Administration of Il-18bp by Gene Therapy Reduces Inflammation and Prevents Joint Destruction by Downregulation of Mmp9 In Rat Aia: Role ff Mmp9 In Bone and Joint Destruction in Arthritis

Background and objectives Interleukin 18 (IL-18) is a pleiotropic cytokine involved in rheumatoid arthritis (RA) pathogenesis. This study was carried out to evaluate the effi cacy of IL-18 binding protein (IL-18BP) gene therapy in the rat adjuvant- induced arthritis (AIA) model and to decipher the mechanisms by which IL-18BP delivery lessens bone destruction.Materials and methods Arthritis was induced in female Lewis rat by Mycobacterium butyricum and the mRNA expression of IL-18 and IL-18BP was determined in the joints. In a preventive study, rats were divided into an adenovirus producing IL-18BP-Fc (AdmIL-18BP-Fc) group (n=8) and an adenovirus producing green fl uorescent protein (AdGFP) group (n=7). On day 8 after AIA induction, adenoviruses were injected. Clinical parameters were assessed. At day 18, during maximal arthritis, the rats were euthanized, ankles were collected and x-rays were performed. mRNA and protein were extracted from joints for analysis by quantitative reverse transcriptase-PCR, multiplex, Western blot and zymography.Results The authors observed a decrease in the (IL-18BP/ IL-18) ratio from day 7 to 45. Administration of AdmIL-18BPd-Fc decreased clinical parameters and prevented bone and joint destruction compared to AdGFP administration. IL-18BP delivery reduced the (receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG)) ratio by 70%, the matrix metalloproteinase 9 (MMP9) level by 33% and the tartrate-resistant acid phosphatase (TRAP) level by 44% in the joint homogenates from AdmIL-18BPd-Fc compared to AdGFP treated rats.Conclusions In rat AIA, a decrease in the (IL-18BP/IL-18) ratio was observed. IL-18BP delivery prevented joint and bone destruction by downregulating MMP9, (RANKL/OPG) and TRAP, suggesting a potential benefi t of a similar therapy in RA.Abstract topics Towards novel therapeutic strategies.

Polymorphisms of the " macrophage migration inhibitory factor " gene in infectious diseases

Summary The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has emerged as a central mediator of inflammation and innate immune defense against infections. MIF has been shown to play an important role in the pathogenesis of infectious diseases like sepsis, tuberculosis and autoimmune inflammatory diseases, such as arthritis, inflammatory bowel disease and asthma. Two functional polymorphisms of the MIF gene promoter, a five to eight CATT repeat microsatellite at position -794 and a G/C SNP at position -173, have been associated with increased susceptibility to or severity of autoimmune inflammatory diseases like arthritis, colitis and atopy. The aim of this thesis was to define whether, and if so by which mechanisms, MIF gene polymorphisms influence the susceptibility to or the outcome of one of the most severe and one of the most prevalent infectious diseases: meningococcal sepsis and tuberculosis, respectively. The results of the comparison between 1106 patients suffering from severe meningococcal infections and 434 healthy volunteers showed that carriers of the CATT5-5 genotype were protected from meningococcemia. A transmission disequilibrium test involving 106 families confirmed this association. At baseline and after stimulation with Neisseria meningitidis, the CATT5 MIF promoter drove lower transcriptional activity than the CATT6 or CATT7 alleles in human monocytic cells and whole blood of CATT5-5 healthy individuals tended to produce less MIF than whole blood of CATT6-6 individuals. Beyond, we describe several new MIF gene polymorphisms in Africans. Genotyping the CATT microsatellite and the -173*G/C SNP revealed great genetic diversity in six African ethnic groups. Comparing 471 African tuberculosis cases and 932 matched healthy controls, we observed ethnicity dependent associations of the -173*G/C and the CATT5-8 with susceptibility to or severity of tuberculosis, but confirmation in larger cohorts ìs needed. In conclusion, we report that homozygous carriage of a low expression allele of the MIF gene protects from meningococcal disease. These results support the concept that analyses of MIF genotypes in patients with sepsis may help to classify patients into risk categories and to identify those patients who may benefit from anti-MIF therapeutic strategies.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

Asymmetric and differential gene introgression at a contact zone between two highly divergent lineages of field voles (Microtus agrestis).

