Neutralization of a bothropic PLA2-like protein by caftaric acid, a novel potent inhibitor of ophidian myotoxicity.

Envenoming by snakebite is an important global health issue that has received little attention, leading the World Health Organization to naming it as neglected tropical disease. Several snakebites present serious local symptoms manifested on victims that may not be efficiently neutralized by serum therapy. Phospholipase A2-like (PLA2-like) toxins are present in Viperidae venoms and are responsible for local myotoxic activity. Herein, we investigated the association between BthTX-I toxin and caftaric acid (CFT), a molecule present in plants. CFT neutralized neuromuscular blocking and muscle-damaging activities promoted by BthTX-I. Calorimetric and light-scattering assays demonstrated that CFT inhibitor interacted with dimeric BthTX-I. Bioinformatics simulations indicated that CFT inhibitor binds to the toxin's hydrophobic channel (HCh). According to the current myotoxic mechanism, three different regions of PLA2-like toxins have specific tasks: protein allosteric activation (HCh), membrane dockage (MDoS), and membrane rupture (MDiS). We propose CFT inhibitor interferes with the allosteric activation, which is related to the conformation change leading to the exposure/alignment of MDoS/MDiS region. This is the first report of a PLA2-like toxin fully inhibited by a compound that interacts only with its HCh region. Thus, CFT is a novel candidate to complement serum therapy and improve the treatment of snakebite.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Electrophysiological Characterization of the Antarease Metalloprotease from Tityus serrulatus Venom.

Scorpions are among the oldest venomous living organisms and the family Buthidae is the largest and most medically relevant one. Scorpion venoms include many toxic peptides, but recently, a metalloprotease from Tityus serrulatus called antarease was reported to be capable of cleaving VAMP2, a protein involved in the neuroparalytic syndromes of tetanus and botulism. We have produced antarease and an inactive metalloprotease mutant in a recombinant form and analyzed their enzymatic activity on recombinant VAMP2 in vitro and on mammalian and insect neuromuscular junction. The purified recombinant antarease paralyzed the neuromuscular junctions of mice and of Drosophila melanogaster whilst the mutant was inactive. We were unable to demonstrate any cleavage of VAMP2 under conditions which leads to VAMP proteolysis by botulinum neurotoxin type B. Antarease caused a reduced release probability, mainly due to defects upstream of the synaptic vesicles fusion process. Paired pulse experiments indicate that antarease might proteolytically inactivate a voltage-gated calcium channel.

Effects of conopeptide-containing venom from seven species of Conidae gastropoda on the chick biventer-cervicis nerve-muscle assessed using the ConoServer database.

BACKGROUND: Conotoxins in the venom of marine gastropods (genus Conus, family Conidae) have been incriminated in fatal human stingings. Conotoxins are peptides (conopeptides) which target specific classes of ion channels and block receptors involved in neuromuscular transmission. Some conopeptides also block receptors involved in neuropathic pain and one such peptide with an analgesic potency greater than that of morphine is marketed for clinical use. OBJECTIVES: To determine the effects of venom from seven species of Conidae, Conus arenatus, Conus coronatus, Conus ebraeus, Conus lividus, Conus miles, Conus rattus, and Conus textile, collected in the inter-tidal zone of the Indian Ocean, East Africa, on the chick biventer-cervicis nervemuscle preparation and to assess the effects using data on conopeptide content in venom of the species examined reported in the literature and the ConoServer database. RESULTS: Only venom extracts from C. arenatus and C. textile, blocked twitch responses and produced depolarization and contracture of slow fibers of the stimulated chick nerve-muscle preparation. This is the first study showing that venom from C. arenatus is a potent inhibitor of neuromuscular transmission. However, in the case of C. textile, a species associated with fatal human stingings, the inhibitor activity was ~ 3-fold greater. These results are consistent with the occurrence of specific α-conopeptides, namely α-4/6-CtxTxID in C. textile and α-CtxArIB in C. arenatus targeting acetylcholine receptors at the neuromuscular junction. Information extractable from the ConoServer database was of limited value for evaluation of our findings since all the species examined contain numerous conopeptides, the majority of which have not been characterized pharmacologically or for which even the gene superfamily is unknown. Venom from C. textile, C. arenatus, C. coronatus, C. ebraeus, and C. rattus produced an initial facilitation of the twitch response similar to that produced by neostigmine. Venom from C. lividus and C. miles had no effect on twitch responses and did not depolarize slow fibers even at high concentrations. CONCLUSIONS: Using the chick biventer-cervicis nerve-muscle preparation, which contains both twitch and slow muscle fibers, a neuromuscular blocking and muscle depolarizing action could be demonstrated in venom extracts from C. textile, a Conus species associated with fatal human stingings, and C. arenatus. The results are consistent with the known presence of specific α-conopeptides in these species targeting nAChRs. Venom from C. coronatus, C. ebraeus, C. rattus, C. lividus, and C. miles, although purported to contained numerous conopeptides belonging to a variety of pharmacological classes, were either inactive on the preparation or caused only a minor potentiation of the twitch response. Although the ConoServer database provides valuable global data on conopeptide structure, occurrence and properties, it lacks specific information on receptor targets and affinities.

