Modified-Release Phosphatidylcholine (LT-02) for Ulcerative Colitis: Two Double-Blind, Randomized, Placebo-Controlled Trials.

BACKGROUND & AIMS: The aim of this study was to evaluate the efficacy of LT-02, a novel modified-release phosphatidylcholine (PC) formulation, for induction and maintenance of remission in patients with mild to moderate ulcerative colitis (UC) and inadequate response to mesalamine. METHODS: LT-02 was evaluated in a multicenter double-blind, randomized, placebo-controlled study comprising a 12-week induction trial (PCG-2), followed by a 48-week maintenance trial (PCG-4). In PCG-2, patients were randomized 1:1:1 to treatment with 0.8 g LT-02 4 times daily (QID), 1.6 g LT-02 twice daily (BID), or placebo, respectively. All patients continued to take a standard dose of oral mesalamine (≥2.4 g/day). The primary end point in PCG-2 was deep remission. Patients achieving remission at week 12 were randomly assigned 2:1:1 to 1.6 g LT-02 BID, placebo, or 500 mg mesalamine (3 times daily), respectively, in PCG-4; the primary end point was remission at 48 weeks. RESULTS: PCG-2 was terminated early for futility after a prespecified interim analysis; 466 patients (of 762 planned) were randomized. There was no statistically significant difference in deep remission at week 12 (placebo, 13.5%; LT-02 BID, 14.2%; LT-02 QID, 9.7%). In PCG-4, 150 patients (of approximately 400 planned) were randomized. There was no statistically significant difference in remission rates at week 48 (LT-02 BID, 49.3%; mesalamine, 50.0%; placebo, 43.2%). LT-02 was safe. CONCLUSIONS: Despite prior evidence of beneficial effects of PC in phase 2 trials, our induction study with LT-02 in patients with mild to moderate UC was terminated prematurely for futility. Signals of efficacy in maintenance therapy require confirmation in an adequately powered maintenance trial. LT-02 was safe and well-tolerated. CLINICALTRIALS: gov: NCT02280629, NCT02142725.

Elevation of Autoantibodies to Cerebral Proteins in Hepatic Encephalopathy: Another Pathogenic Factor?

BACKGROUND: The pathophysiology of hepatic encephalopathy (HE) is incompletely understood. It remains elusive how the contributing factors of neuronal ammonia accumulation, cell swelling, and inflammation interact. OBJECTIVE: The objective of this study was to find the correlation between neuronal autoantibody levels and the degree of HE as first indication of immune-mediated pathogenesis. METHODS: We investigated serum autoantibody levels of representative brain proteins in patients with HE as well as in an experimental rat model with cirrhosis and HE after carbon tetrachloride exposure. They were examined in relation to presence of HE and the degree of neurological impairment evaluated by quantitative scores. RESULTS: In HE, an increase in all of the examined antibodies was observed in serum. The grade of antibody elevation correlated to the degree of encephalopathy registered by quantitative evaluation of brain dysfunction. CONCLUSION: The degree of HE parallels neuronal autoantibody elevation. In case a causal relationship could finally be established, it adds to the understanding of HE and may open a new perspective for treatment of this handicapping condition by immunosuppressive strategies.

An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1.

Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

Delayed-Release Phosphatidylcholine Is Effective for Treatment of Ulcerative Colitis: A Meta-Analysis.

BACKGROUND: Phosphatidylcholine (PC) is intrinsically missing in intestinal mucus of patients with ulcerative colitis. Topical supplementation with delayed intestinal release PC formulations is assumed to compensate this lack. Three monocenter randomized controlled trials (RCTs) with a 30% PC-containing lecithin were successful, whereas 1 trial with >94% PC-containing lecithin failed. OBJECTIVES: Evaluation of 30% PC-containing lecithin provided in a delayed intestinal release formulation for treatment efficacy of ulcerative colitis was evaluated by meta-analysis of 3 RCTs. METHODS: Meta-analysis of 3 studies was performed using RevMan 5.3 software. Odds ratio (OR) and 95% Cl were calculated for remission, clinical and endoscopic improvement, histology, and life quality. p values <0.05 were accepted as significant. RESULTS: The meta-analysis of 3 RTCs with 160 included patients with ulcerative colitis verified that PC improved the rate of remission (OR = 9.68), as well as clinical (OR = 30.58) and endoscopic outcomes (OR = 36.73). Within the available patient population, also histology and quality of life became better. All effects were significant over placebo. Achieved remission was maintained in a higher percentage of patients under intestinal-release PC formulation than placebo. The profile of adverse events was identical to the placebo population. CONCLUSIONS: A 30% PC-containing lecithin in delayed intestinal release formulation improves clinical and endoscopic outcomes, histologic activity, and quality of life in patients with ulcerative colitis. For the patients, lack of adverse events is an important consideration.

