Viral transport of DNA damage that mimics a stalled replication fork.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

The impact of fibrosis and steatosis on early viral kinetics in HCV genotype 1-infected patients treated with Peg-IFN-alfa-2a and ribavirin.

Summary.  Hepatitis C viral (HCV) kinetics after initiation of interferon-based therapy provide valuable insights for understanding virus pathogenesis, evaluating treatment antiviral effectiveness and predicting treatment outcome. Adverse effects of liver fibrosis and steatosis on sustained virological response have been frequently reported, yet their impacts on the early viral kinetics remain unclear. In this study, associations between histology status and early viral kinetics were assessed in 149 HCV genotype 1-infected patients treated with pegylated interferon alfa-2a and ribavirin (DITTO trial). In multivariate analyses adjusted for critical factors such as IL28B genotype and baseline viral load, presence of significant fibrosis (Ishak stage > 2) was found to independently reduce the odds of achieving an initial reduction (calculated from day 0 to day 4) in HCV RNA of ≥2 logIU/mL (adjusted OR 0.03, P = 0.004) but was not associated with the second-phase slope of viral decline (calculated from day 8 to day 29). On the contrary, presence of liver steatosis was an independent risk factor for not having a rapid second-phase slope, that is, ≥0.3 logIU/mL/week (adjusted OR 0.22, P = 0.012) but was not associated with the first-phase decline. Viral kinetic modelling theory suggests that significant fibrosis primarily impairs the treatment antiviral effectiveness in blocking viral production by infected cells, whereas the presence of steatosis is associated with a lower net loss of infected cells. Further studies will be necessary to identify the biological mechanisms underlain by these findings.

Early neutralizing antibody response against mouse mammary tumor virus: critical role of viral infection and superantigen-reactive T cells.

Infectious mouse mammary tumor virus (MMTV) is a retrovirus that expresses a superantigen shortly after infection of B cells. The superantigen first drives the polyclonal activation and proliferation of superantigen-reactive CD4+ T cells, which then induce the infected B cells to proliferate and differentiate. Part of the MMTV-induced B cell response leads to the production of Abs that are specific for the viral envelope protein gp52. Here we show that this Ab response has virus-neutralizing activity and confers protection against superinfection by other MMTV strains in vivo as soon as 4 to 7 days after infection. A protective Ab titer is maintained lifelong. Viral infection as well as the superantigen-induced T-B collaboration are required to generate this rapid and long lasting neutralizing Ab response. Polyclonal or superantigen-independent B cell activation, on the contrary, does not lead to detectable virus neutralization. The early onset of this superantigen-dependent neutralizing response suggests that viral envelope-specific B cells are selectively recruited to form part of the extrafollicular B cell response and are subsequently amplified and maintained by superantigen-reactive Th cells.

Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system.

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.

Estimating the basic reproductive number from viral sequence data.

Epidemiological processes leave a fingerprint in the pattern of genetic structure of virus populations. Here, we provide a new method to infer epidemiological parameters directly from viral sequence data. The method is based on phylogenetic analysis using a birth-death model (BDM) rather than the commonly used coalescent as the model for the epidemiological transmission of the pathogen. Using the BDM has the advantage that transmission and death rates are estimated independently and therefore enables for the first time the estimation of the basic reproductive number of the pathogen using only sequence data, without further assumptions like the average duration of infection. We apply the method to genetic data of the HIV-1 epidemic in Switzerland.

Fat signal suppression for coronary MRA at 3T using a water-selective adiabatic T2 -preparation technique.

PURPOSE: To improve fat saturation in coronary MRA at 3T by using a spectrally selective adiabatic T2 -Prep (WSA-T2 -Prep). METHODS: A conventional adiabatic T2 -Prep (CA-T2 -Prep) was modified, such that the excitation and restoration pulses were of differing bandwidths. On-resonance spins are T2 -Prepared, whereas off-resonance spins, such as fat, are spoiled. This approach was combined with a CHEmically Selective Saturation (CHESS) pulse to achieve even greater fat suppression. Numerical simulations were followed by phantom validation and in vivo coronary MRA. RESULTS: Numerical simulations demonstrated that augmenting a CHESS pulse with a WSA-T2 -Prep improved robustness to B1 inhomogeneities and that this combined fat suppression was effective over a broader spectral range than that of a CHESS pulse in a conventional T2 -Prepared sequence. Phantom studies also demonstrated that the WSA-T2 -Prep+CHESS combination produced greater fat suppression across a range of B1 values than did a CA-T2 -Prep+CHESS combination. Lastly, in vivo measurements demonstrated that the contrast-to-noise ratio between blood and myocardium was not adversely affected by using a WSA-T2 -Prep, despite the improved abdominal and epicardial fat suppression. Additionally, vessel sharpness improved. CONCLUSION: The proposed WSA-T2 -Prep method was shown to improve fat suppression and vessel sharpness as compared to a CA-T2 -Prep technique, and to also increase fat suppression when combined with a CHESS pulse.