Neuromuscular blocking activity of pinnatoxins E, F and G.

Pinnatoxins are produced by dinoflagellates and belong to the cyclic imine family of toxins. They are fast-acting and highly toxic when administered in vivo in rodent bioassays, causing death by respiratory depression within minutes. Studies have revealed that some cyclic imine toxins cause their toxicity by antagonizing both muscle type and heteromeric and homomeric neuronal nicotinic acetylcholine receptors (nAChRs). Pinnatoxins E, F and G all display potent toxicity in in vivo bioassays, with symptoms of toxicity similar to other cyclic imine toxins. However, very little work has been done on the mechanism of action of these pinnatoxin isomers. Thus the aim of the current study was to investigate the rank order of potency and mechanism of action of pinnatoxins E, F and G. The effects of pinnatoxin E, F and G on in vitro rat hemidiaphragm preparations were investigated using twitch tension and electrophysiological techniques to determine the effects of these toxins on cholinergic transmission at the neuromuscular junction. Pinnatoxins E, F and G all produced concentration-dependent reductions in the nerve evoked twitch response of the rat hemidiaphragm, with IC50 values ranging from 11 to 53 nM and a rank order of potency of F > G > E. Only complete washout of pinnatoxin E was evident, with pinnatoxins F and G displaying slow and incomplete washout profiles. Pinnatoxins F and G also reduced the amplitudes of spontaneous miniature endplate potentials and evoked endplate potentials at the neuromuscular junction, without affecting miniature endplate potential frequency or the resting membrane potential of the muscle fibres. These results show that pinnatoxins E, F and G are all potent neuromuscular blocking agents and cause toxicity by acting as antagonists at muscle type nicotinic acetylcholine receptors.

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom.

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 µl/ml and 118.9 ± 9.3% with 40 µl/ml in PND, and 120.7 ± 7.7% with 40 µl/ml and 114.5 ± 4.4% with 50 µl/ml in BC after 120 min; n = 4-6, mean ± SEM). In PND, the ethanol alone (40 µl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 µl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 µg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 µl/ml) for 30 min before incubation with venom (50 µg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 µg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

A single injection of botulinum toxin decreases the margin of safety of neurotransmission at local and distant sites.

BACKGROUND: We tested the hypothesis that a single injection of botulinum toxin not only has local, but also distant effects on muscle function, biochemistry, and pharmacodynamics of atracurium. METHODS: Botulinum toxin (2.5 U) was injected into the tibialis muscle of anesthetized rats (n = 26). The contralateral side with no injection served to study distant effects. Control animals (n = 25) received a saline injection. Neuromuscular function, pharmacology, and expression of acetylcholine receptors (nAChRs) were evaluated in the tibialis at 0, 4, and 16 days after injection and in comparison with saline- injected controls. RESULTS: On day 4, botulinum toxin caused complete paralysis of the tibialis, while its contralateral side showed a decrease in absolute twitch tension (1.8 N [1.6; 1.9] vs 3.0 N [2.8; 3.1], Newton, P < 0.05). On day 16, muscle weakness was only present on the toxin-injected side where absolute twitch tension was decreased (0.6 N [0.6, 0.7] vs 3.4 N [3.1, 3.7], P < 0.05). Tibialis mass was decreased on the toxin-injected side at day 4 (1.46 mg/g [1.43, 1.48] vs 1.74 mg/g [1.72; 1.75], P < 0.05) and on day 16 (0.78 mg/g [0.76, 0.79] vs 1.73 mg/g [1.69; 1.77], P < 0.05). Effects distant from the site of injection were seen on day 16, when muscle atrophy was also present in the adjacent gastrocnemius and soleus muscles. Normalized to tibialis mass, specific twitch tension (tension/g muscle) was reduced on the contralateral side at day 4 and on the toxin-injected side at day 16 in relation to saline controls. At day 16, an increased sensitivity to atracurium was seen on the toxin-injected side, evidenced as a decreased ED(50) (0.23 mg/kg [0.13, 0.33] vs 0.72 mg/kg [0.63, 0.82], P < 0.05) and a lower infusion rate (38 µL/kg/min [32, 43] vs135 µL/kg/min [126, 144], P < 0.05), together with a reduced plasma concentration requirement of atracurium (0.5 µg/mL [0.4, 0.7] vs 4.5 µg/mL [3.8, 5.2], P < 0.05) to achieve a steady state 50% reduction in baseline (absolute) twitch tension. ED(50) of atracurium was also decreased on the contralateral side at day 16 in relation to saline controls. The nAChRs in the tibialis were increased on the toxin-injected side to 123 fmol/mg [115, 131] vs 28 fmol/mg [25, 29] (P < 0.05) in time-matched saline-injected controls at day 4 and to 378 [341, 413] vs 27 fmol/mg [25, 29] (P < 0.05) at day 16. CONCLUSIONS: Botulinum toxin has local and distant effects on muscle. The decrease in specific twitch tension indicates that the muscle atrophy alone cannot explain the functional changes; neuromuscular transmission is also impaired. An increased sensitivity to atracurium on the toxin-injected side, despite up-regulation of nAChRs, seems unique to botulinum toxin.