Cold traps as reliable devices for quantitative determination of SARS-CoV-2 load in aerosols.

Spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a demanding challenge. This is of particular importance in schools and public areas of unavoidable access. New viral mutations may increase infectivity and require even better methods to identify areas of potential hazards. High-throughput SARS-CoV-2 testing and legal restrictions are not effective in order to get the current outbreak under control. The occurrence of new SARS-CoV-2 variants with a higher transmissibility requires efficient strategies for early detection and surveillance. Until today, testing focuses on nasal or pharyngeal mucosa swabs, neglecting the origin of aerosolic transmission, thus failing to detect the spread by carriers of the virus. Therefore, in this study, SARS-CoV-2 RNA levels were determined by quantitative real time PCR in aerosols collected by non-powered cold traps. SARS-CoV-2 spreading kinetics were recorded in indoor hotspots within a high-endemic area. These hotspots included a SARS-CoV-2 isolation unit, an outpatient endoscopy facility, a concert hall, and a shopping mall. For determination of viral presence aerosols were collected by cold traps positioned at different locations in the area of interest over a period of 4-6 h. Indoor SARS-CoV-2 hotspots were found in non-ventilated areas and in zones that are predisposed to a buoyancy (chimney) effect. SARS-CoV-2 RNA in those aerosols reached concentrations of 105 copies/mL, while extensive outdoor air ventilation reliably eliminated SARS-CoV-2 aerosol contamination. The method presented herein is effective for the identification of SARS-CoV-2 indoor hotspots and may help to characterize the spreading kinetics of SARS-CoV-2. Moreover, it can be used for the surveillance of emerging SARS-CoV-2 variants. Due to low costs and easy handling, the procedure might enable efficient algorithms for COVID-19 screening and prevention.

Age and Sex but Not ATP7B Genotype Effectively Influence the Clinical Phenotype of Wilson Disease.

Wilson disease (WD) is an inherited disorder of hepatic copper metabolism with considerable variation in clinical presentations, the most common ones being liver disease and neuropsychiatric disturbances. This study investigated the clinical presentation in relation to mutations in a large cohort of patients with WD. A total of 1,357 patients (702 children, 655 adults; 1,172 index patients, 185 siblings, all with a Leipzig score ≥4, male/female: 679/678) were studied. The age and the symptoms at presentation were used as key phenotypic markers. Index patients were clinically classified as having either hepatic (n = 711) or neurologic disease (n = 461). Seven hundred fifteen (52.7%) patients had a liver biopsy at diagnosis. DNA was sequenced by the Genetic Analyzers ABI Prism 310 (Perkin Elmer) or 3500 (Applied Biosystems). Three hundred ninety-four different mutation combinations were detected. The most frequent mutation was H1069Q (c.3207C>A; allele frequency: 46.9%), followed by P767P-fs (c.2304dupC; 2.85%), P1134P-fs (c.3402delC; 2.8%), and R969Q (c.2755C>T; 2.18%). There was no correlation between mutations and individual clinical manifestation. There was a gender effect in index patients: Hepatic presentation was more common in females (male/female: 328/383) and neurologic presentation in males (259/202; P < 0.001). At diagnosis, 39.5% of children/adolescents (≤18 years) and 58% of adults already had cirrhosis. The presence of cirrhosis did not correlate with the genotype. Conclusion: These findings refine and extend our understanding of the natural history and individual spectrum/manifestations of WD. Initially, there is asymptomatic hepatic involvement, which may progress and become symptomatic. Neurologic symptoms present many years later.

