PT activation of T cells in as far as their susceptibility to treatment with cholera toxin is concerned . Furthermore, an increase of cGMP level resulted in the enhancement of proliferation, whereas a decrease in cGMP level resulted in the inhibition of proliferation.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1412 - 8
Mono(ADP-ribosyl)ation of poly(ADP-ribose)polymerase by cholera toxin; Martinez M et al.; Poly(ADP-ribose)polymerase (PADPRP) was found to be an efficient protein acceptor for the arginine-specific ADP-ribosylation reaction catalyzed by cholera toxin (CT) . The covalent modification of PADPRP was carried out with {32P}2'-dNAD as a selective mono(ADP-ribosyl)ation substrate . Mono(2'-dADP-ribosyl)ated-PADPRP was identified by autoradiographic analysis of the CT reaction products following sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Addition of recombinant ADP-ribosylation factor (rARF), a small GTP-binding protein that stimulates the enzymatic activity of CT, enhanced the mono(2'-dADP-ribosyl)ation of PADPRP in a time- and substrate-dependent manner . In contrast, rARF did not change the ADP-ribose polymerizing activity of PADPRP . Peptide mapping mapping of {32P} labeled (2'-dADP-ribose)-PADPRP, following partial proteolysis with papain, revealed that the DNA-binding domain of PADPRP contained the mono(2'-dADP-ribosyl)ated arginine residue(s) . Our results are consistent with the conclusion that PADPRP is susceptible to arginine-specific mono(ADP-ribosyl)ation catalyzed by CT.

Neurosci Lett, 1991 Dec 16, 134(1), 113 - 7
Activation of locus coeruleus (LC) neurones by cholera toxin: mediation by cAMP-dependent protein kinase; Alreja M et al.; There is evidence that the tonic pacemaker activity of the noradrenergic pacemaker neurones of the locus coeruleus (LC) depends on endogenous cAMP acting via protein kinase A and its phosphorylation pathway . In this study, we tested the effect of cholera toxin, which produces persistent activation of Gs, on LC firing rates . Bath applied cholera toxin (holotoxin) increased LC firing rates after a lag of 50-110 min . Intracellularly applied A-subunit (active-subunit) but not the B-subunit (binding-subunit) of cholera toxin via low resistance patch electrodes mimicked the excitatory actions of bath-applied holotoxin but without its lag period . The effects of both bath-applied and intracellularly applied cholera toxin A-subunit were blocked by intracellular applications of a specific cAMP-dependent protein kinase inhibitor (PKI5-24) . We conclude that persistent activation of Gs by cholera toxin (A-subunit) increases LC firing rates via the cAMP-dependent protein phosphorylation pathway.

MMWR Morb Mortal Wkly Rep, 1991 Dec 13, 40(49), 844 - 5
Cholera associated with imported frozen coconut milk--Maryland, 1991; Update: cholera--Western Hemisphere et al.; The epidemic of cholera that began in Peru in January 1991 continues to spread . Most recently, Bolivia, El Salvador, Honduras, Nicaragua, and Panama were added to the list of countries reporting cholera cases (1-3).

Tidsskr Nor Laegeforen, 1991 Dec 10, 111(30), 3652 - 6
{Some cholera graveyards in western Norway}; Oeding P; The Norwegian provisional regulations from 1832 for the control of cholera included detailed directions for the burial of cholera victims . How these directions for graveyards were followed up has been studied in the context of the cholera epidemic in Western Norway in 1848-49 . In Bergen an ordinary graveyard was used for burying cholera victims at the beginning of the epidemic, while all later burials took place in a graveyard for cholera victims only . 549 victims were buried here . On the island of Sotra the majority of the cholera victims were buried in three cholera graveyards, and further south in the county several small graveyards were planned or laid out . In general, the directions for cholera graveyards were followed . The cholera graveyard in Bergen was levelled over one hundred years ago, while several other cholera graveyards in Western Norway still exist as reminders of the epidemic.

