Toxin Pretreatment Research Articles

The fungal T-2 toxin alters the activation of primary macrophages induced by TLR-agonists resulting in a decrease of the inflammatory response in the pig

T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. The objective of this work was to evaluate the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM). Cytotoxic effects of T-2 toxin on PAM were first evaluated. An IC50 of 19.47 ± 0.9753 nM was determined for the cytotoxicity of T-2 toxin. A working concentration of 3 nM of T-2 toxin was chosen to test the effect of T-2 toxin on TLR activation; this dose was not cytotoxic and did not induce apoptosis as demonstrated by Annexin/PI staining. A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1 beta, TNF-alpha, nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. By contrast, the activation of TLR7 by ssRNA was not modulated by T-2 toxin pre-treatment. In conclusion, our results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases.

Antibody protection against botulinum neurotoxin intoxication in mice.

Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.

The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

'Cross talk' between opioid peptide and adrenergic receptor signaling in isolated rat heart.

Cardiac myocyte sarcolemma contains both catecholamine and opioid peptide receptors (OPRs). Opioid peptides are coreleased with catecholamines from nerve terminals in the heart. We investigated whether OPR stimulation influences the effects of beta-adrenergic receptor (beta-AR) stimulation in the isolated, isovolumic rat heart and whether the mechanism of such an interaction involves both beta-AR subtypes or an alteration in beta-AR-mediated increase in cAMP. Norepinephrine (NE, 10(-7) mol/L) increased peak left ventricular systolic pressure (LVSP) and cAMP more than twofold compared with controls. The delta-OPR agonist leucine-enkephalin (LE, 10(-8) mol/L) markedly inhibited the beta1-AR-induced positive inotropic effect and increase in cAMP but alone had no effect on basal LVSP or basal cAMP levels. The OPR antagonist naloxone 10(-8) mol/L added to LE+NE perfusate reversed the LE-induced decrease in cAMP and LVSP even though naloxone alone had no effect on LVSP and cAMP levels. LE could not counteract the twofold increase in LVSP produced by the nondegradable cAMP analog CPT-cAMP 2.3x10(-5) mol/L or a high concentration of forskolin (10(-7) mol/L) but did reverse the 173+/-11.8% and 135+/-13.6% increases in LVSP stimulated by 10(-8) and 0.5x10(-8) mol/L forskolin, respectively. LE inhibited cAMP production at all concentrations of forskolin (10(-7), 10(-8), and 0.5x10(-8) mol/L). Pertussis toxin (PTX) pretreatment abolished LE effects on beta1-AR stimulation. Zinterol 10(-5) and 10(-6) mol/L, a specific beta2-AR agonist that elicits a cAMP-independent inotropic effect in rat heart, caused 225+/-14% and 182+/-5% increases in LVSP that could not be reversed by addition of LE. Potent, inhibitory "cross talk" between delta-OPR and beta1-AR signaling pathways occurs via a PTX-sensitive G(i/o) protein involved in adenylyl cyclase inhibition in rat heart.

Effect of cAMP on GnRH stimulated LH secretion from individual pituitary gonadotropes

Enhanced responsiveness of the pituitary gland to GnRH is a fundamental step required for secretion of the proestrous LH surge, and is achieved primarily by the actions of ovarian steroid hormones on the gonadotropes. The mechanisms involved are still unclear but the cAMP second messenger pathway can mediate some of these activities. The present study was undertaken to determine the effects of in vitro cholera toxin (CTX; increases cAMP) pretreatment of pituitary cells from proestrous and diestrous 1 rats relative to their LH secretory response to GnRH using a reverse hemolytic plaque assay. The number of gonadotropes that secrete LH in the group treated with CTX increased at low doses of GnRH and also in the absence of the peptide, but decreased at high doses, showing a dual effect: stimulation of some gonadotropes and inhibition of others. The inhibition was achieved within 3 h of pretreatment in proestrous cells but it was not seen until 20 h in diestrous 1 cells. This suggests the existence of at least two subpopulations of gonadotropes, one of which is stimulated by cAMP and another which is inhibited.