Plasma Lipidome, PNPLA3 polymorphism and hepatic steatosis in hereditary hemochromatosis.

BACKGROUND: Hereditary hemochromatosis (HH) is an autosomal recessive genetic disorder with increased intestinal iron absorption and therefore iron Overload. iron overload leads to increased levels of toxic non-transferrin bound iron which results in oxidative stress and lipid peroxidation. The impact of iron on lipid metabolism is so far not fully understood. The aim of this study was to investigate lipid metabolism including lipoproteins (HDL, LDL), neutral (triglycerides, cholesterol) and polar lipids (sphingo- and phospholipids), and PNPLA3 polymorphism (rs738409/I148M) in HH. METHODS: We conducted a cohort study of 54 subjects with HH and 20 healthy subjects. Patients were analyzed for their iron status including iron, ferritin, transferrin and transferrin saturation and serum lipid profile on a routine follow-up examination. RESULTS: HH group showed significantly lower serum phosphatidylcholine (PC) and significantly higher phosphatidylethanolamine (PE) compared to healthy control group. The ratio of PC/PE was clearly lower in HH group indicating a shift from PC to PE. Triglycerides were significantly higher in HH group. No differences were seen for HDL, LDL and cholesterol. Hepatic steatosis was significantly more frequent in HH. PNPLA3 polymorphism (CC vs. CG/GG) did not reveal any significant correlation with iron and lipid parameters including neutral and polar lipids, grade of steatosis and fibrosis. CONCLUSION: Our study strengthens the hypothesis of altered lipid metabolism in HH and susceptibility to nonalcoholic fatty liver disease. Disturbed phospholipid metabolism may represent an important factor in pathogenesis of hepatic steatosis in HH.

Elevation of blood lipids in hepatocyte-specific fatty acid transport 4-deficient mice fed with high glucose diets.

Fatty acid transport protein4 (FATP4) is upregulated in acquired and central obesity and its polymorphisms are associated with blood lipids and insulin resistance. Patients with FATP4 mutations and mice with global FATP4 deletion exhibit skin abnormalities characterized as ischthyosis prematurity syndrome (IPS). Cumulating data have shown that an absence of FATP4 increases the levels of cellular triglycerides (TG). However, FATP4 role and consequent lipid and TG metabolism in the hepatocyte is still elusive. Here, hepatocyte-specific FATP4 deficient (Fatp4L-/-) mice were generated. When fed with chow, these mutant mice displayed no phenotypes regarding blood lipids. However when fed low-fat/high-sugar (HS) or high-fat/high-sugar (HFS) for 12 weeks, Fatp4L-/- mice showed a significant increase of plasma TG, free fatty acids and glycerol when compared with diet-fed control mice. Interestingly, Fatp4L-/- mice under HS diet had lower body and liver weights and they were not protected from HFS-induced body weight gain and hepatic steatosis. Male mutant mice were more sensitive to HFS diet than female mutant mice. Glucose intolerance was observed only in female Fatp4L-/- mice fed with HS diet. Lipidomics analyses revealed that hepatic phospholipids were not disturbed in mutant mice under both diets. Thus, hepatic FATP4 deletion rendered an increase of blood lipids including glycerol indicating a preferential fatty-acid channeling to TG pools that are specifically available for lipolysis. Our results imply a possible risk of hyperlipidemia as a result of abnormal metabolism in liver in IPS patients with FATP4 mutations who consume high-sugar diets.

Pregnancy in Wilson's disease: Management and outcome.

