Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.
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TGF-beta plays an important role in lung fibrosis, which is a major cause of suffering and death seen in pulmonary disease. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of lung fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant human type 5 adenovirus vector, on bleomycin-induced lung fibrosis in mice. C57BL/6 mice with bleomycin-induced lungs received an intratracheal injection of a recombinant adenovirus carrying mice Smad7 cDNA. These mice demonstrated suppression of type I precollagen mRNA, reduced hydroxyproline content, and no morphological fibrotic responses in the lungs when compared with mice administered adenovirus carrying Smad6 cDNA. In addition, we found that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs. These data indicated that gene transfer of Smad7 (but not Smad6) prevented bleomycin-induced lung fibrosis, suggesting that Smad7 may have applicability in the treatment of pulmonary fibrosis. (+info)
IFN-gamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression.
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TNF-related apoptosis-inducing ligand (TRAIL) is able to kill many transformed cells of diverse tissue types. We show that TRAIL is inducible by IFN-gamma, by TNF-alpha, and by infection with human CMV, and has potent antiviral activity in vitro. CMV infection and IFN-gamma also reciprocally modulate TRAIL receptor (TRAIL-R) expression. CMV infection increased the expression of TRAIL-R1 and -R2, whereas IFN-gamma down-regulated the expression of TRAIL-Rs on uninfected fibroblasts. Moreover, IFN-gamma significantly decreased the basal level of NF-kappaB activation, a known survival factor that inhibits apoptosis. Thus, TRAIL selectively kills virus-infected cells while leaving uninfected cells intact, and IFN-gamma potentiates these effects by dynamic modulation of TRAIL and TRAIL-R expression and by sensitizing cells to apoptosis. The regulation of TRAIL and TRAIL-R expression may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis. (+info)
Cytomegalovirus (CMV)-specific T cell immunity after renal transplantation mediates protection from CMV disease by limiting the systemic virus load.
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The role of cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) and T helper cells (Th) in controlling CMV infection, as detected by antigenemia assay and polymerase chain reaction (PCR) in blood leukocytes, and CMV disease was investigated in 20 renal transplant recipients. Within 3 months after transplant, CMV-specific CTL and Th responses were demonstrable in 11 (55%) and 15 (75%) patients, respectively; CMV infection was detected by antigenemia and PCR in 19 (95%) patients each. During the month of first CMV detection, there was an inverse correlation between CTL response and antigenemia at >/=20 positive cells/105 leukocytes (P=.007) but no association with lower antigenemia levels or PCR positivity. CMV disease developed in 7 (35%) patients and was associated with high-level antigenemia but was inversely correlated with detection of CTLs (P=.04). After renal transplantation, CMV-specific CTLs limit the systemic virus load as reflected by antigenemia levels and thereby mediate protection from CMV disease. (+info)
Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child.
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Children with innate immunodeficiencies may be at high risk for early development of ganciclovir-resistant human cytomegalovirus (HCMV) infection after bone marrow transplantation (BMT). For early and frequent monitoring of the occurrence of ganciclovir resistance-associated mutations in codons of the UL97 gene, a panel of previously described restriction assays was expanded for use on codons 591, 592, and 603. This technique enabled detection of suddenly emerging ganciclovir-resistant HCMV after BMT in a 7-year-old child with a T cell defect. Resistance emerged among the isolation of a ganciclovir-sensitive HCMV strain 32 days after transplantation, the first detection of genotypical resistance at day 44, and the isolation of resistant HCMV (ID50>12 microM) at day 54. Simple and yet comprehensive methods for therapy surveillance may be important in this patient group, in which the restriction assays proved useful. (+info)
Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines.
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Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines. (+info)
Interleukin-1 (IL-1) inhibits growth of cytomegalovirus in human marrow stromal cells: inhibition is reversed upon removal of IL-1.
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A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1alpha (IL-1alpha) and IL-1beta completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1beta was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1-treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 +/- 3 v 151 +/- 12/well, respectively; GFP intensity was 535 +/- 165 v 6,516 +/- 652/well, respectively, in 4 separate experiments). At day 21, when IL-1beta-treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1beta, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1beta effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1-induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1beta-treated and nontreated HS-27a cells has identified some candidate molecules. (+info)
Human cytomegalovirus and human herpesvirus 6 genes that transform and transactivate.
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This review is an update on the transforming genes of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6). Both viruses have been implicated in the etiology of several human cancers. In particular, HCMV has been associated with cervical carcinoma and adenocarcinomas of the prostate and colon. In vitro transformation studies have established three HCMV morphologic transforming regions (mtr), i.e., mtrI, mtrII, and mtrIII. Of these, only mtrII (UL111A) is retained and expressed in both transformed and tumor-derived cells. The transforming and tumorigenic activities of the mtrII oncogene were localized to an open reading frame (ORF) encoding a 79-amino-acid (aa) protein. Furthermore, mtrII protein bound to the tumor suppressor protein p53 and inhibited its ability to transactivate a p53-responsive promoter. In additional studies, the HCMV immediate-early protein IE86 (IE2; UL122) was found to interact with cell cycle-regulatory proteins such as p53 and Rb. However, IE86 exhibited transforming activity in vitro only in cooperation with adenovirus E1A. HHV-6 is a T-cell-tropic virus associated with AIDS-related and other lymphoid malignancies. In vitro studies identified three transforming fragments, i.e., SalI-L, ZVB70, and ZVH14. Of these, only SalI-L (DR7) was retained in transformed and tumor-derived cells. The transforming and tumorigenic activities of SalI-L have been localized to a 357-aa ORF-1 protein. The ORF-1 protein was expressed in transformed cells and, like HCMV mtrII, bound to p53 and inhibited its ability to transactivate a p53-responsive promoter. HHV-6 has also been proposed to be a cofactor in AIDS because both HHV-6 and human immunodeficiency virus type 1 (HIV-1) have been demonstrated to coinfect human CD4(+) T cells, causing accelerated cytopathic effects. Interestingly, like the transforming proteins of DNA tumor viruses such as simian virus 40 and adenovirus, ORF-1 was also a transactivator and specifically up-regulated the HIV-1 long terminal repeat when cotransfected into CD4(+) T cells. Finally, based on the interactions of HCMV and HHV-6 transforming proteins with tumor suppressor proteins, a scheme is proposed for their role in oncogenesis. (+info)
Numerous length polymorphisms at short tandem repeats in human cytomegalovirus.
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We show the presence of numerous short tandem repeats in the human cytomegalovirus (HCMV) genome and assess their usefulness as molecular markers. The genome is shown to contain at least 24 microsatellite regions that exhibit length polymorphisms. Insertion-deletion polymorphisms at these short tandem repeats are common (80% of repeats examined are polymorphic among two laboratory strains and 10 clinical isolates). This is the first report of widespread microsatellite length polymorphism in a viral genome. Some regions are highly polymorphic: one was revealed by DNA sequencing to contain length variants at five closely linked sites, which combined resulted in 10 variants for this region among the 12 strains and isolates examined. This study not only provides a new molecular marker system for this virus but also extends our understanding of microsatellite polymorphism in two important ways. First, variable-length repeats in HCMV can be considerably shorter than polymorphic repeats previously found in other organisms. Second, highly variable microsatellite repeats are not confined to prokaryotes and eukaryotes, as previously assumed. This variation provides a useful marker system for distinguishing viral isolates, and similar markers are also likely to be found in other large-genome DNA viruses. (+info)