J Biol Chem, 1991 Dec 5, 266(34), 23053 - 9
Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin; Tsai SC et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro . Five different human ARFs have been identified by cDNA cloning . Northern analysis using ARF 3-specific oligonucleotides identified two mRNAs of 3.7 and 1.2 kilobases (kb) . We report here the complete nucleotide sequence of the 3.7-kb ARF 3 mRNA derived from three overlapping cDNAs isolated from human hippocampus and fetal brain cDNA libraries, as well as the structure of human ARF 3 gene . Sequences of two overlapping genomic clones indicated that the ARF 3 gene spans approximately 18.3 kb and contains five exons and four introns . The conserved amino acid sequences involved in guanine nucleotide binding by ARF 3 are distributed among separate exons, as found in other GTP-binding protein genes . Translation initiates in exon 2 which includes the sequence GXXXXGK that probably participates in phosphate binding and GTP hydrolysis . The sequence DVGG in exon 3 coordinates binding of Mg2+ and the beta-phosphate of GDP . In the ARF 3 gene in contrast to those of other GTP-binding proteins, the sequence NKXD (which is thought to contribute to the specificity of interaction with the guanine ring) is divided between exons 4 and 5 . The latter encodes the COOH-terminal 53 amino acids of ARF 3 and contains greater than 2500 base pairs of untranslated DNA . The sequence AATTAA is 19 bases 5' to the polyadenylation addition site of the 3.7-kb mRNA . Multiple transcription start sites were identified by primer extension and S1 and mung bean nuclease analyses . The 5'-flanking region of exon 1 contains neither a TATA nor a CAAT box, but is high in GC content (greater than 70%) and includes three potential Sp1-binding sites (GC box), consistent with the promoters described for several housekeeping genes . The 1.2-kb ARF 3 mRNA is shown to arise by use of an alternative polyadenylation signal (AACAAA) at nucleotide 1091 within the ARF 3 cDNA.

Eur J Biochem, 1991 Dec 5, 202(2), 635 - 41
Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin; Iiri T et al.; The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine) . The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin {Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M . & Katada, T . (1989) J . Biol . Chem . 264, 21,394-21,400} . This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein . The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows . (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes . The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes . (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes . In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes . (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi . These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin . Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein . This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.

J Neurobiol, 1991 Dec, 22(9), 976 - 88
Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: a cholera toxin-HRP study; Kurz EM et al.; The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior . Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood . Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats . Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure . Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed . Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport . However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone . Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area . These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.

Poult Sci, 1991 Dec, 70(12), 2425 - 8
Interaction of naturally occurring aflatoxins in poultry feed and immunization against fowl cholera; Hegazy SM et al.; A total of 1,175 poultry feed samples originating from different farms were analyzed for aflatoxin . Poor growth rate and reduced egg production were the main complaints . The rate of contamination with aflatoxin ranged from 10 to 54% of all samples . Of samples examined 30.7% proved positive for aflatoxin with a concentration ranging from 1 to 2,000 ppb . Outbreaks of fowl cholera were diagnosed on two farms where aflatoxin was detected in the rations used . The impact of aflatoxin in the feed on the efficacy of immunization against fowl cholera was monitored by a hemagglutination test and the titers of the involved farms were compared with experimental groups fed on aflatoxin-free rations and vaccinated with the same polyvalent fowl cholera bacterin . The antibody titers of the experimental groups were 4 to 15 times higher than those of the involved farms.

Lymphokine Cytokine Res, 1991 Dec, 10(6), 449 - 55
Cholera toxin synergizes LPS- and IL-1 beta-induced PGE2 release: potential amplification systems in cholera; Hill SJ et al.; Incubation of LPS/IL-1 beta and cholera toxin combinations resulted in a synergistic rise of immunoreactive and {3H}PGE2 released from HGFs . This interaction was not dependent on the type of LPS utilized; however, the cholera toxin enzymatic subunit was essential to the synergism . Intracellular cAMP concentrations paralleled PGE2 synthesized in response to LPS and cholera toxin . dBcAMP and forskolin incubated with LPS failed to demonstrate the synergistic rise of PGE2, suggesting elevated cAMP levels alone are insufficient for increased PGE2 synthesis . These findings demonstrate interactions between LPS/IL-1 beta and cholera toxin and support the potential for an expanded role of LPS and IL-1 beta in the pathogenesis of cholera.

