Angiotensin II favors progression to heart failure in diabetic hearts: role of myocardial fatty acid oxidation downregulation

Purpose: Diabetic myocardium is particularly vulnerable to develop heart failure in response to chronic stress conditions including hypertension or myocardial infarction. We have recently observed that angiotensin II (Ang II)-mediated downregulation of the fatty acid oxidation pathway favors occurrence of heart failure by myocardial accumulation of lipids (lipotoxicity). Because diabetic heart is exposed to high levels of circulating fatty acid, we determined whether insulin resistance favors development of heart failure in mice with Ang II-mediated myocardial remodeling.Methods: To study the combined effect of diabetes and Ang II-induced heart remodeling, we generated leptin-deficient/insulin resistant (Lepob/ob) mice with cardiac targeted overexpression of angiotensinogen (TGAOGN). Left ventricular (LV) failure was indicated by pulmonary congestion (lung weight/tibial length>+2SD of wild-type mice). Myocardial metabolism and function were assessed during in vitro isolated working heart perfusion.Results: Forty-eight percent of TGAOGN mice without insulin resistance exhibited pulmonary congestion at the age of 6 months associated with increased myocardial BNP expression (+375% compared with WT) and reduced LV power (developed pressure x cardiac output; -15%). The proportion of mice presenting heart failure was markedly increased to 71% in TGAOGN mice with insulin resistance (TGAOGN/Lepob/ob). TGAOGN/Lepob/ob mice with heart failure exhibited further increase of BNP compared with failing non-diabetic TGAOGN mice (+146%) and further reduction of cardiac power (-59%). Mice with insulin resistance alone (Lepob/ob) did not exhibit signs of heart failure or LV dysfunction. Myocardial fatty acid oxidation measured during in vitro perfusion was markedly increased in non-failing hearts from Lepob/ob mice (+380% compared with WT) and glucose oxidation decreased (-72%). In contrast, fatty acid and glucose oxidation did not differ from Lepob/ob mice in hearts from TGAOGN/Lepob/ob mice without heart failure. However, both fatty acid and glucose oxidation were markedly decreased (-47% and -48%, respectively, compared with WT/Lepob/+) in failing hearts from TGAOGN/Lepob/ob mice. Reduction of fatty acid oxidation was associated with marked reduction of protein expression of a number of regulatory enzymes implied in fatty acid oxidation.Conclusions: Insulin resistance favors the progression to heart failure during chronic exposure of the myocardium to Ang II. Our results are compatible with a role of Ang II-mediated downregulation of fatty acid oxidation, potentially promoting lipotoxicity.

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK.

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.

Is amoxicillin/clavulanic acid sufficient for preemptive antibiotic therapy in type III grade open fractures ?

The risk of infection after type III° open fractures is high (10-50%).Preemptive antibiotic therapy may prevent posttraumatic infection andimprove the outcome. Recommendations about the type and durationof antibiotic vary among the institutions and it remains unclear whethergram-negative bacilli or anaerobs need to be covered. In Europe, themost commonly recommended antibiotic is amoxicillin/clavulanic acid.We retrospectively analyzed microbiology, characteristics and outcomeof patients with open type III° fractures treated at our institution.Methods: Between 01/2005 and 12/2009 we retrospectively includedall type III grade open fractures of the leg at our institution classifiedafter Gustilo into type IIIA, IIIB and IIIC. Demographic characteristics,clinical presentation, microbiology, surgical and antibiotic treatmentand patient outcome were recorded using a standardized case-reportform.Results: 30 cases of patients with type III° open fractures wereincluded (25 males, mean age was 40.5 years, range 17-67 years).27 fractures (90%) were located on the lower leg and 3 (10%) on theupper leg. Microbiology at initial surgery was available for 19 cases(63%), of which 10 grew at least one organism (including 8 amoxicillin/clavulanic acid-resistant gram-negative bacilli [GNB], 7 amoxicillin/clavulanic acid-resistant Bacillus cereus), 11 were culture-negative.Preemptive antibiotics were given in all cases (100%) for an averageduration of 8.5 days (range 1-53 days), the most common antibioticwas amoxicillin/clavulanic acid in 60% (n = 18). 11 cases just receivedpreemptive antibiotic treatment, in 19 of 30 cases the antibiotic therapywas changed and prolonged. Microbiology at revision surgery wasavailable for 25 cases and 22 grew at least one pathogen (including32 amoxicillin/clavulanic acid-resistant gram-negative bacilli and 10amoxicillin/clavulanic acid-resistant Bacillus cereus), 3 were culturenegative.Conclusions: At initial surgery, most common isolated organismswere coagulase-negative staphylococci (43%), Bacillus cereus (23%),and gram-negative bacilli (27%), and others (7%) of which 48% wereresistant to amoxicillin/clavulanic acid. At revision surgery, isolatedorganisms were gram-negative bacilli (64%), Bacillus cereus (20%),and others (16%) of which 88% were resistant to amoxicillin/clavulanicacid. The spectrum of amoxicillin/clavulanic does not cover the mostcommon isolated organisms.FM32

