Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the <em>interferon</em> regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the <em>interferon</em>-mediated transcriptional activation of the <em>interferon</em>-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-<em>3</em>. We further show that ectopic expression of vIRF-1 in NIH <em>3</em>T<em>3</em> cells confers resistance to tumor necrosis factor <em>alpha</em>-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the <em>interferon</em> consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 24<em>3</em>. While no binding between the full-size IRF-<em>3</em> and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 162<em>3</em> to 2414) of the transcriptional coactivator p<em>3</em>00 which could also bind IRF-<em>3</em> and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p<em>3</em>00.

It is hypothesized that the balance of cytokines produced by Th1/Th2 subsets of T helper cells plays an important role in the development of autoimmune diseases. Murine collagen-induced arthritis (CIA) is an example of an autoimmune disease in which immunization with cartilage-derived type II collagen induces, firstly, a T cell response to type II collagen and, secondly, the manifestation of a destructive inflammatory response in affected joints. We have investigated the role of Th1/Th2 responses in the development of CIA by monitoring levels of <em>interferon</em> (IFN)-gamma (a Th1 cytokine) and interleukin (IL)-4 and IL-10 (Th2 cytokines), and IL-1 beta and tumor necrosis factor (TNF) (pro-inflammatory cytokines) produced by cultured draining lymph node cells (LNC) from collagen-immunized DBA/1 mice during the induction phase of arthritis and throughout the time of clinical manifestation and subsequent remission of the disease. Although a transient increase in IL-10 was detected <em>3</em> days after immunization, Th2 cytokine production was found to be almost completely suppressed 6 days after immunization. In contrast, IFN-gamma was detected in LNC cultures as early as 6 days after immunization and the addition of type II collagen to the culture medium resulted in an approximately 10-fold increase in IFN-gamma production, indicating that a predominantly Th1 response had become established by this time. IFN-gamma production by LNC was found to be further increased at the time of clinical manifestation of arthritis and could be up-regulated by co-culture with type II collagen. IL-10 was not detected in LNC cultures at the onset of arthritis and IL-4, although present, was found to be markedly suppressed in LNC cultures containing type II collagen. These findings indicate that Th1 responses are predominant at the time of onset of arthritis and that the activation of collagen-specific Th1 cells may result in suppression of Th2 activity. IFN-gamma production declined progressively during the progression and subsequent remission of arthritis whereas levels of IL-10 increased and low, though persistent, levels of IL-4 were detected throughout this period. High levels of IL-1 beta and TNF-<em>alpha</em> production were detected at the onset of the disease. The role of Th1 responses in the development of CIA was further emphasized by the observation that immunization of mice with type II collagen in incomplete Freund's adjuvant, which normally fails to induce arthritis, resulted in a predominantly Th2 cytokine profile.

<em>Interferon</em> regulatory factors (IRF)-<em>3</em> and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-<em>alpha</em>/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-<em>3</em> or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-<em>alpha</em> levels after WNV infection, those lacking IRF-<em>3</em> or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-<em>3</em>(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-<em>alpha</em> response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either <em>interferon</em> (IFN)-gamma or -<em>alpha</em> and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-<em>3</em> receptor (R)<em>alpha</em>(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-<em>3</em>R<em>alpha</em>(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

Mammalian cells respond to virus infections by eliciting both innate and adaptive immune responses. One of the most effective innate antiviral responses is the production of <em>alpha</em>/beta <em>interferon</em> and the subsequent induction of <em>interferon</em>-stimulated genes (ISGs), whose products collectively limit virus replication and spread. Following viral infection, <em>interferon</em> is produced in a biphasic fashion that involves a number of transcription factors, including the <em>interferon</em> regulatory factors (IRFs) 1, <em>3</em>, 7, and 9. In addition, virus infection has been shown to directly induce ISGs in the absence of prior <em>interferon</em> production through the activation of IRF<em>3</em>. This process is believed to require virus replication and results in IRF<em>3</em> hyperphosphorylation, nuclear localization, and proteasome-mediated degradation. Previously, we and others demonstrated that herpes simplex virus type 1 (HSV-1) induces ISGs and an antiviral response in fibroblasts in the absence of both <em>interferon</em> production and virus replication. In this report, we show that the entry of enveloped virus particles from diverse virus families elicits a similar innate response. This process requires IRF<em>3</em>, but not IRF1, IRF7, or IRF9. Following virus replication, the large DNA viruses HSV-1 and vaccinia virus effectively inhibit ISG mRNA accumulation, whereas the small RNA viruses Newcastle disease virus, Sendai virus, and vesicular stomatitis virus do not. In addition, we found that IRF<em>3</em> hyperphosphorylation and degradation do not correlate with ISG and antiviral state induction but instead serve as a hallmark of productive virus replication, particularly following a high-multiplicity infection. Collectively, these data suggest that virus entry triggers an innate antiviral response mediated by IRF<em>3</em> and that subsequent virus replication results in posttranslational modification of IRF<em>3</em>, such as hyperphosphorylation, depending on the nature of the incoming virus.

