BMC Infect Dis . 2001;1(1):1 . Epub 2001 Feb 02. Endemic and epidemic dynamics of cholera: the role of the aquatic reservoir; Codeco CT; BACKGROUND: In the last decades, attention to cholera epidemiology increased, as cholera epidemics became a worldwide health problem . Detailed investigation of V . cholerae interactions with its host and with other organisms in the environment suggests that cholera dynamics is much more complex than previously thought . Here, I formulate a mathematical model of cholera epidemiology that incorporates an environmental reservoir of V . cholerae . The objective is to explore the role of the aquatic reservoir on the persistence of endemic cholera as well as to define minimum conditions for the development of epidemic and endemic cholera . RESULTS: The reproduction rate of cholera in a community is defined by the product of social and environmental factors . The importance of the aquatic reservoir depends on the sanitary conditions of the community . Seasonal variations of contact rates force a cyclical pattern of cholera outbreaks, as observed in some cholera-endemic communities . CONCLUSIONS: Further development on cholera modeling requires a better understanding of V . cholerae ecology and epidemiology . We need estimates of the prevalence of V . cholerae infection in endemic populations as well as a better description of the relationship between dose and virulence. J Immunol, 2001 Mar 1, 166(5), 3522 - 32 Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis; Boirivant M et al.; Trinitrobenzene sulfonic acid (TNBS)-induced colitis is an IL-12-driven, Th1 T cell-mediated colitis that resembles human Crohn's disease . In the present study, we showed initially that the oral administration of recombinant subunit B of cholera toxin (rCT-B) at the time of TNBS-induced colitis by intrarectal TNBS instillation inhibits the development of colitis or, at later time when TNBS-induced colitis is well established, brings about resolution of the colitis . Dose-response studies showed that a majority of mice (68%) treated with rCT-B at a dose of 100 microg (times four daily doses) exhibited complete inhibition of the development of colitis, whereas a minority (30%) treated with rCT-B at a dose of 10 microg (times four daily doses) exhibited complete inhibition; in both cases, however, the remaining mice exhibited some reduction in the severity of inflammation . In further studies, we showed that rCT-B administration is accompanied by prevention/reversal of increased IFN-gamma secretion (the hallmark of a Th1 response) without at the same time causing an increase in IL-4 secretion . This decreased IFN-gamma secretion was not associated with the up-regulation of the secretion of counterregulatory cytokines (IL-10 or TGF-beta), but was associated with a marked inhibition of IL-12 secretion, i.e., the secretion of the cytokine driving the Th1 response . Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation . From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can conceivably be applied to the treatment of Crohn's disease. J Immunol, 2001 Mar 1, 166(5), 3114 - 21 Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses; Kato H et al.; Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses . It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract . To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed . OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS . Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased . Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice . We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance. Biotechnol Bioeng, 2001 Feb 20, 72(4), 490 - 4 Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity; Wang XG et al.; Cholera toxin B subunit (CTB) mature protein was stably expressed in transgenic tobacco plants under the control of the CaMV 35S promoter and TMV Omega fragment . Fusion of the PR1b signal peptide coding sequence to the CTB mature protein gene increased the expression level by 24-fold . The tobacco-synthesized CTB (tCTB) was purified to homogeneity by a single step of immunoaffinity chromatography . The purified tCTB is predominantly in the form of pentamers with molecular weight identical to the native pentameric CTB, indicating that the PR1b-CTB fusion protein has been properly processed in tobacco cells . Furthermore, by immunodiffusion and immunoelectrophoresis, we have shown that the antigenicity of the purified tCTB is indistinguishable from that of the native CTB protein . Regul Pept, 2001 Apr 2, 98(1-2), 13 - 8 Cholera and pertussis toxins inhibit differently hypothermic and anti-opioid effects of neuropeptide FF; Frances B et al.; In mice pretreated intracerebroventricularly (i.c.v.) with pertussis or cholera toxins, effects of neuropeptide FF (NPFF), on hypothermia and morphine-induced analgesia, were assessed . NPFF and a potent NPFF agonist, 1DMe (0.005-22 nmol) injected into the lateral ventricle decreased morphine analgesia and produced naloxone (2.5 mg x kg(-1), s.c.)-resistant hypothermia after administration into the third ventricle . Cholera toxin (CTX 1 microg, i.c.v.) pretreatment (24 or 96 h before) inhibited the effect of 1DMe on body temperature, but failed to reverse its anti-opioid activity in the tail-flick test . CTX reduced hypothermia induced by a high dose of morphine (8 nmol, i.c.v.) but not the analgesic effect due to 3 nmol morphine . Pertussis toxin (PTX) pretreatment inhibited both morphine-hypothermia and -analgesia but did not modify hypothermia induced by 1DMe . The present results suggest that NPFF-induced hypothermia depends on the stimulation of Gs (but not Gi) proteins . In contrast, anti-opioid effects resulting from NPFF-receptor stimulation do not involve a cholera toxin-sensitive transducer protein. Infect Immun, 2001 Mar, 69(3), 1574 - 80 Human infection with Ascaris lumbricoides is associated with suppression of the interleukin-2 response to recombinant cholera toxin B subunit following vaccination with the live oral cholera vaccine CVD 103-HgR; Cooper PJ et al.; To investigate the potential immunomodulatory effects of concurrent ascariasis on the cytokine response to a live oral vaccine, we measured cytokine responses to cholera toxin B subunit (CT-B) following vaccination with the live oral cholera vaccine CVD 103-HgR in Ascaris lumbricoides-infected subjects randomized in a double-blind study to receive two doses of either albendazole or placebo prior to vaccination and in a group of healthy U.S . controls . Postvaccination cytokine responses to CT-B were characterized by transient increases in the production of interleukin-2 (IL-2; P = 0.02) and gamma interferon (IFN-gamma; P = 0.001) in the three study groups combined; however, postvaccination increases in IFN-gamma were significant only in the albendazole-treated A . lumbricoides infection group (P = 0.008) . Postvaccination levels of IL-2 were significantly greater in the albendazole-treated group compared with the placebo group (P = 0.03) . No changes in levels of Th1 and Th2 cytokines in response to control ascaris antigens were observed over the same period . These findings indicate that vaccination with CVD 103-HgR is associated with a Th1 cytokine response (IL-2 and IFN-gamma) to CT-B, that infection with A . lumbricoides diminishes the magnitude of this response, and that albendazole treatment prior to vaccination was able to partially reverse the deficit in IL-2 . The potential modulation of the immune response to oral vaccines by geohelminth parasites has important implications for the design of vaccination campaigns in geohelminth-endemic areas. Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 201 - 12 Exploration of the GM1 receptor-binding site of heat-labile enterotoxin and cholera toxin by phenyl-ring-containing galactose derivatives; Fan E et al.; Cholera toxin (CT) and the closely related heat-labile enterotoxin of Escherichia coli (LT) are responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality . The B subunits of these heterohexameric AB(5) toxins form a pentameric arrangement which is responsible for binding to the receptor GM1 of the target epithelial cells of the host . Blocking these B pentamer-receptor interactions forms an avenue for therapeutic intervention . Here, the structural characterization of potential receptor-blocking compounds are described based on the previously identified inhibitor m-nitrophenyl-alpha-D-galactoside (MNPG) . The structure of a CTB-MNPG complex confirms that the binding mode of this inhibitor is identical in the two homologous toxins CT and LT and is characterized by a glycosyl linkage geometry that leads to displacement of a well ordered water molecule near the amide group of Gly33 by the O1-substituent of MNPG . This glycosyl geometry is not maintained in the absence of a substituent that can displace this water, as shown by a complex of LTB with p-aminophenyl-alpha-D-galactoside (PAPG) . New compounds were synthesized to investigate the feasibility of maintaining the favorable binding interactions exhibited by MNPG while gaining increased affinity through the addition of hydrophobic substituents complementary to either of two hydrophobic regions of the receptor-binding site . The structural characterization of complexes of LTB with two of these compounds, 3-benzylaminocarbonylphenyl-alpha-D-galactoside (BAPG) and 2-phenethyl-7-(2,3-dihydrophthalazine-1,4-dione)-alpha-D-galactoside (PEPG), demonstrates a partial success in this goal . Both compounds exhibit a mixture of binding modes, some of which are presumably influenced by the local packing environment at multiple crystallographically independent binding sites . The terminal phenyl ring of BAPG associates either with the phenyl group of Tyr12 or with the hydrophobic patch formed by Lys34 and Ile58 . The latter interaction is also made by the terminal phenyl substituent of PEPG, despite a larger ring system linking the galactose moiety to the terminal phenyl . However, neither BAPG nor PEPG displaces the intended target water molecule . Both of the designed compounds exhibit increased affinity relative to the galactose and to PAPG notwithstanding the failure to displace a bound water, confirming that additional favorable hydrophobic interactions can be gained by extending the starting inhibitor by a hydrophobic tail . The insight gained from these structures should allow the design of additional candidate inhibitors that retain both the glycosyl geometry and water displacement exhibited by MNPG and the favorable hydrophobic interactions exhibited by BAPG and PEPG. Vaccine, 2001 Feb 8, 19(13-14), 1652 - 60 Effects of intranasal administration of cholera toxin (or Escherichia coli heat-labile enterotoxin) B subunits supplemented with a trace amount of the holotoxin on the brain; Hagiwara Y et al.; Effects of intranasal administration of cholera toxin (CT) {or Escherichia coli heat-labile enterotoxin (LT)} B subunits supplemented with a trace amount of the holotoxin, CTB* or LTB*, on the brain were examined in BALB/c mice by comparing with those of the intracerebral injection . Intracerebral injection of CTB* at doses more than 10 microg/mouse caused significant body weight loss and dose-dependent death within 7 days, with localization of conjugates of horseradish peroxidase with CTB (HRP-CTB) in the ventricular system and in the perineural space of olfactory nerves of the nasal mucosa 3 h after injection . Intracerebral injection of CTB* at doses less than 3 microg/mouse (or LTB* at doses less than 22.7 microg/mouse) did not cause any significant body weight loss for 7 days, with localization of HRP-CTB in the brain but not in the nasal mucosa . On the other hand, intranasal administration