Hypertonic saline releases the attached small intestinal cystic fibrosis mucus

Summary: Hypertonic saline inhalation has become a cornerstone in the treatment of cystic fibrosis (CF), but its effect on CF mucus is still not understood. In CF, mucus stagnates in the airways, causing mucus plugging, and forming a substrate for bacterial invasion. Using horizontal Ussing-type chambers to allow easy access to the tissue, we have recently shown that the small intestinal mucus of CF mice is attached to the epithelium and not freely movable as opposed to normal mucus, thus pointing to a similarity between the CF mucus in the ileum and airways. In the same type of system, we investigated how hypertonic saline affects mucus thickness, attachment and penetrability to fluorescent beads the size of bacteria in ileal explants from the cystic fibrosis transmembrane conductance regulator mutant (ΔF508) mouse, in order to characterize how this common therapy affects mucus properties. Hypertonic saline (1.75-5%) detached the mucus from the epithelium, but the mucus remained impenetrable to beads the size of bacteria. This approach might be used to test other mucolytic interventions in CF

New Generation ENaC Inhibitors Detach Cystic Fibrosis Airway Mucus Bundles via Sodium/Hydrogen Exchanger Inhibition

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Mucus detachment by host metalloprotease meprin \beta requires shedding of its inactive pro-form, which is abrogated by the pathogenic protease RgpB

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease

TRPV1 in Brain Is Involved in Acetaminophen-Induced Antinociception

Background: Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular overthe- counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV1) in vitro. Pharmacological activation of TRPV1 in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV1 in the brain contributes to the analgesic effect of acetaminophen. Methodology/Principal Findings: Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV1 knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E-2 (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV1-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV1 in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test. Conclusions: This study shows that TRPV1 in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV1 in the brain

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn=3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics simulations (MDS), Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (>0.5%) inhibited biofilm formation, demonstrating a significant reduction in both biomass and biofilm height (17.8 vs. 5.5 µm; P <0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of sugar residues, and extracellular (e)DNA (P <0.05) with a corresponding increase in nanoparticle diffusion (P<0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MDS. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections

Design and modelling of an active balancing device for washing machines

In this thesis the possibility of actively balancing the spin drum in a washing machine is examined. Due to the fact that the unbalance in washing machines is highly variable, e.g. by water leaving the laundry during spin drying, active balancing is required where the balancing system continuously is changing according to how much unbalance there is to counterbalance. The idea is to use a concept which with the help of an eccentric mechanism displaces the spin drum during spin drying, thus counteract the unbalance which arises when the laundry has been unevenly distributed before spin drying. In the thesis a preliminary study in rotor dynamics has been done to examine what kind of different techniques there are for active balancing whereupon a development of other concepts to be compared to the eccentric mechanism was done. Primarily to find out if the concept already exists but also to examine if there are other concepts that could be comparable to or even better than the eccentric mechanism. The eccentric mechanism was considered to be the most potential concept. A design including the mechanical parts of the eccentric mechanism has been dimensioned to be able to use in a physical prototype and to make it possible to execute a spin drying speed of 2000 rpm without excessive vibrations. The eccentric mechanism was modelled in Pro/ENGINEER and its behaviour has been studied in a computerised model of ASKO Cylinda washing machine in ADAMS/View. Through analysis in the virtual model it was found that the model is comparable to a physical machine and the vibrations caused by the unbalance can be strongly reduced by the use of the eccentric mechanism

Bioactive Lipids in Nociception

This thesis focuses on bioactive lipids as (1) metabolites of the widely used antipyretic and analgesic drug acetaminophen and (2) activators of the ion channel TRPV1, an important downstream target for inflammatory mediators, in the phospholipase C (PLC)/TRPV1 signaling pathway. Evidence is presented for a fatty acid amide hydrolase (FAAH)-dependent fatty acid conjugation of p-aminophenol, a known acetaminophen metabolite, to form the potent TRPV1 activator AM404 in the central nervous system. We show that acetaminophen is able to reduce the content of prostanoids in brain and kidney, but this effect is independent of FAAH. While ibuprofen produces a similar reduction as acetaminophen of the PGE2 level in brain, only acetaminophen displays antinociceptive activity in animal tests of acute non-inflammatory pain, indicating that other mechanisms than central cyclooxygenase inhibition must be sought to explain the analgesic effect of acetaminophen in these tests. Knowledge of the mechanisms of action of acetaminophen may help to develop analogs with improved analgesic activity. Many TRP ion channels, including TRPV1, are regulated by PLC-coupled surface receptors. After PLC-dependent cleavage of phosphatidylinositol bisphosphate into diacylglycerol (DAG) and inositoltrisphosphate, diacylglycerol may be further metabolized to monoacyl-glycerols, such as 2-arachidonoylglycerol (2-AG) and 2-oleoylglycerol by the action of DAG lipase. 2-Arachidonoylglycerol and 1-AG activate both native TRPV1 on sensory nerve fibers in rodent mesenteric arteries and heterologously expressed rat and human TRPV1. The effects of 2-AG and the metabolically stable analog noladin ether were almost absent in TRPV1 gene-deficient mice. Stimulation of isolated rat dorsal root ganglia with the inflammatory mediators bradykinin and ATP led to an increase in the level of 2-AG, whereas the levels of anandamide and 2-oleoylglycerol were unaffected. Extensive metabolism of d8-2-AG and d5-1-AG, in contrast to d8-anandamide, was demonstrated in homogenates of rat mesenteric arteries. The monoacylglycerol lipase inhibitor MAFP prevented the metabolism of these lipids and enhanced the TRPV1-mediated vascular effects of 2-AG and 1-AG, but not anandamide. The existence of a regulated biosynthesis and enzymatic degradation of 2-AG and 1-AG in TRPV1-containing tissues is compatible with a physiological role for these monoacylglycerols as messengers in the PLC/TRPV1 signaling cascade. Bioactive lipids, with TRPV1 activity, can be formed via drug metabolism and after stimulation of PLC-coupled surface receptors