Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats

BACKGROUND & AIMS: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. METHODS: In bile duct-incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels. RESULTS: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. CONCLUSIONS: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.     

Metaanalysis of the efficacy of sublingual immunotherapy in the treatment of allergic asthma in pediatric patients, 3 to 18 years of age

BACKGROUND: Recent studies have documented the efficacy and safety of sublingual immunotherapy (SLIT) in patients with rhinitis, but the value of this treatment in those with asthma is still debated. We evaluated the efficacy of SLIT in the treatment of allergic asthma in children by a metaanalysis of randomized, double-blind, and placebo-controlled (DBPC) clinical trials. METHODS: Electronic databases were searched up to May 31, 2006, for randomized DBPC trials assessing SLIT in pediatric cases of asthma. Effects on primary outcomes (ie, symptom scores and concomitant use of rescue medication) were calculated with standardized mean differences (SMDs) using the random-effects model. We performed the metaanalysis using a statistical software package (RevMan, 4.2.8; The Cochrane Collaboration; Oxford, UK), and we followed the recommendations of the Cochrane Collaboration and the Quality of Reporting of Metaanalyses guidelines. RESULTS: Seventy-three articles were identified and reviewed. Nine studies, all published after 1990, fulfilled the selection criteria. A total of 441 patients had a final assessment and were included in the analysis. Two hundred thirty-two patients received SLIT, and 209 patients received placebo. The results of the present analysis demonstrated a relevant heterogeneity due to widely differing scoring systems. Overall, there was a significant reduction in both symptoms (SMD - 1.14; 95% confidence interval [CI], - 2.10 to - 0.18; p = 0.02) and medication use (SMD, - 1.63; 95% CI, - 2.83 to - 0.44; p = 0.007) following SLIT. CONCLUSION: SLIT with standardized extracts reduces both symptom scores and rescue medication use in children with allergic asthma compared with placebo.     

A pulmonary embolism treated with the Angiojet technique in a patient with double outlet right ventricle

A 24-year-old man known to have a double outlet right ventricle, status post-modified Fontan procedure at age 10, taking coumadin after having a stroke one year prior, presented with a massive pulmonary embolism and hemodynamic instability. Locally delivered tPA was unsuccessful, and the pulmonary artery thrombus was finally removed with a thrombectomy catheter (Angiojet thrombectomy); the patient recovered soon thereafter. This is the first report of the successful use of a thrombectomy catheter in a patient with pulmonary emboli and an occluded Fontan conduit.     

Capillarity-driven assembly of two-dimensional cellular carbon nanotube foams

Capillary forces arising during the evaporation of liquids from dense carbon nanotube arrays are used to reassemble the nanotubes into two-dimensional contiguous cellular foams. The stable nanotube foams can be elastically deformed, transferred to other substrates, or floated out to produce free-standing macroscopic fabrics. The lightweight cellular foams made of condensed nanotubes could have applications as shock-absorbent structural reinforcements and elastic membranes. The ability to control the length scale, orientation, and shape of the cellular structures and the simplicity of the assembly process make this a particularly attractive system for studying pattern formation in ordered media.     

Animal and human bite injuries: a 5-year retrospective study in a large urban public hospital in Venezuela

Introduction

Animal bite injuries to the head and neck regions are an important public health problem. Most of these bites are from dogs. A 10-year retrospective study was undertaken to determine the prevalence of animal and human bites.

Materials and methods

This retrospective study was done from January 2011 to December 2016 and included 387 patients with a mean age of 21.51 years. Data collection included age, sex, days of hospitalization, lesion type, and clinical management.

Results

Majority of patients were in age group of 21–29 years, followed by 31–55 years. Out of the total 281 patients, 42 patients (51.60%) were males and 34 patients (48.40%) were females. Mean hospital stay was 7.2 days with a minimum of 5 days and a maximum of 12 days. Surgical management included cleansing and primary closure of the wound.

Conclusions

We concluded that the use of empiric antibiotic prophylaxis is essential for management of facial animal bite, and the antibiotic of first choice is amoxicillin-clavulanic acid. The aim of immediate surgical repair (< 6 h) is to avoid infections. The persistence of dog bite is public health problem in Venezuela.

