Investigation of a foodborne outbreak of Shigella sonnei in Ireland and Northern Ireland, December 2016: the benefits of cross-border collaboration and commercial sales data.

OBJECTIVES: To describe a cross-border foodborne outbreak of Shigella sonnei that occurred in Ireland and Northern Ireland (NI) in December 2016 whilst also highlighting the valuable roles of sales data and international collaboration in the investigation and control of this outbreak. STUDY DESIGN: A cross-border outbreak control team was established to investigate the outbreak. METHODS: Epidemiological, microbiological, and environmental investigations were undertaken. Traditional analytical epidemiological studies were not feasible in this investigation. The restaurant chain provided sales data, which allowed assessment of a possible increased risk of illness associated with exposure to a particular type of heated food product (product A). RESULTS: Confirmed cases demonstrated sole trimethoprim resistance: an atypical antibiogram for Shigella isolates in Ireland. Early communication and the sharing of information within the outbreak control team facilitated the early detection of the international dimension of this outbreak. A joint international alert using the European Centre for Disease Control's confidential Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD) did not reveal further cases outside of the island of Ireland. The outbreak investigation identified that nine of thirteen primary case individuals had consumed product A from one of multiple branches of a restaurant chain located throughout the island of Ireland. Product A was made specifically for this chain in a food production facility in NI. S. sonnei was not detected in food samples from the food production facility. Strong statistical associations were observed between visiting a branch of this restaurant chain between 5 and 9 December 2016 and eating product A and developing shigellosis. CONCLUSIONS: This outbreak investigation highlights the importance of international collaboration in the efficient identification of cross-border foodborne outbreaks and the value of using sales data as the analytical component of such studies.

The discovery of a potent Nav1.3 inhibitor with good oral pharmacokinetics.

In this article, we describe the discovery of an aryl ether series of potent and selective Nav1.3 inhibitors. Based on structural analogy to a similar series of compounds we have previously shown bind to the domain IV voltage sensor region of Nav channels, we propose this series binds in the same location. We describe the development of this series from a published starting point, highlighting key selectivity and potency data, and several studies designed to validate Nav1.3 as a target for pain.

Contribution of both nitric oxide and a change in membrane potential to acetylcholine-induced relaxation in the rat small mesenteric artery.

1. Acetylcholine stimulated repolarization and relaxation in isolated segments of rat small mesenteric artery (D100 = 325 +/- 9 microns) in which the smooth muscle cells were depolarized and contracted by submaximal concentrations of noradrenaline (0.75-2.5 microM). There was no significant difference either in the time taken to initiate relaxation or hyperpolarization, or for these parameters to reach maximum in response to acetylcholine. 2. The nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester (L-NAME, 100 microM) reduced the pD2 for acetylcholine-induced relaxation from 7.5 to 7 and depressed the maximum relaxation from 89% to 68% in tissues stimulated with noradrenaline. The pD2 for smooth muscle repolarization in these experiments was also reduced (7.4 to 6.6) but the maximum change in membrane potential in response to acetylcholine was unaltered. The increase in potential now clearly preceded relaxation by 3.7 s (to initiation) and 4.7 s (to maximum). 3. In the presence of noradrenaline and a raised potassium concentration (25 mM), the repolarization to acetylcholine was markedly attenuated. Simultaneous tension measurements also revealed a marked reduction in the maximal relaxation to acetylcholine, but the pD2 was unchanged at 7.4. 4. The residual relaxation recorded in the absence of marked repolarization (in the presence of noradrenaline and 25 mM potassium) was abolished by the addition of 100 microM L-NAME. 5. Nitric oxide gas in solution (0.2-2.2 microM; NOg) relaxed artery segments precontracted with noradrenaline. The magnitude of relaxation to NOg was not altered in the presence of noradrenaline and 25 mM potassium. 6. These data provide additional evidence that acetylcholine-evoked endothelium-dependent increases in membrane potential provide a major mechanism for smooth muscle relaxation in the mesenteric artery.They also show that voltage-dependent and independent (initiated by NO) mechanisms can both contribute to relaxation, and suggest that NO may modulate the increase in membrane potential or the release of a hyperpolarizing factor.

