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Background: Growing evidence suggests that an altered microbiota composition contributes to the pathogenesis and clinical features in celiac disease (CD). We performed a comparative analysis of the gut microbiota in adulthood CD to evaluate whether: (i) dysbiosis anticipates mucosal lesions, (ii) gluten-free diet restores eubiosis, (iii) refractory CD has a peculiar microbial signature, and (iv) salivary and fecal communities overlap the mucosal one. Methods: This is a cross-sectional study where a total of 52 CD patients, including 13 active CD, 29 treated CD, 4 refractory CD, and 6 potential CD, were enrolled in a tertiary center together with 31 controls. A 16S rRNA-based amplicon metagenomics approach was applied to determine the microbiota structure and composition of salivary, duodenal mucosa, and stool samples, followed by appropriate bioinformatic analyses. Results: A reduction of both &alpha, and &beta, diversity in CD, already evident in the potential form and achieving nadir in refractory CD, was evident. Taxonomically, mucosa displayed a significant abundance of Proteobacteria and an expansion of Neisseria, especially in active patients, while treated celiacs showed an intermediate profile between active disease and controls. The saliva community mirrored the mucosal one better than stool. Conclusion: Expansion of pathobiontic species anticipates villous atrophy and achieves the maximal divergence from controls in refractory CD. Gluten-free diet results in incomplete recovery. The overlapping results between mucosal and salivary samples indicate the use of saliva as a diagnostic fluid.

The UK37′ index has proven to be a robust proxy to estimate past sea surface temperatures (SSTs) over a range of time scales, but like any other proxy, it has uncertainties. For instance, in reconstructions of the Last Glacial Maximum (LGM) in the northern North Atlantic, UK37′ indicates higher temperatures than those derived from foraminiferal proxies. Here we evaluate whether such warm glacial estimates are caused by the advection of reworked alkenones in ice-rafted debris (IRD) to deep-sea sediments. We have quantified both coccolith assemblages and alkenones in sediments from glaciogenic debris flows in the continental margins of the northern North Atlantic, and from a deep-sea core from the Reykjanes Ridge. Certain debris flow deposits in the North Atlantic were generated by the presence of massive ice-sheets in the past, and their associated ice streams. Such deposits are composed of the same materials that were present in the IRD at the time they were generated. We conclude that ice rafting from some locations was a transport pathway to the deep sea floor of reworked alkenones and pre-Quaternary coccolith species during glacial stages, but that not all of the IRD contained alkenones, even when reworked coccoliths were present. We speculate that the ratio of reworked coccoliths to alkenone concentration might be useful to infer whether significant reworked alkenone inputs from IRD did occur at a particular site in the glacial North Atlantic. We also observe that alkenones in some of the debris flows contain a colder signal than estimated for LGM sediments in the northern North Atlantic. This is also clear in the deep-sea core studied where the warmest intervals do not correspond to the intervals with large inputs of reworked coccoliths or IRD. We conclude that any possible bias to UK37′ estimates associated with reworked alkenones is not necessarily towards higher values, and that the high SST anomalies for the LGM are unlikely to be the result of a bias caused by IRD inputs.

Background: Adenosine affects the tone and reactivity of airways by activating specific membrane receptors, named A1, A2a, A2b and A3. It affects cellular activities either directly by regulating membrane ion exchanges and polarization, or indirectly by modifying neurotransmitter release. Objectives: We assessed the effect of A1 and A3 receptor activation on electrically induced nonadrenergic, noncholinergic (NANC) relaxations in the guinea pig isolated trachea and the localization of A1 and A3 receptors in tracheal inhibitory neurons. Methods: NANC responses at 3 Hz were evaluat- ed in the presence of 2-chloro-N6-cyclopentyladenosine (CCPA), a selective A1 agonist, and 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective A3 agonist, before and after the administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 antagonist, or 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino[1,2,4]triazolo[1,5-c])quinazoline (MRS 1220), a selective A3 antagonist, respectively. For immunohistochemistry, tissues were exposed to antibodies to HuC/D, a general neuronal marker, neuronal nitric oxide synthase (nNOS), and A1 or A3 adenosine receptors and processed by indirect immunofluorescence. Results: CCPA (10 nM–3 μM) inhibited NANC relaxations. DPCPX (10 nM) failed to antagonize the effect of CCPA, but inhibited per se NANC relaxations (range 0.1–100 nM). CCPA (10 nM–10 μM) contracted unstimulated tracheal preparations, an effect antagonized by 10 nM DPCPX, with a pKB value of 8.43. Cl-IB-MECA (10 nM–3 μM) inhibited NANC relaxations through a mechanism antagonized by MRS 1220 (100 nM). A1- and A3-positive neurons containing nNOS were detected in tracheal sections. Conclusions: Enogenous adenosine may induce airway hyperresponsiveness by inhibiting NANC relaxations via A1 and A3 receptors.

