Linking epigenetics to lipid metabolism: focus on histone deacetylases.

A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

The symptomatic phases of many inflammatory diseases are characterized by migration of large numbers of neutrophils (PMN) across a polarized epithelium and accumulation within a lumen. For example, acute PMN influx is common in diseases of the gastrointestinal system (ulcerative colitis, Crohn's disease, bacterial enterocolitis, gastritis), hepatobiliary system (cholangitis, acute cholecystitis), respiratory tract (bronchial pneumonia, bronchitis, cystic fibrosis, bronchiectasis), and urinary tract (pyelonephritis, cystitis). Despite these observations, the molecular basis of leukocyte interactions with epithelial cells is incompletely understood. In vitro models of PMN transepithelial migration typically use N-formylated bacterial peptides such as fMLP in isolation to drive human PMNs across epithelial monolayers. However, other microbial products such as lipopolysaccharide (LPS) are major constituents of the intestinal lumen and have potent effects on the immune system. In the absence of LPS, we have shown that transepithelial migration requires sequential adhesive interactions between the PMN beta2 integrin CD11b/CD18 and JAM protein family members. Other epithelial ligands appear to be abundantly represented as fucosylated proteoglycans. Further studies indicate that the rate of PMN migration across mucosal surfaces can be regulated by the ubiquitously expressed transmembrane protein CD47 and microbial-derived factors, although many of the details remain unclear. Current data suggests that Toll-like receptors (TLR), which recognize specific pathogen-associated molecular patterns (PAMPs), are differentially expressed on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. Exposure of epithelial TLRs to microbial ligands has been shown to result in transcriptional upregulation of inflammatory mediators whereas ligation of leukocyte TLRs modulate specific antimicrobial responses. A better understanding of these events will hopefully provide new insights into the mechanisms of epithelial responses to microorganisms and ideas for therapies aimed at inhibiting the deleterious consequences of mucosal inflammation.

Failure of neutrophil migration toward infectious focus in severe sepsis: a critical event for the outcome of this syndrome

Sepsis is a systemic inflammatory response commonly caused by bacterial infection. We demonstrated that the outcome of sepsis induced by cecal ligation and puncture (CLP) correlates with the severity of the neutrophil migration failure towards infectious focus. Failure appears to be due to a decrease in the rolling and adhesion of neutrophil to endothelium cells. It seems that neutrophil migration impairment is mediated by the circulating inflammatory cytokines, such as TNF-alpha and IL-8, which induce the nitric oxide (NO) production systemically. It is supported by the fact that intravenous administration of these cytokines reduces the neutrophil migration induced by different inflammatory stimuli, and in severe sepsis the circulating concentrations of the cytokines and chemokines are significantly increased. Moreover, the neutrophil migration failure and the reduction in the rolling/adhesion were not observed in iNOS-/- mice and, aminoguanidine prevented this event. We also demonstrated that the failure of neutrophil migration is a Toll-4 receptor (TLR4) dependent mechanism, since it was not observed in TLR4 deficient mice. Furthermore, it was also observed that circulating neutrophils obtained from septic patients present failure of neutrophil chemotaxis toward fMLP, IL-8, and LTB4 and an increased in sera concentrations of NO3 and cytokines. In conclusion, we demonstrated that, in sepsis, failure of neutrophil migration is critical for the outcome and that NO is involved in the process.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Etude d'administration contrôlée de cannabis et profils cinétiques des cannabinoïdes dans les fluides biologiques

