Antiviral proteins encoded by the <em>interferon</em> (IFN)-stimulated genes provide a front-line defense against viral infections. In particular, PKR, RNase L, and Mx are considered to be the principal proteins through which IFNs mount an antiviral state. To determine whether alternative antiviral pathways exist, RNase L-/- mice and PKR-/- mice were crossed onto an Mx1(-/-) background to generate a strain of triply deficient (TD) mice. After infections with encephalomyocarditis virus, the TD mice died <em>3</em>-4 days earlier than infected, wild-type mice. However, there was an IFN dose-dependent increase in survival times after encephalomyocarditis virus infections for both the TD and wild-type mice. Mice that were deficient for PKR or RNase L showed intermediate survival times between those of the TD and wild-type mice. Surprisingly, cultured embryonic fibroblasts lacking RNase L, PKR, or both proteins were still able to mount a substantial residual antiviral response against encephalomyocarditis virus or vesicular stomatitis virus after IFN-<em>alpha</em> treatments. These results confirm the antiviral functions of RNase L and PKR in vivo but also provide unequivocal evidence for the existence of novel, innate immune pathways against viruses.

OBJECTIVE

Standard therapy of patients with chronic hepatitis C virus (HCV) infected with HCV genotype-2 or -<em>3</em> is the combination of pegylated <em>interferon</em>-<em>alpha</em> and ribavirin for 24 weeks. Whether shorter treatment durations are possible for these patients without compromising sustained virologic response rates is unknown.

METHODS

Patients chronically infected with HCV-2 (n = <em>3</em>9), HCV-2/<em>3</em> (n = 1), or HCV-<em>3</em> (n = 11<em>3</em>) were treated with peg<em>interferon</em>-<em>alpha</em>-2a (180 microg/wk) plus ribavirin 800-1200 mg/day. HCV RNA was quantitatively assessed after 4 weeks. Patients with a rapid virologic response (HCV RNA below 600 IU/mL) were randomized for a total treatment duration of 16 (group A) or 24 weeks (group B). All patients with HCV RNA>> or =600 IU/mL at week 4 (group C) were treated for 24 weeks. End-of-treatment and sustained virologic response were assessed by qualitative RT-PCR (sensitivity 50 IU/mL).

RESULTS

Only 11 of 15<em>3</em> patients (7%) were allocated to group C. End-of-treatment and sustained virologic response rates were 94% and 82%, (group A), 85% and 80% (group B), and 7<em>3</em>% and <em>3</em>6% (group C), respectively. In patients infected with genotype HCV-<em>3</em> and high viral load (>800,000 IU/mL), a significant lower sustained virologic response rate was found than in patients infected with HCV-<em>3</em> and a viral load lower or equal to 800,000 IU/mL (59% vs 85%, respectively; P = .00<em>3</em>).

CONCLUSIONS

In HCV-2 and -<em>3</em> (low viral load)-infected patients who have a rapid virologic response, treatment for 16 weeks with peg<em>interferon</em>-<em>alpha</em>-2a and ribavirin is sufficient. In patients infected by HCV-<em>3</em> (high viral load), longer treatment may be necessary.

Plasmacytoid predendritic cells (pDCs) are the main producers of type I <em>interferon</em> (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-<em>3</em> kinase (PI<em>3</em>K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI<em>3</em>K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI<em>3</em>K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI<em>3</em>K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor <em>alpha</em> and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI<em>3</em>K delta subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI<em>3</em>K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor kappaB activation was preserved. Thus, PI<em>3</em>K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.

