A prospective cohort and extended comprehensive-cohort design provided insights about the generalizability of a pragmatic trial: the ProtecT prostate cancer trial.

OBJECTIVES: Randomized controlled trials (RCTs) deliver robust internally valid evidence but generalizability is often neglected. Design features built into the Prostate testing for cancer and Treatment (ProtecT) RCT of treatments for localized prostate cancer (PCa) provided insights into its generalizability. STUDY DESIGN AND SETTING: Population-based cluster randomization created a prospective study of prostate-specific antigen (PSA) testing and a comprehensive-cohort study including groups choosing treatment or excluded from the RCT, as well as those randomized. Baseline information assessed selection and response during RCT conduct. RESULTS: The prospective study (82,430 PSA-tested men) represented healthy men likely to respond to a screening invitation. The extended comprehensive cohort comprised 1,643 randomized, 997 choosing treatment, and 557 excluded with advanced cancer/comorbidities. Men choosing treatment were very similar to randomized men except for having more professional/managerial occupations. Excluded men were similar to the randomized socio-demographically but different clinically, representing less healthy men with more advanced PCa. CONCLUSION: The design features of the ProtecT RCT provided data to assess the representativeness of the prospective cohort and generalizability of the findings of the RCT. Greater attention to collecting data at the design stage of pragmatic trials would better support later judgments by clinicians/policy-makers about the generalizability of RCT findings in clinical practice.

Mechanisms of TNFα regulation in uveitis: focus on RNA-binding proteins.

Tumour necrosis factor-α (TNFα) is a key mediator of inflammation and plays a crucial role during the early phase of a host's defence against bacterial, viral and parasitic infections. Persistent production of TNFα occurs in many autoimmune inflammatory diseases, including uveitis, and this is associated with significant tissue damage. Although uveitis represents a phenotypically heterogeneous group of intraocular inflammatory conditions, they have in common raised levels of TNFα in both serum and aqueous humour. Supporting a critical role for TNF activity during uveitis are reports that serum levels of TNFα correlate with disease status as well as the increasing evidence of therapeutic success of anti-TNF agents. TNFα is an archetypal pleiotropic cytokine and when acting systemically acute release may cause profound physiological decompensation. Yet, conversely, at tissue sites TNFα plays important roles governing homeostasis and during chronic inflammation regulating immune responses through control of, for example, macrophage-T cell functions. In a murine model of CD4(+) T cell mediated non-infectious uveitis, experimental autoimmune uveitis (EAU), activation of infiltrating macrophages mediates tissue damage. In EAU, whilst both T cells and macrophages generate TNFα, tissue damaging macrophage activation is dependent upon TNF receptor 1 (p55). TNFα protein production is controlled at the level of transcription, pre-mRNA processing, mRNA stability, translation and retention at the plasma membrane. The p38 MAP kinase and MAPKAP-2 pathway are involved in the post-transcriptional regulation of TNFα and are targeted by a functionally divergent group of cytokines including IL-10 and TGFß1. Common to many cytokines, TNFα mRNA 3' untranslated region (UTR) contains an AU-rich element (ARE), which drives repression by mRNA-binding proteins (RBPs). These include tristetraprolin (TTP), T cell antigen-1 (TIA-1), TIA-1-related protein (TIAR), human antigen R (HuR) and fragile-X-related protein 1 (FXR1). Disruption of several RBPs can dysregulate TNFα protein production and has, in some cases, been shown to exacerbate chronic inflammatory disease both in mice and in humans. Given that TNFα is central to clearance of infections, yet during chronic inflammation results in tissue damage, understanding the role that RBPs play in the control of TNFα may give rise to opportunities to not only develop targeted therapy for autoimmunity but also redress homeostasis without compromise and risking infection. The study of mRNA stability remains essential for the understanding of intracellular regulatory pathways and molecular mechanisms of pathology for infection, inflammation and degeneration.

Fragile X-related protein FXR1 controls post-transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-alpha production by transforming growth factor-beta1.

Tumour necrosis factor-alpha (TNF-alpha) is a key mediator of inflammation in host defence against infection and in autoimmune disease. Its production is controlled post-transcriptionally by multiple RNA-binding proteins that interact with the TNF-alpha AU-rich element and regulate its expression; one of these is Fragile X mental retardation-related protein 1 (FXR1). The anti-inflammatory cytokine transforming growth factor-beta1 (TGF-beta1), which is involved in the homeostatic regulation of TNF-alpha, causes post-transcriptional suppression of lipopolysaccharide (LPS)-induced TNF-alpha production. We report here that this depends on FXR1. Using RAW 264.7 cells and bone marrow-derived macrophages (BMDMphi) stimulated with LPS and TGF-beta1, we show that TGF-beta1 inhibits TNF-alpha protein secretion, whereas TNF-alpha mRNA expression remains unchanged. This response is recapitulated by the 3'-UTR of TNF-alpha, which is known to bind FXR1. TGF-beta1 induces FXR1 with a pattern of expression distinct from that of tristetraprolin, T-cell intracellular antigen 1, or human antigen R. When FXR1 is knocked down, TGF-beta1 is no longer able to inhibit LPS-induced TNF-alpha protein production, and overexpression of FXR1 suppresses LPS-induced TNF-alpha protein production. Targeting the p38 mitogen-activated protein kinase pathway of LPS-treated cells with small molecule inhibitors can induce FXR1 protein and mRNA expression. In summary, TGF-beta1 opposes LPS-induced stabilization of TNF-alpha mRNA and reduces the amount of TNF-alpha protein, through induction of expression of the mRNA-binding protein FXR1.