Soluble syndecan-1 and glycosaminoglycans in preeclamptic and normotensive pregnancies.

Preeclampsia, an important cause of maternal and fetal morbidity and mortality, is associated with increased sFLT1 levels and with structural and functional damage to the glycocalyx contributing to endothelial dysfunction. We investigated glycocalyx components in relation to preeclampsia in human samples. While soluble syndecan-1 and heparan sulphate were similar in plasma of preeclamptic and normotensive pregnant women, dermatan sulphate was increased and keratan sulphate decreased in preeclamptic women. Dermatan sulphate was correlated with soluble syndecan-1, and inversely correlated with blood pressure and activated partial thromboplastin time. To determine if syndecan-1 was a prerequisite for the sFlt1 induced increase in blood pressure in mice we studied the effect of sFlt1 on blood pressure and vascular contractile responses in syndecan-1 deficient and wild type male mice. The classical sFlt1 induced rise in blood pressure was absent in syndecan-1 deficient mice indicating that syndecan-1 is a prerequisite for sFlt1 induced increase in blood pressure central to preeclampsia. The results show that an interplay between syndecan-1 and dermatan sulphate contributes to sFlt1 induced blood pressure elevation in pre-eclampsia.

Creation of forest edges has a global impact on forest vertebrates.

Forest edges influence more than half of the world's forests and contribute to worldwide declines in biodiversity and ecosystem functions. However, predicting these declines is challenging in heterogeneous fragmented landscapes. Here we assembled a global dataset on species responses to fragmentation and developed a statistical approach for quantifying edge impacts in heterogeneous landscapes to quantify edge-determined changes in abundance of 1,673 vertebrate species. We show that the abundances of 85% of species are affected, either positively or negatively, by forest edges. Species that live in the centre of the forest (forest core), that were more likely to be listed as threatened by the International Union for Conservation of Nature (IUCN), reached peak abundances only at sites farther than 200-400 m from sharp high-contrast forest edges. Smaller-bodied amphibians, larger reptiles and medium-sized non-volant mammals experienced a larger reduction in suitable habitat than other forest-core species. Our results highlight the pervasive ability of forest edges to restructure ecological communities on a global scale.

Randomised clinical trial: the efficacy of gut-directed hypnotherapy is similar to that of the low FODMAP diet for the treatment of irritable bowel syndrome.

BACKGROUND: A low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) diet is effective in treating irritable bowel syndrome (IBS). AIM: To compare the effects of gut-directed hypnotherapy to the low FODMAP diet on gastrointestinal symptoms and psychological indices, and assess additive effects. METHODS: Irritable bowel syndrome patients were randomised (computer-generated list), to receive hypnotherapy, diet or a combination. Primary end-point: change in overall gastrointestinal symptoms across the three groups from baseline to week 6. Secondary end-points: changes in psychological indices, and the durability of effects over 6 months. RESULTS: Of 74 participants, 25 received hypnotherapy, 24 diet and 25 combination. There were no demographic differences at baseline across groups. Improvements in overall symptoms were observed from baseline to week 6 for hypnotherapy [mean difference (95% CI): -33 (-41 to -25)], diet [-30 (-42 to -19)] and combination [-36 (-45 to -27)] with no difference across groups (P = 0.67). This represented ≥20 mm improvement on visual analogue scale in 72%, 71% and 72%, respectively. This improvement relative to baseline symptoms was maintained 6 months post-treatment in 74%, 82% and 54%. Individual gastrointestinal symptoms similarly improved. Hypnotherapy resulted in superior improvements on psychological indices with mean change from baseline to 6 months in State Trait Personality Inventory trait anxiety of -4(95% CI -6 to -2) P < 0.0001; -1(-3 to 0.3) P = ns; and 0.3(-2 to 2) P = ns, and in trait depression of -3(-5 to -0.7) P = 0.011; -0.8(-2 to 0.2) P = ns; and 0.6(-2 to 3) P = ns, respectively. Groups improved similarly for QOL (all p ≤ 0.001). CONCLUSIONS: Durable effects of gut-directed hypnotherapy are similar to those of the low FODMAP diet for relief of gastrointestinal symptoms. Hypnotherapy has superior efficacy to the diet on psychological indices. No additive effects were observed.

Review article: gut-directed hypnotherapy in the management of irritable bowel syndrome and inflammatory bowel disease.