Marine algal pinnatoxins E and F cause neuromuscular block in an in vitro hemidiaphragm preparation.

Members of the cyclic imine group of toxins, gymnodimine and spirolides, have been found to be potent antagonists of both muscle type and neuronal nicotinic acetylcholine receptors. These toxins exhibit fast acting toxicity in vivo, causing death within minutes by respiratory depression. This toxicity is shared by the novel cyclic imine pinnatoxins E and F, produced by marine dinoflagellates and recently isolated from New Zealand shellfish. However, there is currently very little data available regarding the mechanism of action for any of the pinnatoxins, and no data at all on the novel pinnatoxins E and F. The aim of the current study was to investigate potential antagonism of nicotinic acetylcholine receptors by pinnatoxins E and F using two in vitro tissue preparations. Compound muscle action potentials elicited by stimulation of the phrenic nerve were recorded from the hemidiaphragm in order to test effects on muscle type heteromeric nicotinic receptors, while effects on α7 homomeric neuronal nicotinic receptors were investigated by recording gamma oscillations in response to tetanic stimulation of the CA1 region of the hippocampus. Both a crude extract containing a mixture of pinnatoxins E and F, as well as pure pinnatoxin F, had no effect on gamma oscillation spectral density or spike count at any concentrations. Conversely, at these same concentrations, both crude and pure pinnatoxin caused an almost complete abolition of nerve-evoked hemidiaphragm action potential responses, without any effect on electrically-evoked (direct) responses. This neuromuscular block could not be reversed by neostigmine. These results show that pinnatoxins E and F block neuromuscular transmission and suggest that observed in vivo muscle paralysis by pinnatoxin is due to selective antagonism of muscle type nicotinic acetylcholine receptors.

Cysteine reversal of the novel neuromuscular blocking drug CW002 in dogs: pharmacodynamics, acute cardiovascular effects, and preliminary toxicology.

BACKGROUND: CW002 is a neuromuscular blocking drug that is inactivated by endogenous L-cysteine. This study determined the exogenous L-cysteine dose-response relationship for CW002 reversal along with acute cardiovascular effects and organ toxicity in dogs. METHODS: Six dogs were each studied four times during isoflurane-nitrous oxide anesthesia and recording of muscle twitch, arterial pressure, and heart rate. CW002 (0.08 mg/kg or 9 x ED95) was injected, and the time to spontaneous muscle recovery was determined. CW002 was then administered again followed 1 min later by 10, 20, 50, or 100 mg/kg L-cysteine (1 dose/experiment). After twitch recovery, CW002 was given a third time to determine whether residual L-cysteine influenced duration. Preliminary toxicology was performed in an additional group of dogs that received CW002 followed by vehicle (n = 8) or 200 mg/kg L-cysteine (n = 8). Animals were awakened and observed for 2 or 14 days before sacrificing and anatomic, biochemical, and histopathologic analyses. RESULTS: L-cysteine at all doses accelerated recovery from CW002, with both 50 and 100 mg/kg decreasing median duration from more than 70 min to less than 5 min. After reversal, duration of a subsequent CW002 dose was also decreased in a dose-dependent manner. Over the studied dose range, L-cysteine had less than 10% effect on blood pressure and heart rate. Animals receiving a single 200-mg/kg dose of L-cysteine showed no clinical, anatomic, biochemical, or histologic evidence of organ toxicity. CONCLUSION: The optimal L-cysteine dose for rapidly reversing the neuromuscular blockade produced by a large dose of CW002 in dogs is approximately 50 mg/kg, which has no concomitant hemodynamic effect. A dose of 200 mg/kg had no evident organ toxicity.

