Chronic infection with hepatitis C virus (HCV) affects 170 million people worldwide and is the leading cause of cirrhosis in North America. Although the recommended treatment for chronic infection involves a 48-week course of peg<em>interferon</em>-<em>alpha</em>-2b (PegIFN-<em>alpha</em>-2b) or -<em>alpha</em>-2a (PegIFN-<em>alpha</em>-2a) combined with ribavirin (RBV), it is well known that many patients will not be cured by treatment, and that patients of European ancestry have a significantly higher probability of being cured than patients of African ancestry. In addition to limited efficacy, treatment is often poorly tolerated because of side effects that prevent some patients from completing therapy. For these reasons, identification of the determinants of response to treatment is a high priority. Here we report that a genetic polymorphism near the IL28B gene, encoding <em>interferon</em>-lambda-<em>3</em> (IFN-lambda-<em>3</em>), is associated with an approximately twofold change in response to treatment, both among patients of European ancestry (P = 1.06 x 10(-25)) and African-Americans (P = 2.06 x 10(-<em>3</em>)). Because the genotype leading to better response is in substantially greater frequency in European than African populations, this genetic polymorphism also explains approximately half of the difference in response rates between African-Americans and patients of European ancestry.

Hepatitis C virus (HCV) infects <em>3</em>% of the world's population. Treatment of chronic HCV consists of a combination of PEGylated <em>interferon</em>-<em>alpha</em> (PEG-IFN-<em>alpha</em>) and ribavirin (RBV). To identify genetic variants associated with HCV treatment response, we conducted a genome-wide association study of sustained virological response (SVR) to PEG-IFN-<em>alpha</em>/RBV combination therapy in 29<em>3</em> Australian individuals with genotype 1 chronic hepatitis C, with validation in an independent replication cohort consisting of 555 individuals. We report an association to SVR within the gene region encoding interleukin 28B (IL28B, also called IFNlambda<em>3</em>; rs8099917 combined P = 9.25 x 10(-9), OR = 1.98, 95% CI = 1.57-2.52). IL28B contributes to viral resistance and is known to be upregulated by <em>interferons</em> and by RNA virus infection. These data suggest that host genetics may be useful for the prediction of drug response, and they also support the investigation of the role of IL28B in the treatment of HCV and in other diseases treated with IFN-<em>alpha</em>.

OBJECTIVE

A randomized, phase III trial demonstrated superiority of sunitinib over interferon alfa (IFN-alpha) in progression-free survival (primary end point) as first-line treatment for metastatic renal cell carcinoma (RCC). Final survival analyses and updated results are reported.

METHODS

Seven hundred fifty treatment-naïve patients with metastatic clear cell RCC were randomly assigned to sunitinib 50 mg orally once daily on a 4 weeks on, 2 weeks off dosing schedule or to IFN-alpha 9 MU subcutaneously thrice weekly. Overall survival was compared by two-sided log-rank and Wilcoxon tests. Progression-free survival, response, and safety end points were assessed with updated follow-up.

RESULTS

Median overall survival was greater in the sunitinib group than in the IFN-alpha group (26.4 v 21.8 months, respectively; hazard ratio [HR] = 0.821; 95% CI, 0.673 to 1.001; P = .051) per the primary analysis of unstratified log-rank test (P = .013 per unstratified Wilcoxon test). By stratified log-rank test, the HR was 0.818 (95% CI, 0.669 to 0.999; P = .049). Within the IFN-alpha group, 33% of patients received sunitinib, and 32% received other vascular endothelial growth factor-signaling inhibitors after discontinuation from the trial. Median progression-free survival was 11 months for sunitinib compared with 5 months for IFN-alpha (P < .001). Objective response rate was 47% for sunitinib compared with 12% for IFN-alpha (P < .001). The most commonly reported sunitinib-related grade 3 adverse events included hypertension (12%), fatigue (11%), diarrhea (9%), and hand-foot syndrome (9%).

CONCLUSIONS

Sunitinib demonstrates longer overall survival compared with IFN-alpha plus improvement in response and progression-free survival in the first-line treatment of patients with metastatic RCC. The overall survival highlights an improved prognosis in patients with RCC in the era of targeted therapy.

Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(<em>alpha</em>) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic <em>interferon</em>-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-<em>3</em> (GATA-<em>3</em>)(+)) and regulatory forkhead box P<em>3</em> (Foxp<em>3</em>)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD<em>3</em>-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp<em>3</em>(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.