     

Post-traumatic Stress Symptoms and Exacerbations in COPD Patients

Background: Post-Traumatic Stress Disorder (PTSD) is a common psychological consequence of exposure to traumatic stressful life events. During COPD exacerbations dyspnea can be considered a near-death experience that may induce post-traumatic stress symptoms. The aim of this study was to evaluate the relationship between COPD exacerbations and PTSD- related symptoms. Method: Thirty-three in-patients with COPD exacerbations were screened for the following: PTSS (Screen for Posttraumatic Stress Symptoms), anxiety (Beck Anxiety Inventory) and depression (Beck Depression Inventory). Patients had a median age of 72 years and 72.7% were female. Results: Mean FEV1 and FVC were 0.8±0.3 (37.7 ± 14.9% of predicted) and 1.7 ± 0.6 (60 ± 18.8% of predicted), respectively with a mean exacerbation of 2.9 episodes over the past year. Post-traumatic stress symptoms related to PTSD were found in 11 (33.3%) patients (SPTSS mean score 4.13 ± 2.54); moderate to severe depression in 16 (48.5%) (BDI mean score 21.2 ± 12.1) and moderate to severe anxiety in 23 (69.7%) (BAI mean score 23.5 ± 12.4). In a linear regression model, exacerbations significantly predicted post-traumatic stress symptoms scores: SPTSS scores increased 0.9 points with each exacerbation (p = 0.001). Significant correlations were detected between PTSD-related symptoms and anxiety (rs = 0.57; p = 0.001) and PTSD symptoms and depression (rs = 0.62; p = 0.0001). In a multivariable analysis model, two or more exacerbation episodes led to a near twofold increase in the prevalence ratio of post-traumatic stress symptoms related to PTSD(PR1.71; p = 0.015) specially those requiring hospitalization (PR 1.13; p = 0.030) Conclusion: PTSD symptoms increase as the patient's exacerbations increase. Two or more exacerbation episodes lead to a near twofold increase in the prevalence ratio of post-traumatic symptomatology. Overall, these findings suggest that psychological domains should be addressed along with respiratory function and exacerbations in COPD patients.     

Role of microRNAs 221/222 on Statin Induced Nitric Oxide Release in Human Endothelial Cells

Background

Nitric oxide (NO) has been largely associated with cardiovascular protection through improvement of endothelial function. Recently, new evidence about modulation of NO release by microRNAs (miRs) has been reported, which could be involved with statin-dependent pleiotropic effects, including anti-inflammatory properties related to vascular endothelium function.

Objective

To evaluate the effects of cholesterol-lowering drugs including the inhibitors of cholesterol synthesis, atorvastatin and simvastatin, and the inhibitor of cholesterol absorption ezetimibe on NO release, NOS3 mRNA expression and miRs potentially involved in NO bioavailability.

Methods

Human umbilical vein endothelial cells (HUVEC) were exposed to atorvastatin, simvastatin or ezetimibe (0 to 5.0 μM). Cells were submitted to total RNA extraction and relative quantification of NOS3 mRNA and miRs -221, -222 and -1303 by qPCR. NO release was measured in supernatants by ozone-chemiluminescence.

Results

Both statins increased NO levels and NOS3 mRNA expression but no influence was observed for ezetimibe treatment. Atorvastatin, simvastatin and ezetimibe down-regulated the expression of miR-221, whereas miR-222 was reduced only after the atorvastatin treatment. The magnitude of the reduction of miR-221 and miR-222 after treatment with statins correlated with the increment in NOS3 mRNA levels. No influence was observed on the miR-1303 expression after treatments.

Conclusion

NO release in endothelial cells is increased by statins but not by the inhibitor of cholesterol absorption, ezetimibe. Our results provide new evidence about the participation of regulatory miRs 221/222 on NO release induction mediated by statins. Although ezetimibe did not modulate NO levels, the down-regulation of miR-221 could involve potential effects on endothelial function.     

