CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury.

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte-endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing.

The β-interferon scaffold attachment region confers high-level transgene expression and avoids extinction by epigenetic modifications of integrated provirus in adipose tissue-derived human mesenchymal stem cells.

Because of their abundance and ease of isolation, multilineage differentiation, and paracrine and immunoregulatory capabilities, genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) might combine cell- and gene therapy-based strategies for efficacious tissue repair/regeneration. In this report, we aimed to analyze and influence the long-term dynamics of transgene expression in ASCs transduced with different gammaretroviral vector configurations incorporating the human β-interferon scaffold attachment region (IFN-SAR) and/or chicken 5'HS4 β-globin insulator sequences. In our undifferentiated ASC culture model, naked retroviral vectors experienced EGFP transgene extinction correlating with increases in both H3 histone deacetylation and CpG dinucleotide methylation within the 5' long terminal repeat-primer-binding site proviral region.

Characterization of mesenchymal stem cells isolated from the rabbit fetal liver.

Physiological attributes of mesenchymal stem cells (MSCs) including straightforward manipulation, multilineage differentiation, immunoregulation, and tropism for injury settings render them ideal therapeutic agents for tissue repair/regeneration. Nevertheless, further studies in suitable animal models of disease are needed to translate the potential of MSCs into clinical applications. We report here the isolation and preliminary characterization of MSCs from fetal rabbit liver (fl-MSCs).

Changes in the expression profile of the meiosis-involved mismatch repair genes in impaired human spermatogenesis.

DNA mismatch repair (MMR) genes have been described to participate in crossover events during meiotic recombination, which is, in turn, a key step of spermatogenesis. This evidence suggests that MMR family gene expression may be altered in infertile men with defective sperm production. In order to determine the expression profile of MMR genes in impaired human spermatogenesis, we performed transcript levels analysis of MMR genes (MLH1, MLH3, PMS2, MSH4, and MSH5), and other meiosis-involved genes (ATR, HSPA2, and SYCP3) as controls, by real-time reverse transcription-polymerase chain reaction in testis from 13 patients with spermatogenic failure, 5 patients with primary germ cell tumors, and 10 controls with conserved spermatogenesis.

The p.Arg258Gly mutation in intracellular loop 2 of CFTR is associated with CFTR-related disorders.

Missense mutations account for approximately 50% of the mutations described in the CFTR gene. However, their proportion is higher in CFTR-related disorders (CFTR-RD) than in cystic fibrosis (CF), suggesting a different mutational spectrum. The uncertainty surrounding many of these mutations prevents suitable genetic counseling.

Boundary sequences stabilize transgene expression from subtle position effects in retroviral vectors.

Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-beta SAR and/or 5'HS4 beta-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability.

Inhibiting the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway with a regulator of calcineurin-derived peptide without affecting general calcineurin phosphatase activity.

Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506.

Novel SLC7A7 large rearrangements in lysinuric protein intolerance patients involving the same AluY repeat.

Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y(+)LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y(+)LAT-1 protein, is mutated in LPI patients.

CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling.

The CD40-CD154 dyad seems to play a prominent role fostering the immune-inflammatory response triggered by endothelial cell (EC)-T-cell communication. To delineate comprehensively the involvement of CD40 (TNFRSF5) in EC activation, we combined RNAi-mediated CD40 knockdown with comparative genome-wide transcriptional profiling of ECs interacting with (CD154+) T cells. We report the initiation of a profound stress response in ECs upon CD40-CD154 engagement through early up-regulation of, among others, the major proinflammatory NF-kappaB and MAPK/SAPK pathways and their associated transcription factors.

N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.

Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior.

Slc7a9 knockout mouse is a good cystinuria model for antilithiasic pharmacological studies.

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b(0,+)AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage.

Mutations of SYCP3 are rare in infertile Spanish men with meiotic arrest.

No functional SYCP3 exonic SNP was found in infertile Spanish patients with meiosis arrest, suggesting that SYCP3 mutations very likely are not relevant in Spain in genetic susceptibility to meiosis arrest, just as in other studied European populations.

RCAN3, a novel calcineurin inhibitor that down-regulates NFAT-dependent cytokine gene expression.