A positive interaction between inhibitors of protein synthesis and cefepime in the fight against methicillin-resistant Staphylococcus aureus.

Quinupristin-dalfopristin (Q-D) synergizes with cefepime for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Here, we studied whether the synergism was restricted to MRSA and if it extended to non-beta-lactam cell wall inhibitors or to other inhibitors of protein synthesis. Three MRSA and two methicillin-susceptible S. aureus (MSSA) strains were tested, including an isogenic pair of mecA (-)/mecA (+) S. aureus Newman. The drug interactions were determined by fractional inhibitory concentration (FIC) indices and population analysis profiles. The antibacterial drugs that we used included beta-lactam (cefepime) and non-beta-lactam cell wall inhibitors (D-cycloserine, fosfomycin, vancomycin, teicoplanin), inhibitors of protein synthesis (Q-D, erythromycin, chloramphenicol, tetracycline, linezolid, fusidic acid), and polynucleotide inhibitors (cotrimoxazole, ciprofloxacin). The addition of each protein inhibitor to cefepime was synergistic (FIC ≤ 0.5) or additive (FIC > 0.5 but < 1) against MRSA, but mostly indifferent against MSSA (FIC ≥ 1 but ≤ 4). This segregation was not observed after adding cotrimoxazole or ciprofloxacin to cefepime. Population analysis profiles were performed on plates in the presence of increasing concentrations of the cell wall inhibitors plus 0.25 × minimum inhibitory concentration (MIC) of Q-D. Cefepime combined with Q-D was synergistic against MRSA, but D-cycloserine and glycopeptides were not. Thus, the synergism was specific to beta-lactam antibiotics. Moreover, the synergism was not lost against fem mutants, indicating that it acted at another level. The restriction of the beneficial effect to MRSA suggests that the functionality of penicillin-binding protein 2A (PBP2A) was affected, either directly or indirectly. Further studies are necessary in order to provide a mechanism for this positive interaction.

Antibiotic treatment of experimental endocarditis due to methicillin-resistant Staphylococcus epidermidis.

The natural history and treatment of experimental endocarditis due to heterogeneous and homogeneous methicillin-resistant Staphylococcus epidermidis was investigated. Amoxicillin/clavulanate or vancomycin were administered for 3 days via a computerized pump to mimic human drug kinetics in animals. After challenge with the minimum inoculum producing 90% of infections (ID90), bacteria in the vegetations grew logarithmically for 16 h. Then, bacterial densities stabilized (at approximately 10(8) cfu/g) and growth rates sharply declined. Both regimens cured &gt; or = 60% of endocarditis (due to heterogeneous or homogeneous bacteria) when started 12-16 h after infection, although the bacterial densities in the vegetations had increased by 20 times in between. In contrast, treatment started after 24 h failed in most animals, while bacterial densities had not increased any more. Thus, while both regimens were equivalent, the therapeutic outcome was best predicted by growth rates in the vegetations, not by bacterial densities. These observations highlight the importance of phenotypic tolerance developing in vivo.

In vitro prevention of the emergence of daptomycin resistance in Staphylococcus aureus and enterococci following combination with amoxicillin/clavulanic acid or ampicillin.

Daptomycin is bactericidal against meticillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate-resistant S. aureus (GISA) and vancomycin-susceptible and -resistant enterococci. However, selection for daptomycin-resistant derivatives has occasionally been reported during therapy in humans. Here we evaluate whether selection for daptomycin-resistant S. aureus or enterococci could be prevented in vitro by combining daptomycin with amoxicillin/clavulanic acid, ampicillin, gentamicin or rifampicin. Six strains of S. aureus (four MRSA and two GISA) and four strains of enterococci (two Enterococcus faecalis and two Enterococcus faecium) were serially exposed in broth to two-fold stepwise increasing concentrations of daptomycin alone or in combination with a fixed concentration [0.25x minimum inhibitory concentration (MIC)] of either of the second agents. The daptomycin MIC was examined after each cycle. Exposure to daptomycin alone gradually selected for S. aureus and enterococci with an increased MIC. Gentamicin did not prevent the emergence of daptomycin-resistant bacteria. Rifampicin was also unable to prevent daptomycin resistance, although resistance was slightly delayed. In contrast, amoxicillin/clavulanic acid or ampicillin prevented or greatly delayed the selection of daptomycin-resistant mutants in S. aureus and enterococci, respectively. Addition of amoxicillin/clavulanic acid or ampicillin to daptomycin prevents, or greatly delays, daptomycin resistance in vitro. Future studies in animal models are needed to predict the utility of these combinations in humans.