Zentralbl Veterinarmed B, 1991 Dec, 38(10), 764 - 72
Clinical, post mortem and virological findings after simultaneous inoculation of pigs with hog cholera and bovine viral diarrhoea virus; Dahle J et al.; The clinical course, post mortem lesions as well as virological and serological results after simultaneous intranasal inoculation of pigs with bovine viral diarrhoea virus (BVDV) and hog cholera virus (HCV) are described . Five groups of four weaners received constant doses of BVDV strain OSLOSS/2482 and tenfold decreasing doses of HCV strain ALFORT/187 . Doses of 1,000 and 100 TCID50 of HCV in groups A and B of pigs led to fever and severe clinical signs in all animals of two groups, whereas at higher dilution of inoculum two, three or four animals survived without any clinical signs in the respective groups (C-E) . Leucocyte samples taken from febrile animals and from normal pigs on five consecutive days were inoculated into both fetal calf kidney (FCK) and PK (15) cell cultures . Virus isolates were differentiated with BVDV and HCV specific monoclonal antibodies . HCV viraemia was detected in febrile animals exclusively, and BVDV viraemia occurred in not affected animals on days 3 to 7 post inoculation . Neutralizing antibodies (nab) against BVDV appeared before HCV nab in surviving animals of groups C and D after receiving low doses of HCV (10 or 1 TCID50) . No BVDV nab were detected in group E that had received such a high dilution of HCV in addition to BVDV that theoretically no HCV was applied.

Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 208 - 12
The effect of cholera toxin on human red cell Ca-ATPase; Romero PJ et al.; The effect of cholera toxin on transport Ca-ATPase was studied in membrane fragments from human red cells . A consistently moderate inhibition was found when fragments were previously incubated with toxin in the presence of beta-NAD but not in its absence of after treatment with non-activated toxin . In calmodulin-free preparations, both Ca affinity and maximal rate of hydrolysis were affected whereas only affinity was altered in calmodulin-deficient membranes.

Biochem Pharmacol, 1991 Nov 6, 42(11), 2141 - 6
Gs alpha availability to cholera toxin-catalysed ADP-ribosylation is decreased in membranes of retinoic acid-treated leukemic cell lines HL-60 and THP-1 . A posttranslational effect; de Cremoux P et al.; Retinoic acid (RA) induces HL-60 and THP-1 leukemic cell lines to differentiate into granulocyte-like and monocyte-like cells . Limited data are available concerning the effects of RA on components of the cyclic AMP pathway in human myeloid leukemic cells . We showed previously a decrease in adenylate cyclase activity in the presence of histamine, prostaglandin E1 and forskolin in RA-treated HL-60 cells as compared to untreated cells . We examined the elements of the signal transduction pathway utilized by RA in the human myeloid cell line HL-60 and the human monocytic cell line THP-1 . We therefore studied the effect of RA on the activity of the stimulatory G-protein (Gs) . We demonstrate that addition of RA to two human myeloid leukemia cell lines, HL-60 and THP-1, does not induce a reduction of the 2 subunit of Gs (Gs alpha) RNA or Gs alpha protein in the plasma membrane but leads to a rapid decrease in the cholera toxin (CTX)-catalysed ADP-ribosylation of Gs alpha . In addition, this effect seems to be specific to RA, since there was no modification in Gs alpha ADP-ribosylation in the membranes of cells treated with dimethyl sulfoxide (DMSO), another inducer of differentiation in HL-60 cells.

Infect Immun, 1991 Nov, 59(11), 4266 - 70
Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity; Burnette WN et al.; Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus . Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity . We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity . This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.

J Virol Methods, 1991 Nov-Dec, 35(2), 227 - 36
Rapid detection of hog cholera virus in tissues by the polymerase chain reaction; Liu ST et al.; A rapid method for the detection of hog cholera virus (HCV) in infected tissues, using polymerase chain reaction (PCR) was developed . Total RNA isolated from HCV-infected tissues was reverse transcribed with AMV reverse transcriptase and the resulting complementary DNA was amplified by Taq DNA polymerase in the presence of two HCV-specific primers . The amplified DNA fragment was detected by agarose gel electrophoresis . The sensitivity of this method was at 10(4) TCID50 of HCV . The sensitivity increased approximately 1000-fold when the DNA was reamplified with a set of nested primers . DNA sequencing analysis of the PCR products revealed that the HCV sequence amplified from a local field isolate was highly homologous to the HCV Alfort strain . This method may be useful for pathological and epidemiological studies of HCV in pigs.

J Med Virol, 1991 Nov, 35(3), 192 - 7
Similar subclass antibody responses after intranasal immunization with UV-inactivated RSV mixed with cholera toxin or live RSV; Reuman PD et al.; To determine the effect of cholera toxin as a mucosal adjuvant on the class and subclass antibody response to RSV, mice were immunized intranasally with different doses of live RSV or UV-inactivated RSV mixed with cholera toxin . A single 10(6) pfu dose of live RSV and a single 50 micrograms dose of UV-inactivated RSV mixed with cholera toxin produced comparable serum IgG and respiratory secretion IgG and IgA antibody titers . Subclass antibody titers to whole RSV were also comparable between these two immunizing regimens . A predominance of IgG2a subclass to whole RSV was found for both regimens . The quantity of serum total IgG antibody to glycoprotein F or glycoprotein G did not differ between these regimens . The serum IgG subclass antibody response to both glycoprotein F and G was also not significantly different between regimens . Cholera toxin as a mucosal adjuvant can stimulate class and subclass antibody responses to UV-inactivated RSV that are similar in quantity and distribution to those after live RSV infection.