BACKGROUND: Gut-directed hypnotherapy is being increasingly applied to patients with irritable bowel syndrome (IBS) and to a lesser extent, inflammatory bowel disease (IBD). AIM: To review the technique, mechanisms of action and evidence for efficacy, and to identify gaps in the understanding of gut-directed hypnotherapy as a treatment for IBS and IBD. METHODS: A review of published literature and a systematic review of clinical trials in its application to patients with IBS and IBD were performed. RESULTS: Gut-directed hypnotherapy is a clearly described technique. Its potential mechanisms of action on the brain-gut axis are multiple with evidence spanning psychological effects through to physiological gastrointestinal modifications. Six of seven randomised IBS studies reported a significant reduction (all P < 0.05) in overall gastrointestinal symptoms following treatment usually compared to supportive therapy only. Response rates amongst those who received gut-directed hypnotherapy ranged between 24% and 73%. Efficacy was maintained long-term in four of five studies. A therapeutic effect was also observed in the maintenance of clinical remission in patients with ulcerative colitis. Uncontrolled trials supported the efficacy and durability of gut-directed hypnotherapy in IBS. Gaps in understanding included to whom and when it should be applied, the paucity of adequately trained hypnotherapists, and the difficulties in designing well controlled-trials. CONCLUSIONS: Gut-directed hypnotherapy has durable efficacy in patients with IBS and possibly ulcerative colitis. Whether it sits in the therapeutic arsenal as a primary and/or adjunctive therapy cannot be ascertained on the current evidence base. Further research into efficacy, mechanisms of action and predictors of response is required.

Randomised clinical trial: gluten may cause depression in subjects with non-coeliac gluten sensitivity - an exploratory clinical study.

BACKGROUND: Current evidence suggests that many patients with self-reported non-coeliac gluten sensitivity (NCGS) retain gastrointestinal symptoms on a gluten-free diet (GFD) but continue to restrict gluten as they report 'feeling better'. AIM: To investigate the notion that a major effect of gluten in those with NCGS is on mental state and not necessarily on gastrointestinal symptoms. METHODS: Twenty-two subjects (24-62 years, five male) with irritable bowel syndrome who had coeliac disease excluded but were symptomatically controlled on a GFD, undertook a double-blind cross-over study. Participants randomly received one of three dietary challenges for 3 days, followed by a minimum 3-day washout before crossing over to the next diet. Challenge gluten-free food was supplemented with gluten (16 g/day), whey (16 g/day) or not supplemented (placebo). End-points included mental state as assessed by the Spielberger State Trait Personality Inventory (STPI), cortisol secretion and gastrointestinal symptoms. RESULTS: Gluten ingestion was associated with higher overall STPI state depression scores compared to placebo [M = 2.03, 95% CI (0.55-3.51), P = 0.010] but not whey [M = 1.48, 95% CI (-0.14 to 3.10), P = 0.07]. No differences were found for other STPI state indices or for any STPI trait measures. No difference in cortisol secretion was identified between challenges. Gastrointestinal symptoms were induced similarly across all dietary challenges. CONCLUSIONS: Short-term exposure to gluten specifically induced current feelings of depression with no effect on other indices or on emotional disposition. Gluten-specific induction of gastrointestinal symptoms was not identified. Such findings might explain why patients with non-coeliac gluten sensitivity feel better on a gluten-free diet despite the continuation of gastrointestinal symptoms.

Comparison of preserved vascular allografts using glycerol and University of Wisconsin solution in a goat carotid artery transplantation model.

BACKGROUND: Prosthetic grafts have poor patency rates in peripheral arterial reconstructions. Glycerol (GL)-preserved grafts are an alternative. The aim of this study was to examine patency, graft morphology and function of GL-preserved allografts in a goat carotid artery animal model. METHODS: The first group (n = 7) underwent bilateral replacement of the carotid artery by a carotid allograft that was preserved in GL for 1 week. In the second group (n = 5), a carotid artery allograft that was preserved in University of Wisconsin solution (UW) for 48 h was used. In the third group (n = 5), the jugular vein (autologous vein, AU) was used. The follow-up was 3 months. RESULTS: One UW graft and 1 GL graft occluded in the first 24 h postoperatively. Three-month primary patency rates for GL, UW and AU grafts were 93, 100 and 80%, respectively (p = 0.39). Graft diameter was increased in UW allografts (p < 0.005), whereas GL allografts remained unchanged. After explantation, GL allografts demonstrated contraction and relaxation capacity and lower intimal thickness (p < 0.001). CONCLUSION: GL preservation has proven to be a feasible method for arterial allograft transplantation in a large animal model with decreased intimal hyperplasia and renewed functional capability.

LPS differentially affects vasoconstrictor responses: a potential role for RGS16?

The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to alpha1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 microg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (alpha1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.

Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3)).

BACKGROUND AND PURPOSE: Recently, some ligands targeting the sphingosine-1-phosphate receptor subtype 3 (S1P(3)) have become available. The characterization of these compounds was mainly based on one functional read-out system, although S1P(3) receptors are known to activate different signal transduction pathways. Therefore, this study pharmacologically characterizes these compounds using different assays. EXPERIMENTAL APPROACH: Using CHO-FlpIn cells expressing the human S1P(3) receptor the potencies and maximal effects of S1P, FTY720-P, VPC23019, VPC23153 and VPC24191 were determined in three different assays [inhibition of cAMP accumulation, elevation of intracellular calcium concentrations ([Ca(2+)](i)) and S1P(3) receptor internalization]. KEY RESULTS: All compounds tested inhibited forskolin-induced cAMP accumulation, increased [Ca(2+)](i) and induced S1P(3) receptor internalization but with different potencies and maximal effects. S1P was the most potent compound in all assays followed by FTY720-P. The VPC compounds were generally less potent than S1P and FTY720-P. Regarding the maximal effects, all compounds except VPC23153, behaved as full agonists in the cAMP accumulation assay. In the calcium assay, FTY720-P, VPC23019 and VPC24191 displayed partial and VPC23153 weak partial agonist activity, relative to S1P. Interestingly, treatment with the G(i) inactivator Pertussis toxin, did not affect S1P-induced [Ca(2+)](i) elevations but inhibited those in response to the other compounds, by about 50%. CONCLUSIONS AND IMPLICATIONS: This study demonstrated differential response patterns at the S1P(3) receptor for a range of ligands. These differences could indicate the presence of functional selectivity at this receptor as FTY720-P and the VPC compounds seemed to signal predominantly via G(i)- whereas S1P activated G(i) and G(q)-coupled pathways.

Quantifying the biodiversity value of tropical primary, secondary, and plantation forests.

Biodiversity loss from deforestation may be partly offset by the expansion of secondary forests and plantation forestry in the tropics. However, our current knowledge of the value of these habitats for biodiversity conservation is limited to very few taxa, and many studies are severely confounded by methodological shortcomings. We examined the conservation value of tropical primary, secondary, and plantation forests for 15 taxonomic groups using a robust and replicated sample design that minimized edge effects. Different taxa varied markedly in their response to patterns of land use in terms of species richness and the percentage of species restricted to primary forest (varying from 5% to 57%), yet almost all between-forest comparisons showed marked differences in community structure and composition. Cross-taxon congruence in response patterns was very weak when evaluated using abundance or species richness data, but much stronger when using metrics based upon community similarity. Our results show that, whereas the biodiversity indicator group concept may hold some validity for several taxa that are frequently sampled (such as birds and fruit-feeding butterflies), it fails for those exhibiting highly idiosyncratic responses to tropical land-use change (including highly vagile species groups such as bats and orchid bees), highlighting the problems associated with quantifying the biodiversity value of anthropogenic habitats. Finally, although we show that areas of native regeneration and exotic tree plantations can provide complementary conservation services, we also provide clear empirical evidence demonstrating the irreplaceable value of primary forests.

BML-241 fails to display selective antagonism at the sphingosine-1-phosphate receptor, S1P(3).

BACKGROUND AND PURPOSE: The thiazolidine carboxylic acid, BML-241, has been proposed as a lead compound in development of selective antagonists at the sphingosine-1-phosphate receptor (S1P3), based on its inhibition of the rise in intracellular calcium concentrations ([Ca2+]i) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML-241 for the S1P(3) receptor in more detail. EXPERIMENTAL APPROACH: Chinese hamster ovary (CHO) cells stably transfected with the S1P3, S1P2 or alpha(1A)-adrenoceptors were used to investigate the effect of BML-241 on increases in [Ca2+]i mediated via different receptors. CHO-K1 cells were used to study ATP-induced [Ca2+]i elevations. Effects on S1P3 -mediated inhibition of forskolin-induced cAMP accumulation and on binding to alpha(1A)-adrenoceptors were also investigated. In addition, the effect of BML-241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath. KEY RESULTS: High concentrations of BML-241 (10 microM) inhibited the rise in [Ca2+]i induced by S1P3 and S1P2 receptor stimulation; lower concentrations were ineffective. This high concentration of BML-241 also inhibited [Ca2+]i increases via P2 (nucleotide) receptor or alpha(1A)-adrenoceptor stimulation. Moreover, BML-241 (10 microM) inhibited alpha(1)-adrenoceptor-mediated contraction of rat mesenteric artery but did not displace [3H]-prazosin from alpha(1A)-adrenoceptors in concentrations up to 100 microM. BML-241 (10 microM) did not affect the S1P3 -mediated decrease of forskolin-induced cAMP accumulation. CONCLUSIONS AND IMPLICATIONS: We conclude that BML-241 is a low potency, non-selective inhibitor of increases in [Ca2+]i, rather than a specific antagonist at the S1P3 receptor.