Secondary contact zones have the potential to shed light on the mode and rate at which reproductive isolation accumulates during allopatric speciation. We investigated the population genetics of a contact zone between two highly divergent lineages of field voles (Microtus agrestis) in the Swiss Jura mountains. To shed light on the processes underlying introgression, we used maternally, paternally, and bi-parentally inherited markers. Though the two lineages maintained a strong genetic structure, we found some hybrids and evidence of gene flow. The extent of introgression varied with the mode of inheritance, being highest for mtDNA and absent for the Y chromosome. In addition, introgression was asymmetric, occurring only from the Northern to the Southern lineage. Both patterns seem parsimoniously explained by neutral processes linked to differences in effective sizes and sex-biased dispersal rates. The lineage with lower effective population size was also the more introgressed, and the mode-of-inheritance effect correlated with the male-biased dispersal rate of microtine rodents. We cannot exclude, however, that Haldane's effect contributed to the latter, as we found a marginally significant deficit in males (the heterogametic sex) among hybrids. We propose a possible demographic scenario to account for the patterns documented, and empirical extensions to further investigate this contact zone.

Partial gene deletion of endothelial nitric oxide synthase predisposes to exaggerated high-fat diet-induced insulin resistance and arterial hypertension.

Nitric oxide (NO) plays a major role in the regulation of cardiovascular and metabolic homeostasis, as evidenced by insulin resistance and arterial hypertension in endothelial NO synthase (eNOS) null mice. Extrapolation of these findings to humans is difficult, however, because eNOS gene deficiency has not been reported. eNOS gene polymorphism and impaired NO synthesis, however, have been reported in several cardiovascular disease states and could predispose to insulin resistance. High-fat diet induces insulin resistance and arterial hypertension in normal mice. To test whether partial eNOS deficiency facilitates the development of insulin resistance and arterial hypertension during metabolic stress, we examined effects of an 8-week high-fat diet on insulin sensitivity (euglycemic clamp) and arterial pressure in eNOS(+/-) mice. When fed a normal diet, these mice had normal insulin sensitivity and were normotensive. When fed a high-fat diet, however, eNOS(+/-) mice developed exaggerated arterial hypertension and had fasting hyperinsulinemia and a 35% lower insulin-stimulated glucose utilization than control mice. The partial deletion of the eNOS gene does not alter insulin sensitivity or blood pressure in mice. When challenged with nutritional stress, however, partial eNOS deficiency facilitates the development of insulin resistance and arterial hypertension, providing further evidence for the importance of this gene in linking metabolic and cardiovascular disease.

Secreted subtilisin gene family in Trichophyton rubrum.

Secreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T. rubrum and other fungi revealed a presumed ancestral lineage comprising T. rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T. rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T. rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry.

The role of gene duplication in the origin and evolution of new biological functions

