Étude in vitro de l’implication des cytokines de type Th17 dans la fibrose hépatique

Introduction: L’activation des cellules stellaires hépatiques (CSHs) est un point clé du processus de fibrose hépatique. Les lymphocytes T CD4+ intra-hépatiques sont une source majeure de cytokines anti-inflammatoires comme l’IL-10 et pro-inflammatoire (IL-17A), hépatoprotectrice (IL-22) produites par les Th17. Les Th17 sont impliqués dans de nombreuses pathologies inflammatoires mais l’effet de ces cellules sur les CSHs n’est pas encore élucidé. Objectif: Comprendre le rôle des cytokines de type Th17 dans le processus d’activation des CSHs. Méthodes: La lignée de CSHs humaine LX2 a été stimulée par l’IL-17A ou l’IL-22 puis comparée à des cellules traitées par le TGF-b et le tampon phosphate salin (PBS). L’activation des CSHs a été évaluée en examinant les molécules profibrotique alpha-smooth muscle actin (a-SMA), collagène de type I (COL1A1) et inhibiteur produits par les tissus des métalloprotéases matricielles I (TIMP-I) par q-PCR. L’expression protéique a été validée par immunobuvardage ou coloration au rouge de picro Sirius. L’expression membranaire de l’IL-10Rb, du TGF-b-RII et de l’IL-17RA a été mesurée par cytométrie en flux. Résultats: L’IL-17A et l’IL-22 n’activent pas les cellules LX2, car aucune induction d’a-SMA, de COL1A1 et de TIMP-I n’a été observée. Cependant, l’IL-17A et l’IL-22 sensibilisent les CSHs à l’action du TGF-b, tel que démontré par une forte expression et production d’a-SMA, collagène type I et TIMP-I. L’IL-17A, mais pas l’IL-22, induit la surexpression à la surface cellulaire du TGF-b-RII et inhibe partiellement la baisse d’expression du TGF--RII après stimulation au TGF-b. Conclusion: Nos résultats démontrent une fonction pro-fibrotique de l’IL-17A et de l’IL-22, car les deux cytokines sensibilisent les CSHs à l’action du TGF-b. L’IL-17A agit via la surexpression et la stabilisation du TGF-b-RII tandis que l’IL-22 agit probablement par des mécanismes intracellulaires.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Influence of SCARB1 polymorphisms on serum lipids of hypercholesterolemic individuals treated with atorvastatin

Background: The SR-BI is a key component on the cholesterol metabolism. Polymorphisms in the SR-BI gene (SCARB1) were related with variations on plasma lipoprotein profile and other risk factors for cardiovascular disease. We tested the relationship of 3 SCARB1 single nucleotide polymorphisms (SNPs) with hypercholesterolemia in a Brazilian population and whether these variants can influence lipid-lowering response to atorvastatin. Methods: c.4G>A, c.726+54C>T and c.1050C>T SNPs and serum concentrations of lipid and apolipoproteins were evaluated in 147 hypercholesterolemic (HC) and 185 normolipidemic (NL) unrelated Brazilian subjects. HC patients were treated with atorvastatin (10 mg/day/4 weeks). Results: Frequencies of SCARB1 polymorphisms were similar between the HC and NL groups (p>0.05). The T allele for c.726+54C>T was associated with higher LDL-c in NL and with higher apoB and apoB/apoAI in HC (p<0.05). HC individuals carrying c.1050C allele carriers (CC and CT genotypes) had lower change of total cholesterol, LDL-c, apoB and apoB/apoAI ratio (p<0.05) than the TT genotype carriers in response to atorvastatin. Conclusion: The SCARB1 polymorphisms are related with variations in serum lipids in the Brazilian population and c.1050C>T SNP is associated with lipid-lowering atorvastatin response. (C) 2010 Elsevier B.V. All rights reserved.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The <i>Saccharomyces cerevisiae RAD1i> and human <i>XPFi> genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An <i>Arabidopsis thalianai> gene (<i>AtRAD1i>) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct <i>AtRAD1i> cDNAs from <i>A. thalianai> leaf tissue RNA. One of the isolates (<i>AtRAD1-1i>) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (<i>AtRAD1-2, AtRAD1-3i>) differ slightly in size due to variations at the 5&prime; end of exon 6 or the 3&prime; end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the <i>AtRAD1-1i> and <i>AtRAD1-2i> but not <i>AtRAD1-3i> transcripts in bud and root tissue, and to a fourth transcript (<i>AtRAD1-4i>), having both alterations identified in <i>AtRAD1-2i> and <i>AtRAD1-3i>, in root tissue. However, the low frequency of detection of <i>AtRAD1-3i> and <i>AtRAD1-4i> makes the significance of these tissue-specific patterns unclear. The predicted <i>AtRad1-2i>, <i>AtRad1-3i> and <i>AtRad1-4i> proteins lack part of the region likely required for endonuclease complex formation. Expression of <i>AtRAD1-2i> and <i>AtRAD1-3i> in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

