We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce <em>alpha</em>/beta <em>interferon</em> not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the <em>alpha</em>virus Sindbis virus, a virus shown to normally induce <em>interferon</em>. We investigated whether the inhibition of <em>interferon</em> synthesis by CSFV involved a block in <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF<em>3</em> to the nucleus; however, constitutive shuttling of IRF<em>3</em> was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF<em>3</em> was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF<em>3</em> promoter-luciferase reporter constructs, we demonstrate that loss of IRF<em>3</em> was due to an inhibition of transcription of the IRF<em>3</em> gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of <em>interferon</em> and loss of IRF<em>3</em>. We used cell lines expressing the CSFV N-terminal protease (Npro) to show that this single viral protein, unique to pestiviruses, inhibited <em>interferon</em> production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF<em>3</em> was lost from Npro-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where <em>interferon</em> synthesis is prevented by inhibiting transcription of IRF<em>3</em> in CSFV-infected cells.

Pegylated <em>interferon</em> (PEG-IFN) has become standard therapy for hepatitis C virus (HCV) infection. We evaluated whether PEG-IFN pharmacodynamics and pharmacokinetics account for differences in treatment outcome and whether these parameters might be predictors of therapeutic outcome. Twenty-four IFN-naïve, HCV/human immunodeficiency virus-coinfected patients received PEG-IFN <em>alpha</em>-2b (1.5 microg/kg) once weekly plus daily ribavirin (1000 or 1200 mg) for up to 48 weeks. HCV RNA and PEG-IFN <em>alpha</em> concentrations were obtained from samples collected frequently after the first <em>3</em> PEG-IFN doses. We modeled HCV kinetics incorporating pharmacokinetic and pharmacodynamic parameters. Although PEG-IFN concentrations and pharmacokinetic parameters were similar in sustained virological responders (SVRs) and nonresponders (NRs), the PEG-IFN <em>alpha</em>-2b concentration that decreases HCV production by 50% (EC50) was lower in SVRs compared with NRs (0.04 vs. 0.45 microg/L [P = .014]). Additionally, the median therapeutic quotient (i.e., the ratio between average PEG-IFN concentration and EC50 [C/EC50]), and the PEG-IFN concentration at day 7 divided by EC50 (C(7)/EC50) were significantly increased in SVRs compared with NRs after the first (10.1 vs. 1.0 [P = .012], 2.8 vs. 0.<em>3</em> [P = .007], respectively) and second (14.0 vs. 1.1 [P = .016], 5.4 vs. 0.4 [P = .02], respectively) PEG-IFN doses. All <em>3</em> parameters may be used to identify NRs. In conclusion, PEG-IFN concentrations and pharmacokinetic parameters do not differ between SVRs and NRs. In contrast, pharmacodynamic measurements-namely EC50, the therapeutic quotient, and C(7)/EC50--are different in coinfected SVRs and NRs. These parameters might be useful predictors of treatment outcome during the first month of therapy.

BACKGROUND

The current study was conducted to determine whether the addition of interferon-alpha (IFN-alpha) to treatment with radiation therapy and carmustine (BCNU) improves time to disease progression or overall survival in patients with high-grade glioma.

METHODS

Patients with anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma multiforme, or gliosarcoma received radiation therapy plus BCNU as initial therapy. Subsequently, patients without tumor progression at the completion of radiation therapy were stratified by age, extent of surgery, tumor grade and histology, Eastern Cooperative Oncology Group performance status, and treating institution, and then were randomly assigned to receive either BCNU alone (200 mg/m(2) on Day 1) or BCNU (150 mg/m(2) on Day 3) plus IFN--alpha (12 million U/m(2) on Days 1-3, Weeks 1, 3, and 5) every 7 weeks for a maximum of 6 cycles.

RESULTS

Of the 383 patients enrolled in the study, 275 eligible patients were randomized. There was no significant difference with regard to time to disease progression or overall survival between the two groups. Patients receiving IFN-alpha experienced more fever, chills, myalgias, and neurocortical symptoms including somnolence, confusion, and exacerbation of neurologic deficits. Cox multivariate regression models confirmed known favorable prognostic variables including younger age, Grade 3 tumor (according to World Health Organization criteria), and greater extent of surgery. Cox and classification and regression tree analysis models also demonstrated that a normal baseline Folstein mini-mental status examination (MMSE) score was associated with better prognosis.

