Effect of Cyclic Nucleotides on DNA Synthesis in Mouse Lymphoid Cells

The effect of eight cyclic purine and cyclic pyrimidine nucleotides on DNA synthesis on mouse lymphoid cells was investigated. Two out of eight compounds tested, namely 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) as well as 3',5'-cyclic guanosine monophosphate (3',5'-cGMP), stimulate thymidine incorporation in all types of lymphocytes tested. The stimulatory activity of the cyclic guanosine nucleotides as well as the effects of lectins could be antagonized by 3',5'-cyclic adenosine monophosphate (3',5'-cAMP). 2',3'-cGMP seems to stimulate preferentially mature T-cells while 3',5'-cGMP preferentially acts on B-cells.     

Selective Destruction of Skin Permeability Activity in Cholera Toxin: Effect of Acid on Cholera Enterotoxin

The effect of treatment of purified cholera enterotoxin with acid at pH 2.0 for 1 h followed by neutralization of the acid on the biological activity of the toxin has been studied. It was found that the material loses 85 to 96% of its skin permeability activity and this loss is neither reversed nor increased after 28 days of storage at 5 to 10 C. Other biological properties such as diarrheagenic activity, lipolysis-stimulating activity, and the lethal dose for mice remain unaffected by acid treatment. These observations indicate that the skin permeability activity is more sensitive to relatively minor changes in molecular structure than are the diarrheal, lipolytic, and mouse lethality activities.     

Oral Immunization of Dogs with Purified Cholera Toxin, Crude Cholera Toxin, or B Subunit: Evidence for Synergistic Protection by Antitoxic and Antibacterial Mechanisms

The immunogenicity and safety of purified cholera toxin (CT), its B subunit, and a crude culture filtrate of toxigenic Vibrio cholerae (CrT) were compared in dogs immunized orally and challenged with virulent V. cholerae. CT and CrT caused marked protection in two- or three-dose regimens. Protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea in most dogs; this protection was proportional to the peak antitoxin response in jejunal mucosa and lasted at least 15 weeks. In contrast, minimum protective doses of CrT contained much less cholera toxin, caused very mild diarrhea in only 21% of the dogs, and evoked protection that was greater than predicted from the modest jejunal antitoxin response. B subunit caused smaller jejunal antitoxin responses than did similar doses of CT and was poorly protective, the 50% protective dose being >40-fold greater than that of CT. Two observations indicated that protection due to CrT involved synergy between antibacterial and antitoxic immune responses. First, the 50% protective dose of CrT was 24-fold and >36-fold smaller than the 50% protective doses of its CT and non-CT antigenic components, respectively, when tested separately. Second, protection was greater in CrT-immunized dogs than in CT-immunized dogs for a given mucosal antitoxin response. Low doses of CrT evoked serotype-specific protection, indicating that the serotype-specific O somatic antigen contributed significatly to antibacterial protection. These results suggest that a simple, effective, nonliving oral vaccine for cholera based on combined antibacterial and antitoxic immunity can probably be achieved. However, further studies are needed to determine how a protective antitoxic response can be evoked without causing diarrhea during immunization.     

Cholera Toxin,Ganglioside Receptors and the Immune Response

Cholera toxin activates plasma membrane adenylate cyclase in all mammalian cell types. The structure-function relationship of the toxin has recently been clarified, and the cell membrane receptor identified. This information has made cholera toxin the “agent of choice” for studies in many biological systems of the possible regulatory role of adenylate cyclase/cyclic AMP.

This article describes briefly our current knowledge about the toxin and its receptor. It then reviews recent research which has revealed that cholera toxin has strong modulating influences on the proliferation of normal and malignant lymphocytes as well as on the initiation and expression of immune responses. The toxin has been found to inhibit DNA synthesis of B and T lymphocytes in vitro without inducing cell death and also to inhibit the in vivo proliferation of a virus transformed lymphoma cell line. It seems to decrease antibody secretion from plasma cells in vitro, and might also interfere with the release of other soluble immunological mediator substances. In vivo cholera toxin induces a transient involution of the spleen and a more prolonged lymphocyte depletion of the thymus in mice; these effects appear to be mediated through the adrenal glands. The toxin inhibits allograft rejection, and either stimulates or suppresses antibody formation depending on the timing of the toxin in relation to the antigen administration. It increases the capacity of the spleen cells to induce graft-vs-host reactions and the “allogeneic effect” on antibody production. An inhibitory effect on a normal suppressor population among the spleen cells has been identified. The findings illustrate the complex effects induced on the immune system by the probably most discriminative investigative tool available for stimulation of the adenylate cyclase/cyclic AMP system.     