Wilson's disease (WD) is a rare inherited disorder of copper metabolism causing toxic hepatic and neural copper accumulation. Clinical symptoms vary widely, from asymptomatic disease to acute liver failure or chronic liver disease with or without neuropsychiatric symptoms. Continuation of specific medical treatment for WD is recommended during pregnancy, but reports of pregnancy outcomes in WD patients are sparse. In a retrospective, multicenter study, 282 pregnancies in 136 WD patients were reviewed. Age at disease onset, age at conception, and WD-specific treatments were recorded. Maternal complications during pregnancy, rate of spontaneous abortions, and birth defects were analyzed with respect to medical treatment during pregnancy. Worsening of liver function tests was evident during 16 of 282 (6%) pregnancies and occurred in undiagnosed patients as well as in those under medical treatment. Liver test abnormalities resolved in all cases after delivery. Aggravation of neurological symptoms during pregnancy was rare (1%), but tended to persist after delivery. The overall spontaneous abortion rate in the study cohort was 73 of 282 (26%). Patients with an established diagnosis of WD receiving medical treatment experienced significantly fewer spontaneous abortions than patients with undiagnosed WD (odds ratio, 2.853 [95% confidence interval, 1.634-4.982]). Birth defects occurred in 7 of 209 (3%) live births. CONCLUSION: Pregnancy in WD patients on anticopper therapy is safe. The spontaneous abortion rate in treated patients was lower than that in therapy-naïve patients. Although the teratogenic potential of copper chelators is a concern, the rate of birth defects in our cohort was low. Treatment for WD should be maintained during pregnancy, and patients should be monitored closely for hepatic and neurological symptoms. (Hepatology 2018;67:1261-1269).

Shear-Enhanced Dynamic Adhesion of Lactobacillus rhamnosus GG on Intestinal Epithelia: Correlative Effect of Protein Expression and Interface Mechanics.

The oral uptake of probiotic microorganisms as food additives is one widely used strategy to sustain and improve the homeostasis of intestinal microbiota that protect the intestinal epithelia from attack by pathogenic bacteria. Once delivered to the ileum and colon, probiotics must adhere and form colonies on mucus that coats the surface of intestinal epithelial cells. Although an increasing amount of knowledge about the genetic and molecular level mechanisms of probiotics-mucus interactions has been accumulated, little is known about the physicochemical aspects of probiotics-mucus interactions under physiological shear in intestines. In this study, we established well-defined models of intestinal epithelial cell monolayers based on two major constituents of gut epithelia, enterocytes and goblet cells. First, the formation of a polarized cell monolayer sealed by tight junctions was monitored by transepithelial electrical resistance over time. The establishment of tight junctions and secretion of mucus proteins (mucin) was confirmed by immunofluorescence staining. In the next step, we measured the elasticity of cell monolayer surfaces by indentation using particle-assisted atomic force microscopy. The effective elastic modulus of goblet cell-like cells was 30 times smaller compared to that of enterocyte-like cells, which can be attributed to the secretion of a 3 µm thick mucin layer. As probiotics, we used Lactobacillus rhamnosus GG (LGG), which is one of the most widely used strains as food additives. To investigate the dynamic adhesion of LGG to the intestine model surface, we transferred the epithelial cell monolayer into a microfluidic chamber. A distinct difference in dynamic adhesion between two cell types was observed, which could be attributed to the difference in the mucin expression amount. Remarkably, we found that the dynamic LGG adhesion is enhanced by the increase in shear stress, showing a maximum binding efficiency at 0.3 Pa. Finally, we examined the persistence of LGG adhesion by a stepwise increase in the shear stress exerted on adherent LGG, demonstrating that LGG could withstand high shear stress even beyond that of physiological stress. The obtained results present a large potential to quantitatively understand the influence of engineered foods and probiotics on the homeostasis of microbiota on the surface of intestinal epithelia.

Association between serum IgG level and clinical course in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis is a chronic cholestatic liver disease. The pathomechanism is still not fully understood, but there is evidence that immune-mediated processes may contribute to disease progression. METHODS: We studied the prognostic relevance of serum immunoglobulin G (IgG) elevated above the upper limit of normal as a marker for immune activation at initial diagnosis and its influence on transplantation-free survival in a well-defined cohort of PSC patients. RESULTS: The final study cohort comprises of 148 PSC patients. Elevated IgG levels were found in 66 patients (44.6%). Apart from their younger age at first diagnosis, there was no significant difference between patients with or without elevated IgG levels. The presence of a concomitant inflammatory bowel disease, an autoimmune hepatitis or immunosuppressive medication was equally distributed between both groups. Patients with elevated IgG levels reached the combined endpoint (34 (59.6%) vs. 23 (40.4%); p = 0.004) significantly more often and had reduced transplantation-free survival (Log-rank: 24.0 (10.2-37.9) vs. 14.0 (8.5-19.5); p < 0.05). Cox regression analysis including age, gender, presence of IBD, presence of dominant stricture (DS), Mayo Risk Score (MRS), immunosuppression, biochemical response to UDCA and elevated IgG-levels confirmed MRS (p = 0.03), DS (p = 0.04), biochemical response (p = 0.04) and elevated IgG level (p = 0.04) as independent risk factors for reduced transplantation-free survival. CONCLUSION: We identified elevated serum IgG levels at first diagnosis as an independent risk factor for reduced transplant free-survival in patients with PSC.