CONCLUSIONS

IFN-alpha does not appear to improve time to disease progression or overall survival in patients with high-grade glioma and appears to add significantly to toxicity. The baseline MMSE score may serve as an independent prognostic factor and warrants further investigation.

We have shown by site-directed mutagenesis that the sequence between positions -69 and -40 of the mouse <em>alpha</em> A-crystallin gene is crucial for tissue-specific gene expression in a transfected mouse lens epithelial cell line transformed with the early region of simian virus 40. Gel retardation experiments with synthetic oligodeoxynucleotides revealed a mouse lens nuclear protein which bound specifically to the palindromic sequence 5'-GGGAAATCCC-<em>3</em>' at positions -66 to -57 in the <em>alpha</em> A-crystallin promoter. By screening a bacteriophage lambda gt11 expression library of the transformed lens cells, we isolated a 2.5-kilobase-pair cDNA encoding a fusion protein which bound to this sequence and to the regulatory element of the major histocompatibility complex (MHC) class I gene. This cDNA hybridized to a 10-kilobase-pair polyadenylated RNA present in many different tissues, including lens. It encoded a protein, tentatively called <em>alpha</em> A-CRYBP1, containing at least two zinc fingers. <em>alpha</em> A-CRYBP1 is either homologous or very similar to the human nuclear proteins MBP-1 (Baldwin et al., Mol. Cell. Biol. 10:1406-1414, 1990), PRDII-BFI (Fan and Maniatis, Genes Dev. 4:29-42, 1990), and HIV-EP1 (Maekawa et al., J. Biol. Chem. 264:14591-1459<em>3</em>, 1989), which bind to regulatory elements of the MHC class I, beta <em>interferon</em>, and human immunodeficiency virus genes, respectively. Our results suggest that the lens-specific <em>alpha</em> A-crystallin, MHC class I, beta <em>interferon</em> and other genes have a similar cis-acting DNA regulatory motif that shares <em>alpha</em> A-CRYBPI, MBP-1, PRDII-BF1, HIV-EP1, or other closely related proteins as trans-acting factors.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-<em>3</em>-dodecanoyloxytetradecanoylamino]-<em>3</em>-O [(R)<em>3</em>-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-<em>3</em>-O-[(R) -<em>3</em>-hydroxytetradecanoyl]-2-[(R)-<em>3</em>-hydroxytetradecanoylamino]-<em>alpha</em> -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, <em>interferon</em>- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

This study was undertaken in an effort to understand the role of cytokines in T lymphocyte trafficking into inflamed synovium and in the potential enhancement of antigen presentation by human synovial fibroblasts. We found that interleukin-1 beta (IL-1 beta), tumor necrosis factor <em>alpha</em> (TNF <em>alpha</em>), and <em>interferon</em>-gamma (IFN gamma) each increased the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) on human synovial fibroblasts in a dose- and time-dependent manner. Maximal ICAM-1 expression occurred within 8 hours of induction, with the following order of efficacy: IFN gamma greater than TNF <em>alpha</em> greater than IL-1 beta. The number of cells bearing the ICAM-1 antigen also increased, from a basal level of approximately <em>3</em>0% to more than 8<em>3</em>% after cytokine induction (for all <em>3</em> cytokines). ICAM-1 expression rapidly decreased following cytokine removal. The expression of lymphocyte function-associated antigen <em>3</em> was also examined, but it was not changed by any of the <em>3</em> cytokines. Class I major histocompatibility complex antigen expression was increased modestly by all <em>3</em> cytokines, and expression was maximal by 24 hours after treatment. Only IFN gamma induced HLA class II antigen expression, and this expression persisted for up to 6 days following removal of the lymphokine. IL-6 and granulocyte-macrophage colony-stimulating factor had no effect on any of the parameters examined. Our data support an interactive role for inflammatory cytokines and the expression of adhesion ligands and HLA antigens by human synovial fibroblasts in the pathogenesis of synovial inflammation in rheumatoid arthritis.