Cholera Toxin, Ganglioside Receptors and the Immune Response

Cholera toxin activates plasma membrane adenylate cyclase in all mammalian cell types. The structure-function relationship of the toxin has recently been clarified, and the cell membrane receptor identified. This information has made cholera toxin the “agent of choice” for studies in many biological systems of the possible regulatory role of adenylate cyclase/cyclic AMP.

This article describes briefly our current knowledge about the toxin and its receptor. It then reviews recent research which has revealed that cholera toxin has strong modulating influences on the proliferation of normal and malignant lymphocytes as well as on the initiation and expression of immune responses. The toxin has been found to inhibit DNA synthesis of B and T lymphocytes in vitro without inducing cell death and also to inhibit the in vivo proliferation of a virus transformed lymphoma cell line. It seems to decrease antibody secretion from plasma cells in vitro, and might also interfere with the release of other soluble immunological mediator substances. In vivo cholera toxin induces a transient involution of the spleen and a more prolonged lymphocyte depletion of the thymus in mice; these effects appear to be mediated through the adrenal glands. The toxin inhibits allograft rejection, and either stimulates or suppresses antibody formation depending on the timing of the toxin in relation to the antigen administration. It increases the capacity of the spleen cells to induce graft-vs-host reactions and the “allogeneic effect” on antibody production. An inhibitory effect on a normal suppressor population among the spleen cells has been identified. The findings illustrate the complex effects induced on the immune system by the probably most discriminative investigative tool available for stimulation of the adenylate cyclase/cyclic AMP system.     

Promotion of Colonization and Virulence by Cholera Toxin Is Dependent on Neutrophils

The innate immune response to Vibrio cholerae infection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenic V. cholerae El Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenic V. cholerae infection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.     

Mucosally induced Immunological Tolerance, Regulatory T Cells and the Adjuvant Effect by Cholera Toxin B Subunit

Induction of peripheral immunological tolerance by mucosal administration of selected antigens (Ags) ('oral tolerance') is an attractive, yet medically little developed, approach to prevent or treat selected autoimmune or allergic disorders. A highly effective way to maximize oral tolerance induction for immunotherapeutic purposes is to administer the relevant Ag together with, and preferably linked to the non-toxic B subunit protein of cholera toxin (CTB). Oral, nasal or sublingual administration of such Ag/CTB conjugates or gene fusion proteins have been found to induce tolerance with superior efficiency compared with administration of Ag alone, including the suppression of various autoimmune disorders and allergies in animal models. In a proof-of-concept clinical trial in patients with Behcet's disease, this was extended with highly promising results to prevent relapse of autoimmune uveitis. Tolerization by mucosal Ag/CTB administration results in a strong increase in Ag-specific regulatory CD4⁺ T cells, apparently via two separate pathways: one using B cells as APCs and leading to a strong expansion of Foxp3⁺ Treg cells which can both suppress and mediate apoptotic depletion of effector T cells, and one being B cell-independent and associated with development of Foxp3^- regulatory T cells that express membrane latency-associated peptide and transforming growth factor (TGF-β) and/or IL-10. The ability of CTB to dramatically increase mucosal Ag uptake and presentation by different APCs through binding to GM1 ganglioside (which makes most B cells effective APCs irrespective of their Ag specificity), together with CTB-mediated stimulation of TGF-β and IL-10 production and inhibition of IL-6 formation may explain the dramatic potentiation of oral tolerance by mucosal Ags presented with CTB.     

