Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation: Focus on the role of Akt/mTOR and Wnt/ß-catenin pathways on adipogenesis.

Adipose-derived mesenchymal stem cells (ASCs) represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. A better understanding of the underlying mechanisms regulating ASC biology might represent the chance to modulate their in vitro characteristics and differentiation potential for regenerative medicine purposes. Herein, we investigated the effects of the demethylating agent 5-azacytidine (5-aza) on proliferation, clonogenicity, migration, adipogenic differentiation and senescence of ASCs, to identify the molecular pathways involved. Through functional assays, we observed a detrimental effect of 5-aza on ASC self-renewal capacity and migration, accompanied by actin cytoskeleton reorganization, with decreased stress fibers. Conversely, 5-aza treatment enhanced ASC adipogenic differentiation, as assessed by lipid accumulation and expression of lineage-specific markers. We analyzed the involvement of the Akt/mTOR, MAPK and Wnt/ß-catenin pathways in these processes. Our results indicated impairment of Akt and ERK phosphorylation, potentially explaining the reduced cell proliferation and migration. We observed a 5-aza-mediated inhibition of the Wnt signaling pathway, this potentially explaining the pro-adipogenic effect of the drug. Finally, 5-aza treatment significantly induced ASC senescence, through upregulation of the p53/p21 axis. Our data may have important translational implications, by helping in clarifying the potential risks and advantages of using epigenetic treatment to improve ASC characteristics for cell-based clinical approaches.

Treatment of the Mayer-Rokitansky-Küster-Hauser syndrome with autologous in vitro cultured vaginal tissue: descriptive study of long-term results and patient outcomes.

OBJECTIVE: Evaluating sexual function and quality of life (QoL) in patients treated with a modified Abbé-McIndoe technique using in vitro cultured autologous vaginal mucosa. DESIGN: Descriptive study. SETTING: Policlinico Umberto I, Sapienza University of Rome. POPULATION: From 2006 to 2016, 39 women affected by Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) underwent vaginoplasty at our centre using a modified Abbé-McIndoe technique with in vitro cultured autologous vaginal tissue. METHODS: For each patient, vaginal tissue was obtained by full-thickness biopsy of the vaginal vestibule. Following enzymatic dissociation, cells were cultured for 2-3 weeks before the transplant. MAIN OUTCOME MEASURES: Each patient completed two validated questionnaires to quantify sexual function and QoL: the Female Sexual Function Index (FSFI), administered at 12, 36, and 60 months, and the Psychological General Well Being Index (PGWBI) administered at 0, 6, and 36 months after surgery. RESULTS: Twelve months after surgery, 29 patients were engaging in regular sexual activity. The FSFI test results showed a satisfactory sexual function compared to the general population, with median values of 25.85 (range 4.6-30.5) at 12 months, 27.2 (range 4.4-33.6) at 36 months, and 29.6 (range 23.9-33.6) at 60 months. The PGWBI questionnaire showed a median score of 420.5 (range 108-540) before surgery, and 459 (range 252-533) at the 60-month follow-up. CONCLUSIONS: Vaginoplasty performed with the use of autologous vaginal tissue, besides ensuring a long-term satisfying sex life, helps in achieving an improvement in QoL that is maintained over time. TWEETABLE ABSTRACT: Vaginoplasty using in vitro vaginal tissue ensures a satisfactory sexual function and improves quality of life.

Assessment of genetic diversity and yield performance in Jordanian barley (Hordeum vulgare L.) landraces grown under Rainfed conditions.