Cytokines are thought to cause the depression of cytochrome P-450 (CYP)-associated drug metabolism in humans during inflammation and infection. We have examined the role of five cytokines, i.e., interleukin-1 beta, interleukin-4, interleukin-6, tumor necrosis factor-<em>alpha</em>, and <em>interferon</em>-gamma, on the expression of CYP1A2, CYP2C, CYP2E1, CYP<em>3</em>A, and epoxide hydrolase in primary human hepatocyte cultures. Steady state P-450 and epoxide hydrolase mRNA levels, as well as ethoxyresorufin-O-deethylase and nifedipine oxidation activities, which are mainly supported by CYP1A1/1A2 and CYP<em>3</em>A, respectively, were measured. Interleukin-1 beta, interleukin-6, and tumor necrosis factor-<em>alpha</em> were found to be the most potent depressors of P-450 enzymes. After <em>3</em> days of treatment, both mRNA levels and enzyme activities were depressed, typically by at least 40%, whatever the cytokine and the enzyme considered. <em>Interferon</em>-gamma also suppressed CYP1A2 and CYP2E1 mRNA levels and ethoxyresorufin-O-deethylase activity but had no effect on CYP<em>3</em>A and epoxide hydrolase mRNAs. In addition, interleukin-4 had the opposite effect, compared with other cytokines, on CYP2E1 mRNA, which was increased up to 5-fold; ethoxyresorufin-O-deethylase and nifedipine oxidation activities were not significantly affected. These results provide the first demonstration that various cytokines act directly on human hepatocytes to affect expression of major P-450 genes and that a wide range of responses can be observed among the enzymes for a given cytokine, suggesting that different regulatory mechanisms may be involved.

<em>Interferons</em> (IFNs) have established antitumor action; the mechanism underlying this effect is, however, not yet clear. To probe the possible contribution of inhibition of angiogenesis, we have assessed angiogenesis in the mouse initiated by either human or murine tumor cell lines. Whether test cells were inoculated in the dermis or tumor fragments were grafted onto the cornea, tumor-induced angiogenesis (TIA) was inhibited by IFNs. TIA was also inhibited by the potent IFN inducer polyriboinosinic-polyribocytidylic acid. The effect of IFN was species specific; human IFNs inhibited human tumors and mouse IFNs inhibited murine tumors. This effect suggested that in contrast to other angiogenesis inhibitors, IFNs modulated the signal for angiogenesis produced by the tumor cells. Tumor cells treated in vitro with homologous IFN were significantly (P less than 0.005) less competent to initiate angiogenesis than were untreated cells. Inhibition of angiogenesis was achieved whether vascular response was assessed 1 or <em>3</em> days after tumor cell inoculation, suggesting that antiangiogenesis activity was independent of the antiproliferative effects of IFNs. To further substantiate this, L1210 leukemia cells, resistant to the antiproliferative effects of IFNs, were treated with 500 units/ml IFN-beta. IFN had no effect on their proliferation, but in four separate experiments, L1210R cells were impaired in their ability to induce angiogenesis. Thus, inhibition of TIA by IFNs was species specific, occurred at least partly by modulation of the signal inducing angiogenesis, and was expressed in the absence of antiproliferative effects. IFNs also inhibited immunologically induced angiogenesis, whether initiated by allogeneic lymphocytes (LIA) or by the mouse's own T-cells in response to an exogenous antigen (sheep RBC). LIA was markedly suppressed by treatment of host mice with homologous IFN-beta. For example, mean vessel counts induced by allogeneic mouse lymphocytes were decreased from 22.8 +/- 1.4 (SE) to 12.5 +/- 0.8 (P less than 0.0001); mouse IFN-beta had no corresponding effect on xenogeneic human lymphocytes (mean vessel counts decreased to 21.7 +/- 2.6 from 22.7 +/- 2.0). Treatment with human IFN-<em>alpha</em>, -beta, or -gamma in vitro or host mice in vivo reduced the ability of inoculated human peripheral blood lymphocytes to initiate xenogeneic LIA. Inhibition of LIA required a lower dose and/or a shorter incubation period than that needed to modulate TIA. Treatment of the donor of the allogeneic spleen cells in vivo with murine IFN or inducers also resulted in lesser LIA.(ABSTRACT TRUNCATED AT 400 WORDS)