Expression analyses of three members of the AtPHO1 family reveal differential interactions between signaling pathways involved in phosphate deficiency and the responses to auxin, cytokinin, and abscisic acid.

The PHO1 protein is involved in loading inorganic phosphate (Pi) to the root xylem. Ten genes homologous to AtPHO1 are present in the Arabidopsis thaliana (L.) Heyn genome. From this gene family, transcript levels of only AtPHO1, AtPHO1;H1 and AtPHO1;H10 were increased by Pi-deficiency. While the up-regulation of AtPHO1;H1 and AtPHO1;H10 by Pi deficiency followed the same rapid kinetics and was dependent on the PHR1 transcription factor, phosphite only strongly suppressed the expression of AtPHO1;H1 and had a minor effect on AtPHO1;H10. Addition of sucrose was found to increase transcript levels of both AtPHO1 and AtPHO1;H1 in Pi-sufficient or Pi-deficient plants, but to suppress AtPHO1:H10 under the same conditions. Treatments of plants with auxin or cytokinin had contrasting effect depending on the gene and on the Pi status of the plants. Thus, while both hormones down-regulated expression of AtPHO1 independently of the plant Pi status, auxin and cytokinin up-regulated AtPHO1;H1 and AtPHO1;H10 expression in Pi-sufficient plants and down-regulated expression in Pi-deficient plants. Treatments with abscisic acid inhibited AtPHO1 and AtPHO1;H1 expression in both Pi-sufficient and Pi-deficient plants, but increased AtPHO1;H10 expression under the same conditions. The inhibition of expression by abscisic acid of AtPHO1 and AtPHO1;H1, and of the Pi-starvation responsive genes AtPHT1;1 and AtIPS1, was dependant on the ABI1 type 2C protein phosphatase. These results reveal that various levels of cross talk between the signal transduction pathways to Pi, sucrose and phytohormones are involved in the regulation of expression of the three AtPHO1 homologues.

