Membrane Of Epithelial Cells Research Articles

Evidence-based pathogenesis and treatment of ulcerative colitis: A causal role for colonic epithelial hydrogen peroxide.

In this comprehensive evidence-based analysis of ulcerative colitis (UC), a causal role is identified for colonic epithelial hydrogen peroxide (H2O2) in both the pathogenesis and relapse of this debilitating inflammatory bowel disease. Studies have shown that H2O2 production is significantly increased in the non-inflamed colonic epithelium of individuals with UC. H2O2 is a powerful neutrophilic chemotactic agent that can diffuse through colonic epithelial cell membranes creating an interstitial chemotactic molecular “trail” that attracts adjacent intravascular neutrophils into the colonic epithelium leading to mucosal inflammation and UC. A novel therapy aimed at removing the inappropriate H2O2 mediated chemotactic signal has been highly effective in achieving complete histologic resolution of colitis in patients experiencing refractory disease with at least one (biopsy-proven) histologic remission lasting 14 years to date. The evidence implies that therapeutic intervention to prevent the re-establishment of a pathologic H2O2 mediated chemotactic signaling gradient will indefinitely preclude neutrophilic migration into the colonic epithelium constituting a functional cure for this disease. Cumulative data indicate that individuals with UC have normal immune systems and current treatment guidelines calling for the suppression of the immune response based on the belief that UC is caused by an underlying immune dysfunction are not supported by the evidence and may cause serious adverse effects. It is the aim of this paper to present experimental and clinical evidence that identifies H2O2 produced by the colonic epithelium as the causal agent in the pathogenesis of UC. A detailed explanation of a novel therapeutic intervention to normalize colonic H2O2, its rationale, components, and formulation is also provided.

Histological and immunohistochemical study of gilthead seabream tongue from the early stage of development: TRPV4 potential roles

BackgroundTaste buds, the morphofunctional units for taste perception, transduce gustatory stimuli using G protein-coupled receptors and a complex arrangement of ion channels, among which TRPV4, a member of the TRP superfamily. Studies on taste buds development on gilthead seabream are unknown, and the TRPV4 expression on fish taste cells studies were conducted only on zebrafish. MethodsIn our study, we have investigated the histological features of the gilthead seabream tongue dorsal surface from the earliest stage of development using Masson trichrome with aniline blue staining. Additionally, the TRPV4 expression pattern was studied by means of immunohistochemical labeling and quantitative RT-PCR. ResultsWe have recorded for the first time on gilthead seabream lingual dorsal surface the presence of, stage-specific, three types of taste buds: type I, type II and type III in larvae, juveniles and adults respectively. At 40 days post-hatching, taste buds were mature-looking. TRPV4 expression was detected in a subpopulation of taste cells of larvae, juveniles, and adults. Furthermore, TRPV4 was expressed in the basal epithelial cells of the tongue at the larvae and juvenile stage, while this expression pattern was more diffused within all the epithelial cell layers in the adult. ConclusionOur findings presume a taste sensory role of TRPV4 in the three stage-specific taste buds and oral epithelia of gilthead seabream. In addition to its sensory role on the epithelial cell layers, we hypothesize that TRPV4 is implicated in epithelial cells differentiation and membrane protection.

Effect of Excessive Serotonin on Pharmacokinetics of Cephalexin after Oral Administration: Studies with Serotonin-Excessive Model Rats.

Serotonin (5-HT) is important for gastrointestinal functions, but its role in drug absorption remains to be clarified. Therefore, the pharmacokinetics and oral absorption of cephalexin (CEX) were examined under 5-HT-excessive condition to understand the role of 5-HT. 5-HT-excessive rats were prepared by multiple intraperitoneal dosing of 5-HT and clorgyline, an inhibitor for 5-HT metabolism, and utilized to examine the pharmacokinetics, absorption behavior and the intestinal permeability for CEX. Higher levels of 5-HT in brain, plasma and small intestines were recognized in 5-HT-excessive rats, where the oral bioavailability of CEX was significantly enhanced. The intestinal mucosal transport via passive diffusion of CEX was significantly increased, while its transport via PEPT1 was markedly decreased specifically in the jejunal segment, which was supported by the decrease in PEPT1 expression on brush border membrane (BBM) of intestinal epithelial cells. Since no change in antipyrine permeability and significant increase in FITC dextran-4 permeability were observed in 5-HT-excessive rats, the enhanced permeability for CEX would be attributed to the opening of tight junction, which was supported by the significant decrease in transmucosal electrical resistance. In 5-HT-excessive rats, furthermore, total body clearance of CEX tended to be larger and the decrease in PEPT2 expression on BBM in kidneys was suggested to be one of the reasons for it. 5-HT-excessive condition enhanced the oral bioavailability of CEX in rats, which would be attributed to the enhanced permeability across the intestinal mucosa via passive diffusion through the paracellular route even though the transport via PEPT1 was decreased.