To better understand the dynamics of hepatitis C virus and the antiviral effect of <em>interferon</em>-<em>alpha</em>-2b (IFN), viral decline in 2<em>3</em> patients during therapy was analyzed with a mathematical model. The analysis indicates that the major initial effect of IFN is to block virion production or release, with blocking efficacies of 81, 95, and 96% for daily doses of 5, 10, and 15 million international units, respectively. The estimated virion half-life (t1/2) was, on average, 2.7 hours, with pretreatment production and clearance of 10(12) virions per day. The estimated infected cell death rate exhibited large interpatient variation (corresponding t1/2 = 1.7 to 70 days), was inversely correlated with baseline viral load, and was positively correlated with alanine aminotransferase levels. Fast death rates were predictive of virus being undetectable by polymerase chain reaction at <em>3</em> months. These findings show that infection with hepatitis C virus is highly dynamic and that early monitoring of viral load can help guide therapy.

Members of the nuclear factor kappa B (NF-kappaB) family of dimeric transcription factors (TFs) regulate expression of a large number of genes involved in immune responses, inflammation, cell survival, and cancer. NF-kappaB TFs are rapidly activated in response to various stimuli, including cytokines, infectious agents, and radiation-induced DNA double-strand breaks. In nonstimulated cells, some NF-kappaB TFs are bound to inhibitory IkappaB proteins and are thereby sequestered in the cytoplasm. Activation leads to phosphorylation of IkappaB proteins and their subsequent recognition by ubiquitinating enzymes. The resulting proteasomal degradation of IkappaB proteins liberates IkappaB-bound NF-kappaB TFs, which translocate to the nucleus to drive expression of target genes. Two protein kinases with a high degree of sequence similarity, IKK<em>alpha</em> and IKKbeta, mediate phosphorylation of IkappaB proteins and represent a convergence point for most signal transduction pathways leading to NF-kappaB activation. Most of the IKK<em>alpha</em> and IKKbeta molecules in the cell are part of IKK complexes that also contain a regulatory subunit called IKKgamma or NEMO. Despite extensive sequence similarity, IKK<em>alpha</em> and IKKbeta have largely distinct functions, due to their different substrate specificities and modes of regulation. IKKbeta (and IKKgamma) are essential for rapid NF-kappaB activation by proinflammatory signaling cascades, such as those triggered by tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>) or lipopolysaccharide (LPS). In contrast, IKK<em>alpha</em> functions in the activation of a specific form of NF-kappaB in response to a subset of TNF family members and may also serve to attenuate IKKbeta-driven NF-kappaB activation. Moreover, IKK<em>alpha</em> is involved in keratinocyte differentiation, but this function is independent of its kinase activity. Several years ago, two protein kinases, one called IKKepsilon or IKK-i and one variously named TBK1 (TANK-binding kinase), NAK (NF-kappaB-activated kinase), or T2K (TRAF2-associated kinase), were identified that exhibit structural similarity to IKK<em>alpha</em> and IKKbeta. These protein kinases are important for the activation of <em>interferon</em> response factor <em>3</em> (IRF<em>3</em>) and IRF7, TFs that play key roles in the induction of type I <em>interferon</em> (IFN-I). Together, the IKKs and IKK-related kinases are instrumental for activation of the host defense system. This Review focuses on the functions of IKK and IKK-related kinases and the molecular mechanisms that regulate their activities.

Induction of the <em>interferon</em> (IFN)-<em>alpha</em>/beta gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-<em>3</em> are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-<em>alpha</em>/beta gene expression levels are reduced and the spectrum of the IFN-<em>alpha</em> mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-<em>alpha</em>/beta mRNA induction can be achieved by coexpressing both IRF-<em>3</em> and IRF-7. These results demonstrate the essential and distinct roles of thetwo factors, which together ensure the transcriptional efficiency and diversity of IFN-<em>alpha</em>/beta genes for the antiviral response.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with <em>interferon</em>-<em>alpha</em> (IFN<em>alpha</em>), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFN<em>alpha</em> treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFN<em>alpha</em> target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFN<em>alpha</em>/beta receptor (IFNAR), STAT1 (ref. <em>3</em>) or Sca-1 (ref. 4) are insensitive to IFN<em>alpha</em> stimulation, demonstrating that STAT1 and Sca-1 mediate IFN<em>alpha</em>-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFN<em>alpha</em> and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFN<em>alpha</em> are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFN<em>alpha</em> pathway in HSCs impairs their function, acute IFN<em>alpha</em> treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFN<em>alpha</em> on leukaemic cells, and raise the possibility for new applications of type I <em>interferons</em> to target cancer stem cells.

Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-<em>alpha</em>, and <em>interferon</em> (IFN)-gamma. IL-1beta and/or TNF-<em>alpha</em> plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-<em>3</em> activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.

Activation of naive CD4(+) T-helper cells results in the development of at least two distinct effector populations, Th1 and Th2 cells. Th1 cells produce cytokines (<em>interferon</em> (IFN)-gamma, interleukin (IL)-2, tumour-necrosis factor (TNF)-<em>alpha</em> and lymphotoxin) that are commonly associated with cell-mediated immune responses against intracellular pathogens, delayed-type hypersensitivity reactions, and induction of organ-specific autoimmune diseases. Th2 cells produce cytokines (IL-4, IL-10 and IL-1<em>3</em>) that are crucial for control of extracellular helminthic infections and promote atopic and allergic diseases. Although much is known about the functions of these two subsets of T-helper cells, there are few known surface molecules that distinguish between them. We report here the identification and characterization of a transmembrane protein, Tim-<em>3</em>, which contains an immunoglobulin and a mucin-like domain and is expressed on differentiated Th1 cells. In vivo administration of antibody to Tim-<em>3</em> enhances the clinical and pathological severity of experimental autoimmune encephalomyelitis (EAE), a Th1-dependent autoimmune disease, and increases the number and activation level of macrophages. Tim-<em>3</em> may have an important role in the induction of autoimmune diseases by regulating macrophage activation and/or function.

<em>Alpha</em>/beta <em>interferon</em> immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and <em>interferon</em> promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream <em>interferon</em> regulatory factor <em>3</em> activation and <em>interferon</em>-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.

BACKGROUND

The hyper-IgE syndrome (or Job's syndrome) is a rare disorder of immunity and connective tissue characterized by dermatitis, boils, cyst-forming pneumonias, elevated serum IgE levels, retained primary dentition, and bone abnormalities. Inheritance is autosomal dominant; sporadic cases are also found.

METHODS

We collected longitudinal clinical data on patients with the hyper-IgE syndrome and their families and assayed the levels of cytokines secreted by stimulated leukocytes and the gene expression in resting and stimulated cells. These data implicated the signal transducer and activator of transcription <em>3</em> gene (STAT<em>3</em>) as a candidate gene, which we then sequenced.

RESULTS

We found increased levels of proinflammatory gene transcripts in unstimulated peripheral-blood neutrophils and mononuclear cells from patients with the hyper-IgE syndrome, as compared with levels in control cells. In vitro cultures of mononuclear cells from patients that were stimulated with lipopolysaccharide, with or without interferon-gamma, had higher tumor necrosis factor alpha levels than did identically treated cells from unaffected persons (P=0.00<em>3</em>). In contrast, the cells from patients with the hyper-IgE syndrome generated lower levels of monocyte chemoattractant protein 1 in response to the presence of interleukin-6 (P=0.0<em>3</em>), suggesting a defect in interleukin-6 signaling through its downstream mediators, one of which is STAT<em>3</em>. We identified missense mutations and single-codon in-frame deletions in STAT<em>3</em> in 50 familial and sporadic cases of the hyper-IgE syndrome. Eighteen discrete mutations, five of which were hot spots, were predicted to directly affect the DNA-binding and SRC homology 2 (SH2) domains.

CONCLUSIONS

Mutations in STAT<em>3</em> underlie sporadic and dominant forms of the hyper-IgE syndrome, an immunodeficiency syndrome involving increased innate immune response, recurrent infections, and complex somatic features.