Recurrent human herpes simplex lesions are infiltrated by macrophages and CD4 and CD8 lymphocytes, which secrete cytokines and chemokines. Vesicle fluid was examined by ELISA for the presence of cytokines and beta (C-C) chemokines. On the first day of the lesion, high concentrations of interleukin (IL)-1beta, and IL-6, moderate concentrations of IL-1<em>alpha</em> and IL-10, and low concentrations of IL-12 and beta chemokines were found; levels of macrophage inflammatory protein (MIP)-1beta were significantly higher than levels of MIP-1<em>alpha</em> and RANTES. At day <em>3</em>, the concentrations of IL-1beta, IL-6, and MIP-1beta were lower, whereas the levels of IL-10, IL-12, and MIP-1<em>alpha</em> remained similar, and the level of tumor necrosis factor-<em>alpha</em> was now detectable. Herpes simplex virus infection of keratinocytes in vitro stimulated production of beta chemokines followed by IL-12 and then IL-10, IL-1<em>alpha</em>, IL-1beta, and IL-6, indicating a potential role for these events in early recruitment, activation, and <em>interferon</em>-gamma production of CD4 cells in herpetic lesions.

<em>Interferon</em> (IFN) <em>alpha</em> induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFN<em>alpha</em> also induced phosphoinositide <em>3</em>-kinase (PI<em>3</em>K) activity. Pharmacological inhibition of PI<em>3</em>K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFN<em>alpha</em>-induced apoptosis. A PI<em>3</em>K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFN<em>alpha</em> treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFN<em>alpha</em> stimulation was not affected by abrogating PI<em>3</em>K/mTOR pathway. Neither was the expression of several IFN<em>alpha</em> target genes affected, nor the ability of IFN<em>alpha</em> to protect against virus-induced cell death affected by inhibition of the PI<em>3</em>K/mTOR pathway. These data demonstrate that an intact PI<em>3</em>K/mTOR pathway is necessary for the ability of IFN<em>alpha</em> to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFN<em>alpha</em>-induced effect.

<em>Interferon</em>-gamma (IFN-gamma) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-gamma on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in <em>3</em>T<em>3</em>-L1 adipocytes. Although IFN-gamma regulated the expression of several adipocyte transcription factors, IFN-gamma treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-gamma also resulted in a decrease of both CCAAT/enhancer-binding <em>alpha</em> and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARgamma mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARgamma in the presence of IFN-gamma. Our studies clearly demonstrated that the IFN-gamma-induced loss of PPARgamma protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-gamma also inhibited the transcription of PPARgamma, which was accompanied by a decrease in PPARgamma mRNA accumulation. In addition, exposure to IFN-gamma resulted in a substantial increase in STAT 1 expression and a small increase in STAT <em>3</em> expression. IFN-gamma treatment of <em>3</em>T<em>3</em>-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-gamma treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARgamma.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like <em>3</em> (APOBEC<em>3</em>) proteins are encapsidated by assembling HIV-1 virions and edit viral cDNA in the next round of infection. Using <em>alpha</em> <em>interferon</em> (IFN-α)-treated monocyte-derived macrophages, we show that infrequent editing of HIV-1 reverse transcripts can also be mediated by APOBEC<em>3</em> proteins supplied by the targets of infection. Based on the local sequence contexts of these mutations and the established characteristics of APOBEC<em>3</em> protein expression in myeloid cells, we speculate that APOBEC<em>3</em>A may be responsible for a substantial proportion of this activity.