From a series of benzamide derivatives, roflumilast (<em>3</em>-cyclo-propylmethoxy-4-difluoromethoxy-N-[<em>3</em>,5-di-chloropyrid-4-yl]-benzamide) was identified as a potent and selective PDE4 inhibitor. It inhibits PDE4 activity from human neutrophils with an IC(50) of 0.8 nM without affecting PDE1 (bovine brain), PDE2 (rat heart), and PDE<em>3</em> and PDE5 (human platelets) even at 10,000-fold higher concentrations. Roflumilast is almost equipotent to its major metabolite formed in vivo (roflumilast N-oxide) and piclamilast (RP 7<em>3</em>401), however, more than 100-fold more potent than rolipram and Ariflo (cilomilast; SB 207499). The anti-inflammatory and immunomodulatory potential of roflumilast and the reference compounds was investigated in various human leukocytes using cell-specific responses: neutrophils [N-formyl-methyl-leucyl-phenylalanine (fMLP)-induced formation of LTB(4) and reactive oxygen species (ROS)], eosinophils (fMLP- and C5a-induced ROS formation), monocytes, monocyte-derived macrophages, and dendritic cells (lipopolysaccharide-induced tumor necrosis factor-<em>alpha</em> synthesis), and CD4+ T cells (anti-CD<em>3</em>/anti-CD28 monoclonal antibody-stimulated proliferation, IL-2, IL-4, IL-5, and <em>interferon</em>-gamma release). Independent of the cell type and the response investigated, the corresponding IC values (for half-maximum inhibition) of roflumilast were within a narrow range (2-21 nM), very similar to roflumilast N-oxide (<em>3</em>-40 nM) and piclamilast (2-1<em>3</em> nM). In contrast, cilomilast (40-<em>3</em>000 nM) and rolipram (10-600 nM) showed greater differences with the highest potency for neutrophils. Compared with neutrophils and eosinophils, representing the terminal inflammatory effector cells, the relative potency of roflumilast and its N-oxide for monocytes, CD4+ T cells, and dendritic cells is substantially higher compared with cilomilast and rolipram, probably reflecting an improved immunomodulatory potential. The efficacy of roflumilast in vitro and in vivo (see accompanying article in this issue) suggests that roflumilast will be useful in the treatment of chronic inflammatory disorders such as asthma and chronic obstructive pulmonary disease.

In this study microglial cells isolated from brain cell cultures of newborn mice were characterized and investigated for morphology, their responses to growth factors and their functional properties. The microglial cells were phagocytic, contained nonspecific esterase activity and expressed Fc (IgG1/2b) and type-<em>3</em> complement receptors. Scanning electron microscopy revealed that in analogy to brain tissue two types of microglial cells are present in the cultures: the ameboid and the ramified type which both display similar appearance by transmission electron microscopy. Interleukin <em>3</em> and the granulocyte-macrophage colony-stimulating factor were potent growth factors for the cultured microglial cells. The cells were negative for class II antigens (Ia) of the major histocompatibility antigen complex. However, upon treatment with <em>interferon</em>-gamma (IFN-gamma) microglial cells became Ia+ and functioned as antigen-presenting cells when tested on ovalbumin-specific Ia-restricted helper T cells. Furthermore, microglial cells exposed to IFN-gamma and endotoxin developed tumor cell cytotoxicity and produced tumor necrosis factor <em>alpha</em>. Taken together, microglial cells share the characteristics of cells of the macrophage lineage.