Chemokines and their receptors are important elements for the selective attraction of various subsets of leukocytes. To better understand the selective migration of functional subsets of T cells, chemokine receptor expression was analyzed using monoclonal antibodies, RNase protection assays, and the response to distinct chemokines. Naive T cells expressed only CXC chemokine receptor (CXCR)4, whereas the majority of memory/activated T cells expressed CXCR<em>3</em>, and a small proportion expressed CC chemokine receptor (CCR)<em>3</em> and CCR5. When polarized T cell lines were analyzed, CXCR<em>3</em> was found to be expressed at high levels on T helper cell (Th)0s and Th1s and at low levels on Th2s. In contrast, CCR<em>3</em> and CCR4 were found on Th2s. This was confirmed by functional responses: only Th2s responded with an increase in [Ca2+]i to the CCR<em>3</em> and CCR4 agonists eotaxin and thymus and activation regulated chemokine (TARC), whereas only Th0s and Th1s responded to low concentrations of the CXCR<em>3</em> agonists IFN-gamma-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig). Although CCR5 was expressed on both Th1 and Th2 lines, it was absent in several Th2 clones and its expression was markedly influenced by interleukin 2. Chemokine receptor expression and association with Th1 and Th2 phenotypes was affected by other cytokines present during polarization. Transforming growth factor beta inhibited CCR<em>3</em>, but enhanced CCR4 and CCR7 expression, whereas <em>interferon</em> <em>alpha</em> inhibited CCR<em>3</em> but upregulated CXCR<em>3</em> and CCR1. These results demonstrate that chemokine receptors are markers of naive and polarized T cell subsets and suggest that flexible programs of chemokine receptor gene expression may control tissue-specific migration of effector T cells.

Virus-responsive signaling pathways that induce <em>alpha</em>/beta <em>interferon</em> production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>), thereby inducing <em>interferon</em> in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an <em>interferon</em>-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF<em>3</em> by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an <em>interferon</em>-responsive gene, may play a key role in <em>interferon</em>-based therapies for the treatment of HCV infection.

The <em>interferon</em> regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the <em>alpha</em> beta <em>interferon</em> (IFN-<em>alpha</em>/beta) gene promoters, as well as the <em>interferon</em>-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-<em>3</em> was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-<em>3</em> is expressed constitutively in a variety of tissues, and the relative levels of IRF-<em>3</em> mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-<em>3</em> is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-<em>3</em>. A combination of IRF-<em>3</em> deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids <em>3</em>95 and 407; point mutation of residues Ser-<em>3</em>96 and Ser-<em>3</em>98 eliminated virus-induced phosphorylation of IRF-<em>3</em> protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-<em>3</em>, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-<em>3</em> that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-<em>3</em> was prevented by mutation of the IRF-<em>3</em> Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-<em>3</em> with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-<em>3</em>96 and Ser-<em>3</em>98 in IRF-<em>3</em> abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-<em>3</em> alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates <em>interferon</em> regulatory factor (IRF)-<em>3</em>, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)<em>3</em> and 4 activate these pathways to induce IFN-<em>alpha</em>/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma <em>interferon</em>-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR<em>3</em> (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR<em>3</em>. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or <em>interferon</em>-<em>alpha</em>/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor <em>3</em> (TLR<em>3</em>), its adaptor TRIF, and induction of <em>interferon</em>-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR<em>3</em>-RNA complex. Choroidal endothelial cells from people expressing the TLR<em>3</em> coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR<em>3</em>, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals <em>interferon</em> regulatory factor <em>3</em> activation to induce the expression of <em>interferon</em>-<em>alpha</em>/beta and antiviral/<em>interferon</em>-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome <em>3</em>' non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered <em>interferon</em> and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.

OBJECTIVE

Interferon alfa-2b (IFN alpha-2b) exhibits antitumor activity in metastatic melanoma and on this basis has been evaluated as an adjuvant therapy following surgery for deep primary (T4) or regionally metastatic (N1) melanoma.

METHODS

A randomized controlled study of IFN alpha-2b (Schering-Plough, Kenilworth, NJ) administered at maximum-tolerated doses of 20 MU/m2/d intravenously (i.v.) for 1 month and 10 MU/m2 three times per week subcutaneously (SC) for 48 weeks versus observation, was conducted by the Eastern Cooperative Oncology Group (ECOG) in 287 patients.

RESULTS

A significant prolongation of relapse-free survival (P = .0023, one-sided) and prolongation of overall survival (P = .0237, one-sided) was observed with IFN alpha-2b therapy in this trial, which is now mature with a median follow-up time of 6.9 years. The impact of treatment on relapse rate is most pronounced early during the treatment interval. The overall benefit of treatment in this trial was analyzed stratified by tumor burden and the presence or absence of microscopic nonpalpable and palpable regional lymph node metastasis. The benefit of therapy with IFN alpha-2b was greatest among node-positive strata. Toxicity of IFN alpha-2b required dose modification in the majority of patients, but treatment at>> or = 80% of the scheduled dose was feasible in the majority of patients through the IV phase of treatment, and for more than 3 months of SC maintenance therapy. Discontinuation of treatment due to toxicity was infrequent after the fourth month of therapy.