The protopypic long pentraxin <em>3</em> (PTX<em>3</em>) is a unique, humoral pattern-recognition receptor, which plays a nonredundant function in innate resistance to pathogens. Dendritic cells (DC) of myelomonocytic origin, but not plasmacytoid DC, are a major source of PTX<em>3</em> in response to Toll-like receptor (TLR) engagement. The present study was designed to explore the regulation of PTX<em>3</em> production in DC. PTX<em>3</em> production was induced by TLR ligands, CD40 ligand, and interleukin (IL)-1beta and was suppressed by dexamethasone, 1<em>alpha</em>, 25-dihydroxivitamin D<em>3</em>, and prostaglandin E2. It was unexpected that lipopolysaccharide (LPS)-stimulated PTX<em>3</em> production was enhanced by IL-10 and inhibited by IL-4 and <em>interferon</em>-gamma (IFN-gamma). Enhancement of PTX<em>3</em> production by IL-10 was also evident when Pam<em>3</em> Cys-Ser-(Lys)4.<em>3</em>HCl, a TLR2-TLR1 agonist, polyionisicpolycytidylic acid, a TLR<em>3</em> agonist, and IL-1beta were used as stimuli. The effect of IL-10 was blocked by an anti-IL-10 monoclonal antibody (mAb) or an anti-IL-10 receptor <em>alpha</em> mAb, which also reduced the LPS-induced production. Thus, production of PTX<em>3</em> in DC is subjected to a distinct regulatory network, with inhibition by IFN-gamma and enhancement by IL-10. The amplification by IL-10 of production of a nonredundant component of fluid-phase innate immunity mirrors the IL-10 stimulatory function on B cells in adaptive immunity. As PTX<em>3</em> is also an extracellular matrix component, IL-10-enhanced PTX<em>3</em> production may play a role in orchestration of tissue remodeling in chronic inflammation.

OBJECTIVE

To assess the clinical-effectiveness and cost-effectiveness of pegylated interferon-alpha combined with ribavirin in the treatment of chronic hepatitis C.

METHODS

Electronic databases, reference lists of retrieved reports, and the industry submissions to the National Institute for Clinical Excellence.

METHODS

Sources were rigorously searched and studies were selected that met the inclusion criteria of being randomised controlled trials (RCTs) involving comparisons between pegylated interferon-alpha plus ribavirin and non-pegylated interferon plus ribavirin (two trials) or pegylated interferon alone and non-pegylated interferon alone (four trials). The primary outcome in all trials was sustained virological response (SVR) at follow-up. The trials were generally of good quality, although reporting of methodological details could have been more thorough in places. A cost-effectiveness model followed a hypothetical cohort of 1000 individuals with chronic hepatitis C over a 30-year period.

RESULTS

In the two trials that tested pegylated interferon plus ribavirin against non-pegylated interferon plus ribavirin the combined percentage of sustained virological response was 55%. The relative risk (RR) for remaining infected was reduced by 17% for pegylated interferon plus ribavirin compared with non-pegylated interferon plus ribavirin. Response to therapy varied according to viral genotype. Patients with genotype 1 had the lowest levels of sustained virological response and patients with genotype 2 or 3 had the highest. In the four trials that evaluated pegylated interferon monotherapy against non-pegylated interferon the combined sustained virological response rates were 31% for pegylated interferon and 14% for non-pegylated interferon. The RR for remaining infected with hepatitis C was reduced by 20% with the use of pegylated interferon. Patients with genotype 1 had the lowest levels of sustained virological response. There were also variations in sustained virological response according to other prognostic variables such as baseline viral load. Regimens involving pegylated interferon appear to be fairly well tolerated. Adverse events were been reported, but they did not differ substantially from levels of adverse events in regimens involving non-pegylated interferon. The incremental discounted cost per QALY for comparing no active treatment to 48 weeks of dual therapy with pegylated interferon and ribavirin (PEG + RBV) was 6045 pounds sterling. When moving from 48 weeks of dual therapy with non-pegylated interferon and ribavirin (IFN + RBV) to 48 weeks of dual therapy with PEG + RBV the figure was 12,123 pounds sterling. Subgroup analyses for dual PEG + RBV therapy demonstrated that the most favourable incremental discounted cost per QALY estimates were for patients infected with genotypes 2 and 3, and with low baseline viral load (3921 pounds sterling) compared with no active treatment. Results of one-way sensitivity analyses showed that the estimates varied according to differences in SVRs, drug costs and discount rates. In general estimates remained under 30,000 pounds sterling per QALY. The incremental discounted cost per QALY when moving from no active treatment to 48 weeks of monotherapy with pegylated interferon was 6484 pounds sterling. When moving from 48 weeks of monotherapy with IFN to 48 weeks of monotherapy with PEG the figure was 8404 pounds sterling. As with dual therapy, the lowest incremental cost per QALY was for patients with genotypes 2 and 3 and low baseline viral load, in the range 2641-4194 pounds sterling. The highest estimates were for patients with genotype 1 and high baseline viral load, around 30,000 pounds sterling.