Regulation of PI hydrolysis and cAMP formation by muscarinic M3 receptor in guinea pig gallbladder.

Carbachol (10(-8)-10(-3) M) produced two distinct biochemical responses in the guinea pig gallbladder smooth muscle: simulation of phosphoinositide (PI) hydrolysis and inhibition of forskolin-mediated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The mean effective dose (ED50) concentration (1.6 x 10(-5) M) of carbachol-mediated stimulation of PI hydrolysis was 145 times greater than the ED50 concentration (1.1 x 10(-7) M) of carbachol mediated inhibition of cAMP formation. The inhibitory effect of carbachol on cAMP formation was antagonized by the pretreatment of pertussis toxin. To determine whether these two biochemical responses were mediated by the same or different subtypes of muscarinic receptors, the relative potencies of muscarinic receptor antagonists were calculated by Schild analysis. The M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) exhibited inhibitory constant (Ki) values at 0.3 and 1.2 nM in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. The corresponding Ki values for pirenzepine, a muscarinic M1 antagonist, were 11 and 130 nM. The corresponding Ki values for AF-DX 116, a muscarinic M2 antagonist, were 34 and 450 nM. Thus 4-DAMP was 37x and 108x more potent than pirenzepine in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. In addition, compared with AF-DX 116, 4-DAMP was 113x and 375x more potent in reducing stimulation of PI hydrolysis and inhibition of cAMP formation. Cholecystokinin (CCK) octapeptide (10(-10)-(10-6) M) caused a significant increase of PI hydrolysis but had no inhibitory effects on cAMP formation evoked by forskolin (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Pertussis toxin stimulation of catecholamine release from adrenal medullary chromaffin cells: Mechanism may be by direct activation of L-type and G-type calcium channels

We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca2+]o-dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca(2+)-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca(2+)-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K+]o, nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba2+]o. All these data are consistent with the concept that PTX may act on Ca2+ channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca2+ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca2+ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca2+ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca2+ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca(2+)-channel conductance (6 nS/cell at 2.6 mM [Ca2+]o). PTX evoked the activation of a new class of Ca(2+)-selective channels (5 pS in 25 mM [Ca2+]pipet), which are rather insensitive to membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells

R. V. Considine, J. K. Bielicki, L. L. Simpson and J. R. Sherwin. Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells. Toxicon 28, 13–19, 1990.—Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma × glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10 −8 M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10 −10 M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10 −8 or 10 −10 M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10 −7−10 −10 M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.

Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin.

The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.

Control of aldosterone production by angiotensin II is mediated by two guanine nucleotide regulatory proteins.

The involvement of guanine nucleotide regulatory proteins in the steroidogenic response of the adrenal glomerulosa to angiotensin II (AII) was investigated by analyzing the effects of Bordetella pertussis toxin (PT) on several aspects of AII action. These included receptor binding, stimulation of aldosterone production and GTPase activity, inhibition of cAMP production, and attenuation of the aldosterone response at high angiotensin concentrations. Pretreatment of glomerulosa cells with PT abolished the inhibitory effects of both AII and somatostatin (SRIF) on ACTH-stimulated cAMP production. Under the same incubation conditions, the stimulation of aldosterone secretion by submaximal and maximal steroidogenic concentrations of AII was completely unaffected by the toxin. However, the attenuation of steroid responses seen with supramaximal concentrations of AII was abolished. In addition, the ability of SRIF to inhibit AII-stimulated steroid production was markedly reduced by PT treatment. The binding of [125I]AII to high affinity sites in intact cells and particulate fractions, and modulation of the binding by guanine nucleotides, were unaffected by toxin pretreatment, even under conditions where a 40-41K protein was completely ADP ribosylated. In contrast, the toxin substantially diminished the binding of [125I]Tyr0-SRIF to SRIF receptors in glomerulosa cells (by 50% after 5 h and by 90% after 20 h). These results indicate that Ni or a similar protein probably mediates the inhibition of cAMP formation by AII and the attenuation of the steroid response by high concentrations of AII as well as the inhibitory actions of SRIF in the adrenal glomerulosa cell. Furthermore, the lack of effect of PT on AII binding and stimulation of GTPase activity suggests the existence of an additional pertussis-insensitive guanine nucleotide-regulatory protein that is activated by lower concentrations of AII and mediates the stimulation of aldosterone production.