Proton pump inhibitors exert their preventive and healing effects on gastropathy induced by nonsteroidal anti-inflammatory drug (NSAIDs) by a dual action: the antisecretory and the antioxidant effect. The latter was investigated by using esomeprazole against indomethacin-induced gastric mucosa lesions in rats and assessed by a histomorphometric analysis. Treatment by intragastric gavage were 1% methocel as vehicle; esomeprazole 10, 30, or 60 micromol/kg; indomethacin 100 micromol/kg; and esomeprazole 10, 30, or 60 micromol/kg plus indomethacin 100 micromol/kg. The evaluation of glutathione (GSH) levels and respiratory chain complex activities [nicotinamide adenine dinucleotide, reduced (NADH)-ubiquinone oxidoreductase, succinate dehydrogenase, cytochrome C reductase, cytochrome oxidase] was performed in the isolated gastric mucosa. Esomeprazole (10-60 micromol/kg) dose dependently reversed, up to complete recovery, the inhibitory effect of indomethacin on GSH levels (approximately 60% inhibition) and mitochondrial enzyme activities (inhibition ranging from 60% to 75%). Indomethacin-induced mucosal injuries were reduced by esomeprazole. Thus, in addition to inhibiting acid secretion, the gastroprotective effect of esomeprazole can be ascribed to a reduction in gastric oxidative injury.

HISTORY An 81-year-old man, previously good health, suddenly developed confusion and rapidly progressive severe tetraparesis. INVESTIGATIONS Peripheral blood and bone marrow revealed marked eosinophilia: allergic, parasitic, neoplastic or vasculitic causes were excluded. Magnetic resonance imaging demonstrated multiple lesions in the cortical and subcortical white matter. DIAGNOSIS, TREATMENT AND COURSE The findings indicated idiopathic hypereosinophilic syndrome involving the central nervous system and treatment with high doses of glucocorticoids was started. After a stormy course almost complete recovery occurred. CONCLUSION Idiopathic hypereosinophilic syndrome can rarely manifest itself a an isolated severe subacute encephalopathy.

Background Constipation is extremely common in patients with Parkinson's disease (PD) and has been described in PD animal models. In this study, we investigated whether a PD-like degeneration of dopaminergic neurons of the substantia nigra can influence peristalsis in colonic segments of rats by impacting on enteric dopaminergic transmission. Methods Male, Sprague–Dawley rats received a unilateral injection of neurotoxin 6-hydroxydopamine (6-OHDA), or saline, into the medial-forebrain-bundle. Peristaltic activity was recorded in isolated colonic segments, in baseline conditions and following exposure to combinations of D2 receptor (DRD2) agonist sumanirole and antagonist L-741626. Dopamine levels and DRD2 expression were assessed in the ileum and colon of animals. We also investigated the involvement of the dorsal motor nucleus of the vagus (DMV) — a potential relay station between central dopaminergic denervation and gastrointestinal (GI) dysfunction — by analyzing cytochrome c oxidase activity and FosB/DeltaFosB expression in DMV neurons. Key Results We observed profound alterations in the response of colonic segments of 6-OHDA lesioned animals to DRD2 stimulation. In fact, the inhibition of colonic peristalsis elicited by sumanirole in control rats was absent in 6-OHDA-lesioned animals. These animals also showed reduced DRD2 expression in the colon, along with elevation of dopamine levels. No significant changes were detected within the DMV. Conclusions & Inferences Our results demonstrate that selective lesion of the nigrostriatal dopaminergic pathway subverts the physiological response of the colon to dopaminergic stimulation, opening new perspectives in the comprehension and treatment of GI dysfunctions associated with PD.