Type I <em>interferon</em> (IFN-<em>alpha</em>/beta) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-<em>3</em>) and induction of <em>interferon</em> regulatory factor IRF-<em>3</em>-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-<em>3</em> results in enhanced lethality. However, IRF-<em>3</em> was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7(-/-) mice. Compared to congenic wild-type mice, IRF-7(-/-) mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-<em>alpha</em> gene expression and protein production were reduced and viral titers were increased in IRF-7(-/-) primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-beta response remained largely intact. Our data suggest that the early protective IFN-<em>alpha</em> response against WNV occurs through an IRF-7-dependent transcriptional signal.

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, <em>3</em> log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and <em>interferon</em>-inducible protein (IP)-10. We found that eotaxin-2 and I-<em>3</em>09 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-<em>alpha</em> treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.

Fifty-nine patients with prior hepatitis B virus infection underwent orthotopic liver transplantation. During the first 2 mo, mortality was not significantly different in the hepatitis B virus-infected group (25.5%) vs. a hepatitis B virus-immune control group (21%). Beyond 2 mo, the mortality, rate of graft loss, need for retransplantation and incidence of abnormal liver function were significantly higher in the hepatitis B virus-infected group. Treatment of the hepatitis B virus infection was attempted with passive immunization, combined active and passive immunization, <em>alpha</em>-<em>interferon</em> or nothing. The clinical outcome was not significantly influenced by any of these therapies. However, of the patients who lived more than 60 days, 6 of 22 treated with active plus passive immunization were cleared of HBsAg, something achieved once in 16 patients treated with <em>alpha</em>-<em>interferon</em>, never in <em>3</em> patients with passive immunization only and once in 4 patients with no therapy. In patients with recurrent hepatitis B virus infection, the pace of hepatitis development in the graft appeared to be accelerated, and this was particularly striking in patients who underwent multiple retransplantations at progressively shorter intervals. None of the patients who became HBsAg-negative had HBeAg preoperatively.

OBJECTIVE

To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(<em>3</em>) (1,25[OH](2)D(<em>3</em>)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology.

METHODS

Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD<em>3</em>/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(<em>3</em>), dexamethasone (DEX), and 1,25(OH)(2)D(<em>3</em>) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry.

RESULTS

The presence of 1,25(OH)(2)D(<em>3</em>) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(<em>3</em>) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(<em>3</em>), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA.

CONCLUSIONS

These data indicate that 1,25(OH)(2)D(<em>3</em>) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I <em>interferon</em> (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-<em>alpha</em>/beta exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-beta, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor <em>alpha</em>. Among structural and nonstructural proteins encoded by EV71, the <em>3</em>C protein is capable of inhibiting IFN-beta activation by virus and RIG-I. Nevertheless, EV71 <em>3</em>C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 <em>3</em>C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of <em>interferon</em> regulatory factor <em>3</em>. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with <em>3</em>C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the <em>3</em>C protein may contribute to the pathogenesis of EV71 infection.

Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are detected by the RNA helicase RIG-I, initiating downstream signaling and <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) expression that establish an antiviral state. We demonstrate here that the hepatitis C virus (HCV) <em>3</em>' untranslated region (UTR) RNA has greater activity as an immune stimulator than several flavivirus UTR RNAs. We confirmed that the HCV <em>3</em>'-UTR poly(U/UC) region is the determinant for robust activation of RIG-I-mediated innate immune signaling and that its antisense sequence, poly(AG/A), is an equivalent RIG-I activator. The poly(U/UC) region of the fulminant HCV JFH-1 strain was a relatively weak activator, while the antisense JFH-1 strain poly(AG/A) RNA was very potent. Poly(U/UC) activity does not require primary nucleotide sequence adjacency to the 5'ppp, suggesting that RIG-I recognizes two independent RNA domains. Whereas poly(U) 50-nt or poly(A) 50-nt sequences were minimally active, inserting a single C or G nucleotide, respectively, into these RNAs increased IFN-beta expression. Poly(U/UC) RNAs transcribed in vitro using modified uridine 2' fluoro or pseudouridine ribonucleotides lacked signaling activity while functioning as competitive inhibitors of RIG-I binding and IFN-beta expression. Nucleotide base and ribose modifications that convert activator RNAs into competitive inhibitors of RIG-I signaling may be useful as modulators of RIG-I-mediated innate immune responses and as tools to dissect the RNA binding and conformational events associated with signaling.

This is a phase II clinical trial investigating the safety and efficacy of intravenous vaccination with mature autologous dendritic cells (DCs) pulsed ex vivo with a liver tumor cell line lysate (HepG2) in patients with advanced hepatocellular carcinoma (HCC). HCC is an attractive target for immunotherapy as evidenced by an active recruitment of tumor-infiltrating lymphocytes that are capable of lysing autologous tumor cells in ex vivo studies. DCs are the most potent antigen-presenting cells, with the capacity to take up, process, and present tumor antigens to T cells and stimulate an immune response, thus providing a rational platform for vaccine development. Thirty-five patients with advanced HCC and not suitable for radical or loco-regional therapies received a maximum of six DC vaccinations each at <em>3</em>-week intervals. In total, 1<em>3</em>4 DC infusions were administered with no significant toxicity and no evidence of autoimmunity. Twenty-five patients who received at least three vaccine infusions were assessed clinically for response. The radiologically determined disease control rate (combined partial response and stable disease>>or=<em>3</em> months) was 28%. In 17 patients the baseline serum <em>alpha</em>-fetoprotein (AFP) was>>or= 1,000 ng/mL; in four of these patients, it fell to (<em>3</em>0% of baseline following vaccination. In one patient there was a radiological partial response associated with a fall in AFP to <10% of baseline. Immune responses were assessed using an ELIspot assay of <em>interferon</em>-gamma (IFN-gamma) release. In several cases there was induction of T cell responses to the vaccine and/or AFP following vaccination.

CONCLUSIONS

Autologous DC vaccination in patients with HCC is safe and well tolerated with evidence of antitumor efficacy assessed radiologically and serologically, with generation of antigen-specific immune responses in some cases.

The direct interaction between antigen specific CD8(+) CD28(-) T suppressor cells (T(S)) with dendritic cells (DC) results in the tolerization of DC by inducing the upregulation of immunologlobulin like transcript <em>3</em> (ILT<em>3</em>) and ILT4. We show here that such tolerogenic DC anergize alloreactive CD4(+) CD45RO(+) CD25(+) T cells converting them into regulatory T cells (T(R)), which in turn, continue the cascade of suppression by tolerizing other DC. Interleukin 10 (IL-10) and <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) also induce ILT<em>3</em> and ILT4 upregulation in DC, rendering them tolerogenic. This implies a common mechanism of DC-mediated suppression. This finding and the observation that in organ allograft recipients quiescence is associated with the presence in the circulation of donor-specific T(S) and T(R) emphasize the importance of the cross talk between tolerogenic DC and T cells in suppression of the immune response.

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5' end and a hepatitis delta virus ribozyme at the <em>3</em>' end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10(8) copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by <em>alpha</em> <em>interferon</em>. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

BACKGROUND

Pilot studies suggested that more patients with chronic hepatitis C virus (HCV) infection had a sustained virological response when treated with the combination of interferon alpha-2b and ribavirin than with interferon alpha-2b alone. We investigated the biochemical and virological responses and safety of treatment with interferon alpha-2b and ribavirin compared with interferon alpha-2b alone.

METHODS

In this double-blind trial 100 patients were randomly assigned to treatment with interferon alpha-2b (3 MU three times a week) in combination with ribavirin (1000 or 1200 mg per day) or placebo for 24 weeks and then followed up for a further 24 weeks. A further follow-up was done 1 year after active treatment stopped. The primary endpoint was the sustained virological response, defined as no detectable HCV RNA by PCR at both week 24 and week 48. Retrospectively, the baseline HCV-RNA load was analysed as a predictor of a sustained virological response. Data were analysed by intention to treat.

