pRb is an obesity suppressor in hypothalamus and high‐fat diet inhibits pRb in this location

pRb is frequently inactivated in tumours by mutations or phosphorylation. Here, we investigated whether pRb plays a role in obesity. The Arcuate nucleus (ARC) in hypothalamus contains antagonizing POMC and AGRP/NPY neurons for negative and positive energy balance, respectively. Various aspects of ARC neurons are affected in high‐fat diet (HFD)‐induced obesity mouse model. Using this model, we show that HFD, as well as pharmacological activation of AMPK, induces pRb phosphorylation and E2F target gene de‐repression in ARC neurons. Some affected neurons express POMC; and deleting Rb1 in POMC neurons induces E2F target gene de‐repression, cell‐cycle re‐entry, apoptosis, and a hyperphagia‐obesity‐diabetes syndrome. These defects can be corrected by combined deletion of E2f1. In contrast, deleting Rb1 in the antagonizing AGRP/NPY neurons shows no effects. Thus, pRb‐E2F1 is an obesity suppression mechanism in ARC POMC neurons and HFD‐AMPK inhibits this mechanism by phosphorylating pRb in this location.     

Developmental methylation program and concerted expression of Stx11 in mouse tissues

The human 6q24 region is involved in growth and development, transient neonatal diabetes (TND), cancer, and metabolic dysfunction. To further characterize this region, the developmental status of DNA methylation and expression of Zac1 and Stx11 genes located within the mouse 10A1 region ortholog of human 6q24 were determined. In mice, imprinted Zac1 and Stx11 were highly expressed at the end of fetal development but downregulated at 4 and 11 weeks in brain, pancreas, and heart. Postnatal Zac1 downregulation was independent from promoter methylation of the expressed allele, suggesting the mediation of age-dependent chromatin remodeling. Stx11 nonpromoter CpG island was methylated de novo from E18 to 1 year with tissue-specific kinetics. The high conservation in vertebrates of Stx11 CpG2 is suggestive of an important regulatory function in age-related regional epigenetic state and/or chromatin configuration. Stx11 alleles were unequally expressed in F1 mice tissues, reflecting the influence of cis-regulatory factors on its expression. These data suggest the presence of a methylation domain and a coordinated gene expression pattern in multiple tissues. Methylation variation and allelic regulation of expression may underlie genetic diversity and contribute to disease susceptibility at the 6q24 locus in humans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cost-Effectiveness of Dolutegravir in HIV-1 Treatment-Experienced (TE) Patients in France

Objectives

To evaluate the cost-effectiveness of a new generation integrase inhibitor (INI), dolutegravir (DTG), in France, in treatment-experienced (TE) and INI-naïve HIV-infected adults with at least two classes resistance compared to raltegravir (RAL), by adapting previously published Anti-Retroviral Analysis by Monte Carlo Individual Simulation (ARAMIS) model.

Methods

ARAMIS is a microsimulation Markov model with a lifetime time horizon and a monthly cycle length. Health states are defined as with or without opportunistic infection and death. In the initial cohort, efficacy and safety data were derived from a phase III study comparing DTG to RAL. Antiretroviral treatment algorithms, accounting for patient history, were based on French guidelines and experts opinion. Costs are mainly including treatment costs, routine HIV and opportunistic infection care, and death. Utilities depend on CD4+ cell count and the occurrence of opportunistic infections.

Results

The ARAMIS model indicates in the TE population that DTG compared to RAL over a life time is associated with 0.35 additional quality-adjusted life years (QALY; 10.75 versus 10.41) and additional costs of €7,266 (€390,001 versus €382,735). DTG increased costs are mainly related to a 9.1-month increase in life expectancy for DTG compared with RAL, and consequently a longer time spent on ART. The incremental cost-effectiveness ratio (ICER) for DTG compared with RAL is €21,048 per QALY gained. About 83% and 14% of total lifetime costs are associated with antiretroviral therapy and routine HIV care respectively. Univariate deterministic sensitivity analyses demonstrate the robustness of the model.

Conclusion

DTG is cost-effective in the management of TE INI naive patients in France, from a collective perspective. These results could be explained by the superior efficacy of DTG in this population and its higher genetic barrier to resistance compared to RAL. These data need to be confirmed with longer-term real life data.     