BACKGROUND: Barley (Hordeum vulgare L.) is a major cereal crop, which is cultivated under variable environmental conditions and abiotic stresses in marginal areas around the globe. In this study, we evaluated 150 Jordanian landraces obtained from ICARDA Gene Bank and four local checks for yield and yield components related-traits in two locations across Jordan for three growing seasons under rainfed conditions. The study aims to identify superior Jordanian barley genotypes under dry conditions, to understand the genotype × environment (G × E) interactions, to analyze stability parameters and to identify markers associated with yield and yield components under rainfed conditions. RESULTS: The barley accessions exhibited significant variation for all traits studied. Three accessions with high yield, cultivar superiority and stability under specific environments were identified with accession G69 is the highest yielding and superior for Madaba and overall environments and G144 is the highest yielding at Ramtha. Accession G123 was high yielding in all environments and was stable across different environments. At the genetic level, the Jordanian landraces were found to be diverse with a clustering that was based on row-type. The GWAS analysis identified 77 significant markers-traits associations for multiple traits including grain yield (GY) with three significant QTLs located at 1H, 2H and 7H, which seem important for dry environments. CONCLUSION: Utilizing Jordanian barley landraces can effectively improve and adapt the current barley cultivars for cultivation under environmental stresses in dry regions. Utilization of markers associated with important agronomical traits and their incorporation in breeding using marker assisted selection can improve barley tolerance to drought stress.

Urinary (1)H-NMR-based metabolic profiling of children with NAFLD undergoing VSL#3 treatment.

BACKGROUND: Nowadays, non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in children. Our recent clinical trial demonstrated that dietary and VSL#3-based interventions may improve fatty liver by ultrasound and body mass index (BMI) after 4 months. OBJECTIVES: As in this short-term trial, as in others, it is impracticable to monitor response to therapy or treatment by liver biopsy, we aimed to identify a panel of potential non-invasive metabolic biomarkers by a urinary metabolic profiling. METHODS: Urine samples from a group of 31 pediatric NAFLD patients, enrolled in a VSL#3 clinical trial, were analyzed by high-resolution proton nuclear magnetic resonance spectroscopy in combination with analysis of variance-Simultaneous Component Analysis model and multivariate data analyses. Urinary metabolic profiles were interpreted in terms of clinical patient feature, treatment and chronology pattern correlations. RESULTS: VSL#3 treatment induced changes in NAFLD urinary metabolic phenotype mainly at level of host amino-acid metabolism (that is, valine, tyrosine, 3-amino-isobutyrate or ß-aminoisobutyric acid (BAIBA)), nucleic acid degradation (pseudouridine), creatinine metabolism (methylguanidine) and secondarily at the level of gut microbial amino-acid metabolism (that is, 2-hydroxyisobutyrate from valine degradation). Furthermore, some of these metabolites correlated with clinical primary and secondary trial end points after VSL#3 treatment: tyrosine and the organic acid U4 positively with alanine aminotransferase (R=0.399, P=0.026) and BMI (R=0.36, P=0.045); BAIBA and tyrosine negatively with active glucagon-like-peptide 1 (R=-0.51, P=0.003; R=-0.41, P=0.021, respectively). CONCLUSIONS: VSL#3 treatment-dependent urinary metabotypes of NAFLD children may be considered as non-invasive effective biomarkers to evaluate the response to treatment.

Variation at the vernalisation genes Vrn-H1 and Vrn-H2 determines growth and yield stability in barley (Hordeum vulgare) grown under dryland conditions in Syria.

KEY MESSAGE: Spring growth in barley controlled by natural variation at Vrn-H1 and Vrn-H2 improved yield stability in marginal Syrian environments. The objective of the present study was to identify QTL influencing agronomic performance in rain-fed Mediterranean environments in a recombinant inbred line (RIL) population, ARKE derived from the Syrian barley landrace, Arta and the Australian feed cultivar, Keel. The population was field tested for agronomic performance at two locations in Syria for 4 years with two sowing dates, in autumn and winter. Genotypic variability in yield of the RIL population was mainly affected by year-to-year variation presumably caused by inter-annual differences in rainfall distribution. The spring growth habit and early flowering inherited from the Australian cultivar Keel increased plant height and biomass and improved yield stability in Syrian environments. QTL for yield and biomass coincided with the map location of flowering time genes, in particular the vernalisation genes Vrn-H1 and Vrn-H2. In marginal environments with terminal drought, the Vrn-H1 allele inherited from Keel improved final biomass and yield. Under changing climate conditions, such as shorter winters, reduced rainfall, and early summer drought, spring barley might thus outperform the traditional vernalisation-sensitive Syrian landraces. We present the ARKE population as a valuable genetic resource to further elucidate the genetics of drought adaptation of barley in the field.