ROLES OF ACID-SENSING ION CHANNELS (ASICS) IN PERIPHERAL NEUROPEPTIDE SECRETION AND PAIN

Acid-sensing ion channels (ASICs) are non-voltage-gated sodium channels activated by an extracellular acidification. They are widely expressed in neurons of the central and peripheral nervous system. ASICs have a role in learning, the expression of fear, in neuronal death after cerebral ischemia, and in pain sensation. Tissue damage leads to the release of inflammatory mediators. There is a subpopulation of sensory neurons which are able to release the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Neurogenic inflammation refers to the process whereby peripheral release of the neuropeptides CGRP and SP induces vasodilation and extravasation of plasma proteins, respectively. Our laboratory has previously shown that calcium-permeable homomeric ASIC1a channels are present in a majority of CGRP- or SP-expressing small diameter sensory neurons. In the first part of my thesis, we tested the hypothesis that a local acidification can produce an ASIC-mediated calcium-dependant neuropeptide secretion. We have first verified the co-expression of ASICs and CGRP/SP using immunochemistry and in-situ hybridization on dissociated rat dorsal root ganglion (DRG) neurons. We found that most CGRP/SP-positive neurons also expressed ASIC1a and ASIC3 subunits. Calcium imaging experiments with Fura-2 dye showed that an extracellular acidification can induce an increase of intracellular Ca2+ concentration, which is essential for secretion. This increase of intracellular Ca2+ concentration is, at least in some cells, ASIC-dependent, as it can be prevented by amiloride, an ASIC antagonist, and by Psalmotoxin (PcTx1), a specific ASIC1a antagonist. We identified a sub-population of neurons whose acid-induced Ca2+ entry was completely abolished by amiloride, an amiloride-resistant population which does not express ASICs, but rather another acid-sensing channel, possibly transient receptor potential vanilloïde 1 (TRPV1), and a population expressing both H+-gated channel types. Voltage-gated calcium channels (Cavs) may also mediate Ca2+ entry. Co-application of the Cavs inhibitors (ω-conotoxin MVIIC, Mibefradil and Nifedipine) reduced the Ca2+ increase in neurons expressing ASICs during an acidification to pH 6. This indicates that ASICs can depolarise the neuron and activate Cavs. Homomeric ASIC1a are Ca2+-permeable and allow a direct entry of Ca2+ into the cell; other ASICs mediate an indirect entry of Ca2+ by inducing a membrane depolarisation that activates Cavs. We showed with a secretion assay that CGRP secretion can be induced by extracellular acidification in cultured rat DRG neurons. Amiloride and PcTx1 were not able to inhibit the secretion at acidic pH, but BCTC, a TRPV1 inhibitor was able to decrease the secretion induced by an extracellular acidification in our in vitro secretion assay. In conclusion, these results show that in DRG neurons a mild extracellular acidification can induce a calcium-dependent neuropeptide secretion. Even if our data show that ASICs can mediate an increase of intracellular Ca2+ concentration, this appears not to be sufficient to trigger neuropeptide secretion. TRPV1, a calcium channel whose activation induces a sustained current - in contrary of ASICs - played in our experimental conditions a predominant role in neurosecretion. In the second part of my thesis, we focused on the role of ASICs in neuropathic pain. We used the spared nerve injury (SNI) model which consists in a nerve injury that induces symptoms of neuropathic pain such as mechanical allodynia. We have previously shown that the SNI model modifies ASIC currents in dissociated rat DRG neurons. We hypothesized that ASICs could play a role in the development of mechanical allodynia. The SNI model was performed on ASIC1a, -2, and -3 knock-out mice and wild type littermates. We measured mechanical allodynia on these mice with calibrated von Frey filaments. There were no differences between the wild-type and the ASIC1, or ASIC2 knockout mice. ASIC3 null mice were less sensitive than wild type mice at 21 day after SNI, indicating a role for ASIC3. Finally, to investigate other possible roles of ASICs in the perception of the environment, we measured the baseline heat responses. We used two different models; the tail flick model and the hot plate model. ASIC1a null mice showed increased thermal allodynia behaviour in the hot plate test at three different temperatures (49, 52, 55°C) compared to their wild type littermates. On the contrary, ASIC2 null mice showed reduced thermal allodynia behaviour in the hot plate test compared to their wild type littermates at the three same temperatures. We conclude that ASIC1a and ASIC2 in mice can play a role in temperature sensing. It is currently not understood how ASICs are involved in temperature sensing and what the reason for the opposed effects in the two knockout models is.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function

Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P.aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E.amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P.fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E.amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Long-term outcome of idiopathic steroid-resistant nephrotic syndrome: a multicenter study.

Long-term outcome of idiopathic steroid-resistant nephrotic syndrome was retrospectively studied in 78 children in eight centers for the past 20 years. Median age at onset was 4.4 years (1.1-15.0 years) and the gender ratio was 1.4. Median follow-up period was 7.7 years (1.0-19.7 years). The disease in 45 patients (58%) was initially not steroid-responsive and in 33 (42%) it was later non-responsive. The main therapeutic strategies included administration of ciclosporine (CsA) alone (n = 29; 37%) and CsA + mycophenolate mofetil (n = 18; 23%). Actuarial patient survival rate after 15 years was 97%. Renal survival rate after 5 years, 10 years and 15 years was 75%, 58% and 53%, respectively. An age at onset of nephrotic syndrome (NS) > 10 years was the only independent predictor of end-stage renal disease (ESRD) in a multivariate analysis using a Cox regression model (P < 0.001). Twenty patients (26%) received transplants; ten showed recurrence of the NS: seven within 2 days, one within 2 weeks, and two within 3-5 months. Seven patients lost their grafts, four from recurrence. Owing to better management, kidney survival in idiopathic steroid-resistant nephrotic syndrome (SRNS) has improved during the past 20 years. Further prospective controlled trials will delineate the potential benefit of new immunosuppressive treatment.

Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-β induction by efficient MAVS cleavage.

BACKGROUND & AIMS: The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-β, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage. METHODS: The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-β was quantified in serum from patients with different NS3-4A RAVs. RESULTS: We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-β induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-β serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs. CONCLUSIONS: Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.

Occurrence and new mutations involved in rifampicin-resistant Propionibacterium acnes strains isolated from biofilm or device-related infections.

We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.