of 10 microg of CTB* caused localization of HRP-CTB in the nasal mucosa but not in the brain 3 h after administration and caused body weight loss even after 30 administrations . Neither any histological changes of brain tissues nor marked changes in serum biochemical parameters were found in mice after the 30 administrations of CTB* or LTB* . These results suggest that 0.1 microg of CTB* or LTB*, which is known to be close to the minimal effective dose as an adjuvant for nasal influenza vaccine in mice and corresponds to 100 microg per person, can be used as a safe nasal adjuvant without adversely affecting the brain. Vet Immunol Immunopathol, 2000 Dec 29, 77(3-4), 191 - 9 Induction of systemic immune responses in sheep by topical application of cholera toxin to skin; Chen D et al.; Cholera (and related) toxins (CT) when applied topically on unbroken skin induce systemic immune responses in mice, a procedure called transcutaneous immunization (TCI) . The current study examined the capacity for TCI to induce systemic immune responses in sheep . Three groups (n=5 per group) were immunized at day 0 (priming) and day 28 (boosting) with 250 microg of CT in water by TCI, with 25 microg of CT in alum by intramuscular injection, or not immunized . Serum samples were taken at days 0, 28, 42, 56 and 70 after immunization for measurement of CT-specific IgG as well as CT-specific IgG1, IgG2, IgA and IgM antibodies by ELISA . After immunization, IgG, IgG1 and IgG2 antibody in immunized groups were significantly higher than in the control group, and boosting further increased these titres . IgG, IgG1 and IgG2 in the injection group were significantly higher than in the TCI group . There was a preponderance of IgG1 antibody, relative to IgG2, in both immunized groups . CT-specific IgA and IgM were detected in both immunized groups . Lymphocyte proliferation to CT was measured at day 90 . A CT-specific lymphocyte proliferative response (stimulation index>2) was detected in all sheep from the injection group, in two sheep from the TCI group and in none of the controls . Results demonstrated that TCI induces primary and secondary antibody responses and specific proliferative responses to CT in sheep. Jpn J Infect Dis, 2000 Oct, 53(5), 181 - 8 Current status of cholera and rise of novel mucosal vaccine; Yamamoto T; Three serious cholera epidemics have threatened the world during the last 10 years . As a countermeasure against such cholera epidemics, three vaccines, CVD 103-HgR, WC/rBS, and Vietnamese WC, showed good performance . CVD 103-HgR is a recombinant attenuated live vaccine for travelers, and its highly safety and protective efficacy have been demonstrated in volunteers in advanced countries . WC/rBS, which consists of heat- and formalin-killed bacteria and cholera toxin B subunit, protects the vaccinees (>5 years old) from cholera for 6 months . Vietnamese WC, a heat- and formalin-killed vaccine, is inexpensive and effective even for 1 to 5-year-old children . Additionally, irradiated WC vaccines and new serotype (O139) vaccines are being developed . Regarding intestinal immunity, secretory IgA has been mainly examined . In addition, mucosal IgG, as induced by the irradiated WC vaccine, should also be investigated . Development of mucosal adjuvant, such as holotoxin-type mutants of cholera toxin and related Escherichia coli heat-labile enterotoxin, has been actively undertaken . Diverse custom-made vaccines may be one countermeasure for the changing situations in endemic countries or areas and for "barriers" against live vaccines in such areas. Am J Physiol Lung Cell Mol Physiol, 2001 Jan, 280(1), L152 - 64 Labeling of vagal motoneurons and central afferents after injection of cholera toxin B into the airway lumen; Perez Fontan JJ et al.; We tested the hypothesis that application of the subunit B of cholera toxin (CTB) to the airway mucosa would produce labeling of neuronal somata and sensory fibers in the medulla oblongata . Using (125)I-CTB as a tracer, we demonstrated first that CTB is transported across the tracheal epithelium, but once in the airway wall, it remains confined to the subepithelial space and lamina propria . Despite the rarity of intrinsic neurons in these areas, intraluminal CTB labeled approximately 10-60 neurons/rat in the nucleus ambiguus and a smaller number of neurons in the dorsal motor nucleus of the vagus . Well-defined sensory fiber terminals were also labeled in the commissural, medial, and ventrolateral subnuclei of the nucleus of the tractus solitarius . Approximately 50 and 90% of the neurons labeled by intraluminal CTB were also labeled by injections of FluoroGold into the tracheal adventitia and lung parenchyma, respectively . These findings demonstrate that a substantial number of medullary vagal motoneurons innervate targets in the vicinity of the airway epithelium . These neurons do not appear to be segregated anatomically from vagal motoneurons that project to deeper layers of the airway wall or lung parenchyma. Shock, 2000 Dec, 14(6), 640 - 5 Local attenuation of systemically mediated splanchnic vasoconstriction during shock due to cholera; Huang AH et al.; Hypovolemic shock, most often due to hemorrhage, is typically associated with intense splanchnic vasoconstriction . This can be severe enough to impair the functional and structural integrity of the gastrointestinal tract . Paradoxically, with cholera the structure of the gastrointestinal tract is preserved, and the intestine continues to secrete fluid delivered to it in the circulating blood in spite of severe hypovolemic shock . This suggests that splanchnic blood flow is maintained at higher levels in hypovolemic shock due to cholera than in hypovolemic shock due to hemorrhage . Our hypothesis is that cholera toxin in the intestinal lumen activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction . Blood flow to an isolated ileal segment in situ in the anesthetized rabbit was measured continuously (ultrasound transit-time volume flow probe) for 5 to 6 h after instillation of cholera toxin into the isolated intestinal lumen . Norepinephrine was infused selectively into the mesenteric artery supplying the segment to elicit local responses uncomplicated by compensatory changes secondary to systemic effects of norepinephrine . Baseline vascular conductance increased gradually and became significantly greater in cholera toxin experiments than in vehicle experiments 5 h after treatment (P < 0.035) . Animals treated with cholera toxin were less responsive to norepinephrine than vehicle treated animals were (P < 0.05) and became more so over time (P < 0.001) . Our conclusion is that cholera toxin activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction, at least in part by reducing vascular responsiveness to a systemic vasoconstrictor, norepinephrine. J Biol Chem, 2001 Mar 23, 276(12), 9182 - 8 Epub 2000 Dec 11. Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism; Shogomori H et al.; A number of studies have demonstrated that cholera toxin (CT) is found in detergent-insoluble, cholesterol-enriched domains (rafts) in various cells, including neurons . We now demonstrate that even though CT is associated with these domains at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism, at both early and late stages of neuronal development . CT transport to the Golgi apparatus, and its subsequent degradation, is inhibited by hypertonic medium (sucrose), and by chlorpromazine; the former blocks clathrin recruitment, and the latter causes aberrant endosomal accumulation of clathrin . Moreover, both internalization of the transferrin receptor (Tf-R), which occurs via a clathrin-dependent mechanism, and CT internalization, are inhibited to a similar extent by sucrose . In contrast, the cholesterol-binding agents filipin and methyl-beta-cyclodextrin have no effect on the rate of CT or Tf-R internalization . Finally, once internalized, CT becomes more detergent-soluble, and chlorpromazine treatment renders internalized CT completely detergent-soluble . We propose two models to explain how, despite being detergent-insoluble at the cell surface, CT is nevertheless internalized via a raft-independent mechanism in hippocampal neurons. Int J Med Microbiol, 2000 Oct, 290(4-5), 447 - 53 Immune modulation by the cholera-like enterotoxin B-subunits: from adjuvant to immunotherapeutic; Williams NA; Cholera toxin (Ctx) and its close relative, Escherichia coli heat-labile enterotoxin (Etx) have long been established as potent mucosal and systemic adjuvants . Problems arising from their inherent toxicity have, however, precluded human use . Here we describe findings which demonstrate that contrary to the established dogma the non-toxic B-subunit of Etx (EtxB) is a highly potent mucosal adjuvant capable of potentiating protective immunity to viral infection . The mechanisms which underlie this activity arise from an ability to trigger specific signaling processes in lymphocyte populations which modulate differentially their activation, differentiation and survival . The elucidation of these properties has led to the further use of EtxB as an agent capable of preventing the establishment of autoimmune diseases . The basis for these activities and their potential applicability to human therapies are discussed. Int J Med Microbiol, 2000 Oct, 290(4-5), 403 - 8 Floating cholera toxin into epithelial cells: functional association with caveolae-like detergent-insoluble membrane microdomains; Badizadegan K et al.; In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and likely endoplasmic reticulum (ER) (Lencer et al., J . Cell Biol . 131, 951-962 (1995)) . We have recently found that the toxin's apical membrane receptor ganglioside GM1 acts specifically in this signal transduction pathway, likely by coupling CT with caveolae or caveolae-related membrane domains (lipid rafts) (Wolf et al., J . Cell Biol . 141, 917-927 (1998)) . Work in progress shows that 1) cholesterol depletion uncouples the CT-GM1 receptor complex from signal transduction, a characteristic of lipid rafts; 2) the GM1 acyl chains rather than the carbohydrate head groups appear to account for the structural basis of ganglioside specificity in toxin trafficking; and 3) intestinal epithelial cells obtained from normal adult humans exhibit lipid rafts which differentiate between CT-GM1 and LTIIb-GD1a complexes and which contain caveolin 1. Sante, 2000 Jul-Aug, 10(4), 277 - 86 {One-year assessment of the cholera epidemic in Madagascar, from March 1999 to March 2000}; Champetier de Ribes G et al.; The seventh cholera pandemic reached Madagascar in March 1999, 30 years after its appearance in East Africa . Two waves of infection were observed during the first year . The second wave was the stronger of the two . It occurred in the warm rainy season and spread over six provinces of the country . The incidence of cholera was from 0.1% to 2% and the hospital case fatality rate was between less than 2% and 6%, depending on the geographical area and the period . This outbreak has raised the awareness of communities and their leaders with respect to the environmental, practical and cultural factors that increase the risk of diseases transmitted by feces. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14662 - 7 Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system; Jobling MG et al.; The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide . This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs) . We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively . Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system . We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6 . Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT . These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF . Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity . These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo. Hepatology, 2000 Dec, 32(6), 1357 - 69 Effect of cholera toxin and cyclic adenosine monophosphate on fluid-phase endocytosis, distribution, and trafficking of endosomes in rat liver; Van Dyke RW; In prior studies, we showed that cholera (CTX) and pertussis toxins (PTX) increase rat liver endosome acidification . This study was performed to characterize the effects of these toxins and cyclic adenosine monophosphate (cAMP) on endosome ion transport, fluid-phase endocytosis (FPE), and endosome trafficking in liver . In control liver, more mature populations of endosomes acidified progressively more slowly, but both toxins and cAMP caused retention of an early endosome acidification profile in maturing endosomes . CTX caused a density shift in endosomes, and all agents increased net FPE at time points from 5 to 60 minutes . By confocal microscopy, fluorescent dextrans first appeared in small vesicles at the hepatocyte sinusoidal membrane and trafficked rapidly to the pericanalicular area, near lysosomes and the trans-Golgi network (TGN) . Prolonged exposure to these agents caused redistribution of many labeled vesicles to the perinuclear region, colocalized with markers of both early (EEA1 and transferrin receptor) and late (LAMP1) endosomes . We conclude that cAMP is the common agent that disrupted normal maturation and trafficking of endosomes and increased net FPE, in part via decreased diacytosis. J Neurosci Methods, 2000 Nov 15, 103(1), 83 - 90 Tracer-toxins: cholera toxin B-saporin as a model; Llewellyn-Smith IJ et al.; We have shown previously that retrogradely-transported cholera toxin B (CTB)-saporin has eliminated sympathetic preganglionic neurons by 7 days after injection (Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F., Minson, J.B., 1999 . NeuroReport 10, 307) . To ascertain whether this tracer-toxin can kill other types of neurons that transport CTB retrogradely with a similar time course, we injected CTB-saporin into the facial nerves of rats and allowed them to survive for 7 days . Facial motoneurons were counted ipsilateral and contralateral to the injected nerves in sections of perfused medulla processed to reveal immunoreactivity for choline acetyltransferase (ChAT) . There was a statistically significant decrease in the number of ChAT-immunoreactive neurons ipsilateral to the injected nerve in three out of nine rats . Inadequate injections were probably the reason that most rats showed no decrease in motoneurons numbers after treatment with CTB-saporin, since the staining intensity and numbers of facial motoneurons that showed CTB-immunoreactivity varied markedly between rats after retrograde tracing with unconjugated CTB . These results show that CTB-saporin can eliminate motoneurons as well as sympathetic preganglionic neurons, indicate that protocols for the injection of tracer-toxins should be optimized to ensure maximum neuronal death and support our contention that CTB-saporin should kill any central neuron that expresses GM1 ganglioside, the membrane component to which CTB binds. Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 333 - 6 {Induction of protective immune response in mice and rhesus monkeys by immunization with fusion protein of cholera toxin B subunit and multiples of Plasmodium falciparum}; Cao C et al.; Recombinant fusion protein of cholera toxin B subunit (CTB) and poly-valent protective epitopes of plasmodium falciparum was given to i.m . to C57BL/6j mice and rhesus monkeys three times . In rhesus monkeys, high level of antibodies for CTB (1:6400) and malaria epitopes (1:3200) amtobpdoes were elicited as well as the specific CTL activity for P . plasmodium . After the mice were challenged with sporozoites of P . yeolli, about 50% of them were protected from the patent infection . A blood-stage challenge with 10(8) of P . cynomolgi parasite were given to rhesus monkeys, which showed that two animals in control group were patent infection for at least 30 days, in contrast, the two animals immunized were recovered respectively at the day of 11 and 15 after challenges . The results suggested that cholera toxin acts as an effective adjuvent in the development of malaria vaccine. Vet Pathol, 2000 Sep, 37(5), 402 - 8 Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus; Narita M et al.; Eight pigs were inoculated subcutaneously with a highly virulent hog cholera virus (HCV) strain ALD . The infected pigs developed severe illness and became moribund on postinoculation day (PID) 7 or PID 10 . Histologic lesions were characterized by severe generalized vasculitis, necrosis of lymphocytes, and encephalitis . HCV antigen was detected in crypt tonsilar epithelial cells, macrophages, and reticular endothelial cells of lymphoid tissues . Antigen localization corresponded well with histologic lesions . Five pigs were inoculated with less virulent HCV Kanagawa/74 strain and were euthanatized on PID 30 . All five infected pigs recovered from the illness but became stunted . They also had a slight follicular depletion of lymphocytes, histiocytic hyperplasia, and hematopoiesis in the spleen . Less virulent HCV antigen was observed in the tonsils, kidneys, pancreas, adrenal glands, and lungs . Although antigen localization was less associated with histologic lesions, immunoreactivity was stronger than that in the pigs infected with the ALD strain of HCV . An almost complete loss of B lymphocytes was recognized in pigs infected with the ALD strain and was correlated with follicular necrosis in lymphoid tissues . Loss of B lymphocytes was not prominent in the pigs infected with Kanagawa/74 strain . The number of CD4+ and CD8+ T lymphocytes was significantly higher than that in the noninfected control pigs. Yi Chuan Xue Bao, 2000, 27(7), 654 - 7 {Translation initiation function of the regulation element in the operon of cholera toxin A}; Cao C et al.; To demonstrate that there existed translation coupling between cholera toxin A subunit gene and B subunit gene, and give the answer why the expression level of B gene is five times more than that of A gene, alpha report system for the investigation of translation coupling was constructed by using lacZ gene as reporter . Frame-shift mutation was introduced near the C terminal of ctxA gene, and the ribosome would read through its normal stop codon . The report plasmid was constructed and it was found that the expression level of lacZ gene decreased five times after the frame-shift mutation . The translation of cholera toxin B subunit gene was translational coupled with A subunit gene, and was responsible for the differential expression level of the two genes. J Immunol, 2000 Nov 1, 165(9), 4778 - 82 Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues; van Ginkel FW et al.; We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally . Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h . This uptake into olfactory regions was monosialoganglioside (GM1) dependent . Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS . We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons . This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity. Gastroenterology, 2000 Oct, 119(4), 1037 - 44 Neurokinin 1 and 2 receptors mediate cholera toxin secretion in rat jejunum; Turvill JL et al.; BACKGROUND & AIMS: Substance P, a member of the tachykinin family, is a prosecretory neuropeptide distributed widely throughout the enteric nervous system . Implicated in inflammatory states, its role in enterotoxigenic water and electrolyte secretion is unclear . We assessed the effect of substance P antagonists and neurokinin receptor antagonists on cholera toxin-, Escherichia coli heat-labile enterotoxin (LT)-, and heat-stable enterotoxin (STa)-induced water secretion in an in vivo rat jejunal perfusion model . METHODS: Anesthetized adult male Wistar rats were pretreated with substance P antagonists (D-Pro(2), D-Trp(79), substance P, 0.1-3.0 mg/kg; or CP 96,345/4, 0.3-3 mg/kg) or neurokinin (NK)-1 (sendide, 1.0 mg/kg), NK-2 (GR83074, 1.0 mg/kg), or NK-3 ({Trp(7),betaAla(8)}NKA(4-10), 1.0 mg/kg) receptor antagonists . In a subgroup, extrinsic sensory afferents were ablated by pretreatment with capsaicin . Jejunal perfusion, with a plasma electrolyte solution containing a nonabsorbable marker, was undertaken after exposure to cholera toxin (25 microg), LT (25 microg), STa (200 microg/L), or saline . Results: Cholera toxin-induced water and electrolyte secretion was inhibited by the substance P antagonists and the NK-1 and NK-2 receptor antagonists, but not by the NK-3 receptor antagonist or by pretreatment with capsaicin . Neither LT- nor STa-induced secretions were affected by the pretreatments . CONCLUSIONS: Prosecretory pathways involving NK-1 and NK-2 receptors specifically mediate the actions of cholera toxin in the small intestine. Cochrane Database Syst Rev . 2000;(4):CD000974. Vaccines for preventing cholera; Graves P et al.; BACKGROUND: Oral cholera vaccines are newer alternatives to the parenteral vaccines which have been thought to confer only moderate and short-term immunity . OBJECTIVES: The objective of this review was to assess the effect of cholera vaccines in preventing cases of cholera and preventing deaths . SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, Medline, Embase and reference lists of articles . We handsearched the journal Vaccine, contacted researchers in the field and manufacturers . SELECTION CRITERIA: Randomised and quasi-randomised studies comparing cholera vaccines (killed or live) with placebo, control vaccines or no intervention, or comparing types, doses or schedules of cholera vaccine . We included adults and children irrespective of immune status or special risk category . DATA COLLECTION AND ANALYSIS: Data extraction and assessment of trial quality was done independently by two reviewers . MAIN RESULTS: Twenty-five trials were included . Eighteen efficacy trials of relatively good quality, testing parenteral and oral killed whole cell vaccines and involving over 2.6 million adults, children and infants were included . No randomised efficacy trials of live vaccines were available and therefore this review is restricted to killed vaccines only . Eleven safety trials have been conducted for both types of killed whole cell vaccines and have involved 9,342 people . For killed whole cell vaccines compared to placebo, the relative risk of contracting cholera at 12 months was 0.49, 95% confidence interval 0.41 to 0.59 (random effects model) . This translates to an efficacy of 51%, 95% confidence interval 41% to 59% . Both parenteral and oral administration were relatively efficacious, but significant protection extended into the third year for oral killed whole cell vaccines . Children under 5 were only protected for up to a year, while older children or adults were protected for up to three years . Parenteral killed whole cell vaccines were associated with increased systemic and local adverse effects compared to placebo, while oral killed whole cell vaccines were not . REVIEWER'S CONCLUSIONS: Cholera killed whole cell vaccines appear to be relatively effective and safe . Protection against cholera appears to persist for up to two years following a single dose of vaccine, and for three to four years with an annual booster. Clin Immunol, 2000 Nov, 97(2), 130 - 9 Enhanced immunological tolerance against allograft rejection by oral administration of allogeneic antigen linked to cholera toxin B subunit; Sun JB et al.; A single oral intragastric administration of cholera toxin B subunit (CTB) conjugated to allogeneic thymocytes (ATC, 4 x 