Mol Microbiol, 1991 Nov, 5(11), 2621 - 7
Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins; Moss J et al.; Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively . Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target . Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c . 20 kDa, termed ADP-ribosylation factors or ARFs . In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin . The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding . Six different mammalian ARF genes have been identified . They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding . Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein . Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.

Vaccine, 1991 Nov, 9(11), 825 - 32
The effect of cholera toxin and cholera toxin B subunit on the nasal mucosal membrane; Gizurarson S et al.; The effects of the self-adjuvanting substances, cholera toxin (CT) and cholera toxin B subunit (CTB), on rabbit nasal mucosal membrane, were investigated by using Ussing chambers . The control nasal mucosa (lateral wall), isolated from rabbits and mounted in the chamber, showed transepithelial potential difference, short-circuit current and conductance of -10 mV, 200 microA cm-2 and 20 mS cm-2, respectively . These parameters were compared with mucosa isolated from human inferior conchae, showing that rabbit nasal mucosa may be usable to understand effects on human mucosa . When the mucosa was exposed to various concentrations of CTB and CT, the short-circuit current and conductance of the mucosa increased with increasing concentration . CTB showed gradual increase in the short-circuit current when added in the same molar concentration as the B subunit contained in CT, which caused drastic changes by increasing the current to infinite value . Furthermore, the total amount of transepithelially fluxed CTB, which occurred rapidly after addition to the mucosal side of the chambers, increased with increasing CTB concentration . On the other hand, less flux was observed after addition of CT . These changes could be blocked by addition of ganglioside GM1 . This demonstrates that the effect of CTB on the rabbit mucosal membrane are different from those of CT, although both CT and CTB act specifically on the membrane via the CTB receptor, ganglioside GM1.

Med Hypotheses, 1991 Nov, 36(3), 253 - 8
The cystic fibrosis heterozygote--advantage in surviving cholera?
Rodman DM, Zamudio S.
Cystic fibrosis (CF) is the most common fatal genetic disorder of caucasians . While it has been hypothesized that there is a CF heterozygote advantage which allowed the gene to achieve such high prevalence, the nature of that advantage remains a mystery . The recent identification and sequencing of the CF gene has increased the probability that the CF heterozygote advantage will be discovered . In this hypothesis we review the information which is known about the selection of the CF mutation and its cellular consequences, and present evidence which suggests that resistance to cholera may have been the environmental factor which selected CF heterozygotes over their 'normal' homozygote cohort . Future lines of experimentation and possible clinical applicability to therapy of secretory diarrhea are presented.

Dig Dis Sci, 1991 Nov, 36(11), 1574 - 81
Intestinal mucin secretion in streptozotocin-diabetic rats: lack of response to cholinergic stimulation and cholera toxin; Mantle M et al.; In diabetic rats, intestinal mucin secretion is unusually high compared with that in normal rats . These studies demonstrate that mucin synthesis is also increased in the diabetic intestine . alpha- and beta-adrenergic agonists or antagonists did not affect mucin output in either normal or diabetic animals, suggesting that altered release in diabetes was not due to goblet cells responding abnormally to adrenergic agents . The cholinergic agonist bethanechol caused a dose-dependent and atropine-sensitive increase in mucin secretion from the normal intestine but had no effect on mucin release from diabetic tissue . Atropine alone did not reduce mucin secretion from the diabetic intestine to levels found in normal tissue . Cholera toxin caused an approximately fivefold increase in mucin output from normal rats but had no effect on mucin secretion from diabetic animals . Thus, goblet cell responses to cholinergic stimulation and cholera toxin in the diabetic intestine are markedly impaired . However, loss of cholinergic control does not appear to be responsible for altered baseline mucin secretion in diabetes.

Biochim Biophys Acta, 1991 Oct 21, 1097(3), 193 - 204
Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice; Palmer TM et al.; Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals . In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits . Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals . Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals . Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p{NH}ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22% . Lower concentrations (EC50-60 nM) of p{NH}ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM) . As well as causing activation, p{NH}ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals . However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals . Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p{NH}ppG through a process suggested to be mediated by the abolition of functional Gi activity . The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits . We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p{NH}ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.