Real-World Outcomes of Vedolizumab Therapy in Ulcerative Colitis and Crohn's Disease at a Tertiary Referral Center.

BACKGROUND: Vedolizumab was approved for the therapy of ulcerative colitis and Crohn's disease in mid-2014. Real-world treatment data are necessary for a balanced assessment of its position among other therapeutic options. SUMMARY: Patients with ulcerative colitis or Crohn's disease, initiating vedolizumab therapy at the outpatient clinic for inflammatory bowel diseases at the University Hospital -Heidelberg between June 1, 2014 and August 31, 2016, were recruited based on electronic medical records. The primary study endpoint was response at week 30, while the secondary endpoints were the need for surgery and discontinuation of therapy due to inadequate response, or adverse events. Twenty-five patients with ulcerative colitis (40% anti-tumor necrosis factor α [TNFα] naive) and 28 patients with Crohn's disease (10.7% anti-TNFα naive, 53.6% having undergone at least one intestinal surgery) were enrolled. Among the ulcerative colitis patients, 20% achieved remission, 32% partial response, and 48% were non-responders to vedolizumab. In Crohn's disease, 14.3% of the patients achieved remission, 46.4% partial response, and 39.4% were non-responders. Two patients discontinued vedolizumab therapy due to suspected side effects. Key Message: In a relatively treatment-refractory cohort of inflammatory bowel disease patients, vedolizumab was efficacious in achieving response. However, the majority of the patients were not satisfactorily treated, as they did not reach remission.

Hepatic Encephalopathy Aggravated by Systemic Inflammation.

BACKGROUND: The pathogenesis of hepatic encephalopathy (HE) is only partially understood. Beside ammonia accumulation in brain, a proinflammatory component has been suggested as precipitating event. OBJECTIVES: To evaluate the role of cytokines in cirrhosis for development of HE. METHODS: Pro- and anti-inflammatory cytokine profiles were determined in rats with CCL4-induced cirrhosis and HE as well as in patients with cirrhosis either due to metabolic disorders or chronic hepatitis C virus (HCV) with various grades of concomitant HE and depression. RESULTS: In the rat model and human cirrhosis a proinflammatory cytokine pattern (elevation of interferon gamma, interleukin [IL]-1ß, IL-6) was registered which in humans correlated to the degree of HE and depression. The most prominent elevation of proinflammatory cytokines was observed in chronic HCV as an additional inflammatory stimulus. In all cases of cirrhosis a comparable background activation of anti-inflammatory cytokines (e.g., IL-4) was detected which was interpreted as a physiologic counterbalance mechanism. CONCLUSIONS: The degree of HE and depression correlated with a proinflammatory cytokine pattern. It suggests that beside ammonia elevation, inflammatory cytokines determine occurrence and severity of hepatic encephalopathies. Thus, it can be defined a preferential therapeutic target.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) - an underestimated condition: ferritin light chain variant spectrum in German families.

Background In hereditary hyperferritinaemia-cataract syndrome (HHCS), single nucleic acid alterations in the ferritin light chain (L-ferritin) iron response element (IRE) constitutively derepress ferritin synthesis, resulting in hyperferritinaemia, L-ferritin deposits in the lens of the eye and early bilateral cataract onset. Methods In this study, six German families with putative HHCS were analysed. Clinical diagnosis of HHCS was based on medical history, evaluation of ferritin serum levels, transferrin saturation and clinical ophthalmological examination. Diagnosis was confirmed by polymerase chain reaction (PCR)-based DNA sequencing of the L-ferritin IRE. Results Genetic analysis of the L-ferritin IRE revealed relevant single nucleic acid alterations in each of the affected families. Variants c.-168G > A, c.-168G > U and c.-167C > U were located in the C-bulge region; and variants c.-161C > U and c.-157G > A were located in the hexanucleotide loop of the L-ferritin IRE. Conclusions Family history of hyperferritinaemia and juvenile cataracts are strong indicators of HHCS. Genetic analysis of the L-ferritin IRE is a straightforward procedure to confirm the diagnosis. Accurate diagnosis of hyperferritinaemia can avoid unnecessary treatment by venesection, and focus attention on early cataract detection in offspring at risk.

