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    ABSTRACT: Assessment of transmural scar at the site of latest mechanical activation is relevant to maximize outcomes in cardiac resynchronization therapy (CRT). Few studies have assessed the ability of speckle tracking echocardiography (STE)-derived short-axis strain to identify segmental myocardial scar, defined by contrast-enhanced cardiac magnetic resonance imaging (CMR), in patients referred for CRT. A total of 26 patients with ischemic cardiomyopathy who underwent preprocedure echocardiography and CMR were studied. Extent of transmural scar was assessed using contrast-enhanced CMR and corresponding peak segmental radial and circumferential strains were derived using two-dimensional (2D) STE. Total left ventricle (LV) scar volume was compared with parameters of global strain. CRT response was defined as >15% reduction in LV end systolic volume (LVESV) at 6 months. Speckle tracking short-axis strain analysis was technically possible in over 90% of LV segments. Applying a segmental radial strain cutoff value of 10% distinguished segments with >50% scar area with a high negative predictive value (98%). Global longitudinal strain <-5% predicted CRT response. Two-dimensional STE offers potential to characterize dysfunctional myocardium and define segmental scar offering an integrated imaging approach to guide LV lead placement for CRT.
    Echocardiography 12/2013;
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    ABSTRACT: Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.
    Science 10/2013;
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    ABSTRACT: Functional mitral regurgitation (FMR) is a consequence of mitral annular enlargement, leaflet tethering and reduced co-aptation. The importance of the left atrium (LA) as a cause of mitral regurgitation (MR) is less clear. We applied a co-aptation index using three-dimensional (3D) transoesophageal echocardiography to FMR and MR secondary to LA dilatation (atrial mitral regurgitation, AMR). Seventy-two patients underwent comprehensive 3D echo studies: FMR (n = 19); AMR (n = 33); and 20 controls. We recorded: LV size and function; LA dimensions; mitral annular area (MVA); and leaflet area in early and late systole. MVA fractional change was defined: (MVA late systole - MVA early systole)/MVA late systole × 100%; the co-aptation index was defined: (leaflet area early systole - leaflet area late systole)/leaflet area early systole × 100%. Despite normal LV size and function in AMR, MVA was increased similarly to FMR (AMR 12.86 cm(2) vs. FMR 12.33 cm(2), P = ns; both P < 0.01 vs. controls 8.83 cm(2)), and MVA fractional change similarly reduced (AMR 5.1% vs. FMR 6.3%; P = ns; both P < 0.001 vs. controls 14.6%). The co-aptation index was reduced in both MR groups (FMR 6.6% vs. AMR 7.0%, P = ns; both P < 0.001 vs. controls 19.6%). After multivariate analysis, the co-aptation index (χ(2) = 41.2) and MVA fractional change (χ(2) = 22.1) remained the strongest predictors of MR (both P < 0.001 for the model). A co-aptation index of ≤13% was 96% sensitive and 90% specific for the presence of MR. LA dilatation leads to MVA enlargement, reduced leaflet co-aptation and MR even without LV dilatation. A co-aptation index describes this in vivo. This work provides insights into the mechanism of AMR.
    European heart journal cardiovascular Imaging. 10/2013;
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    ABSTRACT: Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for ER stress and initiate NF-κB signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared to wild-type (MM) controls, and this appears to be mediated by MEK, EGFR and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.
    Human Molecular Genetics 10/2013;
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    ABSTRACT: Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4(+) T cell mediated. These UL138-specific CD4(+) T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4(+) T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4(+) T cell responses included CD4(+) T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4(+) T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4(+) T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4(+) T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.
    PLoS Pathogens 10/2013; 9(10):e1003635.