RESULTS

18 (36%) of the 50 patients in the interferon alpha-2b and ribavirin group had a sustained virological response compared with nine (18%) of the 50 patients in the interferon alpha-2b and placebo group (p = 0.047). At the 1 year follow-up the proportion of patients with a virological response was greater in the interferon alpha-2b and ribavirin group than the interferon alpha-2b and placebo group (42 vs 20%, p = 0.03), respectively. More patients with baseline HCV-RNA concentrations greater than 3 x 10(6) genome equivalents (Eq) per mL had a sustained response with interferon alpha-2b and ribavirin than with interferon alpha-2b and placebo (12/29 vs 1/26, p = 0.009), whereas the sustained response did not differ between the two treatment groups for HCV-RNA amounts less than 3 x 10(6) Eq per mL (6/21 vs 8/24, p = 0.67), respectively.

CONCLUSIONS

More patients with chronic hepatitis C have a sustained virological response with interferon alpha-2b and ribavirin than with only interferon alpha-2b treatment. We suggest that patients with high HCV-RNA loads should be treated with interferon alpha-2b and ribavirin.

<em>Interferons</em> IFN-<em>alpha</em>/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-<em>alpha</em> stimulates phosphorylation and nuclear localization of the 84/91K and 11<em>3</em>K subunits of latent ISGF<em>3</em> (<em>interferon</em>-stimulated gene factor <em>3</em>), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-<em>alpha</em>-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-<em>alpha</em> signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-<em>alpha</em> and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during <em>interferon</em> signalling.

TMC4<em>3</em>5 is a small-molecule inhibitor of the NS<em>3</em>/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC4<em>3</em>5 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS<em>3</em> residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS<em>3</em> positions 4<em>3</em>, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC4<em>3</em>5 inhibitory activity. The change in the 50% effective concentrations (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC4<em>3</em>5 (<10-fold change in EC(50)s), while greater effects were observed for some replicons with mutations at positions 4<em>3</em>, 155, and 156. TMC4<em>3</em>5 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions <em>3</em>6, 54, and 170 ((<em>3</em>-fold change in EC(50)s). Replicons carrying mutations affecting the activity of TMC4<em>3</em>5 remained fully susceptible to <em>alpha</em> <em>interferon</em> and NS5A and NS5B inhibitors. Finally, combinations of TMC4<em>3</em>5 with <em>alpha</em> <em>interferon</em> and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

<em>Interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) and IFN-gamma regulate gene expression by tyrosine phosphorylation of several transcription factors that have the 91-kilodalton (p91) protein of <em>interferon</em>-stimulated gene factor-<em>3</em> (ISGF-<em>3</em>) as a common component. <em>Interferon</em>-activated protein complexes bind enhancers present in the promoters of early response genes such as the high-affinity Fc gamma receptor gene (Fc gamma RI). Treatment of human peripheral blood monocytes or basophils with interleukin-<em>3</em> (IL-<em>3</em>), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-gamma response region (GRR) located in the promoter of the Fc gamma RI gene. Although tyrosine phosphorylation was required for the assembly of each of these GRR binding complexes, only those formed as a result of treatment with IFN-gamma or IL-10 contained p91. Instead, complexes activated by IL-<em>3</em> or GM-CSF contained a tyrosine-phosphorylated protein of 80 kilodaltons. Induction of Fc gamma RI RNA occurred only with IFN-gamma and IL-10, whereas pretreatment of cells with GM-CSF or IL-<em>3</em> inhibited IFN-gamma induction of Fc gamma RI RNA. Thus, several cytokines other than <em>interferons</em> can activate putative transcription factors by tyrosine phosphorylation.

BACKGROUND

Interferon alpha (IFN-alpha) activated cellular signalling is negatively regulated by inhibitory factors, including the suppressor of cytokine signalling (SOCS) family. The effects of host factors such as obesity on hepatic expression of these inhibitory factors in subjects with chronic hepatitis C virus (HCV) are unknown.

OBJECTIVE

To assess the independent effects of obesity, insulin resistance, and steatosis on response to IFN-alpha therapy and to determine hepatic expression of factors inhibiting IFN-alpha signalling in obese and non-obese subjects with chronic HCV.

