Differential regulation of macrophage interleukin-1 (IL-1), IL-12, and CD80-CD86 by two bacterial toxins.
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The ability of innate immune cells to differentially respond to various bacterial components provides a mechanism by which the acquired immune response may be tailored to specific pathogens. The response of innate immune cells to bacterial components provides regulatory signals to cognate immune cells. These signals include secreted cytokines and costimulatory molecules, and to a large extent they determine the quantitative and qualitative nature of the immune response. In order to determine if innate immune cells can differentially respond to bacterial components, we compared the responses of macrophages to two bacterially derived molecules, cholera toxin (CT) and lipopolysaccharide (LPS). We found that CT and LPS differentially regulated the expression of interleukin-12 (IL-12) and CD80-CD86 but not that of IL-1beta. LPS and CT each induced IL-1beta expression in macrophages, while only LPS induced IL-12 and only CT induced CD80-CD86. These differences were markedly potentiated in gamma interferon (IFN-gamma)-treated macrophages, in which LPS potently induced IL-12 and CD80-CD86 expression. In contrast, IFN-gamma treatment had no effect on the expression of IL-1beta. These results define a molecular basis for the differential pathogenicities of bacterial toxins and are relevant to the design of vaccine adjuvants able to selectively induce desired types of immunity. (+info)
Analysis of receptor for Vibrio cholerae El tor hemolysin with a monoclonal antibody that recognizes glycophorin B of human erythrocyte membrane.
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El Tor hemolysin (ETH), a pore-forming toxin secreted by Vibrio cholerae O1 biotype El Tor and most Vibrio cholerae non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes. (+info)
Ganglioside GM(1a) on the cell surface is involved in the infection by human rotavirus KUN and MO strains.
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Rotavirus is the most common cause of severe gastroenteritis in infants and children worldwide. The cell attachment of most animal rotaviruses, which belong to the neuraminidase-sensitive strains, requires sialic acid residues on the host cell membranes. On the other hand, most human rotaviruses are classified as neuraminidase-insensitive strains. The involvement of gangliosides on the host cell surface in human rotavirus infection was investigated by immunostaining analysis of target cells, and by assaying the neutralization of infection by rotavirus and the blocking of target cellular receptors. In host cells (MA104 cells) pretreated with Arthrobacter ureafaciens neuraminidase, which were still infected by human rotaviruses (KUN and MO strains), GM(3) was hydrolyzed markedly by the neuraminidase, while GM(1a) was not hydrolyzed at all. Infection by the rotaviruses was strongly inhibited by exogenous ganglioside GM(1a), but not GA(1). Infection was also inhibited by pretreatment of the MA104 cells with cholera toxin B-subunit, which specifically blocked ganglioside GM(1a) on the plasma membrane. The treatment of MA104 cells with the endoglycoceramidase attenuated human rotavirus infection. From these findings, we concluded that GM(1a) on the plasma membrane of the host cells was involved in the infection by human rotavirus KUN and MO strains. (+info)
Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation. (+info)
Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.
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Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain T(h)1-based autoimmune diseases. We have established a model of aerosol sensitization leading to T(h)2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence T(h)2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4 production were decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens-in conjugated as in unconjugated form-had different effects on the T(h)2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB-and probably also other antigen-delivery systems-strongly depend on the nature of the coupled antigen-allergen. (+info)
An improved method for detecting faecal Vibrio cholerae by PCR of the toxin A gene.
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A method for removing inhibitor(s) of the PCR assay for the direct detection of cholera toxin A gene (ctxA) in human faeces is described. Inhibitors of the PCR were removed by centrifugation and the activity of the remaining inhibitors by dilution. Based on these data, a protocol was developed for pre-treatment of stool specimens for PCR assay, and a simple and rapid protocol was constructed for the diagnostic detection of the ctxA genes in stool specimens in combination with single band detection on gel electrophoresis, dot-blot hybridisation and enrichment culture. This protocol was applied to clinical specimens and showed that the PCR method gave 100% agreement with established culture methods for the detection of cholera toxin-producing Vibrio cholerae O1. This protocol was considered to be useful because of its simplicity and the rapidity of diagnosis. (+info)
Vasopressin and amastatin induce V(1)-receptor-mediated suppression of excitatory transmission in the rat parabrachial nucleus.