Phosphatidylcholine Passes by Paracellular Transport to the Apical Side of the Polarized Biliary Tumor Cell Line Mz-ChA-1.

Phosphatidylcholine (PC) translocation into mucus of the intestine was shown to occur via a paracellular transport across the apical/lateral tight junction (TJ) barrier. In case this could also be operative in biliary epithelial cells, this may have implication for the pathogenesis of primary sclerosing cholangitis (PSC). We here evaluated the transport of PC across polarized cholangiocytes. Therefore, the biliary tumor cell line Mz-ChA-1 was grown to confluency. In transwell culture systems the translocation of PC to the apical compartment was analyzed. After 21 days in culture, polarized Mz-ChA-1 cells revealed a predominant apical translocation of choline containing phospholipids including PC with minimal intracellular accumulation. Transport was suppressed by TJ destruction employing chemical inhibitors and pretreatment with siRNA to TJ forming proteins as well as the apical transmembrane mucin 3 as PC acceptor. Apical translocation was dependent on a negative apical electrical potential created by the cystic fibrosis transmembrane conductance regulator (CFTR) and the anion exchange protein 2 (AE2). It was stimulated by apical application of secretory mucins. The results indicated the existence of a paracellular PC passage across apical/lateral TJ of the polarized biliary epithelial tumor cell line Mz-ChA-1. This has implication for the generation of a protective mucus barrier in the biliary tree.

The Bile Acid-Phospholipid Conjugate Ursodeoxycholyl-Lysophosphatidylethanolamide (UDCA-LPE) Disintegrates the Lipid Backbone of Raft Plasma Membrane Domains by the Removal of the Membrane Phospholipase A2.

The bile acid-phospholipid conjugate ursodeoxycholyl-lysophosphatidylethanolamide (UDCA-LPE) was shown to have anti-inflammatory, antisteatotic, and antifibrotic properties, rendering it as a drug targeting non-alcoholic steatohepatitis (NASH). On a molecular level, it disrupted the heterotetrameric fatty acid uptake complex localized in detergent-resistant membrane domains of the plasma membrane (DRM-PM). However, its mode of action was unclear. Methodologically, UDCA-LPE was incubated with the liver tumor cell line HepG2 as well as their isolated DRM-PM and all other cellular membranes (non-DRM). The membrane cholesterol and phospholipids were quantified as well as the DRM-PM protein composition by Western blotting. The results show a loss of DRM-PM by UDCA-LPE (50 µM) with a 63.13 ± 7.14% reduction of phospholipids and an 81.94 ± 8.30% reduction of cholesterol in relation to mg total protein. The ratio of phospholipids to cholesterol changed from 2:1 to 4:1, resembling those of non-DRM fractions. Among the members of the fatty acid uptake complex, the calcium-independent membrane phospholipase A2 (iPLA2ß) abandoned DRM-PM most rapidly. As a consequence, the other members of this transport system disappeared as well as the DRM-PM anchored fibrosis regulating proteins integrin ß-1 and lysophospholipid receptor 1 (LPAR-1). It is concluded that UDCA-LPE executes its action by iPLA2ß removal from DRM-PM and consequent dissolution of the raft lipid platform.

The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells.

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells. RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66 µm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally. In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.

Nonclassical Interactions of Phosphatidylcholine with Mucin Protect Intestinal Surfaces: A Microinterferometry Study.