CONCLUSIONS

Well-designed RCTs show that patients treated with pegylated interferon, both as dual therapy and as monotherapy, experience higher sustained viral response rates than those treated with non-pegylated interferon. Patients with genotypes 2 and 3 experience the highest response, with rates in excess of 80%. Patients with the harder to treat genotype 1 nevertheless benefit, with up to 46% of patients experiencing an SVR in one of the trials. Pegylated interferon also appears to be relatively cost-effective in both monotherapy and dual therapy, with cost per QALY estimates remaining generally under 30,000 pounds sterling. The most favourable estimates were for patients with genotypes 2 and 3. Pegylated interferon is a relatively new intervention in the treatment of hepatitis C and therefore there are areas where further research is needed. These include: efficacies of therapy with PEG-alpha-2a vs PEG-alpha-2b; retreatment of previous non-responders using pegylated interferon; efficacy of treatments and long-term outcomes in patients who have other co-morbidities; prospective tests of rules governing stopping treatment; treating patients with acute hepatitis C; problems that may occur in a minority of patients with hepatitis C, such as cryoglobulinaemia and vasculitis; additional psychological effects on quality of life due to hepatitis C and also on the treatment of children and adolescents with hepatitis C.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective transepithelial Cl- transport. The regulation of CF gene expression is not fully understood. We report that <em>interferon</em>-gamma (IFN-gamma), but not IFN-<em>alpha</em> or -beta, downregulates CFTR mRNA levels in two colon-derived epithelial cell lines, HT-29 and T84, in a time- and concentration (from 0.1 IU/ml)-dependent manner. IFN-gamma has no effect on the transcription rate of the CFTR gene but reduces CFTR mRNA half-life, indicating that it exerts a posttranscriptional regulation of CFTR expression, at least partly, through destabilization of the transcripts. Cells treated with IFN-gamma contain subnormal amounts of 165-kDa CFTR protein. Assays of adenosine <em>3</em>',5'-cyclic monophosphate-stimulated <em>3</em>6Cl- efflux and whole cell currents show that CFTR function is diminished in IFN-gamma-treated cells. IFN-gamma and tumor necrosis factor-<em>alpha</em> synergistically reduce CFTR gene expression. Our results suggest that production of these cytokines in response to bacterial infections and inflammatory disorders may alter transmembrane Cl- transport.

Exquisitely regulated cytokine balance during early pregnancy is thought to be necessary for promoting survival of the fetal allograft. Our previous studies have demonstrated that membrane-bound human leukocyte antigen (mHLA-G) expressed on trophoblasts is one of the key factors in regulating cytokine balance by shifting the Th1/Th2 balance toward Th2 polarization, a favourable milieu for the maintenance of pregnancy. Given that trophoblasts secrete soluble HLA-G (sHLA-G), we examined its biological roles in comparison with mHLA-G. We cultured peripheral blood mononuclear cells (PBMC) with either the HLA-A and -B-deficient B lymphoblast cell line (721.221 cells) or the same cell line transfected with mHLA-G (721.221-G1 cells), in the presence or absence of recombinant sHLA-G. Cytokine concentrations in the culture media were determined by enzyme-linked immunosorbent assay. In contrast to mHLA-G protein, sHLA-G stimulated the release of tumour necrosis factor (TNF)-<em>alpha</em> and <em>interferon</em> (IFN)-gamma, whereas it reduced the release of interleukin (IL)-<em>3</em>, regardless of the presence of the presence of a stimulatory effect of the mHLA-G-expressing cells. Although mHLA-G reduced the release of IL-4, sHLA-G did not have any effect. Conversely, sHLA-G stimulated the release of IL-10 whereas mHLA-G was without effect. These results suggest that sHLA-G regulates the release of cytokines from PBMC chiefly by counterbalancing mHLA-G, and thereby may play a role in maintaining pregnancy.