Mixed model association scans of multi-environmental trial data reveal major loci controlling yield and yield related traits in Hordeum vulgare in Mediterranean environments.

An association panel consisting of 185 accessions representative of the barley germplasm cultivated in the Mediterranean basin was used to localise quantitative trait loci (QTL) controlling grain yield and yield related traits. The germplasm set was genotyped with 1,536 SNP markers and tested for associations with phenotypic data gathered over 2 years for a total of 24 year × location combinations under a broad range of environmental conditions. Analysis of multi-environmental trial (MET) data by fitting a mixed model with kinship estimates detected from two to seven QTL for the major components of yield including 1000 kernel weight, grains per spike and spikes per m(2), as well as heading date, harvest index and plant height. Several of the associations involved SNPs tightly linked to known major genes determining spike morphology in barley (vrs1 and int-c). Similarly, the largest QTL for heading date co-locates with SNPs linked with eam6, a major locus for heading date in barley for autumn sown conditions. Co-localization of several QTL related to yield components traits suggest that major developmental loci may be linked to most of the associations. This study highlights the potential of association genetics to identify genetic variants controlling complex traits.

Hyper-IgM, neutropenia, mild infections and low response to polyclonal stimulation: hyper-IgM syndrome or common variable immunodeficiency?

A young woman presenting respiratory infections, polyarthritis, severe neutropenia, and increased serum IgM was treated with intravenous immunoglobulin (IVIG) with good clinical and laboratory outcome followed by a loss of efficacy. The increased serum IgM associated to recurrent infections and autoimmune manifestations suggested the diagnosis of a hyper-IgM syndrome (HIGMs). The frequency of peripheral T cells, the expression of CD40 on the patients' B cells and CD40L on T cells and the activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase (UNG) at mRNA level was comparable to controls. In contrast, the frequency of B cells was one half of the healthy control and all cells showed an atypical phenotype. Although AID and UNG were normal, class-switch recombination was not very efficient because circulating switched memory were reduced and, once stimulated with CpG, generated less antibody-secreting cells than controls. An increase in serum B Lymphocytes stimulator (BLyS) was also found. The patient presented a peculiar clinical and immunological phenotype fitting for many aspects of both HIGM4 and Common Variable Immunodeficiency (CVID). These findings underline the need to better explore the complex link between these two diseases.

[Inhaled corticosteroids in asthma: which have the best tolerance?]. / Corticoïdes inhalés dans l'asthme: quelle tolérance en 2009?

Asthmatic syndrome, better definition of asthma, is an inflammatory chronic disease, probably the most frequent in pediatrics. An important characteristic of asthma is the bronchial inflammation with a complex network of cells and inflammatory mediators of pivotal importance in the pathogenesis. The long term control and treatment of this disease are cardinal points of the management of asthmatic syndrome. Inhaled corticosteroids (ICS) are the first-line treatment for persistent asthma in children of any age. The adverse events of the inhaled steroids on growth, bone mineralization, and hypothalamic-pituitary adrenal (HPA) axis function are the main concerns for the pediatricians. The long-term effects on growth and bone mineralization are actually reassuring. Good tolerance is achieved on hypothalamic-pituitary adrenal axis function with low to moderate doses in children. Scientific and clinical researches are pointed to find out molecules able to improve clinical efficacy of ICS with better tolerance and higher reduction of adverse events.

Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca(2+)/Mn(2+)-adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease. OBJECTIVES: This study was designed to determine early signalling events that affect epithelial cell growth and differentiation during HHD development. METHODS: Expression of key regulatory signals important for maintaining skin homeostasis were evaluated by Western blot analysis and by reverse transcriptase-polymerase chain reaction in primary keratinocytes obtained from skin biopsies of patients with HHD. Reactive oxygen species accumulation in primary keratinocytes derived from lesional skin of patients with HHD was assessed by dihydrorhodamine 123 (DHR) assay. RESULTS: HHD-derived keratinocytes showed downregulation of both Notch1 and differential regulation of different p63 isoforms. Itch and p63 are co-expressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. We found that the Itch protein was significantly decreased in HHD-derived keratinocytes whereas the expression of its target, c-Jun, remained unaffected. We also found that HHD-derived keratinocytes undergo oxidative stress, which may explain both Notch1 and Itch downregulation. CONCLUSIONS: Our attempt to explore the molecular mechanism underlying HHD indicates a complex puzzle in which multi-hit combinations of altered signal pathways may explain the wide spectrum of defects in HHD.