Infect Immun, 1991 Oct, 59(10), 3630 - 8
Host defense against cholera toxin is strongly CD4+ T cell dependent; Hornqvist E et al.; This study investigates the role of CD4+ T cells in host defense against cholera enterotoxin-induced diarrhea . Antitoxin immunoglobulin A formation and gut protection against cholera toxin (CT) following oral immunizations with CT were evaluated in normal mice and mice that had been depleted of CD4+ T cells by in vivo treatment with specific anti-CD4 monoclonal antibodies . Flow cytometer analysis demonstrated that anti-CD4 monoclonal antibody effectively eliminated CD4+ T cells in the spleen, mesenteric lymph nodes, and Peyer's patches . In contrast, lamina propria lymphocytes demonstrated only some decrease in CD4+ T-cell numbers following antibody treatment . However, CD4 expression of individual lamina propria lymphocytes was strongly down-regulated . Depletion of CD4+ T cells performed prior to oral immunization with CT completely inhibited the ability to respond to CT . No antitoxin production, as detected at the single-cell level by the ELISPOT technique, was found in the spleen, mesenteric lymph nodes, or Peyer's patches, nor did we observe serum antitoxin responses in these mice . Control mice demonstrated strong antitoxin responses in all locations following oral immunization with CT . Anti-CD4 antibody treatment also effectively inhibited the antitoxin immunoglobulin A response in the lamina propria to CT as well as blocked the ability to develop gut protection against CT challenge of ligated intestinal loops after oral CT immunization . Thus, in vivo CD4+ T-cell depletion rendered these mice unable to develop protective immunity in the gut following oral immunization with CT . Moreover, CD4+ T-cell depletion effectively inhibited the antitoxin immune response in the gut lamina propria, mesenteric lymph nodes, Peyer's patches, and spleen when performed prior to both priming and booster immunizations with CT . This study clearly demonstrates the requirement of functional CD4+ T cells in the gut immune system for the development of host defense against CT-induced disease . Our data also reinforce the concept of a strong association between gut protection against CT and local production of neutralizing immunoglobulin A antitoxin.

Infect Immun, 1991 Oct, 59(10), 3651 - 8
Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes; Lucas GP et al.; A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1) . The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT . MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes . Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats . No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb . When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected . There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats . Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2 . This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1991 Oct, 13(5), 332 - 7
{Cloning and expression of the cholera toxin B subunit gene}; Tian J; The CT-B gene-expressing plasmid pXB1 and modified pXB2 were constructed using recombinant DNA techniques . These resulted in high CT-B gene expression in E . coli (280-350 ng/ml), with CT-B accounting for 71.5% of total extracellular protein . The results of CT-B expression kinetics showed that the CT-B product levels reached a peak after 24 h in culture . Certain concentrations (100 micrograms/ml) of MgCl2, L-histidine and asparagine can increase the CT-B product level by 10%-25% . The binding of the expressed CT-B product to GM1 receptor on free or live cell membranes was identified by GM1-ELISA, CHO cytotoxicity test, indirect immunofluorescence, and the rabbit ileal loop test . No toxic response to cells or rabbits was detected.

Eur J Immunol, 1991 Oct, 21(10), 2333 - 9
The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant; Wilson AD et al.; The in vitro production of a variety of cytokines by lymphocytes isolated from spleen mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria (LP) was measured, after oral immunization with keyhole limpet hemocyanin using cholera toxin as an adjuvant . LP responses were characterized by very high levels of interleukin (IL) 4, IL 5 and IL 6 with lower levels of IL 2 and interferon-gamma (IFN-gamma) . The PP had lower levels of IL 4, IL 5 and IL 6 than LP but higher levels of IL 2, IFN-gamma was only present at very low levels in this organ . The MLN had a pattern of cytokine production similar to the PP but did produce IFN-gamma . The spleen produced all cytokines measured except IL 5 . Antibody production was characterized by IgA in the LP and PP but IgG was the dominant isotype in the spleen, the MLN was a poor source of antibody-producing cells . We interpret the results to show that (a) the LP response to cholera toxin/keyhole limpet hemocyanin is dominated by Th2-type cytokines compared to a lower production of Th1 type and (b) that the PP has responses typical of an organ with a high proportion of resting lymphocytes which develop mainly into Th2-types cells . The spleen is less dominated by Th2-type cytokines than the mucosal sites and this difference is paralleled by IgA antibody production at the mucosal sites and IgG antibody dominance in the spleen.