Uptake of gentamicin by bullfrog saccular hair cells in vitro.

Vertebrate sensory hair cells in the inner ear are pharmacologically sensitive to aminoglycoside antibiotics. Although the ototoxicity of aminoglycosides is well known, the route of drug uptake by hair cells and mechanisms of cytotoxicity remain poorly understood. Previously published studies have documented the intracellular distribution of gentamicin using immunocytochemical, electron microscopic, and autoradiographic methods. In this article, we compare the subcellular distribution of fluorescently conjugated gentamicin (gentamicin-Texas Red, GTTR) with immunolabeled gentamicin using confocal or electron microscopy. Gentamicin (detected by postfixation immunocytochemistry) and GTTR were rapidly taken up by hair cells throughout the bullfrog saccular explant in vitro and preferentially in peripheral hair cells. Immunolabeled gentamicin and GTTR were observed at the apical membranes of hair cells, particularly in their hair bundles. GTTR was also identified within a variety of subcellular compartments within hair cells, including lysosomes, mitochondria, Golgi bodies, endoplasmic reticulum, and nuclei, and in similar structures by immunoelectron microscopy. The distributions of GTTR and immunolabeled gentamicin are largely identical and corroborate a variety of published immunocytochemical and autoradiography studies. Thus, GTTR is a valid fluorescent probe with which to investigate the pharmacokinetics and mechanisms of gentamicin accumulation.

The effects of hypochlorite-induced oxidative stress on presynaptic M2-receptors at sympathetic nerve endings in the rat tail artery.

1 It was shown recently that stimulation of cardiac muscarinic M2-receptors revealed an enhanced negative inotropic response in isolated rat left atria after exposure to hypochlorite-induced oxidative stress. This phenomenon was not observed after stimulation of the cardiac A1-receptor, which like the M2-receptor is coupled to Gi-proteins. Since even the contractile response to M3-receptor stimulation was not amplified in the rat portal vein, we hypothesized a M2-receptor specificity of this hypochlorite-induced enhancement. 2 The present study was performed in order to investigate whether the sympathoinhibitory response to presynaptically located M2-receptor stimulation would also be modified after exposure to hypochlorite in the rat tail artery. We applied electrical field stimulation (EFS) in order to mimic sympathetic neurotransmission. 3 EFS increased the vascular tone frequency-dependently (0.3-4 Hz). EFS-induced vasoconstriction could be attenuated by acetylcholine (30 nM-1 microM) in a concentration-dependent manner. Hypochlorite (10 and 100 microM) did not affect the sympathoinhibitory effect of acetylcholine (100 nM). 4 In conclusion, in contrast to cardiac M2-receptors, hypochlorite did not amplify the sympathoinhibitory effects of presynaptic M2-receptors. The different responsiveness between neuronal and cardiac M2-receptors to hypochlorite may be explained by the different G-protein subunits involved in the activation of the underlying signalling cascade.

Oxidative stress impairs the haemodynamic activity of cardiovascular agents with antioxidant properties.

Reactive oxygen species (ROS) are known to be involved in the pathogenesis and progression of various cardiovascular diseases. For some therapeutics like carvedilol and captopril used in the treatment of such diseases antioxidant properties have been proposed to play a role in addition to their haemodynamic activities. It was the aim of the present study to assess whether ROS may affect the molecular integrity and the primary pharmacological actions of compounds with additional antioxidant properties. Accordingly, well-known drugs as mentioned were exposed to ROS, generated by electrolysis and analyzed by means of functional and chemical investigations. For this purpose rat thoracic aortic rings were incubated with either the beta1,2/alpha1-adrenoceptor antagonist carvedilol (100 nM), the alpha1-adrenoceptor antagonist prazosin (5 nM), the thiol-containing ACE-inhibitor captopril (3 microM) or lisinopril (300 nM), an ACE-inhibitor without a thiol moiety. Furthermore, isolated rat left atria were incubated with either carvedilol (14 nM) or with the beta1,2-adrenoceptor antagonist timolol (50 nM). After an incubation period of 15 min, electrolysis was applied to the buffer medium in order to generate ROS. After an additional 15 min, concentration-response curves were constructed for angiotensin I and phenylephrine in thoracic aortic rings incubated with the ACE-inhibitors and the alpha1-adrenoceptor antagonists, respectively. In addition, concentration-response curves were constructed for isoprenaline in presence of the beta1,2-adrenoceptor antagonists in isolated left atria. After exposure to oxidative stress the alpha1- and beta-adrenoceptor blocking activity of carvedilol was significantly impaired, when compared to control conditions. In contrast, the pharmacological effects of prazosin and timolol remained unaffected. The ACE-inhibition by captopril was completely abolished after electrolysis, while the pharmacological action of lisinopril was only slightly reduced. In addition, a complete oxidative degradation of captopril and carvedilol could be demonstrated by using UV/Vis spectroscopy and HPLC/fluorospectroscopy, respectively. From these results we conclude that the haemodynamic therapeutics with additional radical scavenging properties may undergo a chemical modification due to ROS-exposure which results in a loss of pharmacological activity.

