BackgroundStudies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.ResultsWe identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.Conclusions OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.
2Protective CD8 + T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named Lymphocyte Expansion Molecule (LEM) that promoted antigen-dependent CD8 + T cell proliferation, effector function and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results.Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration resulting in the production of pro-proliferative mitochondrial Reactive Oxygen Species (mROS).LEM provides a link between immune activation and the expansion of protective CD8 + T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified CTL. 3Cytotoxic CD8 + T cells (CTL) are a central arm of the immune system responsible for protection from intracellular viruses and cancer because they kill infected or transformed cells (1). Since chronic virus infection (2) and cancer (3) are wide spread diseases it is clear that CTL-immunity often fails. A major reason for this failure is because high viral (4, 5) or tumor (6-8) load results in either deletion or functional inactivation (known as immune exhaustion) of CTL. The result is failure in both short-term CTL immunity and immunological memory because memory CD8 T cell development is blocked (9). Impaired expansion is an important cause of deletion and immune exhaustion and results in the failure to produce sufficient numbers of protective CTL and memory cells (5). Retro mutant mice have increased immunity to chronic viral infectionInfection of wild-type C57BL/6 mice with the clone 13 variant of lymphocytic choriomeningitis virus (LCMV C13) is an established model for human chronic viral infection resulting in a massive viral load that causes both deletion and immune exhaustion of CTL and a block in memory CD8 T cell development (10).We examined the CTL response to LCMV C13 infection after germ-line mutagenesis to identify mutants with enhanced immunity. To this end, 430 third-generation (G3) ethyl-N-nitrosourea (ENU)-induced germ-line mutants were produced in a C57BL/6J background (11). G3 mice were infected with LCMV C13 and after 8 days the level of LCMV-specific CD8 T cells measured in the spleen by staining with a tetramer for the np396 LCMV peptide and flow-cytometry (12). Three independent germ-line transmissible modifications, which resulted in increased levels of LCMV-specific 4 CD8 T cells were isolated, of which one (a semi-dominant) was bred to homozygosity (Fig. S1a). We named this strain Retro.Homozygous Retro mutant mice showed a 10-fold increase in CD8 T cells specific for LCMV np396 peptide compared to wild-type (WT) and a smaller but significant increase in the number of CTL specific for the gp33-LCMV peptide ( Fig. 1 a, b).Compared to WT mice, a smaller percentage of Retr...
The homeostasis of the immune system is tightly controlled by both cell-extrinsic and –intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function.Here we describea deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway,which affects peripheral T-cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosoUreainduced mutation in the last α-helix of DCK, a functionally null proteinhas been generated in the mouse and affects the composition of the hematopoietic system. Both B- and T-lymphocytes development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44highCD62Llow memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notablythe result of a cell-extrinsic driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.
M utations in >50 genes, mostly encoding sarcomeric and Z disc proteins, cause hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) that lead to heart failure.1 Genetic variation in a Z disc gene can cause various forms of cardiomyopathies such as HCM or DCM perhaps because of pleiotropic effects on survival and hypertrophic pathways. 2 The muscle LIM protein (cysteine and glycinerich protein 3 [CSRP3]) is a Z disc protein involved in cardiac mechanosensation that is important for myocyte-specific survival pathways through interactions with other Z disc proteins.3-5 CSRP3 mutations have been found in both HCM and DCM patients, 3,4,6 and we used protein-protein interaction studies to identify new CSRP3-interacting partners that may be important for cardiac myocyte survival or cardiomyopathy. Clinical Perspective on p 652 MethodsAll primers and antibodies used are listed in Tables I to III in the Data Supplement. Tissue culture experiments were performed using primary neonatal and adult rat cardiac myocytes, and animal experiments were performed using conditional knockout (cKO-Zbtb17), overexpressing transgenic (TG-ZBTB17) and double-transgenic Background-Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. Methods and Results-We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurindependent manner. Conclusions-We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene. (TG-ZBTB17/KO-Ppp3cb) protein phosphatase 3, catalytic subunit, β isozyme (calcineurin Aβ, Ppp3cb) animals. The procedures in animal studies have been followed in accordance with institutional guidelines. Human studies have been approved by an institutional review committee, and that the subjects gave informed consent.Yeast 2-hybrid, Western blot, quantitative real-time polymerase chain reaction, and other assays were performed as described elsewhere. [3][4][5]7 Statistical evaluations were performed by nonparametric Mann-Whitney U tests and unpaired Student t tests (more details are available in the Data Supplement). Results CSRP3 Interacts With ZBTB17Yeast 2-hybrid screens using CSRP3 as bait 3 classified α-actinin and telethonin as CSRP3-interacting proteins that are important determinants of cardiac function 5,8 and also identified ZBTB17 ( Figure 1A). ZBTB1...
ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model.MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay.ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA.ConclusionNeutralizing-antibody titers can be used as a surrogate marker.
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