Lack of effect of sumatriptan and UK-14,304 on capsaicin-induced relaxation of guinea-pig isolated basilar artery.

1. The objectives of this study were to assess the effects of sensory neuropeptide antagonists and presynaptically acting receptor agonists on capsaicin-induced relaxations of guinea-pig isolated basilar artery (GPBA). 2. Capsaicin, human alpha-calcitonin gene-related peptide (CGRP) and substance P (SP) caused concentration-related relaxations of GPBA which had been pre-contracted with prostaglandin F2 alpha (PGF2 alpha). Responses to capsaicin were not modified by the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM). 3. The relaxant responses to capsaicin were blocked in a selective manner by ruthenium red (3 microM) and by the CGRP antagonist, CGRP8-37 (1 microM). CGRP8-37 also selectively inhibited the relaxant effects of CGRP. 4. The selective NK1 receptor antagonist, GR82334 (10 microM), inhibited SP-induced relaxations but had little effect on capsaicin-induced relaxations. 5. The 5-HT1 receptor agonist, sumatriptan, produced small contractions of GPBA under conditions of resting tone. In the presence of PGF2 alpha, sumatriptan had no further contractile effect. Sumatriptan (0.3 and 3 microM) did not modify capsaicin-induced relaxations of GPBA. 6. The alpha 2-adrenoceptor agonist, UK-14,304 (0.1 microM), had no effect on basal or PGF2 alpha-induced tone. UK-14,304 did not modify capsaicin-induced relaxations. 7. These results suggest that capsaicin causes relaxation of GPBA via a release of CGRP. This process is amenable to blockade by CGRP8-37 and ruthenium red, but not to modulation by either sumatriptan or UK-14,304.

Characterization of the receptor mediating relaxation to substance P in canine middle cerebral artery: no evidence for involvement of substance P in neurogenically mediated relaxation.

1. The aim of this study was to characterize the neurokinin receptor which mediates relaxation of dog isolated middle cerebral artery by the use of selective agonists and antagonists and to establish whether substance P is involved in the neurogenically mediated relaxant response in this vessel. 2. Substance P caused concentration-related, endothelium-dependent relaxations of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha. The selective NK1 receptor agonists, GR73632 and substance P methyl ester (SPOMe), also caused relaxation with similar maximum effects to those of substance P. GR73632 and SPOMe were approximately 20 times and 6 times less potent respectively than substance P. The selective NK2 and NK3 receptor agonists, GR64349 and senktide, were only weakly active in causing relaxation being at least 425 times and 245 times less potent respectively than substance P. 3. The selective NK1 receptor antagonist, GR82334, was a potent, specific, competitive antagonist of the relaxant effects of substance P. In contrast, the selective NK2 receptor antagonist, R396 (10 microM) had no effect on the response to substance P. 4. Electrical field stimulation of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha, caused neurogenically mediated, non-adrenergic non-cholinergic (NANC) relaxations. These NANC relaxations were unaffected by endothelium removal, GR82334 (10 microM) or by capsaicin (10 microM) treatment. However, the nitric oxide synthesis inhibitor, L-NG-monomethyl arginine methyl ester (L-NMMA) (100 microM) markedly attenuated the response to electrical stimulation. 5. These results suggest that substance P causes relaxation of dog isolated middle cerebral artery via activation of NK1 receptors. However, substance P does not appear to be involved in NANC neurotransmission. In contrast, the marked inhibitory effect of L-NMMA on NANC relaxations implicates nitric oxide in NANC neurotransmission in this vessel.

Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery.

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.

The relative importance of nitric oxide and nitric oxide-independent mechanisms in acetylcholine-evoked dilatation of the rat mesenteric bed.