MoPn-specific T-cell clones were isolated from a T-cell line that was capable of curing chlamydial genital infection by the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) after adoptive transfer. Two clones (designated as 2.14-0 and 2.14-<em>3</em>) were characterized by flow cytometry techniques to be homogenous for L<em>3</em>T4, CD<em>3</em>, and <em>alpha</em>/beta T cell receptor (TcR) T-helper cell markers. The two clones were biovar specific, because they reacted to MoPn but not the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) or C. trachomatis, serovar type E. Cytokine profile analysis, by a combination of bioassays, ELISA, and slot/Northern blotting for specific cytokine messenger RNAs, further revealed that cultures of antigen-stimulated clone 2.14-0 contained interleukin-2 (IL-2), tumor necrosis factor-<em>alpha</em>, and gamma <em>interferon</em> (a T helper 1 cell [Th1] profile). Clone 2.14-<em>3</em> was also positive for gamma <em>interferon</em>, a level much lower than that of clone 2.14-0, and negative for IL-4 secretion, suggesting a Th1 profile as well. The ability of these clones to bring about the resolution of the chronic genital chlamydial infection of nude mice was tested by the adoptive transfer of 10(7) cells of each clone into the mice. By 4 weeks after cell transfer of clone 2.14-0, 81% of recipient nude mice (<em>3</em>0 of <em>3</em>7) resolved the disease. In contrast, clone 2.14-<em>3</em> or a control T-cell clone specific for a heterologous antigen were unable to resolve the infection in 20 recipients in each case, even after 100 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Of <em>3</em>7 pancreases removed at necropsy from patients with type 1 diabetes <em>3</em>4 had residual beta cells. In <em>3</em><em>3</em> of the <em>3</em>4 the beta cells were positive for immunoreactive <em>alpha</em>-<em>interferon</em>, and this finding in islets was related to hyperexpression of class I major histocompatibility complex (MHC) antigens. Of islets showing class I hyperexpression 9<em>3</em>% contained <em>alpha</em>-<em>interferon</em> compared with 0.4% of those showing no hyperexpression. Among 80 control pancreases significant numbers of beta cells containing <em>alpha</em>-<em>interferon</em> were present only in 4 cases of acute infantile viral pancreatitis, known to be caused by Coxsackie-B viral infection in <em>3</em> cases. Chronic viral infection of beta cells may underlie the pathogenesis of some cases of type 1 diabetes.

Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of <em>interferon</em> (IFN)-<em>alpha</em>, IFN-beta, interleukin (IL)-6, IL-18, MIP-1<em>alpha</em>, and MIP-1beta were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in <em>3</em>/<em>3</em> animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, <10% of the cells were positive by immunohistochemistry. In contrast, >90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur independent of viral replication. In further studies with infected PHM, expression of MHC II was remarkably suppressed after 6 days, an additional correlate of immunological dysregulation. In conclusion, our findings suggest that infection of mononuclear phagocytes is critical, triggering a cascade of events involving cytokines/chemokines and oxygen free radicals. It is the consequence of these events rather than direct viral infection that results in much of the observed pathology. Identification of cytokine/chemokine, nitric oxide, and reactive oxygen species involvement in the observed filoviral pathogenesis may lend insight into the rational design of therapeutic countermeasures of filoviral pathogenesis.

Activation of cytosolic phospholipase A(2 )(cPLA(2)) is a prerequisite for the formation of the transcription factor complex <em>interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>) in response to <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>). Here we show that p<em>3</em>8 mitogen-activated protein kinase (MAPK), an activator of cPLA(2), is essential for both IFN-<em>alpha</em> and IFN-gamma signalling. SB20<em>3</em>580, a specific inhibitor of p<em>3</em>8, was found to inhibit ISGF<em>3</em> formation but had no apparent effects on signal transducer and activator of transcription (STAT)1 homodimer formation. Regardless of this, the antiviral activities of both IFN-<em>alpha</em> and IFN-gamma were attenuated by SB20<em>3</em>580. Treatment with either IFN led to rapid and transient activation of p<em>3</em>8. Both IFNs induced STAT1 Ser727 phosphorylation, which was inhibited by SB20<em>3</em>580 but not by an extracellular signal related kinase (ERK)1/2 inhibitor (PD98059). In an inducible <em>3</em>T<em>3</em>-L1 clone, expression of dominant-negative p<em>3</em>8 led to defective STAT1 serine phosphorylation and diminished IFN-gamma-mediated protection against viral killing. Reporter activity mediated by ISGF<em>3</em> or STAT1 homodimer was diminished by SB20<em>3</em>580 and enhanced by a constitutively active mutant of MKK6, the upstream activator of p<em>3</em>8. Therefore, p<em>3</em>8 plays a key role in the serine phosphorylation of STAT1 and transcriptional changes induced by both IFNs.