10(7) cells) under conditions allowing the CTB to bind the complex to GM1 ganglioside receptors was shown to be efficacious in inducing peripheral T cell tolerance associated with significant suppression of both primary and secondary accelerated rejection of heart allografts when tested in mice . Allogeneic in vivo delayed-type hypersensitivity (DTH), in vitro cytotoxicity responses, and mixed lymphocyte reactions (MLR) by T cells from mesenteric lymph nodes (MLN), popliteal lymph nodes (PLN), and spleen were significantly reduced in mice treated with the CTB-ATC conjugate, as were also the numbers of cells in these organs producing IL-2, IFN-gamma, or IL-4 . In contrast, a marked increase in the production of IL-4 in Peyer's patches (PP) and of TGF-beta(1) in PLN was observed . The suppressive potential of T cells from PP and/or MLN after oral treatment with CTB-ATC was further evident by intraperitoneal transfer of such cells from CTB-ATC-treated animals to primed recipients, which led to marked suppression of both allogen-specific DTH and MLR responses . A critical role for PP in inducing peripheral tolerance after oral CTB-ATC treatment was indicated by the absence of tolerance induction in animals whose PP had been destroyed before treatment with CTB-ATC . The results indicate that the protection against allograft rejection by oral treatment with CTB-ATC is mediated by T cells and associated with a strong induction of IL-4 production at mucosal sites and TGF-beta(1) at the effector sites . J Health Popul Nutr, 2000 Jun, 18(1), 49 - 53 Carbachol potentiates cholera toxin-induced secretion in a colonic epithelial cell line (HT29-19A) and rat ileal mucosa in vitro; Mahmood B et al.; Recent studies show that enteric nerves are involved in the action of cholera toxin, both in vivo and in vitro . The aim of this study was to investigate in vitro the influence of carbachol, a cholinergic agonist, on the action of cholera toxin . Cultured HT29-19A cell lines and rat ileal mucosa were used in an Ussing chamber for the measurement of short-circuit current induced by cholera toxin . Cyclic AMP was measured from HT29-19A cell lines by standard radio-immunoassay . Pre-treatment of the HT29-19A cell lines with carbachol potentiated cholera toxin-induced secretory response, and enhanced accumulation of cAMP . Carbachol also potentiated the cholera toxin-secretory response in the rat ileal mucosa, but only following pretreatment with the prostaglandin synthesis inhibitor, indomethacin . There was synergistic interaction between cholera toxin and cholinergic neurotransmitter carbachol on the intestinal epithelium . Cholinergic agonists may play a role in regulating the secretory response to the toxin . Such interaction is masked in the intact tissues in vitro due to the release of prostaglandins during isolation. Bioorg Med Chem Lett, 2000 Oct 2, 10(19), 2197 - 200 Second generation mimics of ganglioside GM1 as artificial receptors for cholera toxin: replacement of the sialic acid moiety; Bernardi A et al.; In a program directed towards the design and synthesis of mimics of ganglioside GM1, the NeuAc recognition domain was replaced by simple hydroxy acids, and the affinity of the new ligands to the cholera toxin was determined by fluorescence spectroscopy . The (R)-lactic acid derivative 4 was found to display the highest affinity of the series (KD = 190 microM). Int Immunol, 2000 Oct, 12(10), 1449 - 57 Oral administration of cholera toxin B subunit conjugated to myelin basic protein protects against experimental autoimmune encephalomyelitis by inducing transforming growth factor-beta-secreting cells and suppressing chemokine expression; Sun JB et al.; The efficacy and mechanism of immunosuppression against experimental autoimmune encephalomyelitis (EAE) by oral low-dose administration of myelin basic protein (MBP) conjugated to cholera toxin B subunit (CTB) were investigated in Lewis rats immunized with MBP together with complete Freund's adjuvant 4 days before the start of treatment . Oral treatment with CTB-MBP conjugate gave almost complete protection against disease, an effect that was totally abrogated by including a low dose of cholera holotoxin (CT) . The protection by CTB-MBP was associated with a dramatic reduction in the number of leukocytes staining for CD4, CD8, IL-2R or MHC class II in the spinal cord as examined by immunohistochemistry . The mRNA expressions of T(h)1 cytokines IFN-gamma, IL-12 and tumor necrosis factor-alpha, as well as of chemokines monocyte chemotactic protein (MCP)-1 and RANTES in the spinal cord were also reduced by 76-94%, as assessed by in situ hybridization . In contrast, transforming growth factor (TGF)-beta mRNA-expressing cells were strongly increased in the spinal cord from animals treated orally with the CTB-MBP conjugate . In the draining peripheral lymph nodes, the number of MBP-specific TGF-beta mRNA-expressing cells was also increased, whereas there was a decrease in cells expressing T(h)1 or T(h)2 cytokine mRNA . Protection against EAE could be transferred by injection of cells from the mesenteric lymph nodes of animals fed with CTB-MBP into naive animals exposed to encephalitogenic T cells . The results indicate that the protective anti-inflammatory effect by oral treatment with CTB-MBP conjugate is, to a large extent, due to the induction of TGF-beta-secreting suppressive-regulatory T cells and to local down-regulation of MCP-1 and RANTES in the spinal cord. Bull Exp Biol Med, 2000 Apr, 129(4), 337 - 9 Lipid peroxidation during experimental cholera intoxication; Ponukalina EV et al.; Cholera intoxication in albino mice was induced by intraperitoneal injection of endotoxin in doses of LD(16), LD(25), and LD(50) and combination of endo- and enterotoxin in doses equivalent to LD(25) . Dose-dependent activation of superoxide dismutase, phasic changes in the contents of MDA and conjugated trienes and dienes, and modulatory influence of enterotoxin on catalase activity in the blood were observed during intoxication. Science, 2000 Sep 8, 289(5485), 1766 - 9 Cholera dynamics and El Niño-Southern Oscillation; Pascual M et al.; Analysis of a monthly 18-year cholera time series from Bangladesh shows that the temporal variability of cholera exhibits an interannual component at the dominant frequency of El Nino-Southern Oscillation (ENSO) . Results from nonlinear time series analysis support a role for both ENSO and previous disease levels in the dynamics of cholera . Cholera patterns are linked to the previously described changes in the atmospheric circulation of south Asia and, consistent with these changes, to regional temperature anomalies. Rev Saude Publica, 2000 Aug, 34(4), 342 - 7 {Cholera and living conditions, Brazil}; Gerolomo M et al.; INTRODUCTION: Factors associated with precarious living and environmental conditions are frequently cited as major obstacles for the control of cholera outbreaks and epidemics . The purposes of the study are to evaluate the contribution of factors associated with the population living conditions and correlate the environmental problems with the onset of cholera and its subsequent impact . METHODS: Using a multiple linear regression by the backward stepwise method, and with the researcher's interaction, the study correlated socioeconomic indicators with cholera incidence rates in some counties of Pernambuco State, Brazil, during the year of 1992 . RESULTS/CONCLUSIONS: The results of the adjusted model showed that the proportion of households without tap water was the variable that contributed the most to the increasing fluctuation of cholera incidence rates . Two other factors, the proportion of households without sewage and the proportion of householders with an income less than or equal to the minimum wage, also revealed a positive association with cholera incidence rates with statistically significant regression coefficients . The proportion of households with no sanitary installations whatsoever showed a negative association with cholera incidence rates, suggesting that sewage disposal, such as open-air sewage ditches, that is not part of the public sewage disposal system, increases the risk of environmental contamination The results indicate that having an adequate tap water supply is of maximum priority for cholera prevention. J Peripher Nerv Syst, 1998, 3(1), 19 - 27 Antibodies to glycolipids and cholera toxin B subunit do not initiate Ca++ signaling in rat Schwann cells; Skoff AM et al.; Antibodies to glycolipids have been implicated in the pathogenesis of several immune-mediated PNS demyelinating diseases . This study focuses on antibodies to galactocerebroside (GalC) and sulfatide and on the B subunit of cholera toxin (CTB), which reacts with GM1 ganglioside, to examine whether these agents have any direct effects on Schwann cells (SC) as measured by Ca++ responses . While surface levels of GalC and sulfatide were markedly upregulated by 8 Br-cAMP treatment, as reported by others, very little expression of surface GM1 ganglioside was detected with or without 8 Br-cAMP treatment . Schwann cells, under either condition, showed no changes in intracellular Ca++ levels when exposed to purified monoclonal antibodies reacting with GalC or sulfatide . Thus upregulation of surface levels of GalC or sulfatide does not lead to antibody-induced Ca++ influx, in contrast to previous findings in mature oligodendrocytes (OLs) exposed to antibodies to GalC . Further, cross-linking with one of the antibodies (R-mAb) did not produce Ca++ responses . No Ca++ responses were elicited by CTB in Schwann cells either with or without 8 Br-cAMP treatment . Since surface binding of CTB was very low and sparsely punctate in Schwann cells +/- 8 Br-cAMP, we tested whether increasing levels of GM1 ganglioside on the surface would lead to induction of a Ca++ signaling pathway, as reported for fibroblasts . GM1 ganglioside on the surface of SC was markedly increased by exposing cells to exogenous GM1 ganglioside, but no Ca++ responses were observed in the treated cells . Thus undifferentiated or partially differentiated SC lack the glycoconjugate-mediated Ca++ signaling pathways found in mature OLs or fibroblasts. Jpn J Infect Dis, 2000 Jun, 53(3), 98 - 106 A proposal for safety standards for human use of cholera toxin (or Escherichia coli heat-labile enterotoxin) derivatives as an adjuvant of nasal inactivated influenza vaccine; Tamura SI et al.; Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) are not only the causative agents of diarrhea but are also strong mucosal adjuvants which enhance immune responses to mucosally coadministered bystander antigens . One of the most promising applications of these toxins would be as mucosal adjuvant of nasal influenza vaccine . In comparison to current inactivated vaccines, the nasal vaccine provides superior cross-protection by inducing production of cross-reacting anti-viral IgA antibodies in the respiratory tract even when the vaccine strain is different from the epidemic strain . On the use of the toxins as mucosal adjuvants in humans, toxicity and allergenicity of the toxins are problems which impinge on safety . To resolve these problems, various approaches have been attempted to produce less toxic and less allergenic CT (or LT) derivatives . We now propose the following standards for human use of safer CT (or LT) derivatives as an adjuvant of a nasal influenza vaccine . Thus, CT (or LT) derivatives can be administered intranasally together with a current inactivated influenza vaccine, provided they meet the following criteria . 1) A single dose of the derivatives, administered intranasally by spraying, should be around 100 Eg/adult in a volume of less than 0.5 ml . 