Patterns of genetic diversity and linkage disequilibrium in a highly structured Hordeum vulgare association-mapping population for the Mediterranean basin.

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT((R)) markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r (2) of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT((R)) markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.

Quantitative trait loci associated with adaptation to Mediterranean dryland conditions in barley.

The objective of the present study was to identify quantitative trait loci (QTL) influencing agronomic performance across rain fed Mediterranean environments in a recombinant inbred line (RIL) population derived from the barley cultivars ER/Apm and Tadmor. The population was tested in four locations (two in Syria and two in Lebanon) during four consecutive years. This allowed the analysis of marker main effects as well as of marker by location and marker by year within location interactions. The analysis demonstrated the significance of crossover interactions in environments with large differences between locations and between years within locations. Alleles from the parent with the higher yield potential, ER/Apm, were associated with improved performance at all markers exhibiting main effects for grain yield. The coincidence of main effect QTL for plant height and yield indicated that average yield was mainly determined by plant height, where Tadmor's taller plants, being susceptible to lodging, yielded less. However, a number of crossover interactions were detected, in particular for yield, where the Tadmor allele improved yield in the locations with more severe drought stress. The marker with the highest number of cross-over interactions for yield and yield component traits mapped close to the flowering gene Ppd-H2 and a candidate gene for drought tolerance HVA1 on chromosome 1H. Effects of these candidate genes and QTL may be involved in adaptation to severe drought as frequently occurring in the driest regions in the Mediterranean countries. Identification of QTL and genes affecting field performance of barley under drought stress is a first step towards the understanding of the genetics behind drought tolerance.

QTLs for straw quality characteristics identified in recombinant inbred lines of a Hordeum vulgare x H. spontaneum cross in a Mediterranean environment.

Barley straw is commonly used as animal feed in many developing countries. Even a small increase in its nutritive value can have a large impact on animal production, and hence, on rural livelihood and human nutrition. Straw quality is strongly affected by environmental factors and is, therefore, difficult to improve with empirical breeding. The objective of this study was to identify molecular markers to facilitate the improvement of straw quality in barley. For this purpose, we have used the genetic linkage map that was already developed for recombinant inbred lines (RILs) of the cross between a Hordeum vulgare cultivar ('Arta') and a H. spontaneum line (H. spontaneum 41-1), covering a total of 890 cM. Straw parameters from RILs grown at Tel Hadya and Breda (ICARDA's research stations) in 2 years (1996/1997 and 1997/1998) were analyzed by NIRS for predicted nutritional characteristics including neutral detergent fiber, acid detergent fiber, lignin, digestible organic matter in dry matter, voluntary intake, crude protein, and straw morphology (the percentage of blades, sheaths, and stems). Localization of QTLs was performed using Windows QTL Cartographer, version 2.0. Seventy-three QTLs were identified, the majority of which (17) in the driest of the four environments. Only six QTLs were identified in two environments; in five cases, one of the two was the wettest environment. This is discussed in relation to the possibility of improving straw quality in favorable environments where yields are higher, rather than in dry environments where straw quality is already relatively good.

QTLs for agronomic traits in the Mediterranean environment identified in recombinant inbred lines of the cross 'Arta' x H. spontaneum 41-1.