Vertebrate innate immunity provides a first line of defence against pathogens such as viruses and bacteria. Viral infection activates a potent innate immune response, which can be triggered by double-stranded (ds)RNA produced during viral replication. Here, we report that mammalian cells lacking the death-domain-containing protein FADD are defective in intracellular dsRNA-activated gene expression, including production of type I (<em>alpha</em>/beta) <em>interferons</em>, and are thus very susceptible to viral infection. The signalling pathway incorporating FADD is largely independent of Toll-like receptor <em>3</em> and the dsRNA-dependent kinase PKR, but seems to require receptor interacting protein 1 as well as Tank-binding kinase 1-mediated activation of the transcription factor IRF-<em>3</em>. The requirement for FADD in mammalian host defence is evocative of innate immune signalling in Drosophila, in which a FADD-dependent pathway responds to bacterial infection by activating the transcription of antimicrobial genes. These data therefore suggest the existence of a conserved pathogen recognition pathway in mammalian cells that is essential for the optimal induction of type I <em>interferons</em> and other genes important for host defence.

Type I <em>interferons</em> (IFN-<em>alpha</em>/beta) affect many aspects of immune responses. Many pathogen-associated molecules, including bacterial lipopolysaccharide (LPS) and virus-associated double-stranded RNA, induce IFN gene expression through activation of distinct Toll-like receptors (TLRs). Although much has been studied about the activation of the transcription factor IRF-<em>3</em> and induction of IFN-beta gene by the LPS-mediated TLR4 signaling, definitive evidence is missing about the actual role of IRF-<em>3</em> in LPS responses in vitro and in vivo. Using IRF-<em>3</em> deficient mice, we show here that IRF-<em>3</em> is indeed essential for the LPS-mediated IFN-beta gene induction. Loss of IRF-<em>3</em> also affects the expression of profile of other cytokine/chemokine genes. We also provide evidence that the LPS/TLR4 signaling activates IRF-7 to induce IFN-beta, if IRF-7 is induced by IFNs prior to LPS simulation. Finally, the IRF-<em>3</em>-deficient mice show resistance to LPS-induced endotoxin shock. These results place IRF-<em>3</em> as a molecule central to LPS/TLR4 signaling.

Dengue fever is an emerging arboviral disease for which no vaccine or antiviral treatment exists and that causes thousands of fatalities each year. To develop an in vivo test system for antidengue drugs, AG129 mice, which are deficient for the <em>interferon</em>- <em>alpha</em> / beta and - gamma receptors, were injected with unadapted dengue virus, resulting in a dose-dependent transient viremia lasting several days and peaking on day <em>3</em> after infection. Additionally, nonstructural protein 1, increased levels of proinflammatory cytokines, and neutralizing IgM and IgG antibodies were found, and mice had splenomegaly. Oral administration of the antiviral compounds 7-deaza-2'-C-methyl-adenosine, N-nonyl-deoxynojirimycin, or 6-O-butanoyl castanospermine significantly reduced viremia in a dose-dependent manner, even after delayed treatment, leading to a reduction of splenomegaly and proinflammatory cytokine levels. The results validate this dengue viremia mouse model as a suitable system for testing antidengue drugs and indicate that antiviral treatment during the acute phase of dengue fever can reduce the severity of the disease.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine model of either acute or chronic infection with B. pseudomallei in Taylor Outbred (TO) mice which mimics many features of the human pathology. Intraperitoneal infection of TO mice at doses of >10(6) CFU resulted in acute septic shock and death within 2 days. In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a <em>3</em>- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested. Resistance to acute infection with B. pseudomallei was absolutely dependent upon the production of gamma <em>interferon</em> (IFN-gamma) in vivo. Administration of neutralizing monoclonal antibody against IFN-gamma lowered the 50% lethal dose from >5 x 10(5) to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h. Neutralization of either tumor necrosis factor <em>alpha</em> or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo. Together, these results provide the first evidence of a host protective mechanism against B. pseudomallei. The rapid production of IFN-gamma within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic.