Background Mediators released in the mucosal milieu have been suggested to be involved in visceral hypersensitivity and abdominal pain in patients with irritable bowel syndrome (IBS). However, their impact on myenteric neurons remains unsettled. Methods Mucosal biopsies were obtained from the descending colon of patients with IBS and controls. Mucosal mast cells were identified immunohistochemically. The impact of spontaneously released mucosal mediators on guinea pig electrically stimulated longitudinal muscle myenteric plexus (LMMP) preparations was assessed in vitro by means of selective receptor antagonists and inhibitors. Key Results Patients with IBS showed an increased mast cell count compared with controls. Application of mucosal mediators of IBS to LMMPs potentiated cholinergic twitch contractions, an effect directly correlated with mast cell counts. Enhanced contractions were inhibited by 50.3% with the prostaglandin D2 antagonist BW A868C, by 31.3% and 39% with the TRPV1 antagonists capsazepine and HC-030031, respectively, and by 60.5% with purinergic P2X antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid. Conversely, the serotonin1-4, histamine1-3, tachykinin1-3 receptor blockade, and serine protease inhibition had no significant effect. Conclusions & Inferences Colonic mucosal mediators from patients with IBS excite myenteric cholinergic motor neurons. These effects were correlated with mast cell counts and mediated by activation of prostanoid receptors, TRPV1, and P2X receptors. These results support the role of mucosal inflammatory mediators and mast cell activation in altered motor function of IBS.

Background. The dopamine D 2 receptor antagonist levosulpiride is a substituted benzamide derivative, whose gastrokinetic properties are exploited clinically for the management of functional dyspepsia. However, for other benzamide derivatives, such as cisapride and mosapride, agonism towards serotonin 5-HT 4 receptors is considered the main mechanism leading to gastrointestinal prokinesia. Aims. To assess whether levosulpiride is able to activate 5-HT 4 receptors in the guinea-pig isolated gastrointestinal tract. Materials and methods. Circular muscle strips from gastric antrum, and colonic longitudinal muscle strips were used to detect electrically stimulated neurogenic contractions. The effect of levosulpiride was assessed in the absence and presence of GR125487, a selective 5-HT 4 receptor antagonist. Furthermore, potential interaction of levosulpiride with 5-HT 3 receptors and tissue cholinesterases was assessed in unstimulated ileal longitudinal muscle-myenteric plexus preparations. Results. Antral and colonic strip contractions were cholinergic/tachykinergic in nature. Micromolar concentrations of levosulpiride potentiated submaximal responses, through a mechanism competitively antagonized by GR125487 (p K B =9.4). In LMMPs, levosulpiride slightly affected contractions caused by the 5-HT 3 receptor agonist 2-methyl-5-HT, and had no effect on contractions to exogenous acetylcholine. Conclusions. Our results indicate that levosulpiride acts as a moderate agonist at the 5-HT 4 receptor. This property, together with antagonism at D 2 receptors, may contribute to its gastrointestinal prokinetic effect.

Patients with Parkinson’s disease develop motor disturbances often accompanied by peripheral autonomic dysfunctions, including gastrointestinal disorders, such as dysphagia, gastric stasis and constipation. While the mechanisms subserving enteric autonomic dysfunctions are not clearly understood, they may involve the enteric dopaminergic and/or nitrergic systems. In the present study, we demonstrate that rats with unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons develop a marked inhibition of propulsive activity compared to sham-operated controls, as indicated by a 60% reduction of daily fecal output at the 4th week of observation. Immunohistochemical data revealed that 6- hydroxydopamine treatment did not affect the total number of HuC/D-positive myenteric neurons in both the proximal and distal segments of ileum and colon. Conversely, in the distal ileum and proximal colon the number of nitrergic neurons was significantly reduced. These results suggest that a disturbed distal gut transit, reminiscent of constipation in the clinical setting, may occur as a consequence of a reduced propulsive motility, likely due to an impairment of a nitric oxide-mediated descending inhibition during peristalsis.

The most widely expressed neuronal nicotinic acetylcholine receptor subtype in chick brain is that containing the α4 and β2 subunits. However, immunoprecipitation and localization studies have shown that some brain areas also contain the α2 and/or α5 subunits, whose role in the definition of receptor properties is still intriguing. Using subunit-specific polyclonal antibodies, we found that the optic lobe is the chick central nervous system region that expresses the highest level of α2-containing receptors. Immunoprecipitation studies of these immunopurified α2-containing receptors labeled with the nicotinic agonist [ 3 H]epibatidine showed that almost all of them contained the β2 subunit and that more than 66% contained the α5 subunit. Western blot analyses of the purified receptors confirmed the presence of the α2, α5, and β2 subunits and the absence of the α3, α4, α6, α7, α8, β3, and β4 subunits. The α2-containing receptors are developmentally regulated: their expression increases 25 times from embryonic day 7 to posthatching day 1 in the optic lobe, compared with an increase of only 5-fold in the forebrain. The α2-containing optic lobe receptors bind [ 3 H]epibatidine ( K d = 29 pM) and a number of other nicotinic agonists with very high affinity and have a pharmacological profile very similar to that of the α4β2 subtype. They form functional cationic channels when reconstituted in lipid bilayers, with pharmacological and biophysical properties different from those of the α4β2 subtype. These channels are activated by nicotinic agonists in a dose-dependent manner and are blocked by the nicotinic antagonist d -tubocurarine.