The regulators of calcineurin (RCAN) proteins, previously known as calcipressins, have been considered to be a well conserved family from yeast to human based on the conservation of their FLISPP motif. Here, after performing a RCAN comparative genomic analysis we propose the existence of a novel functionally closely related RCAN subfamily restricted to vertebrates, the other RCAN proteins being considered only as distantly related members of the family. In addition, while three paralogous RCAN genes are found in vertebrates, there is only one in the other members of Eukarya.

Functional characterization of the calcipressin 1 motif that suppresses calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells.

Inhibition of the calcineurin-NFAT signalling pathway is one of the main challenges for immunosuppression therapy to avoid the severe side effects of the current anticalcineurinic drugs, cyclosporin A and FK506. The members of the calcipressin family are endogenous inhibitors of calcineurin. We describe for the first time that two independent motifs within human calcipressin 1, the ELHA and the PxIxxT motifs, interact with calcineurin in an independent functional manner.

Feasibility of retroviral vector-mediated in utero gene transfer to the fetal rabbit.

Objectives: Successful treatment or prevention of severe hereditary diseases could conceivably be achieved by genetic intervention early in development. Viral vector-mediated fetal gene transfer is proving a valuable tool to test the above concept in relevant animal models. Although the pregnant rabbit is a well-recognized model for fetal therapy, few preclinical assays have used it to validate fetal gene transfer approaches.

Molecular evaluation of CFTR sequence variants in male infertility of testicular origin.

Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non-obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well-defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population.

Expression of mitochondrial genes and transcription estimation in different brain areas in Alzheimer's disease patients.

Accumulation of mitochondrial defects is hypothesised to play a role in the complex pathophysiology of the sporadic form of Alzheimer's disease (SAD). Changes in expression of mtDNA encoded genes have been reported in SAD. However no conclusive results were obtained by using different methodologies and analysing distinct transcripts in a variety of brain areas.

RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells.

The CD40-CD154 dyad has a central role in the development of immune-inflammatory processes. Therefore, disruption of CD40 signaling has the potential to be therapeutically useful in a number of disease indications, including autoimmune syndromes, atherosclerosis, and allograft rejection. Blocking antibodies to CD154 have been successfully employed in experimental animal models, and recently in clinical trials, to prevent or treat these immunologically induced diseases.

Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?

While all patients with cystic fibrosis (CF) have mutations in both CFTR alleles, often only one CFTR change is detected in patients with other lung disorders. The aim of this study was to investigate whether heterozygosity for CFTR mutations could be a determinant risk factor in the development of bronchiectasis in adult patients. We have performed the CFTR gene analysis in a cohort of 55 bronchiectasis adult patients with unknown etiology.

Different CFTR mutational spectrum in alcoholic and idiopathic chronic pancreatitis?

Objective: Cystic fibrosis transmembrane conductance regulator (CFTR) mutations are responsible for cystic fibrosis (CF) and have been postulated as a predisposing risk factor to chronic pancreatitis (CP), but controversial results demand additional support. We have therefore investigated the role of the CFTR gene in a cohort of 68 CP patients.

Methods: We have performed the CFTR gene analysis using 2 screening techniques.

Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice.

DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in Dyrk1A function by gene targeting.

Erratum: Identification of five new mutations of PDS/SLC26A4 in Mediterranean families with hearing impairment.

Pendred syndrome is an autosomal-recessive disorder characterized by congenital sensorineural hearing loss combined with goiter. This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS/SLC26A4) has been mapped to chromosome 7q22-q31 and encodes a chloride-iodide transport protein.

Neurobehavioral development of two mouse lines commonly used in transgenic studies.

The present study was aimed at establishing the differences in the neurodevelopmental profile between two F2 lines derived from two F1 hybrid mouse strains (129 x C57BL/6 and C57BL/6 x SJL). The choice of the given strains was based on the frequent use of these mice in transgenic research. For the neurodevelopment phenotyping, we employed a test battery consisting of 23 somatometric, sensorial and motor tests.

A common frameshift mutation and other variants in GJB4 (connexin 30.3): Analysis of hearing impairment families.

Mutations in GJB1, GJB2, GJB3 and GJB6 are involved in hearing impairment. GJB2, GJB3 and GJB6 are also mutated in patients with hyperproliferative skin disorders. The human GJB4 gene has been deduced in silico and a mutation in a family with erythrokeratodermia variabilis has been reported.

ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis.

The Na+-dependent amino acid transporter named ATB(0) was previously found to be located in 19q13.3 by fluorescence in situ hybridisation. Genetic heterogeneity in the 19q13.