Pharmacodynamics and cardiopulmonary side effects of CW002, a cysteine-reversible neuromuscular blocking drug in dogs.

BACKGROUND: CW002 is a novel neuromuscular blocking drug with a duration dependent on the rate of cysteine adduction to the molecule. The current study characterized the pharmacodynamics and cardiopulmonary side effects of CW002 in dogs. METHODS: In eight beagles, the dose required to produce 95% neuromuscular blockade (ED95) for CW002 was first determined and cysteine reversibility was confirmed. Five to 7 days later, incrementally larger doses were injected starting with 6.25 x ED95 and doubling the dose every 15 min. Before and after injection, blood was obtained for histamine analysis. Systemic and pulmonary arterial pressures, cardiac output, and left ventricular pressure and volume were recorded along with inspiratory pressure and pulmonary compliance. Ventricular contractility and lusitropy were indexed from pressure and volume data. RESULTS: The ED95 for CW002 from pooled data was 0.009 mg/kg. At 3 x ED95, onset time was 2.6 +/- 0.9 min and duration was 47 +/- 9 min. The duration was shortened to 3.7 +/- 0.6 min by 50 mg/kg L-cysteine injected 1 min after CW002. At 25 x ED95, CW002 reduced mean arterial pressure with concomitant decreases in systemic vascular resistance, mean pulmonary artery pressure, cardiac output, contractility, and lusitropy, beginning at 50 x ED95. However, even at a dose of 100 x ED95, the average change in any variable was less than 20%. There were no changes in pulmonary vascular resistance or ventilation mechanics at any dose, and histamine release occurred in only two of eight animals. CONCLUSIONS: CW002 is a potent neuromuscular blocking drug that at doses up to 100 x ED95 produces modest hemodynamic effects that are not associated with bronchoconstriction or consistent histamine release.

Telithromycin blocks neuromuscular transmission and inhibits nAChR currents in vitro.

Telithromycin, a ketolide antibiotic, is reported to exacerbate myasthenia gravis, potentially leading to respiratory failure and death. However, telithromycin is not associated with neuromuscular effects in animal toxicity studies. The objective of this study was to examine the effect of telithromycin on the neuromuscular junction in the isolated rat phrenic nerve-diaphragm preparation and to investigate its postsynaptic effects on the muscle-like nicotinic acetylcholine (ACh) receptors expressed on human TE671 cells. Telithromycin decreased the twitch contraction force of the rat diaphragm muscle in response to phrenic nerve stimulation in a concentration-dependent manner with an IC(50) of 22.3 microM and a maximal inhibition of approximately 70%. The trans-membrane current from the ACh receptors expressed in the TE671 neuromedulloblastoma cells was recorded in the whole-cell patch-clamp configuration. When applied to the TE671 cells, telithromycin caused a dose-dependent inhibition of the nicotinic ACh current with an IC(50) of 3.5 microM and maximal inhibition of nearly 100%. These results indicate that telithromycin inhibits postsynaptic nicotinic ACh receptors in vitro and partially blocks neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. Based on these findings, we propose that exacerbation of myasthenia gravis reported in some patients taking telithromycin results in part from postsynaptic neuromuscular transmission block.

Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model of cognitive deficit like condition of Alzheimer's disease.

Recently, numerous medicinal plants possessing profound central nervous system effects and antioxidant activity have received much attention as food supplement to improve cognitive function against cognitive deficit condition including in Alzheimer's disease condition. Based on this information, the effect of piperine, a main active alkaloid in fruit of Piper nigrum, on memory performance and neurodegeneration in animal model of Alzheimer's disease have been investigated. Adult male Wistar rats (180-220 g) were orally given piperine at various doses ranging from 5, 10 and 20mg/kg BW at a period of 2 weeks before and 1 week after the intracerebroventricular administration of ethylcholine aziridinium ion (AF64A) bilaterally. The results showed that piperine at all dosage range used in this study significantly improved memory impairment and neurodegeneration in hippocampus. The possible underlying mechanisms might be partly associated with the decrease lipid peroxidation and acetylcholinesterase enzyme. Moreover, piperine also demonstrated the neurotrophic effect in hippocampus. However, further researches about the precise underlying mechanism are still required.