Binding of Cholera Toxin to Mucins and Inhibition by Gastric Mucin

The effects of mucin on cholera toxin induced intestinal secretion was studied in the rat small intestine. Gastric mucin inhibited secretion when premixed with cholera toxin on an equal dry-weight basis. Neither salivary nor intestinal mucin inhibited the toxin's action in the intestine. Inhibition by gastric mucin was not reversed by a mucolytic agent or by hexoses or agents that bind hexoses. Plasma and serum did not inhibit the effects of cholera toxin in the intestine. Choleragen was labeled with (14)C and its binding to mucins and ganglioside was studied. Gastric mucin was shown to bind to choleragen, but less binding was observed when choleragen was dissociated into subunits by acid pH. Salivary and intestinal mucins bound intact choleragen but not the subunits at acid pH. Salivary mucin binding was reversed by N-acetyl neuraminic acid and neuraminidase. Ganglioside bound choleragen in both the intact and dissociated forms. Binding to the dissociated form was reversed by wheat germ agglutinin. Gastric mucin and ganglioside competed for binding to choleragen with the binding of intact choleragen greater for ganglioside and the affinity of the subunits greater for gastric mucin. Electrophoresis of labeled choleragen showed uniform labeling of the subunits dissociated by acid pH, but a major part of cholera toxin was found not to be labeled when fractionated with sodium dodecyl sulfate and 2-mercaptoethanol.     

Exogenous Cyclic AMP, Cholera Toxin, and Endotoxin Induce Expression of the Lipopolysaccharide Receptor CD14 in Murine Bone Marrow Cells: Role of Purinoreceptors

Little is known about the mechanisms of lipopolysaccharide (LPS) signaling in immature cells that do not express the LPS receptor CD14 yet. Bone marrow granulocytes do not constitutively express CD14 but can be stimulated by low doses of LPS in the absence of serum and then express an inducible form of LPS receptor (iLpsR). We show that in addition to LPS, cholera toxin (CT) and various cyclic AMP (cAMP) analogs can also induce the expression of iLpsR, which was identified as CD14. Induction was independent of intracellular cAMP. The hypothesis that cAMP analogs act via a cell surface receptor was suggested by the unresponsiveness of trypsin-treated cells to these inducers and by the specific binding of [³H]cAMP to the cells. This binding was not inhibited by LPS or CT but was inhibited by various purine derivatives. However, the receptor involved is not a conventional purinoreceptor since both an agonist and an antagonist of such receptors were able to induce iLpsR expression. The results suggest that cAMP analogs and other purine derivatives induce iLpsR after interaction with an unconventional, trypsin-sensitive, purinoreceptor distinct from LPS and CT receptors.     

Biliary Immune Response to Orally Presented Food Antigen, Ovomucoid, and its Potentiation by Cholera Toxin B Subunit

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

Radioimmunoassay for the Antigenic Determinants of Cholera Toxin and Its Components

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, alpha chain, and gamma chain were iodinated by the chloramine T procedure. Radiolabeling did not significantly alter the polyacrylamide electrophoretic migration patterns of the toxin or its components. Moreover, radiolabeled toxin, B subunit, and alpha chain preparations retained substantial ability to bind to intestinal mucosal homogenates. The minimal amount of antitoxin detectable with radiolabeled toxin was 0.04 antitoxin units/ml. Substitution of radiolabeled B subunit, A subunit, and alpha chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled gamma chain did not bind to the antitoxin preparation. Competitive inhibition studies, with titrated anti-choleragen serum and radiolabeled toxin or components, indicated that the minimum concentration of toxin detectable was 7.0 x 10(-8) mumol/ml at a 90% inhibition level. The A subunit and alpha chain preparations inhibited the binding of the radiolabeled B subunit to antitoxin sites. Conversely, B subunit inhibited the binding of radiolabeled A subunit and alpha chain to antitoxin. The gamma chain did not show any reaction with antitoxin or cross-reaction with either whole toxin or its components. These results strongly suggest that the A subunit and the alpha chain contain antigenic determinant(s) that are common to the B subunit. The B subunit (beta chain) and the alpha chain of cholera toxin may therefore contain region(s) of chemical similarity.     