CONCLUSIONS

IFN alpha-2b prolongs the relapse-free interval and overall survival of high-risk resected melanoma patients. The increment in median disease-free survival (from 1 to 1.7 years) and overall survival (from 2.8 to 3.8 years) that results from this therapy is associated with a 42% improvement in the fraction of patients who are continuously disease-free after treatment with IFN (from 26% to 37%) in comparison to observation. IFN alpha-2b is the first agent to show a significant benefit in relapse-free and overall survival of high-risk melanoma patients in a randomized controlled trial.

Transforming growth factor-beta (TGF-beta)/bone morphogenic protein (BMP) signaling is involved in the vast majority of cellular processes and is fundamentally important during the entire life of all metazoans. Deregulation of TGF-beta/BMP activity almost invariably leads to developmental defects and/or diseases, including cancer. The proper functioning of the TGF-beta/BMP pathway depends on its constitutive and extensive communication with other signaling pathways, leading to synergistic or antagonistic effects and eventually desirable biological outcomes. The nature of such signaling cross-talk is overwhelmingly complex and highly context-dependent. Here we review the different modes of cross-talk between TGF-beta/BMP and the signaling pathways of Mitogen-activated protein kinase, phosphatidylinositol-<em>3</em> kinase/Akt, Wnt, Hedgehog, Notch, and the interleukin/<em>interferon</em>-gamma/tumor necrosis factor-<em>alpha</em> cytokines, with an emphasis on the underlying molecular mechanisms.

The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by <em>interferons</em>. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the <em>alpha</em> subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5' untranslated regions. In addition, PKR affects diverse transcriptional factors such as <em>interferon</em> regulatory factor 1, STATs, p5<em>3</em>, activating transcription factor <em>3</em>, and NF-kappaB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IkappaB and NF-kappaB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results.

Statins, <em>3</em>-hydroxy-<em>3</em>-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are approved for cholesterol reduction, may also be beneficial in the treatment of inflammatory diseases. Atorvastatin (Lipitor) was tested in chronic and relapsing experimental autoimmune encephalomyelitis, a CD4(+) Th1-mediated central nervous system (CNS) demyelinating disease model of multiple sclerosis. Here we show that oral atorvastatin prevented or reversed chronic and relapsing paralysis. Atorvastatin induced STAT6 phosphorylation and secretion of Th2 cytokines (interleukin (IL)-4, IL-5 and IL-10) and transforming growth factor (TGF)-beta. Conversely, STAT4 phosphorylation was inhibited and secretion of Th1 cytokines (IL-2, IL-12, <em>interferon</em> (IFN)-gamma and tumour necrosis factor (TNF)-<em>alpha</em>) was suppressed. Atorvastatin promoted differentiation of Th0 cells into Th2 cells. In adoptive transfer, these Th2 cells protected recipient mice from EAE induction. Atorvastatin reduced CNS infiltration and major histocompatibility complex (MHC) class II expression. Treatment of microglia inhibited IFN-gamma-inducible transcription at multiple MHC class II transactivator (CIITA) promoters and suppressed class II upregulation. Atorvastatin suppressed IFN-gamma-inducible expression of CD40, CD80 and CD86 co-stimulatory molecules. l-Mevalonate, the product of HMG-CoA reductase, reversed atorvastatin's effects on antigen-presenting cells (APC) and T cells. Atorvastatin treatment of either APC or T cells suppressed antigen-specific T-cell activation. Thus, atorvastatin has pleiotropic immunomodulatory effects involving both APC and T-cell compartments. Statins may be beneficial for multiple sclerosis and other Th1-mediated autoimmune diseases.

Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture-induced septic shock. LPS as well as TNF-<em>alpha</em> stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as <em>3</em>0 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-kappaB and <em>interferon</em> regulatory factor <em>3</em> in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2's regulation of cellular glutathione and other antioxidants is critical for optimal NF-kappaB activation in response to LPS and TNF-<em>alpha</em>. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.

We present a novel mechanism by which viruses may inhibit the <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral <em>interferon</em> response by inhibiting the activation of <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>), a key regulator of IFN-<em>alpha</em>/beta gene expression. IRF-<em>3</em> activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the <em>interferon</em> signaling pathway. Inhibition of IRF-<em>3</em> activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-<em>3</em> activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.