Albeit many studies demonstrated that the accumulation of phospholipids in the intestinal mucosal surfaces is essential for the protection of colon epithelia against pathogenic bacteria, the mechanism of interactions between phospholipids and the surface protein mucin is not well understood. In this study, the significance of interfacial interactions between phospholipids and mucin proteins was quantified by the combination of an in vitro intestinal surface model and label-free microinterferometry. The model of intestinal surfaces consists of planar lipid membranes deposited on solid substrates (supported membranes) that display mucin proteins at defined surface densities. Following the quantitative characterization of the systems, we monitored the vertical fluctuation of 10 µm-large particles on model intestinal surfaces by using microinterferometry, and calculated the effective interfacial interaction potentials by analytically solving the Langevin equation. We found that the spring constant of interfacial potentials calculated based on a harmonic approximation increased concomitantly with the increase in surface potentials, indicating the dominant role of electrostatic interactions. Intriguingly, the spring constants of particles coated with phospholipids do not follow electrostatic interactions. The spring constant of particles coated with zwitterionic phosphatidylcholine was larger compared to membranes incorporating positively or negatively charged lipids. Our data suggested the presence of another underlying molecular level interaction, such as phosphocholine-saccharide interactions. The fact that phosphatidylcholine sustains the binding capability to enzymatically degraded mucin suggests that the direct delivery of phosphatidylcholine to the damaged mucus is a promising strategy for the better treatment of patients affected by inflammatory bowel diseases.

Metallothionein is elevated in liver and duodenum of Atp7b(-/-) mice.

Different mutations in the copper transporter gene Atp7b are identified as the primary cause of Wilson's disease. These changes result in high copper concentrations especially in the liver and brain, and consequently lead to a dysfunction of these organs. The Atp7(-/-) mouse is an established animal model for Wilson's disease and characterized by an abnormal copper accumulation, a low serum oxidase activity and an increased copper excretion in urine. Metallothionein (MT) proteins are low molecular weight metal-binding proteins and essential for the zinc homeostasis but also play a role for the regulation of other metals, e.g. copper. However the molecular mechanisms of MT in regard to Atp7b remain still elusive. In this study we investigate the expression of MT in the liver and duodenum of Atp7b(-/-) mice and wildtype mice. Hepatic and duodenal expression of MT was measured by real-time reverse transcription-polymerase chain reaction and post-translational expression was analyzed by immunoblot and immunofluorescence. Expression of MT in liver und duodenum was significantly higher in Atp7b(-/-) mice than in controls. Hepatic and duodenal copper, iron and zinc content were also studied. Compared to control hepatic copper and iron content was significantly higher while hepatic zinc content was significantly lower in Atp7b(-/-) mice. In the duodenum copper and zinc content of Atp7b(-/-) mice was significantly lower than in controls. Duodenal iron content was also lower in Atp7b(-/-) mice, but did not reach statistical significance. The results of our study suggest that metallothionein is elevated in the liver and duodenum of Atp7b(-/-) mice.

Bivalent Ligand UDCA-LPE Inhibits Pro-Fibrogenic Integrin Signalling by Inducing Lipid Raft-Mediated Internalization.

Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo. Herein, we aimed to demonstrate the inhibitory effects of UDCA-LPE on pro-fibrogenic integrin signalling. UDCA-LPE treatment of human embryonic liver cell line CL48 and primary human hepatic stellate cells induced a non-classical internalization of integrin ß1 resulting in dephosphorylation and inhibition of SRC and focal adhesion kinase (FAK). Signalling analyses suggested that UDCA-LPE may act as a heterobivalent ligand for integrins and lysophospholipid receptor1 (LPAR1) and co-immunoprecipitation demonstrated the bridging effect of UDCA-LPE on integrin ß1 and LPAR1. The disruption of either the UDCA-moiety binding to integrins by RGD-containing peptide GRGDSP or the LPE-moiety binding to LPAR1 by LPAR1 antagonist Ki16425 reversed inhibitory functions of UDCA-LPE. The lack of inhibitory functions of UDCA-PE and UDCA-LPE derivatives (14:0 and 12:0, LPE-moiety containing shorter fatty acid chain) as well as the consistency of the translocation of UDCA-LPE and integrins, which co-fractionated with LPE but not UDCA, suggested that the observed UDCA-LPE-induced translocation of integrins was mediated by LPE endocytic transport pathway.