We have recently shown that the inflammatory process during experimental allergic encephalomyelitis (EAE), the animal model of MS, attracts transplanted NPC migration into the inflamed white matter. Here we studied how the proinflammatory cytokines tumor necrosis factor-<em>alpha</em> (TNF<em>alpha</em>) and <em>interferon</em>-gamma (IFNgamma) affect NPC growth, survival, differentiation, and migration. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+, NG2(-) cells, which differentiated into astrocytes, oligodendrocytes, and neurons. NPCs expressed receptors of TNF<em>alpha</em> and IFNgamma but not interleukin-1. TNF<em>alpha</em> and IFNgamma inhibited sphere cell proliferation, determined by [(<em>3</em>)H]thymidine and BrdU incorporation. IFNgamma increased apoptotic cell death (determined by TUNEL stains); this effect partially blocked by TNF<em>alpha</em>. Neither cytokine affected NPC lineage fate, determined by percentage of GFAP+, neurofilament+, and GalC+ cells after differentiation. TNF<em>alpha</em> and IFNgamma increased outward migration of cells from spheres in vitro. Thus, TNF<em>alpha</em> and IFNgamma, key players in MS and EAE, inhibit NPC proliferation and induce their migration.

Demonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-<em>alpha</em> and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-<em>3</em>-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-<em>alpha</em>, IL-6, and IL-8 with an IC(50) = 170 to <em>3</em>20 nM. Prophylactic administration of TPCA-1 at <em>3</em>, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-<em>alpha</em>, and <em>interferon</em>-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at <em>3</em> or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.

Mouse cells carrying the dominant resistance gene Mx develop a more efficient antiviral state toward influenza viruses in response to <em>interferon</em> than do Mx-negative cells. We have identified an Mx gene-associated product by labeling cultured peritoneal macrophages and embryonic cells with [<em>3</em>5S]methionine in the presence or absence of <em>interferon</em>. The radioactive proteins from unfractionated cytoplasmic extracts were separated electrophoretically in two dimensions and were revealed by fluorography. A protein with a Mr of 72,500 and an isoelectric point of 6.<em>3</em> was induced by mouse <em>interferon</em> type I (a mixture of <em>alpha</em> and beta <em>interferons</em>) in cells carrying the gene Mx but not in cells lacking Mx. The induction of this protein could be blocked by actinomycin D. The maximal rate of synthesis was reached in embryonic cells 4-5 hr after treatment with 10(<em>3</em>) reference units of <em>interferon</em> per ml. When the allele Mx (present in the inbred mouse strain A2G) was repeatedly backcrossed on different genetic backgrounds (BALB/c, C57BL/6, A/J), a clear correlation between the inducibility by <em>interferon</em> of this protein and the presence of the allele Mx was observed. The results suggest that this protein induced by the interaction of <em>interferon</em> with Mx plays a role in the selective antiviral state against influenza viruses that is observed in <em>interferon</em>-treated Mx-bearing cells.