Le cannabis est l'une des drogues illégales les plus consommées au monde. Le delta-9- tétrahydrocannabinol (THC) est le composé psychoactif majeur du cannabis et est fréquemment détecté dans le sang de conducteurs impliqués dans un accident de la route, alors qu'il est actuellement interdit de conduire sous l'influence du THC en Suisse comme dans de nombreux pays européens. La détection de ce composé dans le sang est suivie d'une série de mesures pénales et administratives à l'encontre du conducteur impliqué. Cette thèse intitulée « Etude d'administration contrôlée de cannabis et profils cinétiques dans les fluides biologiques » s'inscrit dans une étude toxicologique menée au Centre Hospitalier Universitaire Vaudois (CHUV). Le but de cette étude est d'évaluer les effets du cannabis sur le fonctionnement du cerveau lorsque le consommateur accompli une tâche fondamentale de la conduite automobile. Pour cela, l'imagerie médicale par résonnance magnétique fonctionnelle (IRMf) est combinée à un test informatisé simulant une tâche psychomotrice requise pour une conduite automobile sûre. Il s'agit d'un test de double tâche de poursuite d'une cible au moyen d'un curseur dirigé par un joystick, combiné à une tâche secondaire de détection de signaux routiers. Parallèlement, les profils cinétiques sanguins et salivaires des volontaires ont été déterminés grâce à des prélèvements effectués tout au long de la journée d'étude. Les objectifs principaux de ce travail de thèse sont les suivants : après une revue de la littérature existante sur les techniques d'analyse des cannabinoïdes dans les fluides biologiques, une méthode a été développée et validée pour ces composés dans la salive puis appliquée aux échantillons de l'étude. En parallèle, les échantillons sanguins et urinaires ont été analysés, en partie avec une méthode adaptée de celle développée pour la salive. Pour montrer la versatilité de cette méthode, celle-ci a été employée pour analyser des échantillons de bile qui a l'avantage de contenir des concentrations élevées de métabolites conjugués. Dans un deuxième temps, les dosages effectués sur le sang et la salive ont permis d'établir les profils cinétiques des cannabinoïdes, de déterminer leurs paramètres pharmacocinétiques et de les comparer. Les données obtenues ont montré que la concentration sanguine en acide 11-nor-9- carboxy-delta-9-tétrahydrocannabinol (THCCOOH) pouvait servir à distinguer les fumeurs occasionnels des fumeurs réguliers de cannabis, car elle est significativement plus élevée chez les consommateurs réguliers. De plus, d'autres cannabinoïdes présents dans la salive pourraient servir de marqueurs de consommation récente dans ce fluide biologique. Enfin, que ce soit dans le sang, la salive ou l'urine, des phytocannabinoïdes peuvent être utilisés comme marqueur de consommation de cannabis illégal car ils sont absents des médicaments à base de cannabis utilisant des préparations synthétiques ou purifiées du THC.

Potential value of biliary CEA assay in early detection of colorectal adenocarcinoma liver metastases.

Elevation of the biliary CEA level in patients with liver metastases from colorectal carcinoma has been reported. The aim of this study is to determine the potential value of biliary CEA assay in the early detection of liver metastases. Biliary and serum CEA levels were determined in patients operated on for a colorectal cancer and in control groups. Among 13 patients with liver metastases from colorectal carcinomas, biliary CEA levels were markedly elevated (> 40 ng/ml) in nine, moderately elevated (5-40 ng/ml) in two and normal (arbitrarily defined as < 5 ng/ml) in two. Of 28 patients with primary colorectal carcinoma without detectable hepatic secondaries, three had marked CEA elevation in the bile, 10 had moderate CEA elevation and 15 had normal levels. Among nine patients with non-malignant hepatobiliary pathology, there was one marked biliary CEA elevation, one moderate elevation and seven normal levels. None of the 13 individuals with no identified hepatobiliary pathology had elevated biliary CEA levels. The follow-up of patients with a primary colorectal tumour, no evidence of hepatic secondaries and a biliary CEA elevation is of particular interest. If subsequent appearance of liver metastases is found in such cases, intra-operative biliary CEA assay could be considered a valuable diagnostic test. Further studies will then have to prove the possible benefit of a specific treatment for this group of patients.

Functional late outgrowth endothelial progenitors isolated from peripheral blood of burned patients.

BACKGROUND: Bioengineered skin substitutes are increasingly considered as a useful option for the treatment of full thickness burn injury. Their viability following grafting can be enhanced by seeding the skin substitute with late outgrowth endothelial progenitor cells (EPCs). However, it is not known whether autologous EPCs can be obtained from burned patients shortly after injury. METHODS: Late outgrowth EPCs were isolated from peripheral blood sampled obtained from 10 burned patients (extent 19.6±10.3% TBSA) within the first 24h of hospital admission, and from 7 healthy subjects. Late outgrowth EPCs were phenotyped in vitro. RESULTS: In comparison with similar cells obtained from healthy subjects, growing colonies from burned patients yielded a higher percentage of EPC clones (46 versus 17%, p=0.013). Furthermore, EPCs from burned patients secreted more vascular endothelial growth factor (VEGF) into the culture medium than did their counterparts from healthy subjects (85.8±56.2 versus 17.6±14pg/mg protein, p=0.018). When injected to athymic nude mice 6h after unilateral ligation of the femoral artery, EPCs from both groups of subjects greatly accelerated the reperfusion of the ischaemic hindlimb and increased the number of vascular smooth muscle cells. CONCLUSIONS: The present study supports that, in patients with burns of moderate extension, it is feasible to obtain functional autologous late outgrowth EPCs from peripheral blood. These results constitute a strong incentive to pursue approaches based on using autotransplantation of these cells to improve the therapy of full thickness burns.