1. The relative contribution of nitric oxide (NO) to acetylcholine-induced smooth muscle relaxation was investigated in the rat perfused mesenteric vasculature and in isolated segments of second, third and fourth order arterial branches. 2. The EC50 values and maximal relaxation to acetylcholine were not significantly different in the sequential arterial branches, being approximately 0.05 microM and 85%, respectively. 3. The NO synthase inhibitor L-NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) reduced acetylcholine-evoked endothelium-dependent dilatation and relaxation in the perfused mesenteric bed and in isolated arterial segments. The maximum response to acetylcholine in both preparations was reduced by between 35% to 40% while the EC50 values were increased by 5-6 fold. L-NAME had no effect on basal smooth muscle tone in either case. 4. In contrast, endothelium-dependent dilatation of the perfused mesenteric bed evoked by A23187 (0.002-20 nmol), was unaffected by exposure to L-NAME. The EC50 values and maximal responses elicited by A23187 (20 nmol) before and after exposure to L-NAME were 0.96 +/- 0.5 nmol and 67.0 +/- 7.0% (n = 4), and 0.7 +/- 0.4 nmol and 70.0 +/- 5.0% (n = 4; P > 0.01), respectively. 5. Perfusion of the isolated mesenteric bed with raised K(+)-Krebs buffer (25 mM) had no effect on basal tone, but reduced the amplitude of both acetylcholine- and A23187-evoked dilatation. The maximum responses to acetylcholine (2 micromol) and A23187 (20 nmol) were reduced from 67.5 +/- 7.3% and 65.4+/-8.2% to 18.9 +/-11.0% (n=5; P<0.01) and 13.5 +/-12.0% (n=4; P<0.01), respectively.6. Exposure of the mesenteric bed to L-NAME in the presence of raised K+-Krebs further reduced the maximal response elicited by acetylcholine to only 8.9 +/- 2.8% (n =4; P< 0.01).7. These results indicate that acetylcholine-evoked vasodilatation of the rat mesenteric vasculature is mediated by both NO-dependent and -independent mechanisms. The relative contribution made by these mechanisms does not appear to differ in sequential branches of the mesenteric artery. In contrast,A23187-evoked vasodilatation appears to be mediated predominantly by a NO-independent mechanism which is sensitive to increases in the extracellular potassium concentration and may reflect the action of endothelium-derived hyperpolarizing factor (EDHF).

NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery.

1. The nature and cellular mechanisms that are responsible for endothelium-dependent relaxations resistant to indomethacin and NG-nitro-L-arginine methyl ester (L-NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit. 2. In the presence of the cyclo-oxygenase inhibitor, indomethacin (10 microM), acetylcholine (ACh) induced a concentration- and endothelium-dependent relaxation of PE-induced tone which was more potent than the calcium ionophore A23187 with pD2 values of 7.03 +/- 0.12 (n = 8) and 6.37 +/- 0.12 (n = 6), respectively. The ACh-induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD2 value 7.00 +/- 0.10 and maximal relaxation 99 +/- 3%, n = 6). Bradykinin and histamine (0.01-100 microM) had no effect either upon resting or PE-induced tone (n = 5). 3. In the presence of indomethacin plus the NO synthase inhibitor, L-NAME (30 microM), the response to A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD2 value and the maximal relaxation decreasing to 6.48 +/- 0.10 and 67 +/- 3%, respectively (for both P < 0.01, n = 8). The L-NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n = 8). 4. The Ca(2+)-activated K+ (KCa) channel blockers, tetrabutylammonium (TBA, 1 mM, n = 5) and charybdotoxin (CTX, 0.1 microM, n = 5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI2)-independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 microM, n = 8) or apamin (1-3 microM, n = 6) only partially inhibited the relaxation. 5. Inhibitors of the cytochrome P450 mono-oxygenase, SKF-525A (1-10 microM, n = 6), clotrimazole (1 microM, n = 5) and 17-octadecynoic acid (17-ODYA, 3 microM, n = 7) also reduced the NO/PGI2-independent relaxation response to ACh. 6. In endothelium-denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either KCa channel blockade with apamin (1 microM, n = 5) or CTX (0.1 microM, n = 5), or by the cytochrome P450 mono-oxygenase blockers SKF-525A (10 microM, n = 4) and clotrimazole (10 microM, n = 5). However, the NO-induced response was shifted to the right by LY83583 (10 microM, n = 4), a guanylyl cyclase inhibitor, with the pD2 value decreasing from 6.95 +/- 0.14 to 6.04 +/- 0.09 (P < 0.01). 7. ACh (0.01-100 microM) induced a concentration-dependent relaxation of PE-induced tone in endothelium-denuded arterial segments sandwiched with endothelium-intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 microM) plus L-NAME (30 microM), but abolished by the combination of indomethacin, L-NAME and TBA (1 mM, n = 5). 8. These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450-derived arachidonic acid metabolite, and (c) EDHF-induced relaxation involves the opening of at least two types of KCa channels, whereas NO mediates vasorelaxation via a guanosine 3': 5'-cyclic monophosphate (cyclic GMP)-mediated pathway, in which a cytochrome P450 pathway and KCa channels do not seem to be involved.