霍乱毒素对成年金黄地鼠视网膜节细胞存活的影响

近端切断成年金黄地鼠视神经,视网膜节细胞大量死亡,两周后达９０％。本研究应用荧光逆行示踪标记技术,观察玻璃体内注射霍乱毒素对视神经切断后视网膜节细胞存活的影响。结果表明：①正常视网膜节细胞平均密度为２００７±１１５／ｍｍ２;②切断视神经５、７、１４ｄ后,视网膜节细胞平均密度分别下降至：１１４７±２０９／ｍｍ２、８７３±１２３／ｍｍ２和２０１±７３／ｍｍ２;③给予Ｎａ２ＥＤＴＡ／ＮａＣＬ的对照组在上述各时间组视网膜节细胞平均密度与单纯视神经切断组结果相似;④给予霍乱毒素的实验组在５、７、１４ｄ视网膜节细胞的平均密度分别为１４３６±１５０／ｍｍ２、１１９２±１２９／ｍｍ２和４１３±９０／ｍｍ２,与视神经切断组和Ｎａ２ＥＤＴＡ／ＮａＣＬ对照组相比在各时间组上均存在显著性差异（Ｐ＜０．０５）,结果表明霍乱毒素有提高视神经切断后节细胞存活的作用     

覆盆子提取成分促进淋巴细胞增殖作用及与环核苷酸的关系

本文采用３Ｈ－ＴｄＲ掺入法，研究了覆盆子的四种提取组分在体外对脾脏淋巴细胞ＤＮＡ合成的影响，并从环核苷酸角度探讨其机理。结果表明覆盆子的四种提取组分：水提取液、醇提取液、粗多糖和正丁醇组分均有明显的促进淋巴细胞增殖作用。在没有丝裂原存在时，其增殖指数在３．０左右。在有微量丝裂原辅助下。其增殖指数亦为３．０左右（相对于微量丝裂原组）。在淋巴细胞激活的早期伴有ｃＡＭＰ水平的升高。     

Anti-Oxidative Effects of Theophylline on Human Neutrophils Involve Cyclic Nucleotides and Protein Kinase A

The effects were studied of the non-specific phosphodiesterase inhibitor, theophylline (37.5-300 M), on intracellular levels of cyclic adenosine monophosphate (cAMP) and superoxide generation following exposure of human neutrophils to four different stimuli of neutrophil membrane-associated oxidative metabolism, each of which utilizes a different transductional mechanism to activate NADPH-oxidase, in vitro. Exposure of neutrophils to FMLP (1 M), the calcium ionophore A23187 (1 M), and opsonized zymosan (OZ, 0.5 mg/ml) was accompanied by activation of superoxide production and increased concentrations of intracellular cAMP. Inclusion of theophylline resulted in augmentation of cAMP and inhibition of superoxide production by these stimuli. These negative effects of theophylline on neutrophil superoxide generation were mimicked by dibutyryl cAMP and 8-bromo-cGMP, while the inhibitory activity of all 3 agents was antagonized by the protein kinase A inhibitor KT 5720, but not by the G-kinase inhibitor KT 5823. Unlike FMLP, OZ and A23187, intracellular cAMP levels did not increase in cells activated with phorbol-12-myristate-13-acetate (PMA, 25 ng/ml), while oxidant production activated by this stimulus was insensitive to the inhibitory effects of theophylline. These observations suggest that the beneficial, anti-inflammatory interactions of theophylline with human nuetrophils are related to the phosphodiesterase inhibitory properties of this agent, and are selective for those pro-inflammatory stimuli which elevate cAMP, resulting in enhanced activity of protein kinase A and inhibition of the production of potentially harmful reactive oxidants by these cells.     