1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

To investigate the presence of antibodies in myasthenia gravis (MG) patients, we have developed a new reproducible and sensitive enzyme immunoassay (EIA-AChR), in which a β subunit-specific monoclonal antibody (mAb 73) immobilizes fetal calf acetylcholine receptors (AChRs). We tested 92 MG patients (42 with positive and 50 with negative antibody titers), 60 healthy controls, and 40 controls with other autoimmune diseases. EIA-AChR detected immunoglobulin G (IgG) titers in all of the seropositive samples, with a significant correlation between these and those obtained using the traditional immunoprecipitation method. Moreover, 5 seronegative patients at immunoprecipitation assay were positive at EIA-AChR. EIA-AChR was also useful in revealing: (1) a seropositive patient subpopulation with generalized MG who had Abs directed against α-Bungarotoxin binding sites; and (2) patients with IgM directed against fetal calf AChR (detected in 13 seronegative and 16 seropositive MG patients, and in 6 of the patients with other autoimmune diseases). © 1997 John Wiley & Sons, Inc. Muscle Nerve20: 800–808, 1997

1. In isolated detrusor strips from the guinea-pig urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine (ACh) and adenosine 5'-triphosphate (ATP). 2. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent increase in the amplitude of stimulated detrusor strip contractions. The 5-HT concentration-response curve showed a biphasic profile: the high potency phase was obtained at sub-micromolar concentrations (10-300 nM), while the low potency phase in the range 1-30 microM. The maximum response of the first phase was 30% of the total 5-HT response. 3. Like 5-HT, the 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: 0.3-100 microM), the 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI: 30 nM-3 microM) and the 5-HT4 receptor agonist, 5-methoxytryptamine (5-MeOT: 0.1-30 microM) potentiated, though with lower potency, detrusor contractions. The resulting concentration-response curves were monophasic in nature. 2-Methyl-5-HT had a maximum effect comparable to that of 5-HT. By contrast, the maximal effects of DOI and 5-MeOT were only 20% and 30% of that elicited by 30 microM 5-HT, respectively. 4. The 5-HT3 receptor antagonist, granisetron (0.3 microM) had no effect on the high potency phase, but caused a rightward parallel shift of the low potency phase of the 5-HT curve (pKB = 7.3). Granisetron(0.3 microM) antagonized with comparable affinity (pKB = 7.1) 5-HT-induced responses after pharmacological isolation of 5-HT3 receptors with the 5-HT1/5-HT2 receptor antagonist, methiothepin (0.3 microM) and the 5-HT4 receptor antagonist, GR 125487 (30 nM). Granisetron (0.1, 0.3 and 1 microM) competitively antagonized the potentiating effect of 2-methyl-5-HT with an estimated pA2 of 7.3.5. Methiothepin (0.3 microM) and the 5-HT2A receptor antagonist, ketanserin (0.3 microM) produced a slight inhibition of the first phase of the 5-HT curve. In the presence of ketanserin, an equimolar concentration of methiothepin was ineffective in further reducing the effect of 5-HT. Similarly, the 5-HT4 receptor antagonist, GR 125487 (30 nM) slightly inhibited the first phase of the 5-HT curve. Conversely, this phase was suppressed when detrusor strips were coincubated with ketanserin (or methiothepin) and GR125487.6. In a separate set of experiments, the interactions of 5-HT with either the purinergic or cholinergic components of excitatory neuromuscular transmission were investigated. In the presence of hyoscine(1 microM), 5-HT was mostly effective at sub-micromolar concentrations, while in the presence of the P2-purinoceptor antagonist, suramin (300 microM), 5-HT-induced potentiation was mainly obtained with micromolar concentrations.7. Thus, in electrically stimulated detrusor strips from guinea-pig, 5-HT potentiated excitatory neuromuscular transmission by activating at least three separate neural 5-HT receptors. These include the 5-HT2A and 5-HT4 receptors, which mediate the 5-HT high potency phase mainly by activation of purinergic transmission. On the other hand, the potentiating effect caused by micromolar concentrations of 5-HT mostly involves cholinergic transmission and is mediated by the 5-HT3 receptors.