Description of Evandromyia grimaldii sp. nov. (Diptera: Psychodidae), a new phlebotomine species from the state of Espírito Santo, Southeast Brazil

Phlebotomine sand flies present great species diversity, especially in Brazil, where there are about 300 known species. This work describes a new species of sand fly found in the Brazilian state of Espírito Santo, in the Reserva Biológica de Duas Bocas, municipality of Cariacica. Spermathecae with superficial striations and the common duct longer than the genital fork permit the inclusion of the new species in the subgenus Evandromyia s. str., series rupicola. The new species resemble Ev. rupicola from which it may be distinguished by the aspects of the spermatheca and the length of the genital filament of the male, longer in the new species.

Epidermal growth factor receptors, testosterone levels and parotid gland changes in rats infected with Trypanosoma cruzi

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease.

Background: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. Methods: CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. Results: A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. Conclusions: The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Regulation of the vascular gap junctions in a mouse model of intimal hyperplasia

Objective: Intimal hyperplasia (IH) is one of the leading causes of failure¦after vascular interventions. It involves the proliferation of smooth muscle¦cells (SMCs) and the production of extracellular fibrous matrix. Gap junctional¦communication, mediated by membrane connexins (Cx), participates to the¦control of proliferation and migration. In human and mice vessels, endothelial¦cells (ECs) express Cx37, Cx40 and Cx43, whereas SMCs are coupled by Cx43.¦We previously reported that Cx43 was increased in the SMCs of a human vein¦during the development of IH.¦In our experimental model of mice carotid artery ligation (CAL), luminal¦narrowing occurred by SMCs-rich neointima after 2-4 weeks of ligation.¦This experimental model of mice allows us to decipher the regulation of the¦cardiovascular connexins in the mouse.¦Methods: C57BL/6 mice were anesthetized and the left common carotid artery¦was dissected through a neck incision and ligated near the carotid bifurcation.¦The mice were then euthanized at 7, 14 and 28 days. Morphometric analyses¦were then performed with measurements of total area, lumen and intimal area¦and media thickness. Western blots, immunocytochemistry and quantitative¦RT-PCR were performed for Cx43, Cx40 and Cx37.¦Results: All animals recovered with no symptom of stroke. Morphometric¦analysis demonstrated that carotid ligation resulted in an initial increase (after¦7 days) of the total vessel area followed by its reduction (after 28 days). This¦phenomena was associated with a progressive increase in the intimal area and a¦consecutive decrease of the lumen. The media thickness was also increased after¦14 and 28 days. This neointima formation was associated to a marked increase¦in the expression of Cx43 at both protein and RNA levels. Concomitantly,¦Cx40 and Cx37 protein expression were reduced in the endothelium. This was¦confirmed by en face analyses showing reduced Cx37 and Cx40 levels in the¦endothelial cells covering the lesion.¦Conclusion: This study assessed the regulation of the cardiovascular connexins¦in the development of IH. This model will allow us to characterize the¦involvement of gap junctions in the IH. In turn, this understanding is¦instrumental for the development of new therapeutical tools, as well as for¦the evaluation of the effects of drugs and gene therapies of this disease for which¦there is no efficient therapy available.

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

The role of circadian timing system on drug metabolism and detoxification.

INTRODUCTION: It has been known for a long time that the efficiency and toxicity of drugs change during a 24-h period. However, the molecular mechanisms involved in these processes have started to emerge only recently. AREAS COVERED: This review aims to highlight recent discoveries showing the direct role of the molecular circadian clock in xenobiotic metabolism at the transcriptional and post-transcriptional levels in the liver and intestine, and the different ways of elimination of these metabolized drugs via biliary and urine excretions. Most of the related literature focuses on transcriptional regulation by the circadian clock of xenobiotic metabolism in the liver; however, the role of this timing system in the excretion of metabolized drugs and the importance of the kidney in this phenomenon are generally neglected. The goal of this review is to describe the molecular mechanisms involved in rhythmic drug metabolism and excretion. EXPERT OPINION: Chronopharmacology is used to analyze the metabolism of drugs in mammals according to the time of day. The circadian timing system plays a key role in the changes of toxicity of drugs by influencing their metabolisms in the liver and intestine in addition to their excretion via bile flow and urine.