Hypercholesterolemia and ApoE deficiency result in severe infection with Lyme disease and relapsing-fever Borrelia

The Lyme disease (Borrelia burgdorferi) and relapsing-fever (Borrelia hispanica) agents have distinct infection courses, but both require cholesterol for growth. They acquire cholesterol from the environment and process it to form cholesterol glycolipids that are incorporated onto their membranes. To determine whether higher levels of serum cholesterol could enhance the organ burdens of B. burgdorferi and the spirochetemia of B. hispanica in laboratory mice, apolipoprotein E (apoE)-deficient and low-density lipoprotein receptor (LDLR)-deficient mice that produce large amounts of serum cholesterol were infected with both spirochetes. Both apoE- and LDLR-deficient mice infected with B. burgdorferi had an increased number of spirochetes in the joints and inflamed ankles compared with the infected wild-type (WT) mice, suggesting that mutations in cholesterol transport that result in high serum cholesterol levels can affect the pathogenicity of B. burgdorferi. In contrast, elevated serum cholesterol did not lead to an increase in the spirochetemia of B. hispanica. In the LDLR-deficient mice, the course of infection was indistinguishable from the WT mice. However, infection of apoE-deficient mice with B. hispanica resulted in a longer spirochetemia and increased mortality. Together, these results argue for the apoE deficiency, and not hypercholesterolemia, as the cause for the increased severity with B. hispanica. Serum hyperlipidemias are common human diseases that could be a risk factor for increased severity in Lyme disease.Cholesterol is an essential structural component of the cell membrane of vertebrate animals, and it is required for membrane integrity and fluidity. In addition to being a component of the membrane, cholesterol is the precursor of steroid hormones and bile. In eukaryotic cells, cholesterol and sphingolipids are the main components of membrane microdomains known as lipid rafts. These microdomains are characterized as being more tightly packed than the surrounding bilayer and enriched with proteins involved in signaling (1–3).In the bloodstream of humans and other vertebrates, cholesterol is transported in lipoprotein complexes. Apolipoprotein E (apoE) binds cholesterol for transport through the circulatory system as apoE-containing chylomicrons and very-low-density lipoprotein (VLDL) particles. These apoE–cholesterol particles are internalized through the interaction with the low-density lipoprotein receptors (LDLRs). LDLR is one of the cell-surface receptors in cells that binds to apoE to clear the lipoprotein particles from the blood (4). Both apoE-deficient (apoE⁻) and LDLR-deficient (LDLR⁻) mice show elevated serum cholesterol levels and develop atherosclerotic plaques (5, 6). These mice are the most used mouse models for hyperlipidemia and atherosclerosis research.Lyme disease and relapsing-fever Borrelia have very distinct infection courses and niches in the host. In experimental mouse infections, relapsing-fever borreliae multiply in the blood, reaching high numbers (spirochetemia), until antibodies, mostly of the IgM class, clear the first peak, which is followed by several smaller peaks of antigenically variable organisms. Therefore, infection with Borrelia hispanica, an agent of relapsing fever, can be monitored by direct enumeration of the spirochetes from blood. Conversely, Borrelia burgdorferi, the agent of Lyme disease, does not have an overt spirochetemia, and it is difficult to detect in blood, but invades tissues including the skin, heart, and joints, where it can be quantified by molecular methods.The outer membrane of Borrelia is composed of phospholipids, including phosphatidylcholine and phosphatidylglycerol (7). The borreliae also have cholesterol glycolipids: cholesteryl 6-O-acyl-β-d-galactopyranoside and cholesteryl-β-d-galacto-pyranoside in B. burgdorferi; and 6-O-acylated cholesteryl β-d-glucopyranoside and cholesteryl β-d-glucopyranoside in relapsing-fever Borrelia. They also have noncholesterol glycolipids, monogalactosyl-diacylglycerol in B. burgdorferi and monoglucosyl-diacylglycerol in relapsing-fever Borrelia, as well as many lipoproteins (7–15). The presence of cholesterol is not common among prokaryotes, but it is increasingly being reported in bacterial pathogens other than Borrelia spp., including species of Helicobacter, Mycoplasma, Ehrlichia, Anaplasma, and Brachyspira (16–20).Recently, we demonstrated that B. burgdorferi acquires cholesterol from host cells (21). Cholesterol can remain free in the membrane or can be internalized and glycosylated by undetermined enzymes (22). Subsequently, cholesterol glycolipids are exported to the membrane, where they form lipid rafts (23, 24) that are cholesterol-rich domains with a selective presence of lipoproteins (25). The borreliae require cholesterol for growth and have to recruit it from the host because they cannot synthesize it. In this study, our goal was to determine whether serum hypercholesterolemia could lead to greater yields of bacteria in vivo by providing added cholesterol in a manner that would be accessible to the spirochetes. To this end, we infected apoE⁻ and LDLR⁻ mice that have increased levels of serum cholesterol with B. burgdorferi and B. hispanica. From these experiments, we concluded that infection of apoE⁻ and LDLR⁻ mice with B. burgdorferi resulted in greater severity of infection. In contrast, immune dysfunctions associated with deficiencies in the apoE⁻ mouse model, and not high cholesterol levels, led to increased severity in infection with relapsing-fever Borrelia.     