Abstract : Gene duplication is an essential source of material for the origin of genetic novelty and the evolution of lineage- or species-specific phenotypic traits. The reverse transcription of source gene mRNA followed by the genomic insertion of the resulting cDNA - retroposition - has provided the human genome with a significant number of gene copies during the last ~63 million years (MYA) of primate evolution. We estimated that at least 1 new functional gene (retrogene) per MYA emerged by retroposition in the primate lineage leading to humans. Using a combination of comparative sequencing and evolutionary simulations, we obtained strong evidence of functionality for 7 primate specific retrogenes. Most of these genes are specifically expressed in testis suggesting that retroposition has contributed with genetic raw material necessary for the evolution ofmale-specific functions in primates. We characterized CDC14Bretro (identified in the previous survey) that originated from the retroposition of a cell cycle gene - CDC14B - in the common ancestor of humans and apes. We demonstrate that CDC14Bretro experienced a period of intense positive selection in the African ape ancestor. By virtue of the amino acid substitutions that occurred during this period CDC 14Bretro adapted to a new subcellular compartment in African apes. Further analyses indicate that this subcellular shift reflects the evolution of anew functional role of CDC 14Bretro. Prompted by this result, we used yeast (Saccharomyces cerevisiae) to investigate on a global scale the extent of functional diversification of duplicate genes through the subcellular adaptation of their encoded proteins. We found that duplicate proteins frequently evolved new cellular localization patterns, either by partitioning of ancestral localizations ("sublocalization"), or more frequently by relocalization to previously unoccupied compartments ("neolocalization"). Interestingly, proteins involved in processes with a wider subcellular distribution more frequently evolved new localization patterns suggesting that subcellular localization changes are dependent on progenitor gene functions. Relocated proteins adapted to their new subcellular environments and evolved new functional roles through changes of their physio-chemical properties, expression levels, and interaction partners. Our work suggests an important role of subcellular adaptation for the emergence of new gene functions.

Identification and characterization of the members of the pho1 gene family in moss and rice

Inorganic phosphate (Pi) is one of the main nutrients limiting plant growth anddevelopment in many agro-ecosystems. In plants, phosphate is acquired from the soil by theroots, and is then transferred to the shoot via the xylem. In the model plant Arabidopsisthaliana, PHO1 was previously identified as being involved in loading Pi into the xylem ofroots. AtPHO1, belongs to a multigenic family composed of 10 additional members, namelyAtPHO1;H1 to AtPHO1;10. In this study, we aimed at further investigating the role of thePHO1 gene family in Pi homeostasis in plants, and to this end we isolated and characterizedthe PHO1 members of two main model plants, the moss Physcomitrella patens and the riceOryza sativa.In the bryophyte P. patens, bioinformatic analyses revealed the presence of seven AtPHO1homologues, highly similar to AtPHO1. The seven moss PHO1 genes, namely PpPHO1;1 toPpPHO1;7 appeared to be differentially regulated, both at the tissue level and in response toPi status. However only PpPHO1;1 and PpPHO1;7 were specifically up-regulated upon Pistarvation, suggesting a potential role in Pi homeostasis. We also characterized the responseof P. patens to Pi starvation, showing that higher and lower plants share some commonstrategies to adapt to Pi-deficiency.In the second part, focusing on the monocotyledon rice, we showed the existence of threePHO1 homologues OsPHO1;1 to OsPHO1;3, with the unique particularity of each havingNatural Antisense Transcripts (NATs). Molecular analyses revealed that both the sense andthe antisense OsPHO1;2 transcripts were by far the most abundantly expressed transcripts ofthe family, preferentially expressed in the roots. The stable expression of OsPHO1;2 in allconditions tested, in opposition with the highly induced antisense transcript upon Pistarvation, suggest a putative role for the antisense in regulating the sense transcript.Moreover, mutant analyses revealed that OsPHO1;2 plays a key role in Pi homeostasis, intransferring Pi from the root to the shoot. Finally, complementing the pho1 mutant inArabidopsis, characterized by low Pi in the shoot and reduced growth, with the riceOsPHO1;2 gene revealed a new role for PHO1 in Pi signaling. Indeed, the complementedplants showed normal growth, with however low Pi content.

Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia.

