EP4 receptor stimulation down-regulates human eosinophil function.

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE(2) receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca(2+) mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE(2) and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.

Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes.

Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

Chitosan-graft-6-mercaptonicotinic acid: synthesis, characterization, and biocompatibility.

Thiolated chitosans are relatively new thiolated biopolymers exhibiting mucoadhesive, enzyme inhibitory, and permeation enhancing properties. A drawback, however, is their pH dependent reactivity. The aim of this study was therefore to develop a novel thiolated chitosan showing a non pH dependent reactivity of its thiol groups. For this purpose, 6-mercaptonicotinic acid (6-MNA) was covalently attached to chitosan by a carbodiimide mediated reaction. The obtained conjugate was characterized in vitro by quantification of immobilized thiol groups, cytotoxicity, in situ gelling properties, and disulfide bond formation at different pH. The synthesized conjugate displayed up to 973.80 micromol thiol groups per gram of polymer in reduced form. The polymer was nontoxic and showed in situ gelling properties. Furthermore, disulfide bond formation and therefore gelling properties occurred at various pH ranges. The reactivity of thiol groups was in the same range at pH 3 and pH 6.8. According to these results, chitosan-6 mercaptonicotinic acid seems to have some promising features to be used particularly for mucoadhesive formulations.

Cytotoxicity of nanoparticles independent from oxidative stress.

The use of nano-sized materials offers exciting new options in technical and medical applications. On the other hand, adverse effects on cells have been reported and may limit their use. In addition to physico-chemical parameters such as contamination with toxic elements, fibrous structure and high surface charge, the generation of radical species was identified as key mechanism for cytotoxic action of nanoparticles. The cytotoxic potential of nanoparticles in the absence of radical generation is less well investigated. This study aims to investigate the size-dependent effect of carboxyl polystyrene particles on cells to identify potential adverse effects of these particles. Particles were characterized in different solutions to assess the influence of the medium on size and surface charge. Viability, membrane integrity, apoptosis, proliferation and generation of oxidative stress were investigated. In addition the intracellular localization of the particles was recorded. 20 nm polystyrene particles induced cellular damage by induction of apoptosis and necrosis. These particles generated radicals to the same degree as larger polystyrene particles. Particles were taken up into endosomes and lysosomes in a size-dependent manner. Protein containing solutions led to increases in particle size, decreased cytotoxicity and reduced cellular uptake. It can be concluded that even in the absence of high surface reactivity and not linked to the generation of radicals nano-sized particles may cause cell damage. The mechanism of this damage includes apoptosis, necrosis and inhibition of proliferation.

Immunocytochemical characterization of rat brainstem neurons with vagal afferent input from the stomach challenged by acid or ammonia.

Exposure of the gastric mucosa to backdiffusing acid is signalled to the brainstem via vagal afferents. This study examined whether exposure of the Sprague-Dawley rat stomach to hydrochloric acid (HCl) or ammonium hydroxide (NH4OH), a noxious chemical produced by Helicobacter pylori, activates different vagal afferent pathways as reflected by different circuitries in the medullary brainstem. Two hours after intragastric treatment with HCl or NH4OH the activation of neurons in the nucleus tractus solitarii at the rostrocaudal extension of the area postrema (NTSAP) was visualized by c-Fos immunohistochemistry and their chemical coding characterized by double-labelling immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH4OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTSAP. The number and distribution of NTSAP neurons activated by 0.35 M HCl and 0.3 M NH4OH were similar; the highest number of activated neurons occurring in the medial part of the NTSAP. Some 60% of the NTSAP neurons activated by intragastric HCl and NH4OH stained for the high affinity glutamate transporter EAAC1, while some 30% contained calbindin or neuropeptide Y. Glutamate receptors of the N-methyl-D-aspartate type were found on approximately 50% of the c-Fos-positive cells in the NTSAP, whereas tachykinin NK1, NK2 and NK3 receptors were present on 5-10% of the activated neurons. The similar number and distribution of c-Fos-expressing neurons within the NTSAP and their identical chemical coding indicate that exposure of the rat stomach to backdiffusing concentrations of HCl and NH4OH activates the same vagal afferent-NTSAP pathway.

Stomach-brain communication by vagal afferents in response to luminal acid backdiffusion, gastrin, and gastric acid secretion.

Vagal afferents play a role in gut-brain signaling of physiological and pathological stimuli. Here, we investigated how backdiffusion of luminal HCl or NH(4)OH and pentagastrin-stimulated acid secretion interact in the communication between rat stomach and brain stem. Rats were pretreated intraperitoneally with vehicle or appropriate doses of cimetidine, omeprazole, pentagastrin, dexloxiglumide (CCK(1) receptor antagonist), and itriglumide (CCK(2) receptor antagonist) before intragastric administration of saline or backdiffusing concentrations of HCl or NH(4)OH. Two hours later, neuronal activation in the nucleus of the solitary tract (NTS) and area postrema was visualized by c-Fos immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH(4)OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTS, which was not related to gender, gastric mucosal injury, or gastropyloric motor alterations. The c-Fos response to HCl was diminished by cimetidine and omeprazole, enhanced by pentagastrin, and left unchanged by dexloxiglumide and itriglumide. Pentagastrin alone caused an omeprazole-resistant expression of c-fos, which in the NTS was attenuated by itriglumide and prevented by dexloxiglumide but in the area postrema was reduced by dexloxiglumide and abolished by itriglumide. We conclude that vagal afferents transmit physiological stimuli (gastrin) and pathological events (backdiffusion of luminal HCl or NH(4)OH) from the stomach to the brain stem. These communication modalities interact because, firstly, acid secretion enhances afferent signaling of gastric acid backdiffusion and, secondly, gastrin activates NTS neurons through stimulation of CCK(1) receptors on vagal afferents and of CCK(2) receptors on area postrema neurons projecting to the NTS.