Construction of the small intestine on molecular dynamics simulation and preliminary exploration of drug intestinal absorption prediction

In this study, molecular dynamics simulation was applied to the construction of the small intestinal epithelial cell membrane and prediction of drug absorption. First, we constructed a system of a small intestinal epithelial cell membrane that was close to the real proportion and investigated the effects of temperature, water layer thickness, and ionic strength on membrane properties to optimize environmental parameters. Next, three drugs with different absorptivity, including Ephedrine (EPH), Quercetin (QUE), and Baicalin (BAI), were selected as model drugs to study the ability of drugs through the membrane by the free diffusion and umbrella sampling simulation, and the drug permeation ability was characterized by the free diffusion coefficient D and free energy barrier (△G) in the processes. The results showed that the free diffusion coefficient D and △G orders of the three drugs were consistent with the classical experimental absorption order, indicating that these two parameters could be used to jointly characterize the membrane permeability of the drugs.

Role of SNARE Proteins in the Insertion of KCa3.1 in the Plasma Membrane of a Polarized Epithelium.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Uptake of Functional Ultrasmall Gold Nanoparticles in 3D Gut Cell Models.

Ultrasmall gold nanoparticles (2nm) easily penetrate the membranes of intestinal murine epithelial cells (MODE-K) and colorectal cancer cells (CT-26). They are also taken up by 3D spheroids (400µm) of these cell types and primary gut organoids (500µm). In contrast, dissolved dyes are not taken up by any of these cells or 3D structures. The distribution of fluorescent ultrasmall gold nanoparticles inside cells, spheroids, and gut organoids is examined by confocal laser scanning microscopy. Nanoparticles conjugated with the cytostatic drug doxorubicin and a fluorescent dye exhibit significantly greater cytotoxicity toward CT-26 tumor spheroids than equally concentrated dissolved doxorubicin, probably because they enter the interior of a spheroid much more easily than dissolved doxorubicin. Comprehensive analyses show that the cellular uptake of ultrasmall gold nanoparticles occurs by different endocytosis pathways.

Abstract 5612: A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study

Abstract Monoclonal antibody (mAb) therapies have become the dominant product class within the biopharmaceutical industry mainly due to their intrinsic capacity to bind endogenous immune receptors and targeted antigens. In fact, this kind of therapeutic agent accounts for one fifth of the FDA’s new drug approvals each year. In addition, their stability and specificity make them the ideal scaffold to develop more complex and efficacious drug modalities such as bispecific antibodies and antibody-drug conjugates. However, in order to advance mAb therapies to the clinic, there are a number of parameters that need to be considered during early-stage development. The purpose of this study was to showcase important preclinical characterization and efficacy experiments aimed at assessing the biological activity, binding profile, mechanism of action and in vivo potency of cetuximab, a mAb therapy. Cetuximab targets EGFR, a well characterized receptor present in the epithelial cell membrane that is overexpressed in several cancer types, such as non-small cell lung cancer, breast cancer and colorectal cancer. In normal tissues EGFR activation initiates several intracellular signaling events involved in development and homeostasis. However, when overexpressed, it stimulates the growth, metastasis and invasion of tumors. For this reason, EGFR has been considered an important target for the development of new drugs. Here we measured the binding affinity of cetuximab to two EGFR expressing cancer cell lines (A-431 and A-549) and its off-target binding to a broad range of full-length human proteins employing Retrogenix Cell Microarray Technology. Using the AlphaLISA system, we observed that cetuximab significantly inhibits EGF binding to EGFR. The consequences of cetuximab treatment on EGF binding and the initiation of the signaling cascade were investigated by looking at the phosphorylation status of EGFR via intracellular staining and flow cytometry. Moreover, we tested the ability of cetuximab to induce Antibody Dependent Cellular Cytotoxicity (ADCC) where the target cell lines were co-cultured with freshly isolated NK cells. ADCC was assessed via both flow cytometry and live cell imaging. Lastly, we studied the efficacy of cetuximab in vivo. A tumor progression mouse model generated from A-431 cells and several Patient Derived Xenografts (PDX) mouse models representing a wide variety of cancers were treated with cetuximab and a significant reduction in tumor growth was observed for most of these cancers. The in vivo efficacy correlated directly with the EGFR expression level determined by IHC. With this case study we have generated a complete and valuable preclinical data package that could be used to advance this mAb therapy into the clinic. Moreover, this study serves as the basis for a streamlined workflow for mAb lead optimization and development as well as comparability studies for biosimilars. Citation Format: David Cobeta Lopez, Kerstin Klingner, Marie Carkill, Robert Nunan, Anya Avrutskaya, Paula Miliani de Marval, Amber Blackwell, Sarah Dawson, Donna Barnes, Jim Freeth, Deborah Bruce, Richard Bazin, René McLaughlin, Julia Schueler, Gemma Moiset, Maria L. Vlaming. A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5612.

