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Now showing items 17 - 32 of 6936

  • Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors

    Roback, John D.   Hillyer, Christopher D.   Drew, W. Lawrence   Laycock, Megan E.   Luka, Janos   Mocarski, Edward S.   Slobedman, Barry   Smith, James W.   Soderberg-Naucler, Cecilia   Todd, Deborah S.   Woxenius, Susanne   Busch, Michael P.  

    BACKGROUND: CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors. STUDY DESIGN AND METHODS: A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples. RESULTS: Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB-samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB-samples. CONCLUSION: Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.
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  • Genomic analysis of chimeric human cytomegalovirus vaccine candidates derived from strains Towne and Toledo

    Suarez, Nicolas M.   Lau, Betty   Kemble, George M.   Lee, Ronzo   Mocarski, Edward S.   Wilkinson, Gavin W. G.   Adler, Stuart P.   McVoy, Michael A.   Davison, Andrew J.  

    Human cytomegalovirus (HCMV) is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. In the 1990s, four chimeric viruses were constructed by replacing genome segments of the high passage Towne strain with segments of the low passage Toledo strain, with the goal of obtaining live attenuated vaccine candidates that remained safe but were more immunogenic than the overly attenuated Towne vaccine. The chimeras were found to be safe when administered to HCMV-seronegative human volunteers, but to differ significantly in their ability to induce seroconversion. This suggests that chimera-specific genetic differences impacted the ability to replicate or persist in vivo and the consequent ability to induce an antibody response. To identify specific genomic breakpoints between Towne and Toledo sequences and establish whether spontaneous mutations or rearrangements had occurred during construction of the chimeras, complete genome sequences were determined. No major deletions or rearrangements were observed, although a number of unanticipated mutations were identified. However, no clear association emerged between the genetic content of the chimeras and the reported levels of vaccine-induced HCMV-specific humoral or cellular immune responses, suggesting that multiple genetic determinants are likely to impact immunogenicity. In addition to revealing the genome organization of the four vaccine candidates, this study provided an opportunity to probe the genetics of HCMV attenuation in humans. The results may be valuable in the future design of safe live or replication-defective vaccines that optimize immunogenicity and efficacy.
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  • HIF1 alpha Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming

    Liu, Chun   Ruan, Hongyue   Himmati, Farhan   Zhao, Ming-Tao   Chen, Christopher C.   Makar, Merna   Chen, Ian Y.   Sallam, Karim   Mocarski, Edward S.   Sayed, Danish   Sayed, Nazish  

    Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1 alpha (HIF1 alpha), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1 alpha is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1 alpha regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.
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  • A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation

    Skaletskaya, Anna   Bartle, Laura M.   Chittenden, Thomas   McCormick, A. Louise   Mocarski, Edward S.   Goldmacher, Victor S.  

    We have identified a human cytomegalovirus cell-death suppressor, denoted vICA, encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its activation. vICA does not share significant sequence homology with FLIPs or other known suppressors of apoptosis, suggesting that this protein represents a new class of cell-death suppressors. Notably, resistance to Fas-mediated apoptosis is delayed in fibroblasts infected with viruses that encode mutant vICA, suggesting that vICA suppresses death-receptor-induced cell death in the context of viral infection. Although vICA is dispensable for viral replication in vitro, the common targeting of caspase-8 activation by diverse herpesviruses argues for an important role for this antiapoptotic mechanism in the pathogenesis of viral infection in the host, most likely in avoiding immune clearance by cytotoxic lymphocytes and natural killer cells.
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  • Encyclopedia of Virology || CYTOMEGALOVIRUSES (HERPESVIRIDAE) | General Features (Human)

    Mocarski, Edward S.  

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  • Recombinant Cytomegaloviruses for Study of Replication and Pathogenesis

    Mocarski, Edward S.   Kemble, George W.  

    Systematic mutagenesis of large viral genomes such as those of the cytomegaloviruses requires strategies for identifying relevant functions as well as for detailed analysis of particular genes, A number of genetic markers that have been developed in other biological systems have been useful for insertion mutagenesis in these viruses. Thus far, 57 of the over 227 genes carried by wild-type human cytomegalovirus have been found to be dispensable for growth in cultured cells. Because of the limitations on studying human cytomegalovirus in an animal host, the closely related murine cytomegalovirus has been used as a surrogate for pathogenesis, tissue tropism and latency studies in the laboratory mouse, Genetic analysis of this virus has paralleled work on human cytomegalovirus, and an understanding of genes that specifically impact viral growth in particular organs has emerged from these studies, Strategies for generation of permissive cell lines able to complement essential human cytomegalovirus replication functions have been described, and sets of cosmid clones have been used to generate recombinant viruses, These methods will enable a systematic functional analysis of the genomes of human and animal cytomegaloviruses.
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  • Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming

    Sayed, Nazish   Ospino, Frank   Himmati, Farhan   Lee, Jieun   Chanda, Palas   Mocarski, Edward S.   Cooke, John P.  