Complete mitochondrial DNA sequences of the decapod crustaceans pseudocarcinus gigas (Menippidae) and macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab <i>Pseudocarcinns i>gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp <i>Macrobrachium rosenbergiii> (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of <i>M. rosenbergiii> corresponds with that of the presumed ancestral arthropod gene order, represented by <i>Limulus polyphemusi>, except for the position of the tRNA^Leu(UUR) gene. The<i> Pse. gigasi> gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.

Characterization of haemolytic phospholipase A2 activity in clinical isolates of Campylobacter concisus

A membrane-bound, haemolytic phospholipase A2 (PLA2) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA2 activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA2 activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of <i>pldAi> gene (phospholipase A gene) from <i>Campylobacter colii> amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in <i>C. i>concisus. The detection of haemolytic PLA₂ activity in <i>C.i> concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that <i>C.i> concisus is a possible opportunistic pathogen.

Functional studies on the Wilson copper P-Type ATPase and toxic milk mouse mutant

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the <i>ATP7Bi> gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the <i>Atp7Bi> for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.

Characterization of apolipoprotein E genetic variations in Taiwanese: assocation with coronary heart disease and plasma lipid levels

Apolipoprotein E (apoE, protein; <i>APOEi>, gene) is important in lipoprotein metabolism. Three isoforms, apoE2 (Cys112 Cys158), apoE3 (Cys112 Arg158), and apoE4 (Arg112 Arg158), are present in the general population. This report investigates the frequency distribution of apoE isoforms and the association of <i>APOEi> genotypes with plasma lipid profile and coronary heart disease (CHD) in a population of Taiwan. ApoE isoforms were determined genetically by polymerase chain reaction and <i>HhaIi> restriction enzyme digestion in control and coronary heart disease (CHD) patients. Plasma lipid and lipoprotein concentrations were also determined. The control group exhibited frequencies of 84.6% <i>APOE3i>, 7.9% <i>APOE4i>, 7.5% <i>APOE2i>, 70.6% <i>APOE3E3i>, 14.4% <i>APOE3E4i>, 13.6% <i>APOE2E3i>, and 1.4% <i>APOE2E4i>. Comparable frequencies were observed in the CHD group. In both <i>APOE2i> carrier and <i>APOE3E3i> groups, the CHD patients expressed abnormal lipid profiles while the control group expressed normal lipid profiles. The <i>APOE4i> carriers, however, expressed abnormal lipid profiles in both normal control and CHD groups. Extremely high apoE levels in the hypertriglyceridemic group (TG > 400 mg/dL) seemed to be undesirable and were often observed in CHD patients

A novel zebrafish jak2a V581F model shared features of human JAK2 V617F polycythemia vera

Objective : The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2<i>^V617Fi>) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2^<i>V617Fi> (referred herewith jak2a^<i>V581Fi>) by site-directed mutagenesis and examined its relevance as a model of human PV.

Materials and Methods : Zebrafish embryos at one-cell stage were injected with jak2a^<i>V581Fi> mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.

Results : Injection with jak2a^<i>V581Fi> mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp+ population in dissociated Tg(<i>gata1:gfpi>) embryos. The response was reduced by <i>stat5.1i> morpholino coinjection (control: 4.37% ± 0.08%;<i> jak2ai>^<i>V581Fi> injected: 5.71% ± 0.07%, coinjecting <i>jak2ai>^<i>V581Fi> mRNA and <i>stat5.1i> morpholino: 4.66% ± 0.13%; <i>pi> < 0.01). <i>jak2ai>^<i>V581Fi> mRNA also upregulated <i>gata1i> (1.83 ± 0.08 fold; <i>pi> = 0.005), <i>embryonic α-hemoglobini> (1.61 ± 0.12 fold; <i>pi> = 0.049), and <i>β-hemoglobini> gene expression (1.65 ± 0.13–fold; <i>pi> = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by <i>stat5.1i> morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. <i>jak2ai>^<i>V581Fi> mRNA significantly reduced erythropoietin gene (0.24 ± 0.03 fold; <i>pi> = 0.006) and protein expression (control: 0.633 ± 0.11;<i> jak2ai>^<i>V581Fi> mRNA: 0.222 ± 0.07 mIU/mL; <i>pi> = 0.019).

Conclusion : The zebrafish <i>jak2ai>^<i>V581Fi> model shared many features with human PV and might provide us with mechanistic insights of this disease.