Abstract 1876: ROCKETS Science: A novel processing toolbox for light sheet microscopy reveals unknown binding sites for EpCAM-targeted antibodies

Abstract Summary: We present a novel light sheet fluorescence microscopy (LSFM)-based imaging platform and corresponding set of procedures for greatly improved preclinical investigation of the biodistribution of novel drug candidates by the example of an EpCAM-targeting antibody. Methods: An anti-EpCAM antibody (G.8.8R) was applied intravenously to mice. Whole mice or individual organs were optically cleared following novel protocols, which we termed Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS). The procedures enabled processing of mouse organs and entire bodies for LSFM. For the GI tract, we developed 3D-Swiss rolls, a method that enables fixation, clearing and imaging of intestines in a compact form as a whole. The new procedures allowed us to investigate the biodistribution of G.8.8R in the entire mouse body and highly detailed in individual organs. Results: The ROCKETS concept achieved full transparency of any mouse organ, the entire GI tract and whole mouse bodies. All positive organs were easily identified in scans of whole mice, thereby providing excellent guidance for scanning of positive individual organs at higher magnifications. We detected G8.8R bound to all normal cuboidal and columnar epithelia, as well as in lymphoid organs and were able to reproducibly score binding levels. All reported EpCAM+ tissues in mice were accessed by G8.8R after intravenous application and binding was restricted to basolateral membranes of epithelia, as expected from published expression analyses. Moreover, we detected G8.8R in tissues that were reported EpCAM- or were not investigated, e.g. gustatory papillae of the tongue, choroid plexi in the brain or lingual mucous salivary glands. 3D-Swiss rolls of the GI tract revealed a highly heterogeneous binding pattern in the stomach, while the pattern along the small intestine was overall homogeneous. All binding was restricted to basolateral membranes of epithelial cells. We detected a general gradient of signals, decreasing from the bottom of the crypts to differentiated cells in all tissues, corresponding to described EpCAM downregulation with progressing differentiation. Furthermore, we first describe significantly increased signals at the common bile duct, major and minor duodenal papillae, as well as the mucosa in proximity of Peyer's patches (PP). Conclusions: The developed ROCKETS toolbox allowed for simple preparation of mouse organs and bodies for high-quality LSFM imaging. Our investigations of the highly heterogeneous biodistribution of G8.8R revealed previously unknown EpCAM binding locations, which may have far-reaching implications for EpCAM-targeting therapeutics in general, many of which failed in clinical studies due to dose limiting toxicities. In the future, the developed LSFM-imaging platform may contribute valuable data to preclinical drug development studies of any targeted therapeutic. Citation Format: Joerg P. Mueller, Nils O'Brien, Franz Osl, Frank Herting, Christian Klein, Pablo Umana, Sara Colombetti, Thomas Poeschinger, Andreas Beilhack. ROCKETS Science: A novel processing toolbox for light sheet microscopy reveals unknown binding sites for EpCAM-targeted antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1876.

Interaction mechanism of three egg protein derived ACE inhibitory tri-peptides and DPPC membrane using FS, FTIR, and DSC studies.