Viral-mediated overexpression of mutant huntingtin to model HD in various species.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin (Htt) gene. Despite intensive efforts devoted to investigating the mechanisms of its pathogenesis, effective treatments for this devastating disease remain unavailable. The lack of suitable models recapitulating the entire spectrum of the degenerative process has severely hindered the identification and validation of therapeutic strategies. The discovery that the degeneration in HD is caused by a mutation in a single gene has offered new opportunities to develop experimental models of HD, ranging from in vitro models to transgenic primates. However, recent advances in viral-vector technology provide promising alternatives based on the direct transfer of genes to selected sub-regions of the brain. Rodent studies have shown that overexpression of mutant human Htt in the striatum using adeno-associated virus or lentivirus vectors induces progressive neurodegeneration, which resembles that seen in HD. This article highlights progress made in modeling HD using viral vector gene transfer. We describe data obtained with of this highly flexible approach for the targeted overexpression of a disease-causing gene. The ability to deliver mutant Htt to specific tissues has opened pathological processes to experimental analysis and allowed targeted therapeutic development in rodent and primate pre-clinical models.

Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion.

Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection: this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Encéphalites virales [Viral encephalitis: update]

Suspicion of viral encephalitis should always be considered as a medical emergency and the prognosis depend on both the immune status of the host and the virulence of the virus. Among them, the herpes simplex virus is by far the most important one since it can be associated with severe encephalitis in immunocompetent host, and because a good response to acyclovir can be expected when rapidly initiated. Nevertheless, confirmation of the diagnosis requires exclusion of both metabolic or toxic encephalopathy and inflammatory encephalitis of non-infectious origin. In addition, other germs than viruses can mimic viral encephalitis and must be taken into consideration. The purpose of this review is to update the investigation that should be performed in clinical practice for any patient with suspicion of acute viral encephalitis.

Positive contrast visualization of iron oxide-labeled stem cells using inversion-recovery with ON-resonant water suppression (IRON)

In proton magnetic resonance imaging (MRI) metallic substances lead to magnetic field distortions that often result in signal voids in the adjacent anatomic structures. Thus, metallic objects and superparamagnetic iron oxide (SPIO)-labeled cells appear as hypointense artifacts that obscure the underlying anatomy. The ability to illuminate these structures with positive contrast would enhance noninvasive MR tracking of cellular therapeutics. Therefore, an MRI methodology that selectively highlights areas of metallic objects has been developed. Inversion-recovery with ON-resonant water suppression (IRON) employs inversion of the magnetization in conjunction with a spectrally-selective on-resonant saturation prepulse. If imaging is performed after these prepulses, positive signal is obtained from off-resonant protons in close proximity to the metallic objects. The first successful use of IRON to produce positive contrast in areas of metallic spheres and SPIO-labeled stem cells in vitro and in vivo is presented.

Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways.

BACKGROUND: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. OBJECTIVES: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. RESULTS: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. CONCLUSIONS: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner.

Transmitted drug resistant HIV-1 and association with virologic and CD4 cell count response to combination antiretroviral therapy in the EuroSIDA Study.

OBJECTIVES: To investigate prevalence of transmitted drug-resistant human immunodeficiency virus (TDR) and factors associated with TDR and to compare virological and CD4 count response to combination antiretroviral therapy. METHODS: In this study, 525 mostly chronically infected EuroSIDA patients were included who had genotypic resistance tests performed on plasma samples collected while antiretroviral therapy naive. TDR was defined as at least one resistance mutation from a list proposed for genotypic TDR surveillance. Multivariable logistic regression was used to analyze factors associated with detection of TDR, with virological (viral load<500 copies/mL) and CD4 count response (>or=50% increase) to combination antiretroviral therapy at months 6-12. RESULTS: The overall prevalence of TDR was 11.4%, which was stable over 1996-2004. There were no significant differences in virological suppression (those resistant to at least one drug prescribed versus susceptible), adjusted odds ratio: 0.68 (95% confidence interval: 0.27 to 1.71; P=0.408) or CD4 count response, adjusted odds ratio: 1.65 (95% confidence interval: 0.73 to 3.73; P=0.231). CONCLUSIONS: Prevalence of TDR in antiretroviral-naive patients was found to be in line with other European studies. No significant differences were found in virological and CD4 count response after initiation of first-line combination antiretroviral therapy between resistant and susceptible patients, possibly due to the small number of patients with resistance and consequently low power.