2) CT (or LT) derivatives should retain the properties of the native CT (or LT), i . e., the ability to augment secretory IgA and serum IgG Ab responses to viral surface glycoproteins, when administered intranasally together with an inactivated influenza vaccine . 3) CT (or LT) derivatives should not induce IgE Ab responses to the vaccine, as well as to the CT (or LT) itself . 4) The CT (or LT) should be nontoxic; the toxicity of the derivatives, as determined by the Y-1 adrenal cell assay, should not exceed 1/100 EC(50) of the native CT (or 1/1000 ECi of the native CT) . 5) CT (or LT) derivatives should not cause serious disease in guinea pigs when administered intranasally or intraperitoneally at the dose used in humans (around 100 Eg). Eur J Immunol, 2000 Aug, 30(8), 2394 - 403 Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming; Gagliardi MC et al.; Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co-administered antigens, stimulating predominant Th2-type responses . However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response . We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level . We found that CT induces phenotypic and functional maturation of blood monocyte-derived DC . Indeed, CT-treated DC up-regulate expression of HLA-DR molecules, B7 . 1 and B7.2 co-stimulatory molecules, and are able to prime naive CD4(+)CD45RA(+) T cells in vitro, driving their polarization towards the Th2 phenotype . Furthermore, CT-matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs . Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL-12p70 and TNF-alpha) and chemokine (RANTES, MIP-1alpha and MIP-1beta) secretion by lipopolysaccharide- or CD40 ligand-activated DC . Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC. Gut, 2000 Sep, 47(3), 382 - 6 Effect of vasoactive intestinal polypeptide (VIP) antagonism on rat jejunal fluid and electrolyte secretion induced by cholera and Escherichia coli enterotoxins; Mourad FH et al.; BACKGROUND: The enteric nervous system is important in the pathophysiology of intestinal fluid secretion induced by cholera toxin (CT), Escherichia coli heat labile (LT), and heat stable (STa) toxins . The neurotransmitters involved are not fully elucidated . Vasoactive intestinal polypeptide (VIP), a potent intestinal secretagogue present in the enteric nervous system, is increased after exposure of the cat intestine to CT . Whether VIP is involved in the pathogenesis of cholera and other toxins in not known . AIM: To study in vivo the effect of VIP antagonism on jejunal fluid secretion induced by CT, LT, and STa . METHODS: CT, LT (25 microg), or 0.9% NaCl was instilled in an isolated 25 cm segment of rat jejunum, and the VIP antagonist (VIPa) {4Cl-D-Phe(6), Leu(17)}-VIP (0.2 or 2 microg/kg/min) or 0.9% NaCl was given intravenously . Two hours later, single pass in vivo jejunal perfusion was performed to assess fluid movement . In STa experiments, intravenous VIPa or 0.9% NaCl was given and 30 minutes later the jejunal segment was perfused with a solution containing STa 200 microg/l . RESULTS: VIPa had no effect on basal intestinal fluid absorption . CT induced net fluid secretion (median -68 microl/min/g dry intestinal weight (interquartile range -80 to -56)) which was dose dependently reversed by VIPa (6.2 (-16 to 34) and 29 (17 to 42); p<0.01) . Similarly, LT induced secretion (-63 (-73 to -30)) was attenuated by VIPa (0.2 microg/kg/min) (-15 (-24 to -1); p<0.01) and totally reversed to normal levels by VIPa (2 microg/kg/min) (37 (28-56); p<0 . 01 compared with LT and not significant compared with normal controls) . STa induced secretion (-17 (-19 to -2)) was also reversed by VIPa (12 (9-23) and 14 (0-26); p<0.01) . CONCLUSION: VIP plays an important role in CT, LT, and STa induced intestinal secretion and may be the final putative neurotransmitter in the pathophysiology of these toxins. Clin Exp Immunol, 2000 Aug, 121(2), 283 - 8 Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells; Eriksson K et al.; We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system . CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells . CT however, had a differential effect on naive and activated/memory T cell subsets . Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells . IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e . also in CD45RA+ cells which had maintained proliferative responses . However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies . These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45. Br J Pharmacol, 2000 Aug, 130(7), 1561 - 70 Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis; Sachinidis A et al.; The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth . We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta) . The effect of PDGF-BB on VSMCs growth was assessed by {(3)H}-thymidine incorporation . Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method . Actin mRNA level was quantitated by Northern blotting . Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy . Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions . Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein . Maximal decrease of 70% was observed after 24 h of treatment . CTX also caused a 90% decrease of the actin mRNA level . CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered . Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor . We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs. Biochem Biophys Res Commun, 2000 Aug 11, 274(3), 717 - 21 Effect of cholera toxin on rat liver lysosome acidification; Van Dyke RW et al.; We have shown that cholera toxin and cAMP greatly increase both acidification rates of liver endosomes and the liver and endosome content of fluid-phase endocytosis probes . In this study lysosomes were purified from control and cholera toxin-treated livers that were pulsed with fluorescein conjugated dextran and chased overnight . Cholera toxin-treated livers weighed less, contained less protein and exhibited higher contents of lysosomal marker enzymes, consistent with the catabolic effects of this agent . By contrast to its effects on endosomes, cholera toxin had no consistent or significant effect on lysosome acidification rates, steady-state internal pH or potassium content, proton leak rates or fluorescein-dextran content . We conclude that cholera toxin and cAMP predominantly alter earlier steps of endocytosis but may also increase transfer of probes from lysosomes to bile . Int J Epidemiol, 2000 Aug, 29(4), 764 - 72 Geographical patterns of cholera in Mexico, 1991-1996; Borroto RJ et al.; BACKGROUND: The seventh cholera pandemic has been ongoing in Mexico since 1991 and threatens to become endemic . This paper aims to determine the geographical pattern of cholera in Mexico to define areas at high risk of endemic cholera . METHODS: Ecologic research was conducted based upon the cartography of disease incidence . The 32 Mexican states were grouped into five strata according to the value of the 1991-1996 cumulative incidence rate of cholera . Rate ratios were computed for strata of states classified by geographical situation, urbanization, and poverty level . RESULTS: Cholera incidence was 2.47 times higher in coastal states than in the interior (95% CI : 2.42-2.52) . The disease was negatively associated with urbanization . Incidence in the least urbanized stratum was four times as high as in the most urban stratum (95% CI : 3.9-4.12) . The poorest stratum showed the most remarkable incidence, i.e . 5.9 times higher than the rate in the least poor stratum (95% CI : 5.73-6.04) . CONCLUSIONS: This ecologic research suggests that high poverty level, low urbanization, and southern location are the most important predictors of endemic cholera in Mexican states . It is hypothesized that the natural environment of the coastal plains in southern states may also play a significant role in cholera incidence . Poor communities residing in the southern, predominantly rural, coastal states should be prioritized when it comes to investing in safe water supply facilities, adequate excreta disposal systems and cholera surveillance. Brain Res, 2000 Aug 4, 873(1), 160 - 4 Three novel neural pathways to the lacrimal glands of the cat: an investigation with cholera toxin B subunit as a retrograde tracer; Cheng SB et al.; The distribution of ganglion neurons innervating the lacrimal gland (LG) was investigated following injection of cholera toxin B subunit into the LG of the cat . We report the first evidence that the otic ganglion (OG), and superior vagal and glossopharyngeal ganglia are also the sources of innervation of the LG . LG-innervating neurons in the pterygopalatine ganglion and the OG could be divided into two subpopulations: small and large neurons . They may mediate the vasodilatation and secretion, respectively. Brain Res Brain Res Protoc, 2000 Jul, 5(3), 298 - 304 Double immunocytochemistry for the detection of Fos protein in retrogradely identified neurons using cholera toxin B subunit; Leman S et al.; The focus of this paper was to describe a method combining the neuroanatomical technique of retrograde transport of cholera toxin B subunit (CTB) with the technique of Fos functional labeling . This method allowed us to evaluate whether neurons identified by retrograde tracing were activated following chemical stimulation of another brain area . We have used this method at the light microscopic level to determine whether the stimulation of the rostral ventrolateral medulla activated retrogradely labeled adrenal sympathetic preganglionic neurons in the spinal cord . CTB-containing neurons, Fos immunoreactive neurons and double labeled neurons were observed in spinal autonomic areas . These results suggest that the rostral ventrolateral medulla exerts a descending activation upon identified adrenal preganglionic neurons . The method described in this protocol can be applied for other brain areas in order to establish if a given structure can activate an identified population of neurons linked with a particular target of central or peripheral nervous system. Mol Cells, 2000 Jun 30, 10(3), 325 - 30 Differential effects of cholera toxin and pertussis toxin on the c-fos and c-jun mRNA expression in rat C6 glioma cells; Lee JK et al.; Cholera toxin (CTX) increased c-fos mRNA level whereas it down-regulated the c-jun mRNA level in rat C6 glioma cells . In contrast to the action of CTX, pertussis toxin (PTX) did not affect either c-fos or c-jun mRNA level . The elevated c-fos mRNA level induced by CTX was significantly inhibited by the co-treatment with dexamethasone (DEX) . However, DEX did not affect CTX-induced down-regulation of c-jun mRNA level . Cycloheximide (CHX) increased c-fos and c-jun mRNA levels . CHX caused a super-induction of CTX-induced c-fos mRNA level . Our results suggest that CTX-, but not PTX-, sensitive G-proteins may play an important role for c-fos mRNA up-regulation and c-jun mRNA down-regulation . In addition, DEX appears to have a selective inhibitory action against c-fos mRNA expression regulated by CTX . Ongoing protein synthesis inhibition is required for the superinduction of c-fos, but not c-jun, mRNA induced by CTX. Infect Immun, 2000 Aug, 68(8), 4492 - 7 Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic escherichia coli-cholera toxin B subunit vaccine in Israeli young adults; Cohen D et al.; Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units . Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB) . The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers . However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms . Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively . The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1 . The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens . The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens . Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force. Pharmacol Toxicol, 2000 Jun, 86(6), 270 - 5 Regional differences in neurogenic signal transduction pathway of cholera toxin-induced fluid, electrolyte and serotonin accumulation in the porcine jejunum; Berggreen P et al.; Serotonin, acetylcholine and substance P are mediators involved in the secretory response to cholera toxin in the small intestine . The aim of this study was to investigate the regional difference in the effect of a serotonin receptor type 3 antagonist (ondansetron), a nicotinic receptor antagonist (hexamethonium), and a substance P antagonist (the neurokinin receptor type 1 antagonist, CP 99,994) on the cholera toxin-induced fluid accumulation in the porcine jejunum . A dose-range of cholera toxin (0.32-56.00 microg/loop) was instilled for 4 hr in ligated loops in two regions of the proximal jejunum in 6-8-week-old pigs . Ondansetron (200 microg/kg), hexamethonium (10 mg/kg), CP 99,994 (1 mg/kg), or saline alone (control) were given intravenously 10 min . before cholera toxin instillation . Cardiovascular parameters, blood gas data, net fluid accumulation, serotonin and electrolyte concentration in the accumulated fluid were measured . Cardiovascular and blood gas parameters were within the normal range in all treatments . The apparent maximal response in fluid accumulation was reduced 20% in case of ondansetron, and by 33% using CP 99,994 in the aboral region compared to control, whereas no effect was observed in the oral region . Hexamethonium reduced the apparent maximal secretory response in both the oral and aboral regions by 45% . None of the treatments with antagonists changed the luminal content of serotonin or the electrolyte concentrations in the accumulated fluid . The results demonstrate that the involvement of serotonin receptor type 3 and neurokinin type 1 receptors in the transductional pathway of cholera toxin-induced fluid accumulation vary significantly within the jejunum, while the cholinergic (nicotinic) transmission plays an even role. Br J Pharmacol, 2000 Jul, 130(5), 1031 - 6 The inhibition of cholera toxin-induced 5-HT release by the 5-HT(3) receptor antagonist, granisetron, in the rat; Turvill JL et al.; 1 . The secretagogue 5-hydroxytryptamine (5-HT) is implicated in the pathophysiology of cholera . 5-HT released from enterochromaffin cells after cholera toxin exposure is thought to activate non-neuronally (5-HT(2) dependent) and neuronally (5-HT(3) dependent) mediated water and electrolyte secretion . CT-secretion can be reduced by preventing the release of 5-HT . Enterochromaffin cells possess numerous receptors that, under basal conditions, modulate 5-HT release . 2 . These include basolateral 5-HT(3) receptors, the activation of which is known to enhance 5-HT release . 3 . Until now, 5-HT(3) receptor antagonists (e.g . granisetron) have been thought to inhibit cholera toxin-induced fluid secretion by blockading 5-HT(3) receptors on secretory enteric neurones . Instead we postulated that they act by inhibiting cholera toxin-induced enterochromaffin cell degranulation . 4 . Isolated intestinal segments in anaesthetized male Wistar rats, pre-treated with granisetron 75 microg kg(-1), lidoocaine 6 mg kg(-1) or saline, were instilled with a supramaximal dose of cholera toxin or saline . Net fluid movement was determined by small intestinal perfusion or gravimetry and small intestinal and luminal fluid 5-HT levels were determined by HPLC with fluorimetric detection . 5 . Intraluminal 5-HT release was proportional to the reduction in tissue 5-HT levels and to the onset of water and electrolyte secretion, suggesting that luminal 5-HT levels reflect enterochromaffin cell activity . 6 . Both lidocaine and granisetron inhibited fluid secretion . However, granisetron alone, and proportionately, reduced 5-HT release . 7 . The simultaneous inhibition of 5-HT release and fluid secretion by granisetron suggests that 5-HT release from enterochromaffin cells is potentiated by endogenous 5-HT(3) receptors . The accentuated 5-HT release promotes cholera toxin-induced fluid secretion. Malays J Pathol, 1998 Jun, 20(1), 31 - 3 Evaluation of the cholera spot test: a chromatographic immunoassay for the rapid detection of cholera antigen; Rohani MY et al.; A chromatographic immunoassay cholera antigen detection kit, the Cholera Spot test, was evaluated . The test was found to be specific with a sensitivity of 10(6) cfu/ml for the direct detection of V . cholerae in simulated stool specimens and 10 cfu/ml in simulated cotton-tipped swab specimens after overnight incubation in alkaline peptone water . This enables early recognition of cholera cases and their contacts so that prevention and control measures can be promptly instituted. Clin Exp Allergy, 2000 Jul, 30(7), 1024 - 32 Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice; Rask C et al.; BACKGROUND: Oral tolerance is a long recognized method for inducing systemic immunological tolerance . However, large doses of antigen and frequent administrations are often required . By linking the antigen to the nontoxic mucosa-binding B subunit of cholera toxin (CTB), the required amount can be dramatically reduced . We have previously shown that mucosal administration of small amounts of antigens coupled to CTB can suppress peripheral Th1 cell-reactivity and associated inflammatory immunopathology in both naive and systemically-immunized animals . Induction of oral tolerance by repeated feeding of relatively small doses of antigen has, in some cases been shown to involve the generation of regulatory Th2-like CD4+ T cells, and hence could promote rather than suppress type I immunoglobulin (Ig) E-mediated allergic responses . OBJECTIVES: We examined whether oral prophylactic or therapeutic administration of a model allergen coupled to CTB would modulate allergen-specific IgE responses in high IgE responder Balb/c mice . METHODS: Ovalbumin (OVA) was used as a model allergen . Mice were treated perorally with free or CTB-coupled OVA before or after systemic priming with alum-adsorbed OVA . Allergen-specific IgE levels in serum were measured with the passive cutaneous anaphylaxis test at various time-points . RESULTS: Oral administration of a single low dose of CTB-linked OVA, prior to systemic sensitization and challenge with OVA, suppressed allergen-specific serum IgE antibody responses . Treatment with comparable doses of free OVA was much less effective . Most importantly, oral treatment with CTB-OVA conjugate could also suppress an already initiated IgE antibody response, but to achieve such a 'therapeutic effect', administration of multiple low doses of conjugate over a long time was required . Oral treatment with CTB-OVA conjugate could also effectively suppress antigen-specific Th1-mediated delayed-type hypersensitivity . Thus treatment with a CTB-conjugated model allergen can affect a broad range of T-cell-driven immune responses, even in antigen-experienced animals . CONCLUSION: These results may impact on the development of therapeutic vaccines against type I allergies. Anal Biochem, 2000 May 15, 281(1), 123 - 33 A ganglioside-based assay for cholera toxin using an array biosensor; Rowe-Taitt CA et al.; A rapid assay for cholera toxin (CT) has been developed using a fluorescence-based biosensor . This sensor was capable of analyzing six samples simultaneously for CT in 20 min with few manipulations required by the operator . The biochemical assays utilized a ganglioside-"capture" format: ganglioside GM1, utilized for capture of analyte, was immobilized in discrete locations on the surface of the optical waveguide . Binding of CT to immobilized GM1 was demonstrated with direct assays (using fluorescently labeled CT) and "sandwich" immunoassays (using fluorescently labeled tracer antibodies) . Limits of detection for CT were 200 ng/ml in direct assays and 40 ng/ml and 1 microg/ml in sandwich-type assays performed using rabbit and goat tracer antibodies . Binding of CT to other glycolipid capture reagents was also observed . While significant CT binding was observed to loci patterned with GD1b, Gb3, and Gb4, CT did not bind significantly to immobilized GT1b at the concentrations tested . This is the first description of such a non-antibody-based recognition system in a multi-specific planar array sensor. Brain Res, 2000 Jun 9, 867(1-2), 246 - 9 Differential modulatory roles of cholera toxin and pertussis toxin in the regulation of pain responses induced by excitatory amino acids administered intrathecally in mice; Chung KM et al.; The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in excitatory amino acids induced pain response . Intrathecal (i.t.) injection of glutamate (20 microg), N-methyl-D-aspartic acid (NMDA; 60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 13 ng), and kainic acid (12 ng) showed pain response . Pretreatment with CTX (0.05 and 0.5 microg, i.t.) attenuated pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t . in a dose-dependent manner . On the other hand, i.t . pretreatment with PTX further increased the pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t., especially at the dose of 0.5 microg . Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play opposite roles in modulating the pain response induced by spinally administered . Furthermore, CTX- and PTX-sensitive G-proteins appear to modulate pain response induced by stimuli of both NMDA and non-NMDA glutamate receptors. Microbiol Immunol, 2000, 44(4), 259 - 66 Intranasal sensitization of Japanese cedar pollen by the co-administration of low doses of cholera toxin but not its recombinant B subunit to mice; Hirai T et al.; We evaluated the effects of cholera toxin (CT) and the B subunit of cholera toxin (CTB) on the intranasal sensitization of Japanese cedar pollen (JCP) in mice . JCP suspended in phosphate-buffered saline was administered into the nostrils of mice in combination with varying doses of CT or recombinant CTB(r-CTB) once a week for 5 weeks . Antibody responses specific to sugi basic protein (SBP) were monitored by ELISA for seven weeks . The sensitization of JCP alone did not induce IgG1, IgG2b, IgG2a, IgE or IgA . In contrast, sensitization of JCP in combination with CT (JCP/CT) elicited the prominent production of SBP-specific IgG1 and low levels of IgG2b and IgG2a on Day 49 . IgE production was detected only in the serum of mice which were treated with JCP/CT, and not under any other protocol . Using spleen cells from these mice, cytokine production was examined by ELISA in culture supernatants after they had been stimulated in vitro with major cedar pollen allergens, Cry j 1, Cry j 2 or SBP . Notable responses were an increase of IFN-gamma as well as IL-4 in JCP/CT-sensitized cells stimulated with Cry j 2, but not in those stimulated with Cry j 1 . No significant differences were detected in IL-5 production among the experimental groups . Histopathological examination, however, showed that eosinophil infiltration was evident in the nasal mucosa of the JCP/CT-sensitized mice following challenge with JCP/CT, but weak with BSA/CT or CT alone . Thus, the immunological and histological analyses indicated that the co-administration of a low dose of CT in combination with JCP allows the induction of pollen-allergic states in mice. J Biol Chem, 2000 Sep 29, 275(39), 30240 - 7 A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin; De Wolf MJ; Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH(2)), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation . The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH(2), an inactive analogue of Cbz-Gly-Phe-NH(2) . The effect was reversible and dose- (IC(50) as low as 0.2 mm depending on the cell type) and time-dependent . Adding Cbz-Gly-Phe-NH(2) during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA) . Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced . The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH(2)-treated cells or when Cbz-Gly-Phe-NH(2) was added directly to the assay . The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH(2) and BFA were not additive pointing to a similar mode of action of the two substances . However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH(2) on CT cytotoxicity . Several lines of evidence indicate that a perturbation of intracellular Ca(2+) homeostasis by Cbz-Gly-Phe-NH(2) is not responsible for the inhibitory effect of the dipeptide . The dipeptide had also no effect on the binding of (125)I-CT to cells and even increased its intracellular internalization . In contrast with BFA, Cbz-Gly-Phe-NH(2) did not completely suppress the formation of the catalytically active A(1) fragment from bound CT . The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH(2) cannot be excluded. J Commun Dis, 1999 Mar, 31(1), 49 - 52 An El Tor cholera outbreak in Maldah district, West Bengal; Gupta DN et al.; An outbreak of cholera occurred in Maldah district, West Bengal during July-August 1998 . Attack rate was 34/1000 . Cases were more (59.3%) amongst adults (> 15 years.) . V . cholerae 01 biotype E1 Tor serotype ogawa was isolated as a single pathogen from 52.9% (9/17 samples examined) . All V . cholerae strains belonged to phage type 2 (Basu and Mukherjee scheme) and type 27 (new phage type scheme) . The strains were resistant to co-trimoxazole, furazolidone, ampicillin, streptomycin and nalidixic acid. Biosci Biotechnol Biochem, 2000 Mar, 64(3), 516 - 22 Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology; Oh S et al.; The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected . In addition, a statistical model was developed to optimize the production of CMP . CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment . CMP was further fractionated in an ion-exchange column by FPLC . CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues . The CT binding activity was rapidly lost by a carbohydrase treatment . The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM) . The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min . A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions. Am J Physiol Gastrointest Liver Physiol, 2000 May, 278(5), G789 - 96 Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum; Abely M et al.; The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux . Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers . Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC . In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production . In control tissues, glutamine stimulated sodium absorption and was mainly oxidized . The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane . The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline . Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism . In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism . In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte. Cochrane Database Syst Rev . 2000;(2):CD000974. Vaccines for preventing cholera; Graves P et al.; BACKGROUND: Oral cholera vaccines (either killed whole cell or live recombinant vaccines) are newer alternatives to the parenteral vaccines which have been thought to confer only moderate and short-term immunity . OBJECTIVES: The objective of this review was to assess the effect of cholera vaccines in preventing cases of cholera and preventing deaths . SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, Medline, Embase and reference lists of articles . We handsearched the journal Vaccine, contacted researchers in the field and manufacturers . SELECTION CRITERIA: Randomised and quasi-randomised studies comparing cholera vaccines (killed or live) with placebo, control vaccines or no intervention, or comparing types, doses or schedules of cholera vaccine . We included adults and children irrespective of immune status or special risk category . DATA COLLECTION AND ANALYSIS: Data extraction and assessment of trial quality was done independently by two reviewers . MAIN RESULTS: Thirty-two trials were included . Seventeen efficacy trials of relatively good quality, testing parenteral and oral killed whole cell vaccines and involving over 2 . 6 million adults, children and infants were included . Nineteen safety trials have been conducted for both types of killed whole cell vaccines and for live vaccines and have involved 11,459 people . For all types of vaccines compared to placebo, the relative risk of contracting cholera at 12 months was 0.49, 95% confidence interval 0 . 41 to 0.59 (random effects model) . This translates to an efficacy of 51%, 95% confidence interval 41% to 59% . Both parenteral and oral administration were relatively efficacious, but significant protection extended into the third year for oral killed whole cell vaccines . Children under 5 were only protected for up to a year, while older children or adults were protected for up to three years . Parenteral killed whole cell vaccines were associated with increased systemic and local adverse effects compared to placebo . Oral killed whole cell vaccines or oral live vaccines were not . REVIEWER'S CONCLUSIONS: Cholera killed whole cell vaccines appear to be relatively effective and safe . Live oral recombinant vaccines appear to be safe, but efficacy data are not available . Protection against cholera appears to persist for up to two years following a single dose of vaccine, and for three to four years with an annual booster. Dig Dis Sci, 2000 May, 45(5), 946 - 51 Cholera toxin-induced secretion in rats is reduced by a soluble fiber, gum arabic; Turvill JL et al.; Gum arabic (GA), a soluble fiber with emulsifying properties, enhances intestinal water and electrolyte absorption in normal and secreting rats . Our aim was to assess the effect of GA, 2.5 and 5.0 g/liter, on cholera toxin-induced water and electrolyte secretion in rat jejunum in vivo . After a 2-hr exposure to cholera toxin, jejunal segments of adult rats were perfused in vivo with at plasma electrolyte solution containing GA, 0, 2.5 or 5.0 g/liter . 24Na was used as a marker of sodium influx . Cholera toxin-induced secretion was reduced by GA, 2.5 and 5.0 g/liter . 24Na secretion into the lumen was reduced by GA . GA caused a morphological expansion of intercellular spaces in the villi but not crypts . In conclusion, GA promotes lumen to blood intestinal transport of water and sodium despite cholera toxin activation . These observations support a potential role for GA in enhancing the efficacy of ORS. Anat Embryol (Berl), 2000 Apr, 201(4), 245 - 57 Study of the olivocochlear neurons using two different tracers, fast blue and cholera toxin, in hypothyroid rats; Cantos R et al.; Congenital hypothyroidism results in deafness that is caused by changes in the auditory receptor, including scanty development of the outer hair cells and a lack of synaptogenesis between these cells and the efferent system . although the afferent population is present . The normal efferent innervation of the cochlea originates in the superior olivary complex, arising from efferent neurons belonging to the lateral or to the medial olivocochlear system . In the rat, the former is constituted by neurons located in the lateral superior olivary nucleus, that project to the inner hair cells, while the later originates in the ventral nuclei of the trapezoid body and project to the outer hair cells . The aim of this work is to study the localization, number and morphology of the olivochochlear neurons in congenital hypothyroid animals by means of the injections of the retrograde tracers, either fast blue or cholera toxin, in the cochlea . The mean total number of labeled olivocochlear neurons after injection of fast blue in hypothyroid animals was 1,016, and in control ones was 1,027 . Using cholera toxin, the mean total number of labeled olivocochlear neurons was slightly lower: 863 in hypothyroid animals versus 910 in control ones . Although both tracers showed no significant differences between groups, when the somatic area of the labeled olivocochlear neurons is considered, the size of all of the three different population of cells (lateral olivocochlear neurons, medial olivocochlear neurons and shell neurons) was significantly lower in the hypothyroid rats . This is the first study of the olivocochlear neurons in hypothyroid animals . The conclusion from this work is that in hypothyroid rats the labeled olivocochlear neurons are significantly smaller but that there is not any modification in the localization and number of the labeled olivocochlear neurons, suggesting that thyroid hormones are necessary for the neuronal growth . However, most of the medial olivocochlear neurons do not make contact with their target, so their maintenance suggests that the axons are in contact with other structures of the cochlea. Mol Biol Cell, 2000 May, 11(5), 1645 - 55 High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes; Kenworthy AK et al.; "Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes . However, evidence supporting their existence has been indirect and controversial . In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface . The results of these studies, each based on a single protein, gave conflicting views of rafts . To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types . FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane . However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains . We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface. Vaccine, 2000 Jun 1, 18(24), 2723 - 34 Effective mucosal immunization against respiratory syncytial virus using purified F protein and a genetically detoxified cholera holotoxin, CT-E29H; Tebbey PW et al.; We exploited the powerful adjuvant properties of cholera holotoxin (CT) to create a mucosally administered subunit vaccine against respiratory syncytial virus (RSV) . A genetically detoxified mutant CT with an E to H substitution at amino acid 29 of the CT-A1 subunit (CT-E29H) was compared to wild type CT for toxicity and potential use as an intranasal (IN) adjuvant for the natural fusion (F) protein of RSV . When compared to CT the results demonstrated that: (1) CT-E29H binding to GM1 ganglioside was equivalent, (2) ADP-ribosylation of agmatine was 11.7%, and (3) toxicity was attenuated in both Y-1 adrenal (1.2%) and patent mouse gut weight assays . IN vaccination with F protein formulated with CT-E29H induced serum anti-CT and anti-F protein antibodies that were comparable to those obtained after vaccination with equivalent doses of CT . Vaccinations containing CT-E29H at doses of 0.1 microg were statistically equivalent to 1.0 microg in enhancing responses to F protein . Antigen-specific mucosal IgA and anti-RSV neutralizing antibodies were detected in nasal washes and sera, respectively, of mice that had received F protein and 0.1 or 1.0 microg of CT-E29H . Anti-F protein IgA was not detected in the nasal washes from mice IN vaccinated with 0.01 microg CT-E29H or IM with F protein adsorbed to AlOH adjuvant . In addition, the formulation of purified F protein and CT-E29H (0.1 and 1.0 microg) facilitated protection of both mouse lung and nose from live RSV challenge . Collectively, the data have important implications for vaccine strategies that use genetically detoxified mutant cholera holotoxins for the mucosal delivery of highly purified RSV antigens. Vaccine, 2000 Jun 1, 18(24), 2713 - 22 Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin); Matsuo