Chick central nervous system (CNS) expresses alpha-bungarotoxin (alpha Bgtx) receptors. We have recently reported the purification and characterization of two alpha Bgtx receptor subtypes, alpha 7 and alpha 7-alpha 8 from chick optic lobe (COL). In order to study whether other alpha Bgtx receptor subtypes are present in other areas of the chick CNS, as well as their developmental expression, we used anti-alpha 7 and anti-alpha 8 subunit-specific antibodies to study alpha Bgtx receptors at different developmental stages in COL, brain and retina. We found that only the alpha 7 and alpha 7-alpha 8 subtypes are present at all developmental stages in chick COL and brain, where they represent 90% of all the alpha Bgtx receptors at embryonic day 19 and 1 day post hatching (D1). In chick retina, an alpha 8 subtype representing 50% of all alpha Bgtx receptors at D1 is present in addition to the alpha 7 and alpha 7-alpha 8 subtypes, and the expression of this alpha 8 subtype increases during neurodevelopment.

To evaluate the effect of ammonium persulphate (AP) inhalation on NANC inhibitory (i-NANC) neurotransmitters of guinea pig airways, we exposed eight guinea pigs to AP (1 mg/m3), by aerosol inhalation for 30 minutes daily for three weeks. Control animals inhaled saline aerosol. After the last exposure, the isolated trachea was mounted in an organ bath and electrically stimulated in the presence of hyoscine, piperoxane and propranolol. The i-NANC responses were evaluated as decreases in intraluminal pressure and expressed as area under the curve (AUC, Pa x seconds). The isolated tracheae were treated with a-chymotrypsin, L-NAME, zinc protoporphyrin IX and ODQ, that inhibit the production or action of the single neurotransmitters, like peptides, NO and CO. In the exposed individuals, the NANC relaxations were below 50%, as compared to controls (P0.01). NO and CO were the neurotransmitters responsible for all the i-NANC responses, in similar proportions either in exposed individuals or in controls. In conclusion, ammonium persulphate exposure impairs the i-NANC control of airway tone without specifically affecting any neurotransmitter.

Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.

A case of focal myositis in a healthy 68-year-old woman is described. The patient was admitted for evaluation of a painful soft-tissue mass localised on the medial side of the left thigh, initially misdiagnosed as thrombophlebitis of the v. saphena magna. Laboratory data were normal, in particular sedimentation rate and muscle enzyme levels. After exclusion of venous thrombosis, the mass localised in the left m. gracilis was surgically removed. Histologic examination of the biopsy specimen showed muscle cell necrosis and severe inflammation, with lymphocytic infiltration leading to the diagnosis of focal myositis. This is a rare benign inflammatory pseudotumour of skeletal muscle. The aetiology and pathogenesis of the disease remain unclear. It is most commonly seen in the lower extremities and may mimic thrombophlebitis or soft-tissue neoplasm. Ultrasound and magnetic-resonance scans are helpful, but definitive diagnosis is obtained only by histology. Because recurrent lesions in other skeletal muscles are possible, and a third of patients develop polymyositis, a follow-up of several years is recommended.

The most widely expressed neuronal nicotinic acetylcholine receptor subtype in chick brain is that containing the alpha4 and beta2 subunits. However, immunoprecipitation and localization studies have shown that some brain areas also contain the alpha2 and/or alpha5 subunits, whose role in the definition of receptor properties is still intriguing. Using subunit-specific polyclonal antibodies, we found that the optic lobe is the chick central nervous system region that expresses the highest level of alpha2-containing receptors. Immunoprecipitation studies of these immunopurified alpha2-containing receptors labeled with the nicotinic agonist [(3)H]epibatidine showed that almost all of them contained the beta2 subunit and that more than 66% contained the alpha5 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha2, alpha5, and beta2 subunits and the absence of the alpha3, alpha4, alpha6, alpha7, alpha8, beta3, and beta4 subunits. The alpha2-containing receptors are developmentally regulated: their expression increases 25 times from embryonic day 7 to posthatching day 1 in the optic lobe, compared with an increase of only 5-fold in the forebrain. The alpha2-containing optic lobe receptors bind [(3)H]epibatidine (K(d) = 29 pM) and a number of other nicotinic agonists with very high affinity and have a pharmacological profile very similar to that of the alpha4beta2 subtype. They form functional cationic channels when reconstituted in lipid bilayers, with pharmacological and biophysical properties different from those of the alpha4beta2 subtype. These channels are activated by nicotinic agonists in a dose-dependent manner and are blocked by the nicotinic antagonist d-tubocurarine.

