Frank L. Graham

Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung.

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

Manipulation of Adenovirus Vectors

Adenoviruses have been isolated from a large number of different species (mammalian and fowl) and over 100 different serotypes have been reported, some 43 of them human. The human adenoviruses, particularly types 2, 5, and 12, have been the most extensively characterized, and these viruses have served as valuable tools in the study of the molecular biology of DNA replication, transcription, RNA processing, and protein synthesis in mammalian cells. Seeref. 1 for a general review.

Methods for construction of adenovirus vectors.

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.

A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes

Herpes simplex virus (HSV) infection of human fibroblasts rapidly renders the cells resistant to lysis by HSV-specific CD8+ cytotoxic T lymphocytes (CTLs), which normally recognize cell surface major histocompatibility complex (MHC) class I proteins presenting viral peptides. Within 3 hr of infection with HSV, MHC class I protein complexes are retained in the endoplasmic reticulum (ER)/cis Golgi and show properties of complexes lacking antigenic peptide. The HSV immediate-early protein ICP47 is both necessary and sufficient to block transport of class I proteins and to inhibit lysis by CD8+ CTLs. The target for ICP47 is not known, but since ICP47 does not associate with membranes, it appears that ICP47 inhibits the production or stabilization of antigenic peptides or their translocation into the ER/cis Golgi. Thus, by expressing ICP47, HSV can evade detection by CD8+ T lymphocytes, perhaps explaining the predominance of CD4+ rather than CD8+ HSV-specific CTLs in vivo.

Transfer of Tumor Necrosis Factor-α to Rat Lung Induces Severe Pulmonary Inflammation and Patchy Interstitial Fibrogenesis with Induction of Transforming Growth Factor-β1 and Myofibroblasts

Tumor necrosis factor-alpha is up-regulated in a variety of different human immune-inflammatory and fibrotic pulmonary pathologies. However, its precise role in these pathologies and, in particular, the mechanism(s) by which it may induce fibrogenesis are not yet elucidated. Using a replication-deficient adenovirus to transfer the cDNA of tumor necrosis factor-alpha to rat lung, we have been able to study the effect of transient but prolonged (7 to 10 days) overexpression of tumor necrosis factor-alpha in normal adult pulmonary tissue. We have demonstrated that local overexpression resulted in severe pulmonary inflammation with significant increases in neutrophils, macrophages, and lymphocytes and, to a lesser extent, eosinophils, with a peak at day 7. By day 14, the inflammatory cell accumulation had declined, and fibrogenesis became evident, with fibroblast accumulation and deposition of extracellular matrix proteins. Fibrotic changes were patchy but persisted to beyond day 64. To elucidate the mechanism underlying this fibrogenesis, we examined bronchoalveolar fluids for the presence of the fibrogenic cytokine transforming growth factor-beta1 and tissues for induction of alpha-smooth muscle actin-rich myofibroblasts. Transforming growth factor-beta1 was transiently elevated from day 7 (peak at day 14) immediately preceding the onset of fibrogenesis. Furthermore, there was a striking accumulation of myofibroblasts from day 7, with the most extensive and intense immunostaining at day 14, ie, coincident with the up-regulation of transforming growth factor-beta1 and onset of fibrogenesis. Thus, we have provided a model of tumor necrosis factor-alpha-mediated pulmonary inflammation and fibrosis in normal adult lung, and we suggest that the fibrogenesis may be mediated by the secondary up-regulation of transforming growth factor-beta1 and induction of pulmonary myofibroblasts.

Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.

[75] Assay of transforming activity of tumor virus DNA

Publisher Summary This chapter describes the assay of transforming activity of tumor virus DNA. Several techniques have been developed for assaying biological activity of DNA extracted from mammalian viruses. These methods measure the infectivity of viral nucleic acid preparations in cultured cells. This technique was developed as an assay for infectivity of adenovirus DNA, which also appeared suitable for demonstrating transformation with several viral DNAs. The diethylaminoethanol (DEAE)-dextran and the calcium technique have been used in virus research. A possible reason for the failure to obtain transformation with the DEAE-dextran technique may be the relative toxicity of this compound for many cultured cells. The chapter describes the procedures for assaying transformation of cultured cells by viral DNA or DNA fragments, in particular of adenovirus and SV40 DNA. The only method suitable for the demonstration of transforming activity with these viral DNAs is the calcium technique, therefore, the chapter discusses this method and the modifications developed to enhance its effectiveness.

Host-range mutants of adenovirus type 5 defective for growth in HeLa cells

Abstract Host-range mutants of adenovirus type 5 have been selected on human embryo kidney cells transformed by sheared adeno 5 DNA (293 cells). These mutants grow well on 293 cells, but are restricted on HeLa cells. By complementation tests on HeLa cells, these mutants were classified into two groups, neither of which was found to correspond to any of the 17 temperature-sensitive complementation groups. Members of the two complementation groups of host-range mutants differ in their host range on other cell types: Members of one group grow only on 293 cells, while those of the other group on both normal and transformed human embryo kidney cells. In the second group, cell-associated adeno gene functions are not needed for mutant growth, but, with the other group, it is possible that an adeno gene product made in the transformed cell complements the growth of the mutants. Recombination tests carried out with the host-range mutants and Ad5 temperature-sensitive mutants indicate that the host-range mutations map within the left half of the genetic map and that some (hr 1 and hr 2) probably lie very close to the left end of the map. The results presented in this communication are briefly discussed in relation to recent unpublished work concerning the biochemical characteristics of the mutants and the transformed 293 cells and to the role of adeno genes in transformation.

Human Adenovirus Vectors for Gene Transfer into Mammalian Cells

Publisher Summary The high efficiency of gene transfer mediated by recombinant Adenovirus (Ad) has prompted the construction of dozens of first-generation Ad vectors for high level transgene expression in mammalian cells. These vectors have been designed and employed for uses as diverse as recombinant protein production and characterization, transcriptional analysis, establishment of stably transformed cell lines, and gene therapy. As the Ad vector system holds such huge potential for expression of foreign DNA, particularly in gene therapy regimens, it has been the subject of intense research. This chapter summarizes the advances made in Ad vector design, as well as describes the current status of Ad vector applications for expression analysis and in gene therapy. Because of their ability to infect a wide range of cell types at high efficiencies, recombinant Ads have become powerful tools for the analysis of cis-acting elements involved in transcriptional regulation in different tissues or in response to different stimuli. The insertion of inducible promoters into Ad vectors has facilitated mapping of the various inducer-responsive elements in those promoters. Research on Ad vectors for their potential use in gene therapy has grown enormously. Some of the most encouraging applications of Ad vectors in gene therapy, such as the immunotherapy of cancer, require transient high-level expression of the transgene in target cells that is easily achieved with first-generation Ad vectors. Gene replacement therapies, however, require prolonged expression and/or repeated administration of the vector, both of which have been problematic owing to activation of the host immune system against Ad and Ad-infected cells. These difficulties are being addressed by the development of second-generation vectors and of strategies to modulate the host immune response. These improvements, and no doubt others yet to come, hold promise for the generation of genuinely effective vectors for treatment of genetic disorders and other diseases.

Requirement for Both Shc and Phosphatidylinositol 3′ Kinase Signaling Pathways in Polyomavirus Middle T-Mediated Mammary Tumorigenesis

ABSTRACT Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3′ (PI-3′) kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3′ kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3′ kinase were highly apoptotic, suggesting that recruitment of PI-3′ kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3′ kinase binding site was noted in tumors derived from the strains expressing the PI-3′ kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3′ kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3′ kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.