K et al.; Inactivated influenza vaccine was administered intranasally to BALB/c mice together with an adjuvant (cholera toxin B subunit {CTB} supplemented with a trace amount of the whole toxin, CTB*) and its ability to induce innate immunity and confer protection against influenza was examined . Nasal wash virus titres 3 days after inoculation of homologous viruses were measured as an index of the ability of the vaccine to confer protection in mice immunized with either CTB*-combined vaccine or CTB* alone 1-21 days previously . The results were as follows . (1) Partial but significant reduction of the nasal-wash virus titres (prevention) was detected beginning 3 days after the vaccination, that is, 2 days earlier than the appearance of both virus-specific antibody-forming cells (AFCs) in the nasal-associated lymphoid tissue (NALT) and virus-specific IgA antibody responses in the nasal washes of mice immunized with the CTB*-combined vaccine . (2) The protection, detected on day 3 and peaking on day 5 but lost by day 21, was also conferred in mice immunized with CTB* alone . (3) The non-specific prevention was detected at doses of more than 0.3 microg of CTB*/mouse . (4) The nonspecific protection beginning 3 days after the immunization involved the enhanced expression of cytokine mRNAs (IL-15 and IL-18), considered responsible for natural killer (NK) cell activation, by the non-T cell populations in the NALT . (5) Normal NALT cells, when cultured in vitro with CTB*, secreted IL-1beta within a few hours in culture . These results demonstrate that the CTB*-combined vaccine, when given intranasally into mice, can confer nonspecific protection against influenza beginning 3 days after the vaccination and that CTB* also possessed this ability to confer protection non-specifically and temporarily by inducing the secretion of IL-1beta, one of the most important cytokines that initiates both innate and adaptive immunity, and also NK cell activity. Can J Microbiol, 2000 Mar, 46(3), 283 - 90 Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 admixed with cholera toxin fails to elicit protection against subsequent colonization by the pathogen; Conlan JW et al.; It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen . The present study was conducted to determine whether such an immune response can be experimentally generated in mice . To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin . Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine . Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E . coli O157:H7 as unvaccinated control mice . These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts. Pathobiology, 1999, 67(5-6), 314 - 7 Induction of tolerance in macrophages by cholera toxin B chain; Burkart V et al.; Model systems of human type 1 diabetes have revealed an important role of cellular immune reactions involving macrophages and T cells in the destruction of autologous insulin-producing pancreatic beta cells . Recently, the cholera toxin B chain (CTB) was found to suppress T cell-dependent autoimmune diseases including autoimmune diabetes of nonobese diabetic mice . Therefore, we tested the hypothesis that CTB exerts much of its immunomodulatory activity by targeting macrophages . These studies are reviewed here . Cells of the human monocyte line Mono Mac 6 were exposed to CTB and subsequently tested for proinflammatory immunoreactivity in response to challenge with endotoxin (LPS from Escherichia coli, 10 ng/ml for 5 h) . Incubation of monocytes with CTB (10 microgram/ml) suppressed a later proinflammatory response to LPS as demonstrated by suppression of TNFalpha release from 6.7 +/- 0.7 ng/ml in cultures without CTB preexposure to 1.8 +/- 1.1 ng/ml in CTB-pretreated cells (p < 0.001) . In contrast, the release of IL-10 remained inducible after CTB pretreatment . RT-PCR analysis showed that the suppression of TNFalpha production occurred at the level of mRNA formation . Control experiments excluded a role of possible contamination of CTB by endotoxin or the intact cholera toxin . Tolerance induction was maximal after 5 h of CTB exposure and persisted for 24 h . The suppressive effect of CTB was dose-dependent and no more recognizable at </=1 microgram/ml . Incubation with IL-10- and TGFbeta-neutralizing antibodies during CTB pretreatment prevented tolerization of macrophages . IFNgamma (1,200 U/ml) was found to antagonize actions of CTB . In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred 'silently', i.e . in the absence of a measurable proinflammatory response . In view of the potent instructive role of the innate immune system on T cell responses these findings are important in understanding how CTB prevents the development of autoimmune diabetes and improves tolerance to islet autoantigens . J Cell Biol, 2000 Mar 20, 148(6), 1203 - 12 Cholera toxin is exported from microsomes by the Sec61p complex; Schmitz A et al.; After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism . Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V . cholerae . Export of CTA1 from the microsomes was time- and adenosine triphosphate-dependent and required lumenal ER proteins . By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER . Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes . Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side . Our results suggest that Sec61p complex-mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell. Jpn J Ophthalmol, 2000 Mar, 44(2), 189 - 90 Immunosuppressive effect of cholera toxin B on allergic conjunctivitis model in the guinea Pig Saito K, Shoji J, Inada N, Iwasaki Y, Sawa M. Purpose: To investigate the immunosuppressive effects of mucosal immune therapy in experimental allergic conjunctivitis.Method: We used 11 white Hartrey guinea pigs divided into two groups . Six animals (treated group) received pretreatment with topical instillation of cholera toxin B (4 &mgr;g/30 ml) and ovalbumin (10 &mgr;g/30 ml) . The other group of 5 animals served as control . All the animals received intra-abdominal injection of ovalbumin (100 g/mL) and aluminum hydroxide (5 mg/mL) repeated twice 2 weeks apart . Allergic conjunctivitis was induced by topical instillation of ovalbumin solution (5 mg/mL) 1 week after the above procedure . Result: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation . The allergic reaction was significantly less in score in the treated than in the control group (Mann-Whitney U-test: P <.01) . The clinical findings subsided after 6 hours . The treated group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival epithelium, than in the control group at 6 and 24 hours.Conclusion: Pretreatment with topical cholera toxin B and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis. Bull Soc Pathol Exot, 1999 Dec, 92(5), 349 - 54 {Poland: cholera to typhus, 1831-1950}; Balinska MA; In this article devoted to Poland's direct and indirect role in the elaboration of contemporary international health structures and to her reputation as an epidemic reservoir of Europe, we consider how Poland came to be perceived as the cordon sanitaire of the West . Traditionally seen as upholding Western values, in the 19th and 20th centuries the country became increasingly associated with "Eastern plagues"-cholera and then typhus-coming from Russia and which could spread to the rest of Europe if Poland did not manage to contain them . When Poland was reconstituted as a nation-state in 1918, the new country won international recognition through her successful attempts to contain a typhus epidemic sweeping westwards from Russia . The Polish government convened the first European, League sponsored, health conference following the First World War . A Polish doctor, L . RAJCHMAN, was chosen to head up the League of Nations Health Organisation (forerunner of the WHO) and later (1946) founded UNICEF . Finally, we examine the key issue of exanthematous typhus in both world wars, exemplifying how a disease can come to be "ideologized", in this case by Nazi Germany . Typhus was the pretext used- in the name of "public health"-for segregating Polish citizens of Jewish origin and even killing them . Paradoxically, typhus was in the process of being eradicated when the war began and German policy of mass resettlements, sequestration, and starvation only spurred the epidemic they supposedly wished to control. Int J Infect Dis, 2000, 4(1), 8 - 13 Epidemic cholera in Guinea-Bissau: the challenge of preventing deaths in rural West Africa; Gunnlaugsson G et al.; OBJECTIVES: An epidemiologic investigation was conducted to identify factors associated with cholera mortality in a rural African setting and interventions likely to prevent deaths in future epidemics . METHODS: The authors reviewed surveillance data from rural Biombo, Guinea-Bissau, interviewed family members of persons who died of cholera, and conducted a case-control study in the catchment area of a health center with a high case:fatality ratio (CFR) . RESULTS: Forty-three deaths occurred among the 1169 persons who reported to health centers with cholera during the epidemic (CFR = 3.7%) . Delayed rehydration and over-hydration probably contributed to 10 of these deaths . An additional 19 cholera deaths occurred outside health centers . In the case-control study, persons with cholera who died were 5.4 times (95% CI = 1.0-53.4) more likely to be in poor health or intoxicated at illness onset than persons with cholera who survived . Fatal cases were 6.0 times (95% CI = 1.1-60.8) more likely to not attend the health center than survivors . CONCLUSIONS: The low overall CFR in Biombo, compared to CFRs reported during other epidemics in sub-Saharan Africa, suggests that medical care provided at rudimentary rural health centers prevented numerous deaths . Additional deaths may be prevented by strengthening the infrastructure of health services in the rural areas and by enhanced public education regarding the need for persons with cholera to promptly seek medical care. Biol Reprod, 2000 Mar, 62(3), 775 - 80 Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin; Grondahl C et al.; To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively . Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT) . MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS . Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls . In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis . CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action . In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription. Mol Cells, 1999 Dec 31, 9(6), 609 - 16 Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses; Kang DK et al.; To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli . The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis . To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice . The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice . In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch . Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT . Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization. J Biol Chem, 2000 Feb 4, 275(5), 3231 - 8 Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits; Angstrom J et al.; The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related . However, the carbohydrate binding specificities of the two proteins differ . While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates . The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB . Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained . Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium . A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer . Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta . The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations. J Autoimm