We report the case of a severe relapsing phenprocoumon-induced hepatitis. The first episode of hepatitis was thought to be caused by another drug (Verapamil). The anticoagulation with Phenprocoumon was therefore continued after healing of liver inflammation. The relapse typically developed after a shorter exposition-time supporting the hypothesis of an allergic etiology. Fortunately we didn't find any cross-reaction between Phenprocoumon and Acenocoumarol. The patient could thus be anticoagulated orally without complications. If long term anticoagulation is absolutely essential, it is reasonable to prescribe a different Coumarin-derivate. In the case of a cross-reaction, the therapy should be continued with low-molecular weight heparin.

Organotypic cultures and ileal neuromuscular preparations were used to determine (i) whether endogenous release of opioids by electrical stimulation induces mu receptor endocytosis, and (ii) whether and under which conditions ligand-induced mu receptor endocytosis influences the responsiveness of neurons expressing native mu receptors. In longitudinal muscle-myenteric plexus preparations, electrical stimulation at 20 Hz induced a prominent endocytosis of mu receptors in enteric neurons, indicating endogenous release of opioids. A similar massive endocytosis was triggered by exogenous application of the mu receptor agonist, [D-Ala(2),MePhe(4), Gly-ol(5)] enkephalin, whereas exogenous application of morphine was ineffective. [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin and morphine induced a concentration-dependent inhibition of neurogenic cholinergic twitch contractions to electrical stimulation at 0.1 Hz. beta-Chlornaltrexamine shifted to the right the inhibitory curve of both agonists with a concentration-dependent reduction of the maximum agonist response, which is consistent with the existence of spare mu opioid receptors. Under these conditions, the induction of mu receptor endocytosis by exogenously applied [D-Ala(2), MePhe(4),Gly-ol(5)] enkephalin diminished the inhibitory effect of this agonist on twitch contractions and tritiated acetylcholine release. In contrast, there was no reduction of the inhibitory effect of morphine, which failed to induce mu receptor endocytosis, on neurogenic cholinergic response. These results provide the first evidence for the occurrence of mu receptor endocytosis in neurons by endogenously released opioids and show that agonist-dependent mu receptor endocytosis could serve as a mechanism to regulate mu opioid receptor responsiveness to ligand stimulation when the opioid receptor reserve is reduced.

The case of a 25 year old women with isolated inguinal lymphadenopathy and persistent fever caused by necrotizing inguinal lymphadenitis Kikuchi-Fujimoto is reported. Diagnostic and differential diagnostic aspects of this self-limited disorder with unclear pathogenesis are discussed.

Although the neuronal nicotinic beta3 subunit was cloned several years ago, it has only recently been shown to form heteromeric channels when associated with other nicotinic subunits, and very little information is available concerning its assembly in the native nicotinic receptors of the nervous system. Using subunit-specific antibodies and immunoprecipitation experiments, we have identified the retina as being the chick central nervous system (CNS) area that expresses the highest level of the beta3 subunit. Sequential immunopurification experiments showed that there are at least two populations of beta3-containing receptors in chick retina: in one, the beta3 subunit is associated with the alpha6 and beta4 subunits; in the other more heterogeneous population, the beta3 subunit is associated with the alpha2, alpha3, alpha4, beta2 and beta4 subunits. Both of these receptor populations bind [3H]epibatidine and a number of nicotinic receptor agonists with high affinity (nM) and nicotinic receptor antagonists with a lower affinity (microM). The greatest pharmacological difference between the two populations is the affinity for the alpha-conotoxin MII, which inhibits binding to alpha6-containing receptors and not that to beta3-containing receptors. We also searched for the presence of the beta3 subunit associated with the alpha-bungarotoxin binding subunits alpha7 and/or alpha8 in retina and chick brain. Immunoprecipitation studies using anti-beta3 antibodies did not detect any specific alpha-bungarotoxin labeled receptors, thus, indicating that the beta3 subunit is not present in the alpha-bungarotoxin receptors of these areas.