Evidence that anandamide and EDHF act via different mechanisms in rat isolated mesenteric arteries.

The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB1) antagonist SRI41716A, or mimicked by selective CB1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB1 receptor-independent mechanism and cannabinoid receptor activation apparently does not contribute to EDHF-mediated relaxation in this resistance artery.

Acetylcholine-induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase.

1. Acetycholine-mediated relaxations in phenylephrine-contracted aortas, femoral and mesenteric resistance arteries were studied in vessels from endothelial nitric oxide synthase knock-out (eNOS -/-) and the corresponding wild-type strain (eNOS +/+) C57BL6/SV19 mice. 2. Aortas from eNOS (+/+) mice relaxed to acetylcholine in an endothelium-dependent NG-nitro-L-arginine (L-NOARG) sensitive manner. Aortas from eNOS (-/-) mice did not relax to acetylcholine but demonstrated enhanced sensitivity to both authentic NO and sodium nitroprusside. 3. Relaxation to acetylcholine in femoral arteries was partially inhibited by L-NOARG in vessels from eNOS (+/+) mice, but relaxation in eNOS (-/-) mice was insensitive to a combination of L-NOARG and indomethacin and the guanylyl cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The L-NOARG/ODQ/indomethacin-insensitive relaxation to acetylcholine in femoral arteries was inhibited in the presence of elevated (30 mM) extracellular KCl. 4. In mesenteric resistance vessels from eNOS (+/+) mice, the acetylcholine-mediated relaxation response was completely inhibited by a combination of indomethacin and L-NOARG or by 30 mM KCl alone. In contrast, in mesenteric arteries from eNOS (-/-) mice, the acetylcholine-relaxation response was insensitive to a combination of L-NOARG and indomethacin, but was inhibited in the presence of 30 mM KCl. 5. These data indicate arteries from eNOS (-/-) mice demonstrate a supersensitivity to exogenous NO, and that acetylcholine-induced vasorelaxation of femoral and mesenteric vessels from eNOS (-/-) mice is mediated by an endothelium-derived factor that has properties of an EDHF but is neither NO nor prostacyclin. Furthermore, in mesenteric vessels, there is an upregulation of the role of EDHF in the absence of NO.

Lack of endothelin ETB receptor-mediated smooth muscle hyperpolarization in rat mesenteric resistance arteries.

The presence of functional endothelin ETB receptors was investigated in rat isolated mesenteric resistance arteries. Neither endothelin-3 (0.1-100 nM) nor the endothelin ETB selective agonists sarafotoxin S6c and BQ 3020 (both 1-100 nM) induced any measurable hyperpolarization or relaxation in stimulated (alpha 1-adrenoceptor agonist; phenylephrine) or unstimulated arteries. In both cases, the subsequent addition of acetylcholine (1 microM) hyperpolarized the membrane potential by 10-20 mV and totally reversed any contraction which was present. These results indicate that the endothelin ETB-mediated vasodilatation observed in the intact mesenteric bed does not reflect hyperpolarization of smooth muscle cells in resistance arteries arising from the mesenteric artery.