Two novel missense mutations in hairy-and-enhancer-of-split-7 in a family with spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder with abnormal vertebral segmentation that results in extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Four genes causing a subset of autosomal recessive forms of this disease have been identified: <i>DLL3i> (SCDO1: MIM 277300), <i>MESP2i> (SCDO2: MIM 608681), <i>LFNGi> (SCDO3: MIM609813) and <i>HES7i> (SCDO4). These genes are all essential components of the Notch signalling pathway, which has multiple roles in development and disease. Previously, only a single SCD-causative missense mutation was described in <i>HES7i>. In this study, we have identified two new missense mutations in the <i>HES7i> gene in a single family, with only individuals carrying both mutant alleles being affected by SCD. In vitro functional analysis revealed that one of the mutant <i>HES7i> proteins was unable to repress gene expression by DNA binding or protein heterodimerization.

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of <i>Plasmodium falciparumi>. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in <i>P.falciparumi> blood-stage parasites to disrupt the <i>RAP1i> gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

New insights into the role of MHC diversity in devil facial tumour disease

Devil facial tumour disease (DFTD) is a fatal contagious cancer that has decimated Tasmanian devil populations. The tumour has spread without invoking immune responses, possibly due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Animals from a region in north-western Tasmania have lower infection rates than those in the east of the state. This area is a genetic transition zone between sub-populations, with individuals from north-western Tasmania displaying greater diversity than eastern devils at MHC genes, primarily through MHC class I gene copy number variation. Here we test the hypothesis that animals that remain healthy and tumour free show predictable differences at MHC loci compared to animals that develop the disease.

Only the B biotype of Bemisia tabaci is present on vegetables in São Paulo State, Brazil

Bemisia tabaci (Genn.) é considerada uma das mais importantes pragas em cultivos de hortaliças e ornamentais em todo o mundo. Baseado na análise da seqüência mitocondrial (citocromo oxidase I - mtCOI) foi proposto recentemente que B. tabaci deva ser considerado um complexo críptico de espécies, contendo 11 grupos e 24 espécies. Dois destes grupos: Middle East-Asia Minor e Mediterranean englobam os biótipos B e Q, respectivamente. Avaliou-se a sequência mtCOI de espécimes de B. tabaci coletados em regiões do estado de São Paulo, Brasil. Por PCR-RFLP utilizando-se a enzima Taq I, pôde-se observar somente o padrão típico de clivagem para o biótipo B. Comparando-se com sequências consenso, todas as moscas brancas foram classificadas no grupo Middle East-Asia Minor e puderam ser separadas em quatro haplótipos, indicando prevalência do biótipo B em áreas de pimentão (Capsicum annuum L.), tomate (Solanum lycopersicum L.), cucurbitáceas e berinjela (Solanum melongena L.) do Estado de São Paulo.

Prostaglandin production by human gingival fibroblasts inhibited by triclosan in the presence of cetylpyridinium chloride

Background: the effect of triclosan plus the cationic detergent cetylpyridinium chloride (CPC) was evaluated for prostaglandin inhibition in human gingival fibroblasts. Since triclosan has previously been shown to inhibit proinflammatory cytokine induced prostaglandin E-2 (PGE(2)) production, we wanted to determine if triclosan, in the presence of CPC, could enhance these effects.Methods: Initial studies determined that both triclosan and CPC were cytotoxic to human gingival fibroblasts in concentrations exceeding 1.0 mu g/ml for either agent longer than 24 hours in a tissue culture. Therefore, subsequent studies measuring prostaglandin biosynthesis and cyclooxygenase (COX)-1 and COX-2 mRNA expression were performed in concentrations and times that did not significantly affect cell viability.Results: PGE2 biosynthesis was dose dependently inhibited by both triclosan and triclosan and CPC when challenged by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. At pharmacologically relevant concentrations, triclosan and CPC inhibited ILAP-induced PGE(2) production to a greater extent than triclosan alone (P = 0.02). Moreover, enhanced COX-2 mRNA repression was observed with triclosan and CPC in comparison to triclosan alone in IL-1 beta and TNF-alpha stimulated cells. No effect on COX-I gene expression was observed. Further analysis of cell signaling mechanisms of triclosan and CPC indicates that nuclear factor-kappa B (NF-kappa B) and not p38 mitogen-activated protein kinase (MAPK) signaling may be impaired in the presence of triclosan and CPC.Conclusion: This study indicates that triclosan and CPC are more effective at inhibiting PGE(2) at the level of COX-2 gene regulation, and this combination may offer a potentially better anti -inflammatory agent in the treatment of inflammatory lesions in the oral cavity.