Dexamethasone decreases lethality of rats in acute poisoning with T-2 toxin.

T-2 toxin, a major trichothecene mycotoxin, was administered intravenously to rats. At a dose of 0.75 mg/kg two thirds of the animals died. In animals that received dexamethasone (1.6 mg/kg IV) either 30 min before or 1 h after the toxin, there was a more than fourfold reduction in lethality rate. Dexamethasone injected 3 h after the toxin was less effective. At a lethal dose of T-2 toxin (1 mg/kg IV) pretreatment with dexamethasone only delayed death, whereas lethality rate was barely affected (9/10 vs 10/10 in controls). Dexamethasone markedly reduced the incidence of lung edema and diarrhea. The incidence of hemorrhages, however, was not reduced by dexamethasone. Gastrointestinal bleeding was even more frequent in treated rats than in controls.

Studies on cholera-toxin-induced desensitization of adenylate cyclase in the mouse intestinal mucosa.

Repeated peroral pretreatment ('immunization') with cholera toxin (CT) in mice induces protection against CT as well as against prostaglandin E1 (PGE1), as evaluated by fluid accumulation in intestinal loops. The fluid response to CT is depressed for more than 1 month, while the response to PGE1 is inhibited for 4-7 days after the pretreatment. Immunofluorescence microscopy revealed that neither the binding nor the penetration of CT into the intestinal epithelial cells is affected by the toxin pretreatment. Furthermore, the CT-induced release of mucus in goblet cells is not influenced by the toxin pretreatment. In contrast, the enzyme adenylate cyclase (AC), which mediates the actions of CT and PGE1, shows a long-lasting desensitization to CT, as estimated in mucosal membrane preparations. Chlorpromazine and cycloheximide revert the desensitization to CT as well as to PGE1. The present data suggest that intestinal resistance to CT in the mouse is due to desensitization of the reaction between CT and AC and requires stimulation of AC, as well as an active protein synthesis.

Rat pancreas adenylate cyclase. III. Its role in pancreatic secretion assessed by means of choleara toxin.

1. The role of adenylate cyclase in rat pancreas is further investigated by means of cholera toxin, which is known to activate the enzyme in several tissues. 2. Cholera toxin activates rat pancreatic adenylate cyclase in vitro upon preincubation of tissue slices with the toxin for more than 30 min, but not when it is merely present during the enzyme assay. The maximal effect is reached after 90 min pre-incubation. The half-maximally activating concentration is 3.5 mu-g/ml upon pre-incubation for 90 min. 3. After pre-treatment of pancreatic tissue slices with 2 mu-g/ml cholera toxin, further stimulation of adenylate cyclase activity can be obtained by adding pancreozymin-C-octapeptide, secretin, or fluoride to the assay medium, but the final activity with maximally effective concentrations of the hormones is not higher, and with fluoride even less, than that without the toxin pre-treatment. 4. The in vivo effects of the two hormones and of cholera toxin have been studied after cannulation of the pancreas. Pancreozymin-C-octapeptide (intravenously) markedly stimulates both flow rate and rate of protein secretion. Synthetic secretin (intravenously), in addition to its expected effect on flow rate, slightly stimulates protein secretion, which is not due to a wash-out effect. Cholera toxin, topically applied to the cannulated rat pancreas, causes a steady increase of the flow rate after a delay of 20--30 min. The rate of protein secretion is not affected or slightly decreased by the toxin. Pancreozymin-C-octapeptide, given intravenously 1 h after cholera toxin application, causes the same increase in flow rate and rate of protein secretion as would be expected without cholera toxin treatment. 5. The sodium and potassium levels in the pancreatic fluid after administration of secretin or cholera toxin do not change, while the chloride level decreases in both cases. 6. These observations indicate that the rat pancreas adenylate cyclase activity is a rate-limiting factor in the regulation of water and electrolyte secretion. A possible auxiliary role in the regulation of enzyme secretion cannot yet be excluded.