Avian Dis, 1991 Oct-Dec, 35(4), 718 - 22
Serologic associations between fowl cholera and other diseases; Carpenter TE et al.; As part of a case-control study designed to identify fowl cholera risk factors, 2087 blood samples were collected from 71 California meat-turkey flocks . Samples were tested for antibodies to three mycoplasmas and four viruses pathogenic for turkeys . Flocks that had antibodies to Newcastle disease virus and/or Mycoplasma meleagridis had an increased risk of having an outbreak of fowl cholera . This information should prove useful for fowl cholera control programs in meat turkeys.

Vet Microbiol, 1991 Oct, 29(2), 101 - 8
The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses; Edwards S et al.; A panel of 30 monoclonal antibodies was defined and characterized with respect to the binding capacity in immunoperoxidase assay to different strains of pestivirus . Using the panel it was possible to identify specifically all strains and isolates of hog cholera virus, hog cholera vaccines derived from 'C' strains, and most strains of bovine viral diarrhoea/border disease (BVD/BD) viruses (including those isolated from pigs) . A small proportion of BVD/BD isolates from pigs and ruminants reacted only with the monoclonals specific for pestivirus group antigen . It is recommended that monoclonal typing methods be introduced into official procedures for the diagnosis of hog cholera/classical swine fever.

Lancet, 1991 Sep 28, 338(8770), 791 - 5
Cholera in Africa: lessons on transmission and control for Latin America; Glass RI et al.; In January, 1991, epidemic cholera emerged in Peru and spread to 7 other countries of Latin America . Cholera was introduced 20 years ago to Africa, where it spread rapidly to 30 of the 46 countries of the region and by 1990 accounted for 90% of all cases reported to the World Health Organisation . Many lessons from the cholera epidemic in Africa are relevant to efforts to control the disease in Latin America . Public health practices from the past--quarantine and cordon sanitaire to halt introduction of cholera by travellers, and vaccination and mass chemoprophylaxis to control epidemics--are ineffective in preventing spread of the disease . Cholera can be transmitted not only by contaminated water but also by food . Social phenomena such as mass migrations and burial practices may play a greater role than previously understood . While efforts to prevent the spread of cholera have been ineffective, cholera-associated mortality can be decreased with rehydration therapy . Since the current pandemic is unlikely to retreat soon, new strategies are urgently needed to control the spread of cholera through sanitary and behavioural interventions or improved vaccinesPIP: Latin America had been free of cholera for 70 years until January 1991 when the 7th pandemic of El Tor cholera struck Peru . It killed 1500 people and affected 200,000 people within 6 months . It soon spread to at least 7 other Latin American countries . 20 years earlier the it reached Africa . Foci of infections in Africa included markets, fairs, funerals, and refugee camps . Scientists doubted that vaccination or quarantine would have prevented its introduction into Africa . Yet, in Latin America, public health officials should earnestly reconsider chemoprophylaxis (tetracycline) of family contacts in families with high rates of illness . Presently no such data exist in Latin America . In addition, health workers should test the new oral vaccine in Latin America since there is no preexisting immunity and the people are exposed to high levels of contamination . Little epidemic research was done in Africa to pinpoint modes of transmission so health workers could learn what types of intervention were warranted . It should be done in Latin America, however . As for quarantine, symptomatic and mild to moderate cholera cases can outnumber severe cases as much as 100 to 1, so confining cases would not prevent the spread of the disease . Latin America should broaden diarrheal disease control programs to include adults so they will accept oral rehydration therapy (ORT) . It should be used in mild to moderate dehydration cases and intravenous rehydration therapy for severe cases . If the environmental factors are not known and understood and if feces contaminate water supplies, foods, and fisheries, cholera may become endemic in Latin America . In conclusion prompt disease reporting, surveillance, and implementation of control measures could prevent the endemicity of cholera in Latin America .