Despite the fact that the neuronal chick alpha6 subunit was first cloned several years ago and recently has been shown to form acetylcholine (ACh)-activated channels in heterologous systems, no information is yet available concerning the structure and function of the alpha6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick alpha6 subunit, we performed immunoprecipitation experiments on immunopurified alpha6-containing receptors radiolabeled with the nicotinic agonist [3H]epibatidine (Epi): almost all of the alpha6 receptors contained the beta4 subunit, 51% the beta3 subunit, 42% the alpha3 subunit, and 7.5% the beta2 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha3, beta3, beta2, and beta4 subunits, and the absence of the alpha4, alpha5, and alpha7 subunits. The alpha6-containing receptors bind [3H]Epi (Kd = 35 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epicytisinenicotine1, 1-dimethyl-4-phenylpiperaziniumacetylcholinecarbamylcholine. The alpha6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of alpha-conotoxin MIImethyllycaconitinedihydro-beta-erythroydineMG624d-tubocurarinedecamethoniumhexamethonium. When reconstituted in lipid bilayers, the alpha6-containing receptors form functional cationic channels with a main conductance state of 48 pS. These channels are activated by nicotinic agonists in a dose-dependent manner, and blocked by the nicotinic antagonist d-tubocurarine.

An 81-year-old man, previously good health, suddenly developed confusion and rapidly progressive severe tetraparesis.Peripheral blood and bone marrow revealed marked eosinophilia: allergic, parasitic, neoplastic or vasculitic causes were excluded. Magnetic resonance imaging demonstrated multiple lesions in the cortical and subcortical white matter.The findings indicated idiopathic hypereosinophilic syndrome involving the central nervous system and treatment with high doses of glucocorticoids was started. After a stormy course almost complete recovery occurred.Idiopathic hypereosinophilic syndrome can rarely manifest itself a an isolated severe subacute encephalopathy.

1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosis.

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central mu-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM - 1 microM) inhibited (-log EC50 values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), caused a concentration-dependent (30 nM-1 microM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA2 values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective delta-receptor antagonist) or 30 nM nor-binaltorphimine (a selective kappa-receptor antagonist). These results demonstrate that endomorphins selectively activate mu-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

This article is meant to increase the interest in an often forgotten clinical entity. Cholesterol emboli are in the majority of cases only diagnosed at post-mortem examination. Even though the triad livedo reticularis, renal failure and eosinophilia constitutes its most prominent feature, the variable clinical manifestations of this disorder with multiorgan involvement ("pseudovasculitis") make the search for cholesterol crystals particularly exciting. The discovery of 10 cases of cholesterol emboli over 2 years in a regional hospital's internal medicine department demonstrates that this occurrence is not rarely and that its accurate identification can be particularly relevant. It is important to recognize this disease since it is often iatrogenic, affects elderly people with atherosclerosis of the large vessels and causes high morbidity and mortality.

In the medical unit of our regional hospital we observed eight cases of anticoagulant-related pseudothrombocytopenia within two years. Attention is drawn to this often misdiagnosed laboratory artefact. In every unexpected thrombocytopenia found by automatic cell-counting a simple blood smear should be studied especially for thrombocyte-aggregates and the automatic platelet-count should be repeated using another anticoagulant, before other investigations are undertaken.

In whole segments of rabbit distal colon with mucosa removed, descending reflex relaxations of the circular muscle (descending inhibition) elicited by inflating (0.1-1 ml) an intraluminal balloon, were partially antagonized by 100 microM hexamethonium and the 5-HT3 receptor antagonist, ondansetron (3 microM), and abolished by 1 microM tetrodotoxin. The inhibitory effects of hexamethonium and ondansetron were additive. Conversely, hexamethonium (100 microM) and ondansetron (3 microM) failed to reduce electrically induced non-adrenergic non-cholinergic (NANC) relaxations of colonic circular muscle. It is concluded that interneuronally released acetylcholine and 5-hydroxytryptamine (5-HT) activate descending inhibitory pathways supplying the circular coat, via nicotinic and 5-HT3 receptors, respectively. This evidence suggests a functional involvement of 5-hydroxytryptaminergic transmission in the descending inhibition of rabbit colon.

IMR32 cells express two classes of surface nicotinic receptors: those labelled with high affinity by [125I]neuronal toxin, and those labelled by [125I]alpha-bungarotoxin. Whole-cell patch-clamp recordings indicate that both classes of receptor are able to elicit inward currents that are totally blocked by d-tubocurarine but only partially blocked by alpha-bungarotoxin. In IMR32 cells, nicotine induces an increase in the intracellular level of free Ca2+. This increase, which is also completely blocked by d-tubocurarine and only partially blocked by alpha-bungarotoxin and Cd2+, is due to extracellular calcium influx through both the nicotinic receptors and the voltage-activated Ca2+ channels. By using subunit-specific polyclonal antibodies, we have demonstrated that the alpha-bungarotoxin receptors contain the alpha 7 subunit, but none of the other subunits whose transcripts are present in IMR32 cells. The pharmacological profile of these human alpha 7-containing alpha-bungarotoxin receptors is similar to that observed in the native chick alpha 7 receptor, but there are also some species-specific differences.