METHODS

A total of 145 subjects were analysed to determine host factors associated with non-response to antiviral therapy. Treatment comprised IFN-alpha or peginterferon alpha, either alone or in combination with ribavirin. In a separate cohort of 73 patients, real time-polymerase chain reaction was performed to analyse hepatic mRNA expression. Immunohistochemistry for SOCS-3 was performed on liver biopsy samples from 38 patients with viral genotype 1 who had received antiviral treatment.

RESULTS

Non-response (NR) to treatment occurred in 55% of patients with HCV genotypes 1 or 4 and 22% with genotypes 2 or 3. Factors independently associated with NR were viral genotype 1/4 (p < 0.001), cirrhosis on pretreatment biopsy (p = 0.025), and body mass index>> or = 30 kg/m2 (p = 0.010). Obese subjects with viral genotype 1 had increased hepatic mRNA expression of phosphoenolpyruvate carboxy kinase (p = 0.01) and SOCS-3 (p = 0.047), in comparison with lean subjects. Following multivariate analysis, SOCS-3 mRNA expression remained independently associated with obesity (p = 0.023). SOCS-3 immunoreactivity was significantly increased in obesity (p = 0.013) and in non-responders compared with responders (p = 0.014).

CONCLUSIONS

In patients with chronic HCV viral genotype 1, increased expression of factors that inhibit interferon signalling may be one mechanism by which obesity reduces the biological response to IFN-alpha.

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-<em>3</em> and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I <em>interferon</em>. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-<em>3</em>, or CIS on prototypical TLR target genes such as tumor necrosis factor-<em>alpha</em>. However, we found that TLR-2, TLR-<em>3</em>, TLR-4, and TLR-9 stimulation induced <em>interferon</em>-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-<em>3</em> and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian <em>alpha</em>-2,<em>3</em>- and human <em>alpha</em>-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H<em>3</em>N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H<em>3</em>N2 virus to trigger the type I <em>interferon</em> (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor <em>3</em> (IRF<em>3</em>) nuclear translocation, as well as delayed and reduced production of IFN-beta compared with the H<em>3</em>N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H<em>3</em>N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.

Little is known about the molecular events following severe traumatic brain injury (TBI) in humans and to date there are no efficient therapies for the treatment of patients. In this study, the first of its kind in human tissue, a total of 21 post mortem trauma brain samples were analyzed. The inflammatory response within the brain tissue was explored by measuring the expression of various inflammatory cytokines at the mRNA and protein levels. These mediators were interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-<em>alpha</em>, <em>interferon</em> (IFN)-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study shows for the first time in human brain tissue that 1) pro-inflammatory mediator protein levels are significantly increased in situ following acute brain injury while anti-inflammatory cytokines protein levels remain unchanged; 2) the cerebral inflammatory response begins within minutes of acute TBI, much earlier than previously thought; <em>3</em>) IL-6, IL-8, TNF-<em>alpha</em>, and IL-1beta mRNA levels are significantly increased following injury; 4) the rise in cytokine protein level coincides with increased levels of their mRNAs suggesting that traumatic injury elicits an immediate cerebral inflammatory response. Altogether these data confirm and extend previous observations on the release of cytokines in the cerebrospinal fluid of severe TBI patients. Finally, this study highlights the need to characterize the cell source of cytokines and elucidate their mode of action.

The <em>interferon</em> (IFN) regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the IFN-<em>alpha</em>/beta gene promoters, as well as the IFN-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 IFN-stimulated gene 15 (ISG15) promoter. Several recent studies have focused attention on the unique molecular properties of IRF-<em>3</em> and its role in the regulation of IFN gene expression. 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. Following virus infection, IRF-<em>3</em> is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, located in the carboxy-terminus of IRF-<em>3</em>. Phosphorylation causes the cytoplasmic to nuclear translocation of IRF-<em>3</em>, stimulation of DNA binding, and increased transcriptional activation, mediated through the association of IRF-<em>3</em> with the CBP/p<em>3</em>00 coactivator. The purpose of this review is to summarize recent investigations demonstrating the important role of IRF-<em>3</em> in cytokine gene transcription. These studies provide the framework for a model in which virus-dependent 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.

Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor <em>alpha</em>, and <em>interferon</em> gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 19<em>3</em>9, which targeted specific sequences in the <em>3</em>'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of <em>3</em>5S-labeled proteins. Treatment of cells with ISIS 19<em>3</em>9 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 19<em>3</em>9 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 19<em>3</em>9 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 19<em>3</em>9 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 19<em>3</em>9, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.