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    ABSTRACT: Rotavirus (RV) cores were released from double-layered particles (DLPs) by high concentrations of CaCl2, purified and 'opened' by treatment with EDTA or EGTA. Under appropriate in vitro conditions DLPs have been shown to have transcriptase and 'open cores' replicase activity. Furthermore, it has been demonstrated that transcriptase activity and infectivity of native cores can be restored by transcapsidation with VP6, VP7 and VP4. The missing link for particle reconstitution in vitro has been the manipulation of 'open cores' to become functionally active cores again. The experiments described here were undertaken with the aim of exploring packaging of RV RNAs into opened cores in vitro. Rotavirus cores were opened by approx. 200 uM EGTA, leading to the release of genomic dsRNA. Conversely, RV cores were found to be stable in the presence of minimum concentrations of Ca(2+), Mg(2+), spermidine(3+) and cobalthexamine(3+) of between 40 and 300 uM. Aggregates of purified cores were resolved in the presence of 0.3mM deoxycholate (minimum concentration). Core shells opened with EGTA were reconstituted by the addition of di- or trivalent cations within 2min of the opening procedure. Addition of purified, baculovirus recombinant-expressed VP6 to native and reconstituted cores led to the formation of DLPs or DLP-like particles, which upon transfection into MA104 cells were infectious. The rescued infectivity likely originated in part from unopened and in part from reconstituted cores. Radiolabelled RV (+) ssRNAs could be packaged into reconstituted cores and DLPs, as indicated by resistance to RNase I digestion. The packaging reaction was, however, not RV RNA sequence-specific, since unrelated ssRNAs, such as those transcribed from HIV-2 cDNAs, were also packaged. The kinetics of packaging of homologous and heterologous RNAs were similar, as evidenced by competitive packaging assays. None of the packaged in vitro engineered RNA segments has so far been rescued into infectious virus.
    Virus Research 09/2013;
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    ABSTRACT: The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal β-N-acetylhexosaminidase system. Accumulation of β-hexosaminidases A and B substrates are presumed to cause this fatal condition.An authentic mouse model of Sandhoff disease with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described. We have shown that expression of β-hexosaminidase by intracranial delivery of recombinant adeno-associated viral vectors to young adult Sandhoff mice can prevent many features of the disease and significantly extends lifespan.To investigate the nature of the neurological injury in GM2 gangliosidosis and the extent of its reversibility, we have examined the evolution of disease in the Sandhoff mouse; we have moreover explored the effects of gene transfer delivered at key times during the course of the illness.Here we report greatly increased survival only when the therapeutic genes are expressed either before the disease is apparent or during its early manifestations. However, irrespective of when treatment was administered, widespread and abundant expression of β-hexosaminidase with consequent clearance of glycoconjugates, α-synuclein, and ubiquitinated proteins, and abrogation of inflammatory responses and neuronal loss was observed. We also show that defects in myelination occur in early life and cannot be easily resolved when treatment is given to the adult brain.These results indicate that there is a limited temporal opportunity in which function and survival can be improved - but regardless of resolution of the cardinal pathological features of GM2 gangliosidosis, a point is reached when functional deterioration and death cannot be prevented.
    Human Molecular Genetics 09/2013;
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    ABSTRACT: Newer therapies for systemic vasculitis are required to improve the efficacy and reduce the toxicity associated with current agents. Our understanding of its pathogenesis is increasing with the availability of targeted therapies. B-cell depletion with rituximab is now a licensed therapy for ANCA vasculitis. Targets for therapy currently in development in ANCA associated vasculitis include blockade of complement and pro-inflammatory cytokines, and inhibition of T cell co-stimulation.
    Clinical and Experimental Nephrology 09/2013;
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    ABSTRACT: The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. We found however that PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy could not be identical to its protection against Aβ toxicity.
    Biochimica et Biophysica Acta 08/2013;
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    ABSTRACT: Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immuno-suppressed transplant recipients and late stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34+ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, these previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV seropositive carriers. We show that these in vivo differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show these DCs from healthy HCMV seropositive donors carry HCMV genome and, significantly, are typically positive for viral IE gene expression - in contrast to circulating monocytes which carry genome with an absence of IE expression. Finally, we show HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.
    Journal of Virology 07/2013;
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