Genetic control of ATGL-mediated lipolysis modulates adipose triglyceride stores in leptin-deficient mice

Dissecting the genetics of complex traits such as obesity allows the identification of causal genes for disease. Here, we show that the BALB/c mouse strain carries genetic variants that confer resistance to obesity induced by leptin-deficiency or a high-fat diet (HFD). We set out to identify the physiological and genetic bases underlying this phenotype. When compared with C57BL6/J ob/ob mice (B6), BALB/c ob/ob mice exhibited decreased food intake, increased thermogenic capacity, and improved fat catabolism, each of which can potentially modify obesity. Interestingly, analysis of F1 ob/ob (progeny of B6 ob/+ × BALB/c ob+) mice revealed that obesity resistance in BALB/c ob/ob mice principally relied upon improved fat mobilization. This was mechanistically explained by increased adipose triglyceride lipase (ATGL) content in adipocytes, along with increased lipolysis and fatty acid oxidation. We conducted a genome-wide scan and defined a quantitative trait locus (QTL) on chromosome 2. BALB/c alleles on chromosome 2 not only associated with the obesity resistance phenotype but also supported increased ATGL content in adipose tissue. In summary, our study provides evidence that leptin-independent control of adipocyte lipolysis rates directly modifies the balance of macronutrient handling and is sufficient to regulate fat mass in the absence of alterations in food intake and energy expenditure.-Marcelin, G., S-M. Liu, X. Li, G. J. Schwartz, and S. Chua.     

Host differences in influenza-specific CD4 T cell and B cell responses are modulated by viral strain and route of immunization

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.     

Comparative study of three commerical preparations for the detection of Australia antigen]

2,944 sera from blood donors (420 of them being new donors) were tested in four techniques: Counterelectrophoresis (CEP) and three commercial tests: Ausria II, Auscell and Hepanosticon. Over 10 HBs Ag detected by RIA, 8 were evidenced by the Auscell test and 6 by the Hepanosticon Test. False positive results (approximately 3%) represent a disadvantage to the reverse hemagglutination method; this rate increased considerably in a group of 386 patients sera also tested. The authors conclude that the results obtained with the reverse hemagglutination technique are nearing those of RIA.     

Scoring Methods for Building Genotypic Scores: An Application to Didanosine Resistance in a Large Derivation Set

Background

Several attempts have been made to determine HIV-1 resistance from genotype resistance testing. We compare scoring methods for building weighted genotyping scores and commonly used systems to determine whether the virus of a HIV-infected patient is resistant.

Methods and Principal Findings

Three statistical methods (linear discriminant analysis, support vector machine and logistic regression) are used to determine the weight of mutations involved in HIV resistance. We compared these weighted scores with known interpretation systems (ANRS, REGA and Stanford HIV-db) to classify patients as resistant or not. Our methodology is illustrated on the Forum for Collaborative HIV Research didanosine database (N = 1453). The database was divided into four samples according to the country of enrolment (France, USA/Canada, Italy and Spain/UK/Switzerland). The total sample and the four country-based samples allow external validation (one sample is used to estimate a score and the other samples are used to validate it). We used the observed precision to compare the performance of newly derived scores with other interpretation systems. Our results show that newly derived scores performed better than or similar to existing interpretation systems, even with external validation sets. No difference was found between the three methods investigated. Our analysis identified four new mutations associated with didanosine resistance: D123S, Q207K, H208Y and K223Q.

Conclusions

We explored the potential of three statistical methods to construct weighted scores for didanosine resistance. Our proposed scores performed at least as well as already existing interpretation systems and previously unrecognized didanosine-resistance associated mutations were identified. This approach could be used for building scores of genotypic resistance to other antiretroviral drugs.     

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc

Background

Maraviroc is an HIV entry inhibitor that alters the conformation of CCR5 and is poorly efficient in patients infected by viruses that use CXCR4 as an entry coreceptor. The goal of this study was to assess the capacity of ultra-deep pyrosequencing (UDPS) and different data analysis approaches to characterize HIV tropism at baseline and predict the therapeutic outcome on maraviroc treatment.

Methods

113 patients with detectable HIV-1 RNA on HAART were treated with maraviroc. The virological response was assessed at months 1, 3 and 6. The sequence of the HIV V3 loop was determined at baseline and prediction of maraviroc response by different software and interpretation algorithms was analyzed.

Results

UDPS followed by analysis with the Pyrotrop software or geno2pheno algorithm provided better prediction of the response to maraviroc than Sanger sequencing. We also found that the H34Y/S substitution in the V3 loop was the strongest individual predictor of maraviroc response, stronger than substitutions at positions 11 or 25 classically used in interpretation algorithms.

Conclusions

UDPS is a powerful tool that can be used with confidence to predict maraviroc response in HIV-1-infected patients. Improvement of the predictive value of interpretation algorithms is possible and our results suggest that adding the H34S/Y substitution would substantially improve the performance of the 11/25/charge rule.     

HIV‐1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Ω‐shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir‐resistant viruses. Copyright © 2009 John Wiley & Sons, Ltd.