Background Geleophysic dysplasia (GD, OMIM 231050) is an autosomal recessive disorder characterised by short stature, small hands and feet, stiff joints, and thick skin. Patients often present with a progressive cardiac valvular disease which can lead to an early death. In a previous study including six GD families, we have mapped the disease gene on chromosome 9q34.2 and identified mutations in the A Disintegrin And Metalloproteinase with Thrombospondin repeats-like 2 gene (ADAMTSL2). Methods Following this study, we have collected the samples of 30 additional GD families, including 33 patients and identified ADAMTSL2 mutations in 14/33 patients, comprising 13 novel mutations. The absence of mutation in 19 patients prompted us to compare the two groups of GD patients, namely group 1, patients with ADAMTSL2 mutations (n=20, also including the 6 patients from our previous study), and group 2, patients without ADAMTSL2 mutations (n=19). Results The main discriminating features were facial dysmorphism and tip-toe walking, which were almost constantly observed in group 1. No differences were found concerning heart involvement, skin thickness, recurrent respiratory and ear infections, bronchopulmonary insufficiency, laryngo-tracheal stenosis, deafness, and radiographic features. Conclusions It is concluded that GD is a genetically heterogeneous condition. Ongoing studies will hopefully lead to the identification of another disease gene.

Gene-environment interactions of selected pharmacogenes in arterial hypertension.

Hypertension affects approximately 1 billion people worldwide. Owing to population aging, hypertension-related cardiovascular burden is expected to rise in the near future. In addition to genetic variants influencing the blood pressure response to antihypertensive drugs, several genes encoding for drug-metabolizing or -transporting enzymes have been associated with blood pressure and/or hypertension in humans (e.g., ACE, CYP1A2, CYP3A5, ABCB1 and MTHFR) regardless of drug treatment. These genes are also involved in the metabolism and transport of endogenous substances and their effects may be modified by selected environmental factors, such as diet or lifestyle. However, little is currently known on the complex interplay between environmental factors, endogenous factors, genetic variants and drugs on blood pressure control. This review will discuss the respective role of population-based primary prevention and personalized medicine for arterial hypertension, taking a pharmacogenomics' perspective focusing on selected pharmacogenes.

Long-term cerebral plasticity-related gene expression : a study of the respective role of CBP and CRTC1 in CREB transcriptional activation