Skin necrosis is an uncommon complication associated with coumarin or heparin therapy. The gross appearance and histology of the lesions--a paradoxical thrombosis of skin vessels--are identical. However, skin necrosis is the symptom of two different disorders. Coumarin necrosis may be related to reduced levels of protein C or S during the early stages of anticoagulation. It is possible to continue the warfarin therapy, simultaneously with heparin, if further anticoagulation is still required. In contrast, heparin necrosis is a manifestation of the potentially fatal heparin-associated thrombocytopenia and thrombosis syndrome (HATT). Immediate discontinuation of heparin is mandatory.

We used anti-α7 and anti-α8 subunit specific antibodies to study a-bungarotoxin (α-BTX) receptors at different developmental stages in chick optic lobe and retina. We found that only the α7 and α7-α8 subtypes are present at all developmental stages in chick optic lobe, whereas in addition to these two subtypes, chick retina contains an α8 subtype representing 50% of all α-BTX receptors at 1 day post hatching. By using an immunoaffinity column, we purified these subtypes and studied their biochemical and functional properties by reconstituting them in planar lipid bilayer. The a7 and α7–α8 receptor subtypes are made up of two subunits: a 57000 dalton subunit which binds α-BTX, and a 52000 dalton subunit whose function and structure is unknown. The three subtypes differ in pharmacological and biophysical properties.

Background Mediators released in the mucosal milieu have been suggested to be involved in visceral hypersensitivity and abdominal pain in patients with irritable bowel syndrome (IBS). However, their impact on myenteric neurons remains unsettled. Methods Mucosal biopsies were obtained from the descending colon of patients with IBS and controls. Mucosal mast cells were identified immunohistochemically. The impact of spontaneously released mucosal mediators on guinea pig electrically stimulated longitudinal muscle myenteric plexus (LMMP) preparations was assessed in vitro by means of selective receptor antagonists and inhibitors. Key Results Patients with IBS showed an increased mast cell count compared with controls. Application of mucosal mediators of IBS to LMMPs potentiated cholinergic twitch contractions, an effect directly correlated with mast cell counts. Enhanced contractions were inhibited by 50.3% with the prostaglandin D2 antagonist BW A868C, by 31.3% and 39% with the TRPV1 antagonists capsazepine and HC-030031, respectively, and by 60.5% with purinergic P2X antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid. Conversely, the serotonin1-4, histamine1-3, tachykinin1-3 receptor blockade, and serine protease inhibition had no significant effect. Conclusions & Inferences Colonic mucosal mediators from patients with IBS excite myenteric cholinergic motor neurons. These effects were correlated with mast cell counts and mediated by activation of prostanoid receptors, TRPV1, and P2X receptors. These results support the role of mucosal inflammatory mediators and mast cell activation in altered motor function of IBS.

We attribute a case of acute irreversible renal failure associated with "blue toe syndrome" and eosinophilia to a cholesterol embolism after "successful" treatment of myocardial infarction with fibrinolysis. This case shows that CE can be caused not only by invasive arterial procedures but also by thrombolytic as well as by anticoagulant treatment. In modern medicine, the importance of this often fatal but usually undetected systemic affection is increasing. For patients with serious atherosclerosis it is essential to analyze the risks and benefits before undertaking arterial invasive procedures or fibrinolysis.

A well documented and histologically proven case of cholesterol embolism is discussed, and the recent literature is reviewed. This disorder is usually underdiagnosed and commonly detected only at autopsy. Elderly people with atherosclerotic vascular disease are predominantly affected. Cholesterol embolization can occur spontaneously, but it often results from medical interventions such as arterial invasive procedures, vascular surgery, anticoagulation or thrombolytic therapy. Clinical manifestations are manifold, and two distinct patterns are generally observed: a mild peripheral cutaneous form and a severe visceral form that frequently mimics other systemic diseases. Transient eosinophilia is an important laboratory finding, and it is present in about 80% of the cases. Cholesterol crystals are rarely found in retinal arteries, and premortem diagnosis is established most commonly by biopsy of the muscle, skin or kidney. The role of various therapeutic modalities is still controversial and does not seem to change the course of this frequently fatal disease. The treatment is symptomatic, and the surgical correction of the embolic source is recommended only in the case of peripherally embolizing stenotic lesions. The most effective measures are prevention and the identification of patients at risk. In these patients the aforementioned precipitating events should be avoided, or the potential risk must be carefully weighed against the possible benefits in this particularly fragile group of patients.