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A^Cdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.The eukaryotic cell cycle is a highly coordinated and conserved process. In addition to DNA replication, one of the major requirements for the cell to progress through the cell cycle is the precise duplication of membrane-enclosed organelles and other cellular components before cell division. Knowledge about the mechanisms regulating (membrane) lipid homeostasis during the cell cycle is scarce (1), however several levels of evidence suggest regulation of key enzymes of lipid metabolism in a cell-cycle–dependent manner. The PAH1-encoded phosphatidic acid (PA) phosphatase (Pah1), a key enzyme of triacylglycerol (TG) synthesis that provides the TG precursor diacylglycerol (DG), is phosphorylated and inactivated by the cyclin-dependent kinases Cdc28 and Pho85–Pho80 (2, 3). Kurat et al. showed that Tgl4, next to Tgl3, one of the two major TG lipases in yeast and the ortholog of mammalian ATGL (4, 5), is also phosphorylated by Cdc28. In contrast to Pah1, however, Tgl4 is activated by Cdc28 (6). This inverse regulation of Pah1 and Tgl4 by Cdc28-dependent phosphorylation led to the model by which the TG content oscillates during the cell cycle: On the one hand, TG synthesis serves as a buffer for excess de novo generated fatty acids (FAs), and on the other hand, in times of increased demand—that is, at the onset of bud formation and bud growth—Tgl4-catalyzed lipolysis becomes active to provide TG-derived precursors for membrane lipid synthesis (6).TG and membrane phospholipids share the same intermediates, PA and DG; PA is generated by sequential acylation of glycerol-3-phosphate, reactions that mostly take place in the endoplasmic reticulum (ER) membrane (7). The dephosphorylation of PA to DG by the PAH1-encoded PA phosphatase Pah1 is the major regulator of cellular TG synthesis in yeast (8), similar to its mammalian ortholog, lipin (9). According to this central role, TG content in PAH1-deficient yeast cells is decreased by 70–90%; the source of the residual TG in these mutants is currently unclear. TG synthesis from DG requires one additional acylation step that is catalyzed by the DGA1-encoded acyl-CoA–dependent DG acyltransferase (10, 11) and the phospholipid-dependent acyltransferase, encoded by LRO1 (7). Alternatively, in the presence of the phospholipid precursors, ethanolamine and/or choline, DG may be converted into phospholipids via the Kennedy pathway (7). Thus, net TG synthesis in growing cells is determined by multiple factors, including the availability of FAs, presence of lipid precursors, and the activities of PA phosphatase and the DG acyltransferases. Degradation of TG in yeast is governed by the major lipid droplet (LD)-associated lipases, encoded by TGL3 and TGL4 (4, 12); both enzymes belong to the patatin-domain–containing family of proteins, members of which play a crucial role in lipid homeostasis also in mammals (13). Multiple additional lipases exist in yeast, but their specific function and contribution to TG homeostasis may be restricted to specific growth conditions (7, 14, 15).Absence of lipolysis in mutants lacking TGL3 and TGL4 results in up to threefold elevated levels of TG and reduced levels of phosphatidylcholine and sphingolipids (4, 12, 16, 17), indicating that TG breakdown provides precursors for these lipids or generates some regulatory factors required for their synthesis. The rate of phosphatidylinositol (PI) synthesis after readdition of inositol to inositol-starved cells is reduced by 50% in lipase-deficient cells; the boost of PI synthesis under inositol refeeding conditions is completely abolished