Understanding the interaction of food derived angiotensin converting enzyme (ACE) inhibitory peptides and intestinal epithelial cell membrane may help to improve their absorption. This research aimed to study the molecular interaction of ACE inhibitory tri-peptides ADF, FGR, and MIR with DPPC membrane during absorption process. The DPPC liposome was prepared and characterized, then used as a model membrane. The permeability of tri-peptides across the membrane was investigated using Fluorescence spectroscopy. The effect of tri-peptides on the structure and dynamics of DPPC bilayers was determined using Fourier transform infrared spectroscopy. The effect of tri-peptides on the phase transition temperature in the DPPC membrane was also analyzed using Differential scanning calorimetry. The results showed that ACE inhibitory tri-peptides ADF, FGR, and MIR can penetrate into both the membrane-water interface and hydrophobic region of DPPC bilayer, and the tri-peptide FGR have higher permeability across the membrane.

Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

SummaryTwo-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlAPch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Irbesartan protects against type 2 diabetic nephropathy by regulating miR-93/VEGF and its downstream effectors

Purpose: To investigate the protective effect of irbesartan against type 2 diabetic nephropathy (T2DN), and the mechanism involved.Methods: Wistar rats (n = 48) were used in this study. Diabetes mellitus (DM) was established in the rats via injection of streptozotocin (STZ). Thereafter, the DM rats were randomly divided into diabetic nephropathy (DN) group and irbesartan group, with 16 rats in each group. Rats in the control group (n = 16) received normal saline in place of irbesartan.Results: Total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), as well as protein expression levels of VEGF, FN, collagen IV in DN group were significantly higher in irbesartan group than the corresponding levels in normal control group, while miR-93 protein expression level was significantly lower than that in normal control group (p < 0.05). However, the expression level of mir-93 was significantly higher in irbesartan group than in DN group, while levels of TC, TGs, HDL-C, LDL-C and VEGF, as well as protein levels of FN and collagen IV protein were significantly lower than those in DN group (p < 0.05). There was no obvious change in the renal tissues of the normal control group. In contrast, in the DN group, glomerularcapillary loop hypertrophy, narrow glomerular cavity, thick basement membrane, mesangial matrix, and vacuolar degeneration in renal tubular epithelial cells, were evident. Compared with DN group, the pathological changes in the irbesartan group were significantly mitigated (p < 0.05).Conclusion: Irbesartan protects DM rats against type 2 diabetic nephropathy by regulating mir-93/VEGF and its downstream effector molecules. This provides some ideas for the development of new drugs for the prevention of type 2 diabetic nephropathy.

Treatment With LAU-7b Complements CFTR Modulator Therapy by Improving Lung Physiology and Normalizing Lipid Imbalance Associated With CF Lung Disease.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasians, affecting more than 100,000 individuals worldwide. It is caused by pathogenic variants in the gene encoding CFTR, an anion channel at the plasma membrane of epithelial and other cells. Many CF pathogenic variants disrupt the biosynthesis and trafficking of CFTR or reduce its ion channel function. The most frequent mutation, loss of a phenylalanine at position 508 (F508del), leads to misfolding, retention in the endoplasmic reticulum, and premature degradation of the protein. The therapeutics available for treating CF lung disease include antibiotics, mucolytics, bronchodilators, physiotherapy, and most recently CFTR modulators. To date, no cure for this life shortening disease has been found. Treatment with the Triple combination drug therapy, TRIKAFTA®, is composed of three drugs: Elexacaftor (VX-445), Tezacaftor (VX-661) and Ivacaftor (VX-770). This therapy, benefits persons with CF, improving their weight, lung function, energy levels (as defined by reduced fatigue), and overall quality of life. We examined the effect of combining LAU-7b oral treatment and Triple therapy combination on lung function in a F508deltm1EUR mouse model that displays lung abnormalities relevant to human CF. We assessed lung function, lung histopathology, protein oxidation, lipid oxidation, and fatty acid and lipid profiles in F508deltm1EUR mice.

Identification of the chloride channel, leucine-rich repeat-containing protein 8, subfamily a (LRRC8A), in mouse cholangiocytes.

Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.