Whether there is a pathogenic or protective outcome to chlamydial infection may be defined by the host response. We infected C57BL/6 (C57) and C<em>3</em>H/HeN (C<em>3</em>H) mice with the human biovar of Chlamydia trachomatis, serovar E, and, in select experiments, with the mouse pneumonitis agent of C. trachomatis (MoPn). We compared the courses of infection, histopathology, and host responses that resulted from these infections. The duration of infection with either chlamydial biovar was significantly increased in the C<em>3</em>H strain of mice. The intensity of infection was examined in mice infected with serovar E, and it was significantly increased in the C<em>3</em>H strain. Histopathology revealed the incidence of severe hydrosalpinx to be significantly greater in C<em>3</em>H mice than in C57 mice. In contrast, severe distention of the uterine horns was observed in all infected C57 mice compared to none of the C<em>3</em>H mice infected with serovar E and only 25% of those infected with MoPn. Acute inflammation was significantly increased in the uterine horns of C57 mice compared to that of C<em>3</em>H mice. Examination of antigen-specific responses revealed qualitatively similar responses in the two strains. Determination of gamma <em>interferon</em>- versus interleukin 4- producing cells revealed the predominance of a Th1 response in both strains. Serum enzyme-linked immunosorbent assays for immunoglobulin G1 (IgG1) and IgG2a revealed a predominance of IgG2a antibody in both strains, although the levels of antibody were significantly greater in C<em>3</em>H mice. Lymphocyte proliferation studies revealed increased proliferation in the iliac nodes of both strains at 1 to <em>3</em> weeks after infection. Because of the early eradication of infection observed in the C57 strain, we explored the relative production of tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) in the two strains. TNF-<em>alpha</em> levels were significantly increased in the genital tract secretions of C57 mice compared to that of C<em>3</em>H mice during the first week of infection. Increased TNF-<em>alpha</em> may be beneficial to the host by leading to earlier eradication of infection, thereby preventing infection of the oviduct and thus the major disease sequelae associated with chlamydial infection of the genital tract.

BACKGROUND

Hepatocellular carcinoma (HCC) with portal venous invasion (PVI) has a very poor prognosis, with a median survival of <em>3</em> months and virtually no survival at 1 year. The combination of intraarterial 5-fluorouracil (FU) and systemic <em>interferon</em>-<em>alpha</em> (IFN<em>alpha</em>) was recently reported to be effective against HCC with PVI, but these were small pilot studies.

METHODS

One hundred and sixteen patients with HCC with PVI received IFN<em>alpha</em> (5,000,000 U intramuscularly on Days 1, <em>3</em>, and 5 of each week of treatment) and 5-FU (500 mg into hepatic artery on Days 1-5 of the first and second week of each 4-week cycle). The therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least <em>3</em> cycles, when responses to treatment were evaluated by Eastern Cooperative Oncology Group criteria. The survival rate was compared with that of historical controls (n = 40).

RESULTS

Nineteen (16%) patients showed complete response and another 42 (<em>3</em>6%) showed partial response. Adverse events were limited to nausea and appetite loss. The survival rates at 12 and 24 months among overall patients were <em>3</em>4% and 18%, respectively, in contrast to 15% and 5% among the historical controls. Survival rates at 12 and 24 months were 81% and 59% among complete responders, respectively, and 4<em>3</em>% and 18% among partial responders.

CONCLUSIONS

The combination therapy with 5-FU and IFN was safe, and substantially improved the survival rate among the complete responders. These results provide a rationale for future randomized controlled trials.

Proinflammatory mediators such as cytokines and NO play pivotal roles in various inflammatory diseases. To combat inflammatory diseases successfully, regulation of proinflammatory mediator production would be a critical process. In the present study, we investigated the in vitro effects of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule cytokine production inhibitor, and its mechanism of action. In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppressed lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), and interleukin (IL)-6, with 50% inhibitory concentration (IC50) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC50 values from 11 to <em>3</em><em>3</em> nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 inhibited mRNA expression of IL-6 and TNF-<em>alpha</em> induced by LPS and <em>interferon</em>-gamma in RAW264.7 cells. The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -<em>3</em>, and -9. In addition, IL-1beta-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4.