if de novo FA synthesis is additionally blocked in the lipase mutants by the inhibitor cerulenin (18). These data clearly demonstrate the requirements for TG breakdown, in addition to de novo FA synthesis, to generate precursors for membrane lipids. As a consequence of defective lipolysis, entry of quiescent cells into vegetative growth is significantly delayed; thus, TG breakdown is particularly important for promoting exit from the stationary phase and entry into the gap1 (G1) phase of the cell cycle (4, 6, 19).Progression through the cell cycle is regulated by specific checkpoint pathways that ensure completion of crucial events and execute a halt under nonconducive conditions. Checkpoint mechanisms slow down or arrest the cell cycle to enable cells to fix damage or to obtain the required metabolites before proceeding and are as such important for the integrity of cell division (20–22). According to this critical function in quality control, mutations in checkpoint genes in mammals have been linked to cancer predisposition and progression. The first discovered cell-cycle checkpoint in Schizosaccharomyces pombe that regulates entry into mitosis is executed by the Wee1 kinase (23, 24), which delays mitosis by phosphorylating and inhibiting cyclin-dependent kinase Cdk1 (25). Conversely, the phosphatase Cdc25 promotes entry into mitosis by removing the inhibitory phosphorylation of Cdk1 (26–28). The budding yeast orthologs of Wee1 and Cdc25 are called Swe1 and Mih1, and their key functions in regulating Cdk1 activity are highly conserved (29, 30). Swe1 phosphorylates Cdk1 (encoded by CDC28 in budding yeast) at the tyrosine 19 residue and inhibits its kinase activity (29, 31, 32); the Mih1 phosphatase removes this inhibitory phosphorylation initiating G2/M cell-cycle progression (26). The Swe1 and Cdk1/Cdc28 kinases operate in an autoregulatory loop in which Swe1 is initially phosphorylated and activated by Cdk1/Cdc28 that is associated with mitotic cyclins; subsequently, activated Swe1 phosphorylates and inhibits Cdk1/Cdc28 (33). The initial phosphorylation of Swe1 is opposed by the protein phosphatase 2A (PP2A) with its catalytic subunits Pph21 or Pph22 and the regulatory subunit Cdc55 (PP2A^Cdc55), which sets a threshold, limiting the activation of Swe1 by Cdk1/Cdc28 in early mitosis (34, 35). Loss of the regulatory subunit Cdc55 leads to hyperactivation of Swe1 (35); after the initial phosphorylation of Swe1 in early mitosis, subsequent phosphorylation events trigger full hyperphosphorylation of Swe1 (33), which leads to its ubiquitin-mediated degradation (36, 37). Of note, regulation of Cdk1/Cdc28 by the G1 cyclin Cln2 plays an important role in actin cytoskeleton polarization and the localized delivery of secretory vesicles, which contribute membrane lipids to the developing bud, thus linking cell surface growth to the cell cycle (38).Despite its proposed role as a gap2 phase (G2) checkpoint regulator, we now show that Swe1 kinase is responsible for the G1/S (Gap1/replication phase) cell-cycle delay in mutants defective in TG lipolysis by phosphorylating Cdk1/Cdc28 at tyrosine 19. Deletion of Swe1 in the tgl3 tgl4 lipase mutant restores normal cell-cycle progression; similarly, supplementation of mutant cells with saturated FAs (myristic acid, palmitic acid) or a precursor of sphingolipid synthesis, phytosphingosine (PHS), suppress the cell-cycle delay in the lipase mutants. These data suggest that Swe1 is a lipid-regulated kinase that is activated in the absence of specific lipids, presumably sphingolipids, and halts G1/S transition by phosphorylating Cdk1/Cdc28 in lipase-deficient cells that exit from the G0 phase of the cell cycle.