Nippon Hinyokika Gakkai Zasshi, 1991 Sep, 82(9), 1415 - 23
{The immunosuppressive effect of cholera toxin on rat renal allograft}; Asano T; Immunomodulatory effects of cholera toxin (CT) were investigated in a rat model, and the effects of CT on rat renal allograft (from Lewis rat to WKAH rat) were also examined . The results are: 1) The number of lymphocytes in the thymus, spleen and peripheral blood was remarkably decreased after 7 day administration of CT (0.1 and 0.2 mg/kg/day), but the number of red blood cells and neutrophils was not decreased . 2) CT suppressed mixed lymphocyte reaction (MLR) in a dose dependent fashion, and % suppression reached 97% at the concentration of 10 micrograms/ml . The later the time of addition of CT to MLR, the less became this effect . 3) In the control group, the mean survival time (MST) after transplantation was 8.5 +/- 0.3 (Mean +/- SE) days . CT, given 1 day before transplantation, did not prolong MST . In the group to which CT was given daily for 14 days from the day of transplantation, MST was prolonged with the increase of CT . CT at the dosage of 0.2 mg/kg/day prolonged MST (16.2 +/- 3.2 days) significantly (p less than 0.05), where treated with CT from the 3rd day after transplantation, MST (10.3 +/- 1.3 days) was not significantly prolonged . From the above findings, CT seems to act mainly on the early phase of acute rejection and prolongs rat renal allograft survival.

Infect Control Hosp Epidemiol, 1991 Sep, 12(9), 558 - 62
Cholera, travel, and infection control; Nettleman MD; The spread of cholera is a testimony to our inability to provide a consistently decent standard of living to the peoples of the world . Simply separating human sewage from potable water sources would curtail, if not eliminate, the disease . Even persons who acquire cholera would not die if they were given access to rehydration . American travelers should follow sensible food and water precautions, but need not curtail their travels to countries that have cholera.

Eur J Immunol, 1991 Sep, 21(9), 2087 - 94
The early cellular and humoral immune response to primary and booster oral immunization with cholera toxin B subunit; Lewis DJ et al.; The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers . A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization . Using an immunospot assay, cells spontaneously secreting anti-toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization . There were 105 IgG- and 87 IgA-secreting cells per 2 x 10(6) mononuclear cells 7 days after the first immunization, and 282 IgG- and 413 IgA-secreting cells 5 days after the second immunization . A polyclonal increase in total IgM-secreting cells was observed . Few anti-toxin-secreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti-toxin-secreting cells after oral immunization.

Infect Immun, 1991 Sep, 59(9), 3094 - 100
Differential effect of aging on B-cell immune responses to cholera toxin in the inductive and effector sites of the mucosal immune system; Haq JA et al.; The age-associated primary immune response of B cells from the Peyer's patches (PP), the lamina propria (LP), the mesenteric lymph nodes (MLN), and the spleen of mice following oral immunization with cholera toxin (CTx) was investigated . The induction of immune responses was assessed in 4-, 11-, and 24-month-old, individual C57BL/6J male mice by determining the number and isotype of anti-CTx ELISPOT-forming cells (SFC) in the PP, LPL, MLN, and spleen and the titer and isotype of serum anti-CTx antibody . The data indicate a significant age-associated decline in immunoglobulin G (IgG) and IgA anti-CTx SFC in the LP B cells but only in IgA anti-CTx SFC in the PP . No decline was seen in the anti-CTx SFC response in the MLN and spleen . Peroral immunization of mice with CTx resulted in a serum anti-CTx antibody response which was predominantly of the IgG class in all three age groups of mice tested . There was no age-associated decline in anti-CTx IgM, IgG, or IgA titers in serum . Isoelectric focusing and affinity immunoblotting revealed several distinct new antibody clonotypes in the immune serum of old mice following oral immunization with CTx . The results indicate a loss of immune responsiveness to CTx following oral immunization in senescent PP and LP B cells . The MLN and spleen B-cell responses were found to be refractory to the loss of immune function with aging . These findings suggest a differential effect of aging in the inductive and effector sites of the mucosal immune system, and the loss of antigen-specific IgA responses at mucosal sites may have adverse effects on the host's defense against potential pathogens.

J Virol, 1991 Sep, 65(9), 4705 - 12
Hog cholera virus: molecular composition of virions from a pestivirus; Thiel HJ et al.; Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies . The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14) . An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus . The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55 . All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33 . Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.