We have isolated U6A, a mutant cell line which lacks the STAT2 subunit of the transcription factor <em>interferon</em> (IFN)-stimulated gene factor <em>3</em> (ISGF<em>3</em>). The response of U6A cells to IFN-<em>alpha</em> is almost completely defective, but the response to IFN-gamma is normal. Complementation of U6A cells with a cDNA encoding STAT2 restores the IFN-<em>alpha</em> response, proving that STAT2 is required in this pathway. Binding of IFNs to their receptors triggers tyrosine phosphorylation and activation of the receptors, JAK family kinases, STAT1, and STAT2. In IFN-<em>alpha</em>-treated U6A cells, phosphorylation of the essential tyrosine kinases TYK2 and JAK1 is normal, but the phosphorylation of STAT1 is weak. A mutant STAT2 protein in which the phosphorylated tyrosine at position 690 is changed to phenylalanine does not restore normal phosphorylation of STAT1 in response to IFN-<em>alpha</em>. The dependence of STAT1 phosphorylation on the presence of STAT2 but not vice versa (T. Improta, C. Schindler, C. M. Horvath, I. M. Kerr, G. R. Stark, and J. E. Darnell, Jr., Proc. Natl. Acad. Sci. USA 91:4776-4780, 1994) indicates that in the formation of ISGF<em>3</em>, these two proteins may be phosphorylated sequentially in response to IFN-<em>alpha</em> and that phosphorylated STAT2 may be required to allow unphosphorylated STAT1 to bind to the activated IFN-<em>alpha</em> receptor.

OBJECTIVE

SCH 503034 is a novel and potent oral hepatitis C virus (HCV) protease inhibitor. In this phase Ib study, we assessed safety parameters and virologic response of combination of SCH 503034 plus pegylated (PEG) interferon (IFN) alpha-2b in patients with HCV genotype 1 infections who were previously nonresponders to PEG-IFN-alpha-2b +/- ribavirin therapy.

METHODS

This was a multicenter, open-label, 2-dose level, 3-way crossover, randomized (to crossover sequence) study carried out in 3 medical centers in Europe. Adult patients received SCH 503034 200 mg (n = 14) or 400 mg (n = 12) 3 times daily orally and PEG-IFN-alpha-2b 1.5 microg/kg subcutaneously once each week. Patients received SCH 503034 as monotherapy for 1 week, PEG-IFN-alpha-2b as monotherapy for 2 weeks, and combination therapy for 2 weeks with washout periods between each treatment period.

RESULTS

Combination therapy with SCH 503034 and PEG-IFN-alpha-2b was well tolerated, with no clinically significant changes in safety parameters. Mean maximum log(10) changes in HCV RNA were -2.45 +/- 0.22 and -2.88 +/- 0.22 for PEG-IFN-alpha-2b plus 200 mg and 400 mg SCH 503034, respectively, compared with -1.08 +/- 0.22 and -1.61 +/- 0.21 for SCH 503034 200 mg and 400 mg, respectively, and -1.08 +/- 0.22 and -1.26 +/- 0.20 for PEG-IFN-alpha-2b alone in the 200 mg and 400 mg SCH 503034 groups, respectively.

CONCLUSIONS

SCH 503034 plus PEG-IFN-alpha-2b was well tolerated in patients with HCV genotype 1 nonresponders to PEG-IFN-alpha-2b +/- ribavirin. These preliminary results of antiviral activity of the combination suggest a potential new therapeutic option for this hard-to-treat, nonresponder patient population.

The virus-induced activation of <em>interferon</em> <em>alpha</em>/beta (IFN-<em>alpha</em>/beta) gene transcription is essential for host defense. The IFN-beta promoter is controlled primarily by the virus-inducible enhancer elements, the IRF-Es. Here we show that IRF-<em>3</em>, an IRF family transcription factor, translocates to the nucleus from the cytoplasm upon virus infection in NIH/<em>3</em>T<em>3</em> cells. The nuclear IRF-<em>3</em> is phosphorylated, interacts with the co-activators CBP/p<em>3</em>00, and binds specifically to the IFN-beta IRF-E. Furthermore, overexpression of IRF-<em>3</em> causes a marked increase in virus-induced IFN-beta mRNA expression. Thus, IRF-<em>3</em> is a candidate transcription factor mediating the activation of the IFN-beta gene.