Hepatitis B virus (HBV)-related polyarteritis nodosa (PAN) is a rare disease whose frequency has been decreasing over the past 10 years. We evaluated 41 patients with HBV-related PAN to determine the circumstances leading to infection, the clinical features of vasculitis, the prognostic factors, and the response to therapy. Most patients were first treated briefly with corticosteroids, and all were included in 2 nonrandomized prospective therapeutic trials of an antiviral agent (<em>3</em>5 patients with vidarabine, 6 patients with <em>interferon</em>-<em>alpha</em> 2b) and plasma exchanges. The mean duration of follow-up was 69.6 +/- 44.8 months. At the end of the study, 21 (51.2%) patients had seroconverted to anti-HBeAb and 10 (24.4%) also had seroconverted to anti-HBsAb. In all, 2<em>3</em> (56%) patients no longer expressed serologic evidence of HBV replication. All <em>3</em><em>3</em> (80.5%) patients still alive at the end of follow-up recovered from PAN. Nineteen also recovered from HBV infection and were considered to be cured; 1<em>3</em> patients had persistent HBV infection and were considered to be in clinical recovery; and 1 patient was in remission, maintained with steroid therapy. Eight patients died during the study period; <em>3</em> deaths were directly attributable to PAN. HBV-related PAN is an acute disease, occurring shortly after infection and sharing the characteristics of classic PAN. It is not an antineutrophil cytoplasm antibodies (ANCA)-mediated vasculitis. The outcome was good for patients treated with short-term steroid therapy, antiviral agents, and plasma exchanges. We propose this protocol as the first treatment for HBV-related PAN, because it surpasses the conventional treatment with corticosteroids and cyclophosphamide, which facilitates viral replication and the development of chronic HBV infection.

Cells express a variety of STAT (signal transducer and activator of transcription) transcription factors that are structurally homologous and yet function specifically in response to particular cytokines. The functions of the individual STATs are dependent on distinct protein-protein interactions. STAT1 and STAT2 are activated by tyrosine phosphorylation in response to type I <em>interferons</em>-<em>alpha</em>/beta (IFN-<em>alpha</em>/beta) and subsequently form a multimeric transcription factor designated the IFN-<em>alpha</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>). ISGF<em>3</em> is a unique STAT complex because it also contains a non-STAT molecule, p48, which is a critical DNA-binding component. We provide evidence that STAT2 specifically interacts with p48 in vivo before and after IFN-<em>alpha</em> stimulation. The specificity of ISGF<em>3</em> formation is therefore a result of the distinct nature of the STAT2 molecule. Coimmunoprecipitation assays demonstrate p48 association with STAT2 but not STAT1. Hybrid STAT2. STAT1 molecules were used to identify a region of STAT2 which specifically associates with p48. The region of STAT2 interaction spans an amino-terminal region of two predicted coiled coils. The studies demonstrate the in vivo existence of a STAT2.p48 complex and a distinct STAT2.STAT1 complex after IFN-<em>alpha</em> stimulation. Data suggest that distinct bipartite complexes STAT2.p48 and STAT2.STAT1 translocate to the nucleus and associate on the DNA target site as ISGF<em>3</em>.

SARM (sterile <em>alpha</em>- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF<em>3</em> (<em>interferon</em>-regulatory factor <em>3</em>)-mediated TLR<em>3</em> and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 29<em>3</em> cells, and U9<em>3</em>7 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile <em>alpha</em> motif domains in the autoregulation of SARM activity.

Sindbis virus (SINV) is an <em>alpha</em>virus that causes infection of neurons and encephalomyelitis in adult immunocompetent mice. Recovery can occur without apparent neurological damage. To better define the factors facilitating noncytolytic clearance of SINV in different regions of the central nervous system (CNS) and the roles of innate and adaptive immune responses at different times during infection, we have characterized SINV infection and clearance in the brain, brain stem, and spinal cords of severe combined immunodeficiency (SCID) and C57BL/6 (wild-type [WT]) mice and mice deficient in beta <em>interferon</em> (IFN-beta) (BKO), antibody (muMT), IFN-gamma (GKO), IFN-gamma receptor (GRKO), and both antibody and IFN-gamma (muMT/GKO). WT mice cleared infectious virus by day 8, while SCID mice had persistent virus replication at all sites. For <em>3</em> days after infection, BKO mice had higher titers at all sites than WT mice, despite similar IFN-<em>alpha</em> production, but cleared virus similarly. GKO and GRKO mice cleared infectious virus from all sites by days 8 to 10 and, like WT mice, displayed transient reactivation at 12 to 22 days. muMT mice did not clear virus from the brain, and clearance from the brain stem and lumbar spinal cord was delayed, followed by reactivation. Eighty-one days after infection, muMT/GKO mice had not cleared virus from any site, but titers were lower than for SCID mice. These studies show that IFN-beta is independently important for early control of CNS virus replication, that antiviral antibody is critical for clearance from the brain, and that both antibody and IFN-gamma contribute to prevention of reactivation after initial clearance.

