CFTR-beyond the airways : Recent findings on the role of the CFTR channel in the pancreas, the intestine and the kidneys

With increased longevity of patients suffering from cystic fibrosis, and widespread lung transplantation facilities, the sequelae of defective CFTR in other organs than the airways come to the fore. This minireview highlights recent scientific progress in the understanding of CFTR function in the pancreas, the intestine and the kidney, and explores potential therapeutic strategies to combat defective CFTR function in these organs

The C-terminus of the transmembrane MUC17 mucin binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine

The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes

cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells

<p>Abstract</p> <p>Background</p> <p>The renal (kNBC1) and intestinal (pNBC1) electrogenic Na⁺/HCO₃^-cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. </p> <p>Results</p> <p>Na⁺- and HCO₃^--dependent pH_irecovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO₂/HCO₃^-. Forskolin (10^-5M) had no effect in untransfected cells, but inhibited the pH_irecovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10^-4M), the pH_irecovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na⁺/HCO₃^-cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. </p> <p>Conclusion</p> <p>Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.</p

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells

International audienceA detrimental perceptive consequence of damaged auditory sen-sory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1 −/− mice dis-played OHC hair-bundle shape anomalies in the mid and basal co-chlea, normally tuned to mid-and high-frequency tones, and mild (22–35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the co-chlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1 −/− mice to high-frequency (20–40 kHz

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine

The Ca2+ activated Cl− channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively

Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ('lipid rafts') were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3 - mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs

Разработка мероприятий по улучшению условий труда на примере предприятия ООО "Юргинский машиностроительный завод"

Abstract IL-6 is known to play a crucial role in the pathogenesis of chronic intestinal inflammation by modulating T cell functions. In this study, we investigated the role of gp130, the common signal transducer for all IL-6 cytokines, in a murine model of acute T cell independent colitis to better characterize the impact of gp130 on innate immune cells and the early stages of inflammation. Experimental colitis was induced by dextran sulfate sodium treatment of mice with inducible systemic deletion of gp130 (MxCre/gp130−/−), macrophage/neutrophil-specific gp130-deficiency (LysCre/gp130−/−), or bone marrow chimeric mice and compared with wild-type controls (gp130f/f). Systemic deletion of gp130 (MxCre/gp130−/−) protected mice from severe colitis and wasting and attenuated the mucosal inflammatory infiltrate as well as local cytokine, chemokine, and adhesion molecule expression. Experiments in newly generated macrophage/neutrophil-specific gp130-deleted animals (LysCre/gp130−/−) and gp130 bone marrow chimeric mice, revealed a dual mechanism of proinflammatory effects mediated by gp130. Leukocyte recruitment was impaired in gp130-deleted animals and gp130-deleted recipients of wild-type bone marrow, demonstrating a central role of gp130-dependent signals in nonmyeloid cells for directing leukocytes to sites of inflammation, which was further confirmed in a model of sterile peritonitis. In contrast, macrophage/neutrophil-specific gp130 deficiency delayed and attenuated the disease but only marginally affected the inflammatory infiltrate, indicating a defective activation of mucosal leukocytes. We provide evidence that IL-6 cytokines acting via gp130 are required in the acute stages of intestinal inflammation by modulating the dynamics of innate immune cell recruitment and activation.</jats:p

Open-label extension of a phase 2 trial of risankizumab in patients with moderate-to-severe Crohn's disease

Background: Risankizumab, an anti-interleukin-23 antibody, was superior to placebo in achieving clinical and endoscopic remission at week 12 in a randomised, phase 2 induction study in patients with moderately to severely active Crohn’s disease. The efficacy and safety of extended intravenous induction and/or subcutaneous maintenance therapy with risankizumab was assessed. Methods: Following 12-week, double-blind, randomised, induction treatment comparing 200 mg or 600 mg intravenous risankizumab to placebo every 4 weeks, patients without deep remission, defined as clinical (Crohn’s Disease Activity Index <150) and endoscopic remission (Crohn’s Disease Endoscopic Index of Severity [CDEIS] ≤4 [≤2 for patients with isolated ileitis]), received open-label 600 mg intravenous risankizumab (every 4 weeks) and patients in deep remission underwent washout until week 26 (Period 2). At week 26, patients in clinical remission received maintenance treatment (Period 3) with 180 mg subcutaneous risankizumab (every 8 weeks). Efficacy endpoints included clinical and endoscopic response and remission at weeks 26 (Period 2) and 52 (Period 3) respectively; safety was assessed through both periods. Study registration: ClinicalTrials.gov, NCT02031276. Findings: In Period 2, 101 patients were treated with 600 mg risankizumab resulting in an increase in clinical remission rates at week 26 versus week 12 for all original designated treatment groups: 55% versus 18%, 59% versus 21%, and 47% versus 26% for placebo, 200, and 600 mg risankizumab, respectively. Of the 62 patients receiving maintenance treatment, 54 completed treatment. At week 52, clinical remission was maintained by 71% of patients; endoscopic remission and response (>50% CDEIS reduction from baseline) was achieved by 35% and 55% of patients, respectively, and 29% of patients achieved deep remission. Risankizumab was well tolerated with no new safety signals. Interpretation: Extended induction treatment with open-label intravenous risankizumab was effective in increasing clinical response and remission rates at week 26. Open-label subcutaneous risankizumab maintained remission till week 52 in most patients who were in clinical remission at week 26. Selective blockade of interleukin-23 warrants further evaluation as treatment for Crohn’s disease.Boehringer Ingelhei