Faculty Bibliography

The transcription factor Stat3 is activated through tyrosine phosphorylation by many cytokines and is a fundamental mediator of their signals. In the mammary gland, Stat3 activity increases sharply shortly after weaning, and involution is delayed in mice, that contain a mutant Stat3 lacking 33 amino acids including the key tyrosine residue. We have now generated a more extensive mutation of Stat3 through the deletion of exons 15-21 in mammary epithelium. This resulted in the loss of 245 amino acids including the DNA binding and SH2 domains, and Stat3 protein was undetectable. Pregnancy-mediated mammary development and lactation were normal in these mice. Involution was delayed and, remarkably, Stat3-null mammary epithelium maintained its functional integrity and competence even 6 d after weaning, whereas control mammary tissue was rendered nonfunctional within 2 d. The lack of remodeling and functional stasis of the epithelium correlated with the disruption of proteinase activity. Our data demonstrate that mammary tissue can retain its functional competence in the absence of external lactogenic stimuli and demonstrate a delay in the initiation of the irreversible stage of involution

STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3

Interferons constitute the earliest immune response against viral infection. They elicit antiviral effects as well as multiple biological responses involved in cell growth regulation and immune activation. Because the interferon-induced cellular antiviral response is the primary defense mechanism against viral infection, many viruses have evolved strategies to antagonize the inhibitory effects of interferon. Here, we demonstrate a strategy that Kaposi's sarcoma-associated herpesvirus uses to block virus-mediated induction of type I interferon. We found that a viral immediate-early protein, namely ORF45, interacts with cellular interferon-regulatory factor 7 (IRF-7). In consequence, IRF-7 phosphorylation is inhibited and the accumulation of IRF-7 in the nucleus in response to viral infection is blocked. IRF-7 is a transcription regulator that is responsible for virus-mediated activation of type I interferon genes. By blocking the phosphorylation and nuclear translocation of IRF-7, ORF45 efficiently inhibits the activation of interferon alpha and beta genes during viral infection. Inhibition of interferon gene expression through a viral protein blocking the activation and nuclear translocation of a crucial transcription factor is a novel mechanism for viral immune evasion

Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC). Some NPC components, such as the nucleoporins (Nups) Nup98 and Nup96, are also associated with the nuclear interior. Nup98 is a target of the vesicular stomatitis virus (VSV) matrix (M) protein-mediated inhibition of messenger RNA (mRNA) nuclear export. Here, Nup98 and Nup96 were found to be up-regulated by interferon (IFN). M protein-mediated inhibition of mRNA nuclear export was reversed when cells were treated with IFN-gamma or transfected with a complementary DNA (cDNA) encoding Nup98 and Nup96. Thus, increased Nup98 and Nup96 expression constitutes an IFN-mediated mechanism that reverses M protein-mediated inhibition of gene expression

The anaplastic lymphoma kinase (ALK) gene is characteristically translocated in Anaplastic Large Cell Lymphomas (ALCL) and the juxtaposition of the ALK gene to multiple partners results in its constitutive protein tyrosine kinase activity. We show here that expression of activated ALK induces the constitutive phosphorylation of Stat3 in transfected cells as well as in primary human ALCLs. Furthermore, immunohistochemical studies demonstrate that among distinct human B and T cell lymphomas, activation of Stat3 nuclear translocation is uniquely associated with ALK expression. NPM-ALK also binds and activates Jak3; however, Jak3 is not required for Stat3 activation or for cell transformation in vitro. Moreover, src family kinases are not necessary for NPM-ALK-mediated Stat3 activation or transformation, suggesting that Stat3 may be phosphorylated directly by ALK. To evaluate relevant targets of ALK-activated Stat3, we investigated the regulation of the anti-apoptotic protein Bcl-x(L) and its role in cell survival in NPM-ALK positive cells. NPM-ALK expression caused enhanced Bcl-x(L) transcription, largely mediated by Stat3. Increased expression of Bcl-x(L) provided sufficient anti-apoptotic signals to protect cells from treatment with specific inhibitors of the Jaks/Stat pathway or the Brc-Abl kinase. These studies support a pathogenic mechanism whereby stimulation of anti-apoptotic signals through activation of Stat3 contributes to the successful outgrowth of ALK positive tumor cells

Type I interferon (IFN) stimulates transcription through a heteromeric transcription factor that contains tyrosine-phosphorylated STAT2. We show that STAT2 recruits histone acetyltransferases (HAT) through its transactivation domain, resulting in localized transient acetylation of histones. GCN5, but not p300/CBP or PCAF, is required for STAT2 function. However, GCN5 function is impaired by the transcriptional antagonist, adenovirus E1A oncoprotein. The TFIID component TAFII130 potentiates STAT2 function, but TAFII28 or the HAT activity of TAFII250 do not, and transcriptional induction can proceed independently of the TATA-binding protein, TBP. Moreover, IFN-stimulated transcription was resistant to poliovirus-targeted degradation by TBP, and continued despite host-cell transcriptional shutoff during poliovirus infection. We conclude that a non-classical transcriptional mechanism combats an anticellular action of poliovirus, through a TBP-free TAF-containing complex and GCN5

RATIONALE, AIMS AND OBJECTIVES: This paper describes a recent randomized controlled trial in which 42% of patients receiving ancrod attained a favourable outcome in comparison with 34% of controls. Although the above effect size corresponds to a number needed to treat (to achieve a favourable outcome) of approximately 13, intuition does not necessarily suggest what would be the overall impact of a treatment with this level of efficacy. METHODS: The objective was to evaluate the cost-effectiveness of ancrod. Cost-effectiveness analysis of data from the Stroke Treatment with Ancrod Trial (STAT) trial was carried out. The participants were 495 patients with data on functional status at the conclusion of follow-up. Short-term results were based upon utilization and quality of life observed during the trial; these were merged with expected long-term results obtained through simulation using the Stroke Policy Model. The main outcome measure was incremental cost-effectiveness ratio. RESULTS: Ancrod treatment resulted in both better quality-adjusted life expectancy and lower medical costs than placebo as supported by sensitivity analysis. The cost differential was primarily attributable to the long-term implications of ancrod's role in reducing disability. CONCLUSIONS: If ancrod is even modestly effective, it will probably be cost-effective (and, indeed, cost-saving) as well. The net population-level impact of even modestly effective stroke treatments can be substantial

Interferons (IFN) are potent components of the innate immune response to microbial infection. The genes for type I IFN (IFN-alpha and IFN-beta) are rapidly induced in response to viral infection through a mechanism that involves latent cellular transcription factors that are activated in response to innate recognition of viral components. IFN regulatory factor (IRF) proteins are key to this regulation, and their conversion from latent to active involves virus-induced serine phosphorylation. Differential utilization of distinct IRF proteins by different members of the type I IFN gene family produces a graded induction of gene expression, resulting in tight control of these cytokines through a positive feedback mechanism. Early response to virus causes secretion of a subset of IFN genes through the action of IRF-3 in conjunction with additional transcription factors, such as NF-kappaB and activator protein-1 (AP-1) (c-jun/ATF). This early IFN acts in an autocrine manner to stimulate production of IRF-7, a transcription factor capable of activating the many additional members of the IFN-alpha gene family. The dependence of IRF-7 on virus-induced phosphorylation for its activity insures that IFN production is limited to virus-infected cells. Characterization of the cellular components involved in viral detection and IRF activation will further delineate this vital mechanism of innate immune response