OBJECTIVE

To identify and investigate the kinetic binding properties of interleukin-1 receptors (IL-1R), and examine the abilities of the 2 IL-1 isoforms to stimulate metalloprotease synthesis, in normal and osteoarthritic (OA) chondrocytes.

METHODS

Receptor affinity and density were determined using radioligand binding experiments and flow cytometry. Immunocytochemical analysis and affinity cross-linking studies were performed for characterization of IL-1R.

RESULTS

While no difference in receptor affinity between normal and OA chondrocytes was noted in binding studies (Kd approximately <em>3</em>0 pM), a 2-fold increase in receptor density was found in OA chondrocytes as compared with normal chondrocytes (mean 4,069 sites/cell versus 2,<em>3</em>15 sites/cell). Flow cytometry experiments also showed a significant increase in receptor density in OA cells, as well as an enhancement in the percentage of positive cells in diseased cartilage compared with normal. Binding data for both IL-1 isoforms revealed a single class of binding sites and receptor specificity. Factors such as IL-2, <em>interferon</em>-gamma, tumor necrosis factor <em>alpha</em>, and bovine insulin did not compete with IL-1 beta. By covalent ligand cross-linking and electrophoretic analysis, only type I IL-1R, a protein of 80 kd, was detected on chondrocytes. By immunocytochemical analysis, IL-1R was identified at the cell membrane level, in both normal and OA chondrocytes. The presence of nuclear staining was also observed, but only in OA chondrocytes. Recombinant human IL-1 (<em>alpha</em> and beta) induced the secretion of stromelysin and collagenase in a dose-dependent manner. The IL-1 concentration required for half-maximal metalloprotease stimulation was <em>3</em>-4 times lower in OA chondrocytes than in normal cells.

CONCLUSIONS

These results indicate that OA chondrocytes have a higher sensitivity to the stimulation of metalloprotease synthesis by IL-1 than do normal cells. This could be related to the increased levels of IL-1R expressed in the OA cells. The implications of these findings with regard to the possible roles of IL-1 and IL-1R in the pathogenesis of OA are discussed.

Accumulating data indicate that cytokines, peptides involved in regulation of both physiological and pathological immune responses, are produced predominantly at the site of local antigen stimulation. Cytokine-producing cells were detected at the protein level in human tonsil tissue obtained from children with recurrent tonsillitis or infectious mononucleosis (IM). Concomitant production of 19 different human cytokines, interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), IL-1 beta, IL-1 receptor antagonist (ra), IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6, IL-8, IL-10, IL-1<em>3</em>, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), TNF-beta, <em>interferon</em>-gamma (IFN-gamma) and transforming growth factor-beta 1-<em>3</em> (TGF-beta 1-<em>3</em>), was identified at a single-cell level by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific antibodies (Ab). Fresh frozen sections were fixed with 4% paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the cell-surface membrane and the Golgi complex. The intracellular localization of all cytokines, except IL-1 and IL-1ra, was demonstrated by a characteristic local cytoplasmic perinuclear configuration in producer cells. In addition, the immunoreactivity for certain cytokines (IL-2, IL-4, IL-5, G-CSF and GM-CSF) was expressed on the cell membranes and extended over a large extracellular area encompassing the producer cell. Localization of the cytokine to the Golgi organelle was established by co-staining with a monoclonal antibody (mAb) specific to the Golgi complex. Both the extra- and intracellular cytokine staining reactions could be blocked by preincubation of the cytokine-specific Ab with the corresponding purified natural or recombinant cytokine. A complex cytokine pattern was established in both groups studied, where most T-helper type 1 (Th1) and Th2 lymphokines were expressed in the tonsils but at different frequencies and localizations. Cells expressing IL-4, IL-5, IL-10 and IL-1<em>3</em>, (Th2 response) were evident at higher frequencies in recurrent tonsillitis compared to sections from IM, which were associated with a more pronounced IL-2, IFN-gamma and TNF-beta expression.