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins.

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD(50) test (ip LD(50)). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay. This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD(50), an effect that cannot be detected using the mouse ip LD(50) assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations.

Effects of estrogen on AF64A-induced apoptosis in NG108-15 cells.

In this study, we show that pretreatment with physiological concentrations (1-100 nM) of 17beta-estradiol decreased apoptosis induced by ethylcholine aziridinium (AF64A), a choline toxin, in the cholinergic neuronal cell line NG108-15. These protective effects were observed after short-term (30 min) pretreatment, and were blocked by treatment with an estrogen receptor antagonist and inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK). The protective effects were, however, not reversed by a protein synthesis inhibitor. Furthermore, we examined the effects of 17beta-estradiol on choline uptake in NG108-15 cells. Although choline uptake was inhibited by a selective inhibitor of choline uptake, hemicholinium-3, it was not altered by treatment with 17beta-estradiol. These results indicated that the protective effect of 17beta-estradiol on AF64A-induced apoptosis could be nongenomic, and that this effect may be due to the activation of PI3K/Akt and/or MEK/extracellular signal-regulated kinase (ERK) pathways.

Previous safe administrations of neuromuscular blocking agents do not exonerate from the risk of anaphylactic shock.

We describe a grade IV anaphylactic shock to atracurium. A 34-year-old woman was scheduled for her ninth abdominal surgery. Within the last 10 months, for previous abdominal procedures she had received atracurium, cisatracurium or suxamethonium. No adverse event was reported. In the present case, a cardiac arrest occurred within 1 min after atracurium injection. Anaphylaxis was immediately evoked and treatment started. The diagnosis of anaphylaxis to atracurium was confirmed by the allergological assessment. This case report highlights the fact that patients without any previous adverse events to neuromuscular blocking agents are never exempt from risk of anaphylactic shock, even with a designated low-risk agent of sensitization.

Differential expression of inflammatory and fibrogenic genes and their regulation by NF-kappaB inhibition in a mouse model of chronic colitis.

Fibrosis is a major complication of chronic inflammation, as seen in Crohn's disease and ulcerative colitis, two forms of inflammatory bowel diseases. To elucidate inflammatory signals that regulate fibrosis, we investigated gene expression changes underlying chronic inflammation and fibrosis in trinitrobenzene sulfonic acid-induced murine colitis. Six weekly 2,4,6-trinitrobenzene sulfonic acid enemas were given to establish colitis and temporal gene expression patterns were obtained at 6-, 8-, 10-, and 12-wk time points. The 6-wk point, TNBS-w6, was the active, chronic inflammatory stage of the model marked by macrophage, neutrophil, and CD3(+) and CD4(+) T cell infiltrates in the colon, consistent with the idea that this model is T cell immune response driven. Proinflammatory genes Cxcl1, Ccl2, Il1b, Lcn2, Pla2g2a, Saa3, S100a9, Nos2, Reg2, and Reg3g, and profibrogenic extracellular matrix genes Col1a1, Col1a2, Col3a1, and Lum (lumican), encoding a collagen-associated proteoglycan, were up-regulated at the active/chronic inflammatory stages. Rectal administration of the NF-kappaB p65 antisense oligonucleotide reduced but did not abrogate inflammation and fibrosis completely. The antisense oligonucleotide treatment reduced total NF-kappaB by 60% and down-regulated most proinflammatory genes. However, Ccl2, a proinflammatory chemokine known to promote fibrosis, was not down-regulated. Among extracellular matrix gene expressions Lum was suppressed while Col1a1 and Col3a1 were not. Thus, effective treatment of fibrosis in inflammatory bowel disease may require early and complete blockade of NF-kappaB with particular attention to specific proinflammatory and profibrogenic genes that remain active at low levels of NF-kappaB.

FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4+CD25+ regulatory T cells.

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.

Practical pediatric regional anesthesia.

In children, regional anesthetic techniques are safe and effective adjuncts to general anesthesia and for postoperative pain relief. Application of the techniques described in this article will contribute to improved care for pediatric patients undergoing surgical procedures. The judicious choice of local anesthetics, along with the blockades of targeted nerves, decrease the need for supplemental analgesics in the recovery phase.

Aminoglycoside-nucleic acid interactions: remarkable stabilization of DNA and RNA triple helices by neomycin.

The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.