1.1 AbstractThe treatment of memory disorders and cognitive deficits in various forms of mental retardation may greatly benefit from a better understanding of the molecular and cellular mechanisms of memory formation. Different forms of memory have distinct molecular requirements.Short-term memory (STM) is thought to be mediated by covalent modifications of existing synaptic molecules, such as phosphorylation or dephosphorylation of enzymes, receptors or ion channels. In contrast, long-term memoiy (LTM) is thought to be mediated by growth of new synapses and restructuring of existing synapses. There is extensive evidence that changes in gene expression and de novo protein synthesis are key processes for LTM formation. In this context, the transcription factor CREB (cAMP-response element-binding protein) was shown to be crucial. Activation of CREB requires phosphorylation of a serine residue (Ser-133), and the subsequent recruitment of a coactivator called CREB-binding protein (CBP). Moreover, we have recently shown that another coactivator called CREB Regulated Transcription Coactivator 1 (CRTC1) functions as a calcium- and cAMP-sensitive coincidence detector in neurons, and is involved in hippocampal long-term synaptic plasticity. Given the importance of cAMP and calcium signaling for plasticity-related gene expression in neurons and in astrocytes, we sought to determine the respective involvement of the CREB coactivators CBP and CRTC1 in CREB-mediated transcription.We developed various strategies to selectively interfere with these CREB coactivators in mouse primary neurons and in astrocytes in vitro. However, despite several pieces of evidence implicating CBP and/or CRTC1 in the regulation of neuronal plasticity genes, we could not clearly determine the respective requirement of these coactivators for the activation of these genes. Nevertheless, we showed that calcineurin activity, which is important for CRTC1 nuclear translocation, is necessary for the expression of some CREB-regulated plasticity genes. We associated this phenomena to physiopathological conditions observed in Down's syndrome. In addition, we demonstrated that in astrocytes, noradrenaline stimulates CREB-target gene expression through β-adrenergic receptor activation, intracellular cAMP pathway activation, and CRTC-induced CREB transactivation.Defining the respective role of CREB and its coactivators CBP and CRTC1 in neuronal and astrocytic cultures in vitro sets the stage for future in vivo studies and for the possible development of new therapeutic strategies to improve the treatment of memoiy and cognitive disorders.1.2 RésuméUne meilleure connaissance des mécanismes moléculaires et cellulaires responsables de la formation de la mémoire pourrait grandement améliorer le traitement des troubles de la mémoire ainsi que des déficits cognitifs observés dans différentes formes de pathologies psychiatriques telles que le retard mental. Les différentes formes de mémoire dépendent de processus moléculaires différents.La mémoire à court terme (STM) semble prendre forme suite à des modifications covalentes de molécules synaptiques préexistantes, telles que la phosphorylation ou la déphosphorylation d'enzymes, de récepteurs ou de canaux ioniques. En revanche, la mémoire à long terme (LTM) semble être due à la génération de nouvelles synapses et à la restructuration des synapses existantes. De nombreuses études ont permis de démontrer que les changements dans l'expression des gènes et la synthèse de protéine de novo sont des processus clés pour la formation de la LTM. Dans ce contexte, le facteur de transcription CREB (cAMP-response element-binding protein) s'est avéré être un élément crucial. L'activation de CREB nécessite la phosphorylation d'un résidu sérine (Ser-133), et le recrutement d'un coactivateur nommé CBP (CREB binding protein). En outre, nous avons récemment démontré qu'un autre coactivateur de CREB nommé CRTC1 (CREB Regulated Transcription Coactivator 1) agit comme un détecteur de coïncidence de l'AMP cyclique (AMPc) et du calcium dans les neurones et qu'il est impliqué dans la formation de la plasticité synaptique à long terme dans l'hippocampe. Etant donné l'importance des voies de l'AMPc et du calcium dans l'expression des gènes impliqués dans la plasticité cérébrale, nous voulions déterminer le rôle respectif des coactivateurs de CREB, CBP et CRTC1.Nous avons développé diverses stratégies pour interférer de façon sélective avec les coactivateurs de CREB dans les neurones et dans les astrocytes chez la souris in vitro. Nos résultats indiquent que CBP et CRTC1 sont tous deux impliqués dans la transcription dépendante de CREB induite par l'AMPc et le calcium dans les neurones. Cependant, malgré plusieurs évidences impliquant CBP et/ou CRTC1 dans l'expression de gènes de plasticité neuronale, nous n'avons pas pu déterminer clairement leur nécessité respective pour l'activation de ces gènes. Toutefois, nous avons montré que l'activité de la calcineurine, dont dépend la translocation nucléaire de CRTC1, est nécessaire à l'expression de certains de ces gènes. Nous avons pu associer ce phénomène à une condition physiopathologique observée dans le syndrome de Down. Nous avons également montré que dans les astrocytes, la noradrénaline stimule l'expression de gènes cibles de CREB par une activation des récepteurs β- adrénergiques, l'activation de la voie de l'AMPc et la transactivation de CREB par les CRTCs.Définir le rôle respectif de CREB et de ses coactivateurs CBP et CRTC1 dans les neurones et dans les astrocytes in vitro permettra d'acquérir les connaissances nécessaires à de futures études in vivo et, à plus long terme d'éventuellement développer des stratégies thérapeutiques pour améliorer les traitements des troubles cognitifs.

Linkage arrangement in the vitellogenin gene family of Xenopus laevis as revealed by gene segregation analysis.

Using restriction fragment length polymorphism (RFLP) we have analyzed the segregation of alleles of the different vitellogenin genes of Xenopus laevis. The results demonstrate that the four genes whose expression is controlled by oestrogen, form two linkage groups. The genes A1, A2 and B1 are linked genetically whereas the fourth gene, the gene B2, segregates independently. The possible origin of this unexpected arrangement is discussed.