The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

No interactions between previously associated 2-hour glucose gene variants and physical activity or BMI on 2-hour glucose levels.

Gene-lifestyle interactions have been suggested to contribute to the development of type 2 diabetes. Glucose levels 2 h after a standard 75-g glucose challenge are used to diagnose diabetes and are associated with both genetic and lifestyle factors. However, whether these factors interact to determine 2-h glucose levels is unknown. We meta-analyzed single nucleotide polymorphism (SNP) × BMI and SNP × physical activity (PA) interaction regression models for five SNPs previously associated with 2-h glucose levels from up to 22 studies comprising 54,884 individuals without diabetes. PA levels were dichotomized, with individuals below the first quintile classified as inactive (20%) and the remainder as active (80%). BMI was considered a continuous trait. Inactive individuals had higher 2-h glucose levels than active individuals (^6; = 0.22 mmol/L [95% CI 0.13-0.31], P = 1.63 × 10(-6)). All SNPs were associated with 2-h glucose (^6; = 0.06-0.12 mmol/allele, P ≤ 1.53 × 10(-7)), but no significant interactions were found with PA (P > 0.18) or BMI (P ≥ 0.04). In this large study of gene-lifestyle interaction, we observed no interactions between genetic and lifestyle factors, both of which were associated with 2-h glucose. It is perhaps unlikely that top loci from genome-wide association studies will exhibit strong subgroup-specific effects, and may not, therefore, make the best candidates for the study of interactions.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.

Electron microscopic visualization of protein-DNA interactions at the estrogen responsive element and in the first intron of the Xenopus laevis vitellogenin gene.

Stable protein-DNA complexes can be assembled in vitro at the 5' end of Xenopus laevis vitellogenin genes using extracts of nuclei from estrogen-induced frog liver and visualized by electron microscopy. Complexes at the three following sites can be identified on the gene B2: the transcription initiation site, the estrogen responsive element (ERE) and in the first intron. The complex at the transcription initiation site is stabilized by dinucleotides and thus represents a ternary transcription complex. The formation of the complexes at the two other sites is enhanced by estrogen and is reduced by tamoxifen, an antagonist of estrogen, while this latter effect is reversed by adding an excess of hormone. No sequence homology is apparent between the site containing the ERE and the binding site in intron I and functional tests in MCF-7 cells suggest that these two sites are not equivalent. Finally, we made use of previously characterized deletion mutants of the 5' flanking region of the gene B1, a close relative of the gene B2, to demonstrate that the 13-bp palindromic core element of the ERE is involved in the formation of the complexes observed upstream of the transcription initiation site.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-[6;A46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

The T309G Murine Double Minute 2 Gene Polymorphism Is an Independent Prognostic Factor for Patients with Renal Cell Carcinoma.

The purpose of this study was to evaluate the association of the T309G MDM2 gene polymorphism with renal cell carcinoma (RCC) risk, pathology, and cancer-specific survival (CSS). T309G MDM2 was genotyped in 449 Caucasians, including 240 with RCC and 209 cancer-free controls. The T309G MDM2 genotype was TT in 174 (38.8%), GT in 214 (47.7%), and GG in 61 (13.6%) subjects, without any significant differences between cases and controls on both univariable (p=0.58) and multivariable logistic regression (each p>0.25). Furthermore, T309G MDM2 was not linked with T stage (p=0.75), N stage (p=0.37), M stage (p=0.94), grade (p=0.21), and subtype (p=0.55). There was, however, a statistically significant association of T309G MDM2 with CSS (p=0.022): patients with TT had significantly worse survival than GG/GT (p=0.009), while those with GT and GG had similar outcomes (p=0.92). The 5-year survival rate for patients with TT, GT, and GG was 69.5%, 84.5%, and 89.7%, respectively. On the multivariable analysis, T309G was identified as an independent prognostic factor. The T309G MDM2 polymorphism is an independent prognostic factor for patients with RCC, with the TT genotype being associated with worse prognosis. In this study, there were no significant associations with RCC risk and pathology.

The peroxisome proliferator-activated receptor (PPAR) ^6;/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)^6;/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Ppar^6;/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Ppar^6;/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPAR^6;/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Phenytoin-associated severe hypocalcemia with seizures in a patient with a TSC2-PKD1 contiguous gene syndrome.

We report the case of an inaugural episode of generalized seizures in a 40-year-old male with a history of chronic kidney disease associated with TSC2-PKD1 contiguous gene syndrome. This patient was under prophylactic treatment of phenytoin since 2 years because of a subarachnoid hemorrhage due to a ruptured cerebral aneurysm. Laboratory results revealed therapeutic range of phenytoin levels, but severe hypocalcemia associated with profound vitamin D deficiency that could not be explained by secondary hyperparathyroidism alone. The interaction of phenytoin on the P-450 cytochromes activity has been demonstrated to accelerate the rate of 25-hydroxivitamin D3 and 1α,25-dihydroxivitamin D3 catabolism into inactive metabolites, leading to hypocalcemia. Physicians should be aware of significant phenytoin interactions on vitamin D metabolism which may lead to symptomatic hypocalcemia in patients with chronic kidney disease.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease.