A genetic linkage map has been developed for recombinant inbred lines (RILs) of the cross 'Arta' x Hordeum spontaneum 41-1. One hundred and ninety four RILs, randomly chosen from a population of 494 RILs, were mapped with 189 markers including one morphological trait ( btr = brittle rachis locus). The linkage map extended to 890 cM. Agronomic traits such as grain yield, biological yield, days to heading, plant height, cold tolerance and others were evaluated at the ICARDA research stations Tel Hadya and Breda during the years 1996-97 and 1997-98. QTLs for agronomic traits related to drought resistance were localized. For the most-important character 'plant height under drought stress', QTLs on 2H, 3H and 7H were detected. The 'plant height' QTLs, specially the one on 3H, showed pleiotropic effects on traits such as days to heading, grain yield and biological yield. QTLs were also identified for other traits associated with adaptation to the Mediterranean environment such as cold tolerance, days to heading and tiller number. The identification of QTLs for agronomic traits is a first step to analyze and to dissect complex characters such as adaptation to drought tolerance.

Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan.

In order to examine how molecular polymorphism in barley landraces, sampled from five different ecogeographical regions of Syria and Jordan, is organised and partitioned, genetic variability at 21 nuclear and 10 chloroplast microsatellite loci were examined. Chloroplast polymorphism was detected, with most variation being ascribed to differences between the five regions (Fst 0.45) and to within sites within each region (Fst 0.44). Moreover, the distribution of chloroplast polymorphism is structured and not distributed randomly across the barley landraces sampled. From a total of 125 landrace accessions (five lines from each of five sites from each of five regions) genotyped with 21 SSRs a total of 244 alleles were detected, of which 38 were common to the five regions sampled. Most nuclear variation was detected within sites. Significant differentiation between sites (Fst 0.29) was detected with nuclear SSRs and this partially mirrored polymorphism in the chloroplast genome. Strong statistical associations/interaction was also detected between the chloroplast and nuclear SSRs, together with non-random association (linkage disequilibrium) of alleles at both linked and unlinked SSR loci. These results are discussed in the context of adaptation of landraces to the extreme environment, the concept of 'adapted gene complexes' and the exploitation of landraces in breeding programmes.

Discovery of a new efficient chiral ligand for copper-catalyzed enantioselective Michael additions by high-throughput screening of a parallel library.

A combinatorial library of 125 chiral Schiff base ligands 5 was synthesized with the use of solution-phase parallel synthesis and solid-phase extraction (SPE) techniques to scavenge excess reagents and reaction by-products and avoid chromatography. The synthetic methodology coupled five N-Boc-protected beta-amino sulfonyl chlorides 1a-e with five different amines 2f-j to give 25 N-Boc sulfonamides 3, which were in turn deprotected and coupled with five salicylaldehydes 4p-t to give 125 ligands 5 in good yields and of sufficient purity to be used in ligand-catalyzed reactions. These ligands were tested in the copper-catalyzed conjugate addition of dialkyl zinc to cyclic and acyclic enones. A multisubstrate high-throughput screening of the library was performed with an equimolar mixture of 2-cyclohexenone and 2-cycloheptenone (9 and 10, respectively, 0.2 mmol total), with 5.5 mol% ligand 5 (0.011 mmol) and 5 mol% Cu(OTf)2 (OTf= OSO2CF3) (0.010 mmol) in 1:1 toluene/ hexane at - 20 degrees C. From the screening of the library, 5bhr was identified as the best ligand, which yielded 3-ethylcyclohexanone (12) and 3-ethylcycloheptanone (13) in 82% and 81% ee, respectively, and complete conversions. Under optimized conditions (2.75 mol% 5bhr, 2.5 mol% copper(i) triflate, toluene as reaction solvent), improved results were obtained for 12 (90% ee, 93% yield) and for 13 (91% ee, 95% yield). Selected ligands 5 were also tested in the addition of Me2Zn to 2-cyclohexenone (9, ee up to 79%), of Et2Zn to 2-cyclopentenone (11, ee up to 80%) and to acyclic enones 16 and 17 (ee up to 50%).

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Synthesis and application of novel catalytically active polymers containing 1,4,7-triazacyclononanes.

New polymers containing 1,4,7-triazacyclononanes have been synthesised by means of ring opening metathesis polymerisation (ROMP); their complexes with Mn(II) catalyse the oxidation of simple olefins by hydrogen peroxide.

IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury. METHODS: Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively. RESULTS: The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury. CONCLUSIONS: Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.