Vascular Leakage Through Intraendothelial Channels Induced by Cholera Toxin in the Skin of Guinea Pigs

Guinea pigs were injected intravenously with Evans blue solution ½, 1, 5 and 22 hours after intradermal injection of Vibrio cholerae 569B toxin. Then they were fixed by perfusion with diluted Karnovsky's aldehyde mixture 1½, 5, 10, 15 and 90 minutes later. In some cases, horseradish peroxidase was mixed in the dye solution, and chopped tissue slices were incubated histochemically. Dermal blood vessels from these samples were examined under the electron microscope. The fine structure of the endothelial junctions remained unchanged as compared with controls. However, extended rough surfaced endoplasmic reticulum was observed in the endothelial cells at 30 minutes after the toxin injection, when cutaneous bluing was not marked yet. At 22 hours after toxin injection, endothelial perikarya were filled with prominent Golgi complexes, multivesicular bodies and systems of caveolae and vesicles which coalesced, forming intraendothelial channels after diaphragms disappeared. Endothelial fenestrae were numerous in the attenuated portions.     

Immunological Interrelationships Between Cholera Toxin and the Heat-Labile and Heat-Stable Enterotoxins of Coliform Bacteria

Cholera toxin (CT) and the heat-labile (LT) toxin of Escherichia coli are known to share antigenic properties. The present study examined the immunological relationship of CT and the LT and heat-stable (ST) toxins of E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. The neutralizing capacity of equine CT antiserum and of antiserum raised in rabbits to the LT toxin of the three species of coliform bacteria was evaluated by determining their capacity to inhibit the action of purified CT and semipurified ultrafiltration preparations of the coliform LT and ST toxins in inducing water secretion as assayed by the in vivo marker perfusion technique in the rat jejunum. One milliliter of antiserum to CT and to E. coli and Klebsiella LT completely neutralized the secretory action of each of these three toxins; effective serial dilutions of CT antiserum extended to 1 to 4, whereas those of the antisera to LT were limited to 1 to 2 in most instances. One milliliter of antiserum to E. cloacae LT partially neutralized each of the three coliform LT toxins; serial dilutions were inactive. Antiserum to E. cloacae LT did not neutralize CT. Antiserum to CT and to each of the three coliform LT toxins also had a weak neutralizing effect on the ST toxins of E. coli and Klebsiella, but they did not affect E. cloacae ST. Adsorption of the antiserum to CT and to each of the three LT toxins by incubation with a heat-inactivated preparation of either the homologous or a heterologous LT toxin completely abolished the neutralizing capacity of the antisera towards both LT and ST. These observations indicate that the immunological interrelationship of CT and E. coli LT extends to the LT toxins of Klebsiella and E. cloacae and, further, that these immunological properties are shared to a lesser extent by the ST toxins of E. coli and Klebsiella.     

Efficacy of Intraduodenal,Oral and Parenteral Boosting in Inducing Intestinal Mucosal Immunity to Cholera Toxin in Rats

Considerable effort has been directed toward developing effective mucosal vaccines, especially those targeted to the intestine, and appropriate delivery systems. Numerous studies have demonstrated that direct immunization of the intestinal mucosa is the most efficient route for generating an intestinal IgA reponse. The present study examined the effect of three different routes of secondary immunization (boosting), i.e. intraduodenal, oral and parenteral (subcutaneous) on the intensity of the intestinal mucosal immune response in rats subjected to primary intraduodenal immunization with cholera holotoxin. Specific antibody titers and the relative numbers of antibody-secreting cells in the peripheral blood and antibody-containing cells in the intestinal lamina propria concur that vaccination of the intestinal mucosa directly or in combination with an oral boost yields a more vigorous mucosal immune response in comparison to a parenteral boost.     

Efficacy of Intraduodenal, Oral and Parenteral Boosting in Inducing Intestinal Mucosal Immunity to Cholera Toxin in Rats

Considerable effort has been directed toward developing effective mucosal vaccines, especially those targeted to the intestine, and appropriate delivery systems. Numerous studies have demonstrated that direct immunization of the intestinal mucosa is the most efficient route for generating an intestinal IgA reponse. The present study examined the effect of three different routes of secondary immunization (boosting), i.e. intraduodenal, oral and parenteral (subcutaneous) on the intensity of the intestinal mucosal immune response in rats subjected to primary intraduodenal immunization with cholera holotoxin. Specific antibody titers and the relative numbers of antibody-secreting cells in the peripheral blood and antibody-containing cells in the intestinal lamina propria concur that vaccination of the intestinal mucosa directly or in combination with an oral boost yields a more vigorous mucosal immune response in comparison to a parenteral boost.