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We examined actions of arginine vasopressin (AVP) and amastatin (an inhibitor of the aminopeptidase that cleaves AVP) on synaptic currents in slices of rat parabrachial nucleus using the nystatin-perforated patch recording technique. AVP reversibly decreased the amplitude of the evoked, glutamate-mediated, excitatory postsynaptic current (EPSC) with an increase in paired-pulse ratio. No apparent changes in postsynaptic membrane properties were revealed by ramp protocols, and the inward current induced by a brief application of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid was unchanged after AVP. The reduction induced by 1 microM AVP could be blocked by a V(1) AVP receptor antagonist, [d(CH(2))(5)(1)-O-Me-Tyr(2)-Arg(8)]-vasopressin (Manning compound, 10 microM). Bath application of an aminopeptidase inhibitor, amastatin (10 microM), reduced the evoked EPSC, and AVP induced further synaptic depression in the presence of amastatin. Amastatin's effects also could be antagonized by the Manning compound. Corticotropin-releasing hormone slightly increased the EPSC at 1 microM, and coapplication with AVP attenuated the AVP response. Pretreatment of slices with 1 microg/ml cholera toxin or 0.5 microg/ml pertussis toxin for 20 h did not significantly affect AVP's synaptic action. The results suggest that AVP has suppressant effects on glutamatergic transmission by acting at V(1) AVP receptors, possibly through a presynaptic mechanism involving a pertussis-toxin- and cholera-toxin-resistant pathway. (+info)
Enhanced phenylephrine-induced rhythmic activity in the atherosclerotic mouse aorta via an increase in opening of KCa channels: relation to Kv channels and nitric oxide.
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1. Mice lacking the apolipoprotein E and low density lipoprotein receptor genes (E degrees xLDLR degrees ) develop atherosclerosis. The aim of this study was to investigate changes in endothelium-dependent vasodilation and vasomotion in thoracic aortic rings of E degrees xLDLR degrees mice. 2. K+-induced contractions of the aorta from E degrees xLDLR degrees mice were stronger than those from control mice. The sensitivity of E degrees xLDLR degrees aorta to phenylephrine (PE) was decreased but the maximal contractions were increased. Acetylcholine-induced, but not sodium nitroprusside-induced, relaxations of E degrees xLDLR degrees aorta was decreased. 3. PE induced rhythmic activity in both E degrees xLDLR degrees and control aorta but the amplitude was larger in E degrees xLDLR degrees than in control mice. PE-induced rhythmic activity in both E degrees xLDLR degrees and control aorta was augmented by increase in extracellular Ca2+-concentration, but was abolished by removal of the endothelium, the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester, the guanylate cyclase inhibitor LY-83583, high K+ solution and ryanodine. 4. 4-Aminopyridine, a voltage-dependent potassium (KV) channel blocker, increased basal tension and induced rhythmic activity in E degrees xLDLR degrees aorta but not in control aorta. 5. The Ca2+-activated potassium (KCa) channel blockers tetraethylammonium and charybdotoxin abolished PE-induced rhythmic activity in E degrees xLDLR degrees aorta. 6. In conclusion, opening of Kv channels in E degrees xLDLR degrees mice aorta is reduced and it is susceptible to be depolarized resulting in Ca2+ entry. The vascular smooth muscle is then dependent on compensatory mechanisms to limit Ca2+-entry. Such mechanisms may be decreased sensitivity to vasoconstrictors, or increased opening of KCa channels by NO via a cyclic GMP-dependent mechanism. (+info)