Macrophage migration inhibitory factor (MIF) is an abundantly expressed proinflammatory cytokine playing a critical role in innate immunity and sepsis and other inflammatory diseases. We examined whether functional MIF gene polymorphisms (-794 CATT(5-8) microsatellite and -173 G/C SNP) were associated with the occurrence and outcome of meningococcal disease in children. The CATT(5) allele was associated with the probability of death predicted by the Pediatric Index of Mortality 2 (P=0.001), which increased in correlation with the CATT(5) copy number (P=0.04). The CATT(5) allele, but not the -173 G/C alleles, was also associated with the actual mortality from meningoccal sepsis [OR 2.72 (1.2-6.4), P=0.02]. A family-based association test (i.e., transmission disequilibrium test) performed in 240 trios with 1 afflicted offspring indicated that CATT(5) was a protective allele (P=0.02) for the occurrence of meningococcal disease. At baseline and after stimulation with Neisseria meningitidis in THP-1 monocytic cells or in a whole-blood assay, CATT(5) was found to be a low-expression MIF allele (P=0.005 and P=0.04 for transcriptional activity; P=0.09 and P=0.09 for MIF production). Taken together, these data suggest that polymorphisms of the MIF gene affecting MIF expression are associated with the occurrence, severity, and outcome of meningococcal disease in children.

Genetic polymorphism of CCR5 gene and HIV disease: the heterozygous (CCR5/delta ccr5) genotype is neither essential nor sufficient for protection against disease progression. Swiss HIV Cohort.

Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) deletions in the beta-chemokine receptor 5 (CCR5) gene, which encodes for the major co-receptor for macrophage-tropic HIV-1 entry, have been implicated in resistance to HIV infection and in protection against disease progression, respectively. The CCR5/delta ccr5 genotype was found more frequently in long-term nonprogressors (LTNP) (31.0%) than in progressors (10.6%, p < 0.0001), in agreement with previous studies. Kaplan-Meier survival analyses showed that a slower progression of disease, i.e. higher proportion of subjects with CD4+ T cell counts > 500/microl (p = 0.0006) and a trend toward a slower progression to AIDS (p = 0.077), was associated with the CCR5/delta ccr5 genotype. However, when LTNP were analyzed separately, no significant differences in CD4+ T cell counts (p = 0.12) and viremia levels (p = 0.65) were observed between the wild-type (69% of LTNP) and the heterozygous (31.0%) genotypes. Therefore, there are other factors which play a major role in determining the status of nonprogression in the majority of LTNP. Furthermore, there was no evidence that the CCR5/delta ccr5 genotype was associated with different rates of disease progression in the group of progressors. Taken together, these results indicate that the CCR5/delta ccr5 genotype is neither essential nor sufficient for protection against the progression of HIV disease.

Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome.

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.

Transcriptional regulation by triiodothyronine of the UDP-glucuronosyltransferase family 1 gene complex in rat liver. Comparison with induction by 3-methylcholanthrene.

This study demonstrates that the expression of the phenol UDP-glucuronosyltransferase 1 gene (UGT1A1) is regulated at the transcriptional level by thyroid hormone in rat liver. Following 3,5, 3'-triiodo-L-thyronine (T3) stimulation in vivo, there is a gradual increase in the amount of UGT1A1 mRNA with maximum levels reached 24 h after treatment. In comparison, induction with the specific inducer, 3-methylcholanthrene (3-MC), results in maximal levels of UGT1A1 mRNA after 8 h of treatment. In primary hepatocyte cultures, the stimulatory effect of both T3 and 3-MC is also observed. This induction is suppressed by the RNA synthesis inhibitor actinomycin D, indicating that neither inducer acts at the level of mRNA stabilization. Indeed, nuclear run-on assays show a 3-fold increase in UGT1A1 transcription after T3 treatment and a 6-fold increase after 3-MC stimulation. This transcriptional induction by T3 is prevented by cycloheximide in primary hepatocyte cultures, while 3-MC stimulation is only partially affected after prolonged treatment with the protein synthesis inhibitor. Together, these data provide evidence for a transcriptional control of UGT1A1 synthesis and indicate that T3 and 3-MC use different activation mechanisms. Stimulation of the UGT1A1 gene by T3 requires de novo protein synthesis, while 3-MC-dependent activation is the result of a direct action of the compound, most likely via the aromatic hydrocarbon receptor complex.