Radioprotective effects and mechanism of HL-003 on radiation-induced salivary gland damage in mice

Ionizing radiation (IR) can cause damage to the structure and function of salivary glands. Our research group independently synthesized the ROS scavenger, HL-003. The aim of this study was to explore the protective effects and underlying mechanisms of HL-003 on radiation-induced salivary gland injury. Salivary flow rate measurement, H&E staining, immunohistochemistry, FRAP, TUNEL, and western blotting were used to evaluate the radioprotective effect on salivary glands. The results showed that HL-003 protected the salivary secretion function by protecting the AQP-5 protein, on the salivary epithelial cell membrane, from IR damage. HL-003 reduced oxidative stress in the salivary gland by regulating the expression of ROS-related proteins NOX4, SOD2, and 8-OHdG. Furthermore, HL-003 downregulated the expression of p-p53, Bax, caspase 3, and caspase 9, and upregulated the expression of Bcl-2, suggesting that it could inhibit the activation of p53 to reduce cell apoptosis. In conclusion, HL-003 is an effective radioprotector that prevents damage of the radiation-induced salivary gland.

Multi-functional vesicles improve Helicobacter pylori eradication by a comprehensive strategy based on complex pathological microenvironment

Helicobacter pylori (H. pylori), creating a global infection rate over 50%, presents great challenges in clinical therapies due to its complex pathological microenvironment in vivo. To improve the eradication efficacy, herein we fabricated a pharmaceutical vesicle RHL/Cl-Ch-cal where cholesterol-PEG, calcitriol and first-line antibiotic clarithromycin were co-loaded in the rhamnolipid-composed outer lipid layer. RHL/Cl-Ch-cal could quickly penetrate through gastric mucus layer to reach H. pylori infection sites, and then effectively destroyed the architecture of H. pylori biofilms, killed dispersed H. pylori and inhibited the re-adhesion of residual bacteria (called biofilms eradication tetralogy). Moreover, RHL/Cl-Ch-cal activated the host immune response to H. pylori by replenishing cholesterol to repair lipid raft on the cell membrane of host epithelial cells. Finally, RHL/Cl-Ch-cal killed the intracellular H. pylori through recovering the lysosomal acidification and assisting degradation. In experiments, RHL/Cl-Ch-cal demonstrated prominent anti-H. pylori efficacy in the classical H. pylori-infected mice model. Therefore, the study provides a “comprehensive attack” strategy for anti-H. pylori therapies including biofilms eradication tetralogy, immune activation and intracellular bacteria killing.

Impact of trimethoprim on serum creatinine, sodium, and potassium concentrations in patients taking trimethoprim-sulfamethoxazole without changes in glomerular filtration rate.

Trimethoprim (TMP) inhibits the Na+ /K+ -ATPase present in the basement membrane of distal tubular epithelial cells. However, hyponatremia and hyperkalemia may develop in patients taking TMP-sulfamethoxazole (SMX). In addition, because TMP inhibits drug transporters, such as organic cation transporter 2 and multidrug and toxin extrusion protein 2-K in proximal tubules, reversible increases in the concentration of serum creatinine (SCr), the substrate of these transporters, may occur. Here, we investigated variability in SCr, serum sodium (Na+ ), and serum potassium (K+ ) concentrations after initiation of TMP-SMX treatment and evaluated the risk of hyponatremia and hyperkalemia in patients with increased SCr concentrations without changes in the glomerular filtration rate (GFR). In this retrospective study, all patients aged 20 years or older who received oral TMP-SMX during hospitalization were enrolled. The patients with estimated creatinine (Cr) clearance (eCCr) lower than 30 mL/min were excluded, as were patients taking drugs that were likely to induce renal dysfunction, drugs other than glucocorticoids that were likely to induce electrolyte imbalances, or drugs other than TMP that inhibit tubular Cr secretion. Additionally, those with SCr concentrations elevated more than 30% from baseline or serum blood urea nitrogen concentration levels above 20 mg/dL during follow-up were also excluded. In total, 111 patients were enrolled in the study. The common independent variable affecting the change rate in SCr, Na+ , and K+ concentrations (ΔSCr, ΔNa+ , and ΔK+ ) from baseline to the highest value during the follow-up period (14 days after initiation of TMP-SMX treatment) was the daily dose of TMP. There were significant correlations between ΔSCr and ΔNa+ or ΔK+ (ρ=-0.199, p=0.036 and ρ=0.244, p=0.010, respectively). Kaplan-Meier curves for hyponatremia and hyperkalemia with greater than or equal to grade 1 severity showed different profiles when the TMP dose varied (≤ 160 vs. > 160 mg/day; p=0.005 and 0.008). The cumulative incidences of both adverse effects were 64.7% (median: 7 days) and 29.4% in patients taking more than 160 mg/day TMP and 35.2% and 6.7% in patients taking 160 mg/day TMP or less. Thus, TMP may affect the kinetics of Cr, Na+ , and K+ in the proximal and distal tubules in a dose-dependent without changing the GFR. This study is the first report to demonstrate the degree of changes in SCr, Na+ , and K+ concentrations after initiation of TMP-SMX treatment. If SCr is elevated after initiation of TMP-SMX treatment, clinicians should be aware of decreased Na+ and increased K+ concentrations. TMP may increase the risks of hyponatremia and hyperkalemia in a dose-dependent manner without altering GFR.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