J Clin Lab Immunol, 1991 Sep, 36(1), 33 - 8
Effect of cholera toxin on serum levels of thyrotropin and thyroid autoantibodies in biobreeding/Tokyo (BB/TKY) rats; Iitaka M et al.; The effect of cholera toxin (CT) on the thyroid-pituitary axis and the immune system was examined in Bio-Breeding/Tokyo (BB/TKY) rats, which spontaneously develop insulin-dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT) . Intravenous administration of CT (5 micrograms/100 g body weight) every other week starting at 6 weeks of age resulted in a significant decrease in the serum thyrotropin (TSH) level at 12 and 14 weeks of age when compared with saline treated littermates . CT stimulated rat thyroid cells to proliferate in vitro . Furthermore, serum anti-thyroglobulin antibody (ATA) titers were also significantly decreased in 14-week-old rats treated with CT . In vitro ATA production by spleen cells from BB/TKY rats was inhibited by CT . Antibodies to thyroxine were detected in both CT-treated and control rats . It is of interest that the ratio of W3/25+ helper/inducer cells to OX8+ suppressor/cytotoxic cells was significantly decreased in CT-treated rats . However, there was no significant difference in the incidence of DM and LT between the two groups of rats . The present study showed that CT suppressed ATA production both in vivo and in vitro, and had a stimulatory effect on thyrocytes in BB/TKY rats.

Vet Microbiol, 1991 Sep, 29(1), 1 - 13
Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid; Boye M et al.; The pestiviruses are small enveloped RNA viruses and are causative agents of economically important animal diseases in cattle, swine, sheep and goats worldwide . We used the polymerase chain reaction to amplify one common fragment of several different strains of both hog cholera virus and bovine virus diarrhea virus (BVDV) . The fragment is located at the 5'-end of the genome immediately upstream of the open reading frame . This is a highly conserved region among the different published pestivirus sequences . An internal restriction digest of the amplified fragment with XhoI and PstI was performed in order to confirm specificity of the amplified fragment . The fragment was sequenced for a number of different BVDV strains, and the sequences obtained were compared to those published and used to deduce genetic relationships between strains . Apart from this common fragment we have amplified several other fragments of the Danish BVDV strain Ug59 and obtained specific amplification fragments of the expected size.

J Cell Biochem, 1991 Sep, 47(1), 79 - 89
Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid; Winston JT et al.; Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation . It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding . We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid . In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours . In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes . The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor . While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin . Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid . These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells . Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF) . Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.

Biochim Biophys Acta, 1991 Aug 26, 1067(2), 166 - 70
The ionic channels formed by cholera toxin in planar bilayer lipid membranes are entirely attributable to its B-subunit; Krasilnikov OV et al.; The interaction of cholera toxin with planar bilayer lipid membranes (BLM) at low pH results in the formation of ionic channels, the conductance of which can be directly measured in voltage-clamp experiments . It is found that the B-subunit of cholera toxin (CT-B) also is able to induce ionic channels in BLM whereas the A-subunit is not able to do it . The increase of pH inhibited the channel-forming activity of CT-B . The investigation of pH-dependences of both the conductance and the cation-anion selectivity of the CT-B channel allowed us to suggest that the water pore of this channel is confined to the B-subunit of cholera toxin . The effective diameter of the CT-B channels water pores was directly measured in BLM and is equal to 2.1 +/- 0.2 nm . The channels formed by whole toxin and its B-subunit exhibit voltage-dependent activity . We believe these channels are relevant to the mode of action of cholera toxin and especially to the endosomal pathway of the A-subunit into cells.

Biochem Pharmacol, 1991 Aug 22, 42(6), 1267 - 71
Pertussis and cholera toxins inhibit prostaglandin synthesis in rat astrocyte cultures at distinct metabolic steps; Gebicke-Haerter PJ et al.; The influence of pertussis and cholera toxin-sensitive G-proteins in the prostaglandin synthetic pathway has been investigated . Prostaglandin D2 (PGD2) synthesis was stimulated by the calcium ionophore A23187, the phorbol ester tetradecanoylphorbol acetate (TPA), or by extracellular ATP . Pretreatment of cultures with pertussis toxin (Ptx) resulted in a partial inhibition of PGD2 synthesis in both stimulated and unstimulated cells . A23187-stimulated PGD2 synthesis was affected less than ATP- or TPA-stimulated synthesis . Furthermore, Ptx also inhibited A23187-, ATP-, and TPA-stimulated arachidonic acid release . Basal and stimulated PGD2 synthesis were also inhibited, when cultures were preincubated with cholera toxin (Ctx) . Here, ATP-stimulated synthesis was affected the most . Arachidonic acid release, in contrast, was enhanced by cholera toxin, even without addition of stimuli . These data suggest that regulation of prostaglandin synthesis in rat astrocyte cultures involves Ptx- and Ctx-sensitive G-proteins . Ptx substrates affect events at or proximal to phospholipase A2, whereas Ctx substrates influence events proximal or distal to phospholipase A2.

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