    We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. Stem Cells2017;35:1197-1207 Abstract Video Link:
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  • Inhibition of 2 ',5 '-Oligoadenylate Synthetase Expression and Function by the Human Cytomegalovirus ORF94 Gene Product

    Tan, Joanne C. G.   Avdic, Selmir   Cao, John Z.   Mocarski, Edward S.   White, Kirsten L.   Abendroth, Allison   Slobedman, Barry  

    The human cytomegalovirus (HCMV) ORF94 gene product has been reported to be expressed during both productive and latent phases of infection, although its function is unknown. We report that expression of pORF94 leads to decreased 2',5'-oligoadenylate synthetase (OAS) expression in transfected cells with and without interferon stimulation. Furthermore, the functional activity of OAS was inhibited by pORF94. Finally, we present evidence of OAS modulation by pORF94 during productive HCMV infection of human fibroblasts. This study provides the first identification of a function for pORF94 and identifies an additional means by which HCMV may limit a critical host cell antiviral response.
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  • Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model

    Melnick, Michael   Mocarski, Edward S.   Abichaker, George   Huang, Jing   Jaskoll, Tina  

    Background: Human studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs). Results: We infected E15 SMG explants with mouse CMV (mCMV). Active infection for up to 12 days in vitro results in a remarkable cell and molecular pathology characterized by atypical ductal epithelial hyperplasia, apparent epitheliomesenchymal transformation, oncocytic-like stromal metaplasia, beta-catenin nuclear localization, and upregulation of Nfkb2, Relb, Il6, Stat3, and Cox2. Rescue with an antiviral nucleoside analogue indicates that mCMV replication is necessary to initiate and maintain SMG dysmorphogenesis. Conclusion: mCMV infection of embryonic mouse explants results in dysplasia, metaplasia, and, possibly, anaplasia. The molecular pathogenesis appears to center around the activation of canonical and, perhaps more importantly, noncanonical NF kappa B. Further, COX-2 and IL-6 are important downstream effectors of embryopathology. At the cellular level, there appears to be a consequential interplay between the transformed SMG cells and the surrounding extracellular matrix, resulting in the nuclear translocation of beta-catenin. From these studies, a tentative framework has emerged within which additional studies may be planned and performed.
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  • Proapoptotic Chemotherapeutic Drugs Induce Noncanonical Processing and Release of IL-1 beta via Caspase-8 in Dendritic Cells

    Antonopoulos, Christina   El Sanadi, Caroline   Kaiser, William J.   Mocarski, Edward S.  

    The identification of noncanonical (caspase-1-independent) pathways for IL-1 beta production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1 beta processing and release in murine bone marrow-derived dendritic cells (BMDC) costimulated with TLR4 agonists and proapoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17-kDa) IL-1 beta was nearly equivalent in wild-type (WT) BMDC, Casp1(-/-) Casp11(-/-) BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1 beta was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8(-/-) Rip3(-/-) or Trif(-/-) BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1 beta processing and release in Casp1(-/-) Casp11(-/-) BMDC that was IETD sensitive. These data suggest that TLR4 induces assembly of caspase-8-based signaling complexes that become licensed as IL-1 beta-converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cellular inhibitor of apoptosis protein 1, suggesting suppressive roles for this and likely other inhibitor of apoptosis proteins on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, proapoptotic chemotherapeutic agents stimulate the caspase-8-mediated processing and release of IL-1 beta, implicating direct effects of such drugs on a noncanonical inflammatory cascade that may modulate immune responses in tumor microenvironments.
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  • Cutting Edge: FAS (CD95) Mediates Noncanonical IL-1 beta and IL-18 Maturation via Caspase-8 in an RIP3-Independent Manner

    Bossaller, Lukas   Chiang, Ping-I   Schmidt-Lauber, Christian   Ganesan, Sandhya   Kaiser, William J.   Rathinam, Vijay A. K.   Mocarski, Edward S.   Subramanian, Deepa   Green, Douglas R.   Silverman, Neal   Fitzgerald, Katherine A.   Marshak-Rothstein, Ann   Latz, Eicke  

    Fas, a TNF family receptor, is activated by the membrane protein Fas ligand expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas-induced cytokines, the IL-1 beta family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1 beta cytokines via caspase-1. However, the mechanisms by which Fas regulates IL-1 beta activation remain unresolved. In this article, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells, leading to the maturation of IL-1 beta and IL-18 independently of inflammasomes or RIP3. Hence, Fas controls a novel noncanonical IL-1 beta activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance, and control of infectious diseases. The Journal of Immunology, 2012, 189: 5508-5512.
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  • Reassessing the Organization of the UL42–UL43 Region of the Human Cytomegalovirus Strain AD169 Genome

    Mocarski, Edward S.   Prichard, Mark N.   Tan, Charmain S.   Brown, Janice M.  