VX770 Modulated Bicarbonate Conductance in CFTR‐expressing FRT Cell

The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelia and other tissues, is compromised by genetic mutations that cause cystic fibrosis (CF). Although CFTR is reportedly permeant to both Cl¯ and HCO3¯, high throughput screening studies identified drug scaffolds that enhanced Cl¯ conductance of mutated CFTR by ‘correcting’ processing deficiets and/or ‘potentiating’ aggregate conductance of channels in epithelial cell membranes while being agnostic to HCO3‐. Ongoing work seeks to determine whether mutated forms of CFTR exhibit typical HCO3¯ conductance and to access clinically‐used drugs for their effects on HCO3¯ conductance by wild‐type and mutated CFTR. Fisher rat thyroid (FRT) cells, which form electrically tight epithelial monolayers, express no functionally detectable CFTR and which traffick exogenously expressed CFTR to both the apical and basolateral membranes, were employed to assess transepithelial anion conductance in modified Ussing‐style flux chambers. Initial experiments employing FRT cells expressing human wtCFTR validated that forskolin‐stimulated conductance, which was inhibited by selective CFTR blockers, was proportional to extracellular Cl¯ concentration over the range of ~70‐140 mEq/l. Further, a clear increment in forskolin‐stimulated conductance was observed when HCO3¯ (25 mEq/l) was incorporated into the media. VX770 potentiated forskolin‐stimulated conductance, but had no effect in the absence of forskolin. Foskolin‐stimulated and VX770‐potentiated conductance was compared in base medium (100 mEq/l Cl‐) supplemented with 25 mEq/l HCO3‐ or Cl‐ and bicarbonate (B) conductance was quantified relative to Cl‐ (C) conductance (i.e., gB/gC). Following forskolin exposure the gB/gC ratio for wtCFTR was 0.62±0.20, a value that decreased to 0.11±0.10 following exposure to VX770, suggesting that VX770 may negatively impact gB. Forskolin‐ and VX770‐stimulated conductance across cells expressing F508del CFTR was nearly undetectable unless cells were pretreated with CFTR correctors. Following corrector exposure, forskolin stimulated and VX770 potentiated gC, but gB was virtually undetectable, suggesting that this variant may lack gB. Alternatively, forskolin stimulated little conductance across cells expressing G551D CFTR, but a substantial potentiation with VX770 was observed. Notably, the gB/gC ratio approached 1.0 for both foskolin stimulation and VX770 potentiation. Taken together, these results suggest that CFTR HCO3‐ conductance differs by disease‐associated variant and that gB of some variants are sensitive to VX770. Ongoing experiments are designed to determine the optimal forskolin concentration to maximize VX770 potentiation. Initial outcomes suggest that there is interaction between the CFTR variant and forskolin concentration dependence such that VX770‐potentiated conductance is maximized at different forskolin concentrations. Taken together, results from this investigation suggest that additional or alternative interventions may be required to optimize bicarbonate secretion and pH regulation in CF patients expressing alternative CFTR variants.

Membrane phosphoinositides and renal epithelial cell polarity determination in the Xenopus pronephros in vivo

BackgroundThough they are only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK renal epithelial cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus laevis tadpoles using MCherry‐tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH‐AKT, PH‐PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.MethodsmRNA encoding MCherry‐PH‐AKT or MCherry‐PH‐PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry‐PH‐AKT distribution were assessed by fluorescence microscopy.ResultsIn MDCK cells PH‐AKT and PH‐PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors localizing apically in the proximal portion of the tubule and basolaterally in the distal part. Interestingly, PTEN localization, at least in proximal tubules, is apical and not different than previously reported in cell models. Finally, PH‐AKT staining dramatically re‐distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.ConclusionsThese studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal important differences from observations previously made with in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.