Reactive oxygen species potentiate the negative inotropic effect of cardiac M2-muscarinic receptor stimulation.

The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on the contractile responses of rat isolated left atria to muscarinic receptor stimulation. ROS were generated by means of electrolysis (30 mA, 75 s) of the organ bath fluid. Twenty minutes after the electrolysis period, the electrically paced atria (3 Hz) were stimulated with the adenylyl cyclase activator forskolin (1 microM). Subsequently, cumulative acetylcholine concentration-response curves were constructed (0.01 nM-10 microM). In addition, phosphoinositide turnover and adenylyl cyclase activity under basal and stimulated conditions were measured. For these biochemical experiments we used the stable acetylcholine analogue carbachol. The atria exposed to reactive oxygen species were influenced more potently (pD2 control: 6.2 vs. 7.1 for electrolysis-treated atria, P<0.05) and more effectively (Emax control: 40% vs. 90% reduction of the initial amplitude, P<0.05) by acetylcholine. In contrast, ROS exposure did not alter the responses to adenosine, whose receptor is also coupled via a Gi-protein to adenylyl cyclase. The basal (40% vs. control, P<0.05) as well as the carbachol-stimulated (-85% vs. control, P<0.05) inositol-phosphate formation was reduced in atria exposed to ROS. The forskolin-stimulated adenylyl cyclase activity was identical in both groups but carbachol stimulation induced a more pronounced reduction in adenylyl cyclase activity in the electrolysis-treated atria. Accordingly we may conclude that ROS enhance the negative inotropic response of isolated rat atria to acetylcholine by both a reduction of the positive (inositide turnover) and increase of the negative (adenylyl cyclase inhibition) inotropic components of cardiac muscarinic receptor stimulation. This phenomenon is most likely M2-receptor specific, since the negative inotropic response to adenosine is unaltered by ROS exposure.

Modulation of latent inhibition in the rat by altered dopamine transmission in the nucleus accumbens at the time of conditioning.

Latent inhibition describes a process by which pre-exposure of a stimulus without consequence retards the learning of subsequent conditioned associations with that stimulus. It is well established that latent inhibition in rats is impaired by increased dopamine function and potentiated by reduced dopamine function. Previous evidence has suggested that these effects are modulated via the meso-accumbens dopamine projections. We have now undertaken three experiments to examine this issue directly, especially in the light of one study in which latent inhibition was reported to be unaffected by direct injection of amphetamine into the accumbens. Latent inhibition was studied using the effect of pre-exposure of a tone stimulus on the subsequent formation of a conditioned emotional response to the tone. 6-Hydroxydopamine-induced lesions of dopamine terminals in the nucleus accumbens resulted in potentiation of latent inhibition. Bilateral local injections of the dopamine antagonist haloperidol into the nucleus accumbens (0.5 microg/side) before conditioning also potentiated latent inhibition. Moreover, such injections were able to reverse the disruptive effect of systemic amphetamine (1mg/kg, i.p.) on latent inhibition. Bilateral local injection of amphetamine (5 microg/side) into the nucleus accumbens before conditioning was able to disrupt latent inhibition, provided that it was preceded by a systemic injection of amphetamine (1mg/kg) 24h earlier.We conclude that the attenuation of latent inhibition by increased dopamine function in the nucleus accumbens is brought about by impulse-dependent release of the neurotransmitter occurring at the time of conditioning. The previously reported failure to disrupt latent inhibition with intra-accumbens amphetamine is probably due to impulse-independent release of dopamine. The implications of these conclusions for theories linking disrupted latent inhibition to the attentional deficits in schizophrenia, and to the dopamine theory of this disorder, are discussed.