    A polymorphism in the UL42–UL43 region of the human cytomegalovirus genome has been characterized by nucleotide sequence analysis, revealing a 929-bp insertion following nt 54,612 relative to the published strain AD169-UK genome sequence (M. S. Cheeet al.,1990,Curr. Top. Microbiol. Immunol.154, 125–170). Although AD169-UK exhibited polymorphism in this genomic region, other CMV strains (Towne, Toledo, and AD169-ATCC) carried only the newly characterized longer form. The additional sequence altered the assignment of UL42 and UL43 open reading frames. UL42 decreased in size from 157 to 125 codons, retaining 76 of the previously reported carboxyl terminal codons, and UL43 increased in size from 187 to 423 codons, retaining 185 of the previously reported amino terminal codons. This additional sequence makes UL43 a more conserved betaherpesvirus US22 family member. Only AD169-UK exhibited restriction fragment length polymorphism in this region, suggesting that a deletion occurred during the propagation of this strain in cell culture. The additional sequence should be considered a bona fide part of the cytomegalovirus genome and the AD169 genome size should be corrected to 230,283 bp.
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  • Interplay between systemic inflammation and markers of insulin resistance in cardiovascular prognosis after heart transplantation

    Biadi, Ombretta   Potena, Luciano   Fearon, William F.   Luikart, Helen I.   Yeung, Alan   Ferrara, Rossella   Hunt, Sharon A.   Mocarski, Edward S.  

    Background: Metabolic and immuno-inflammatory risk factors contribute to cardiac allograft vasculopathy (CAV) pathogenesis. Although systemic inflammation, as detected by Greactive protein (CRP), predicts CAV development, the relationship between CRP and markers of metabolic abnormalities remains unexplored.Methods: CRP and the entire metabolic panel were evaluated in 98 consecutive heart transplant recipients at the time of annual coronary angiography, 5.8 years after transplant (range, 1-12 years). A ratio of triglycerides (TG) to high-density lipoproteins (HDL) of 3.0 or more was considered a marker of insulin resistance. CAV prevalence was defined by angiography, and subsequent prognosis was evaluated as incidence of major cardiac adverse events.Results: CRP was higher in the 34 patients with angiographic CAV than in those without CAV (1.10 +/- 0.20 vs 0.50 +/- 0.05 mg/dl, p < 0.001). Patients with insulin resistance had higher CRP concentrations (p = 0.023) and higher CAV prevalence (p = 0.005). High CRP and a TG/HDL of 3.0 or more were independently associated with an increased likelihood of CAV (odds ratio, >= 3.9; P = 0.02) and predicted an increased risk of major cardiac adverse events. The combination of high CRP and a TG/HDL of 3.0 or more identified a subgroup of patients having a 4-fold increased risk for CAV and a 3-fold increased risk for major cardiac adverse events compared with patients with low CRP and normal values for metabolic indicators.Conclusions: Both CRP and insulin resistance, as estimated by TG/HDL, appear to be strong, synergic risk factors for CAV and for major cardiac adverse events. These findings support the hypothesis that in heart transplant recipients, systemic inflammation may be an important mediator of graft vascular injury associated with metabolic syndrome.
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  • Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein,E3

    Koehler, Heather   Cotsmire, Samantha   Langland, Jeffrey   Kibler, Karen V.   Kalman, Daniel   Upton, Jason W.   Mocarski, Edward S.   Jacobs, Bertram L.  

    Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Z alpha) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Z alpha domain of E3 (VACVE3L Delta 83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Z alpha domain-containing DNA sensor, DAI. VACVE3L Delta 83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.
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  • Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis (vol 45, pg 1804, 2007)

    Potena, Luciano   Holweg, Cecile T. J.   Vana, Marcy L.   Bashyam, Leena   Rajamani, Jaya   McCormick, A. Louise   Cooke, John P.   Valantine, Hannah A.   Mocarski, Edward S.  

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  • DAI/ZBP1/DLM-1 Complexes with RIP3 to Mediate Virus-Induced Programmed Necrosis that Is Targeted by Murine Cytomegalovirus vIRA

    Upton, Jason W.   Kaiser, William J.   Mocarski, Edward S.  

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