OBJECTIVE

The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis.

METHODS

To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (<em>3</em>) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases.

RESULTS

Our results show that: (1) tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl(-) and HCO(<em>3</em>)(-) transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (<em>3</em>) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC).

CONCLUSIONS

Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO(<em>3</em>)(-) and Cl(-) secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies.

Human noroviruses (HuNoV) are the major cause of epidemic, nonbacterial gastroenteritis in the world. The short course of HuNoV-induced symptoms has implicated innate immunity in control of norovirus (NoV) infection. Studies using murine norovirus (MNV) confirm the importance of innate immune responses during NoV infection. Type I <em>alpha</em> and beta <em>interferons</em> (IFN-α/β) limit HuNoV replicon function, restrict MNV replication in cultured cells, and control MNV replication in vivo. Therefore, the cell types and transcription factors involved in antiviral immune responses and IFN-α/β-mediated control of NoV infection are important to define. We used mice with floxed alleles of the IFNAR1 chain of the IFN-α/β receptor to identify cells expressing lysozyme M or CD11c as cells that respond to IFN-α/β to restrict MNV replication in vivo. Furthermore, we show that the transcription factors IRF-<em>3</em> and IRF-7 work in concert to initiate unique and overlapping antiviral responses to restrict MNV replication in vivo. IRF-<em>3</em> and IRF-7 restrict MNV replication in both cultured macrophages and dendritic cells, are required for induction of IFN-α/β in macrophages but not dendritic cells, and are dispensable for the antiviral effects of IFN-α/β that block MNV replication. These studies suggest that expression of the IFN-α/β receptor on macrophages/neutrophils and dendritic cells, as well as of IRF-<em>3</em> and IRF-7, is critical for innate immune responses to NoV infection.

Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the <em>alpha</em>-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 <em>alpha</em> kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A2<em>3</em>187 or thapsigargin revealed a 2-<em>3</em>-fold increase in eIF-2 <em>alpha</em> kinase activity without detectable changes in eIF-2 <em>alpha</em> phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 <em>alpha</em> in extracts of pretreated cells, was identified as the <em>interferon</em>-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 <em>alpha</em> than did control extracts. The enhanced dsRNA-dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with <em>interferon</em>. Lower concentrations of dsRNA were required for maximal phosphorylation of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.

Swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) are enzootic viruses causing pulmonary infections in pigs. The first part of this review concentrates on known clinical and pathogenetic features of these infections. SIV is a primary respiratory pathogen; PRCV and PRRSV, on the contrary, tend to cause subclinical infections if uncomplicated but they appear to be important contributors to multifactorial respiratory diseases. The exact mechanisms whereby these viruses cause symptoms and pathology, however, remain unresolved. Classical studies of pathogenesis have revealed different lung cell tropisms and replication kinetics for each of these viruses and they suggest the involvement of different lung inflammatory responses or mediators. The proinflammatory cytokines <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and interleukin-1 (IL-1) have been shown to play key roles in several respiratory disease conditions. The biological effects of these cytokines and their involvement in human viral respiratory disease are discussed in the second part of this review. The third part summarises studies that were recently undertaken in the authors' laboratory to investigate the relationship between respiratory disease in pigs and bioactive lung lavage levels of IFN-<em>alpha</em>, TNF-<em>alpha</em> and IL-1 during single and combined infections with the above viruses. In single SIV infections, typical signs of swine "flu" were tightly correlated with an excessive and coordinate production of the <em>3</em> cytokines examined. PRCV or PRRSV infections, in contrast, were subclinical and did not induce production of all <em>3</em> cytokines. Combined infections with these 2 subclinical respiratory viruses failed to potentiate disease or cytokine production. After combined inoculation with PRCV followed by bacterial lipopolysaccharide, both clinical respiratory disease and TNF-<em>alpha</em>/IL-1 production were markedly more severe than those associated with the respective single inoculations. Taken together, these data are the first to demonstrate that proinflammatory cytokines can be important mediators of viral respiratory diseases in pigs.