Recent studies implicate the <em>interferon</em> (IFN) regulatory factors (IRF) IRF-<em>3</em> and IRF-7 as key activators of the <em>alpha</em>/beta IFN (IFN-<em>alpha</em>/beta) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, IFNA1, and RANTES promoters were stimulated by IRF-<em>3</em> coexpression, whereas the IFNA4, IFNA7, and IFNA14 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-<em>3</em> domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-<em>3</em> and IRF-7, as well as a preferential association of IRF-<em>3</em> with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-<em>3</em> and IRF-7 bound to the 5'-GAAANNGAAANN-<em>3</em>' consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-<em>3</em> binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-<em>3</em> possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-<em>alpha</em>/beta gene expression by IRF-<em>3</em> and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-<em>alpha</em>/beta genes.

2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>). Two IFN-<em>alpha</em>-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-<em>alpha</em>. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than <em>3</em> x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-<em>alpha</em> have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U<em>3</em> and U4 are unresponsive to IFN-<em>alpha</em> and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U<em>3</em>A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (<em>alpha</em>/beta) IFNs. Band-shift complementation assays show that U<em>3</em>A contains the E gamma subunit but does not contain an active E <em>alpha</em> subunit after treatment with IFN-<em>alpha</em>.

OBJECTIVE

To test the hypothesis that an acute exacerbation of mycobacteria-specific Th1 response after HIV infection control by HAART causes immune restoration syndrome (IRS) in HIV-tuberculosis (TB) coinfected patients.

METHODS

Prospective, multicenter study of 19 consecutive untreated HIV-TB coinfected patients included when initiating antimycobacterial therapy and sequentially evaluated during HAART and at time of IRS. IRS was defined according to classical clinical diagnostic criteria. Patients were declared IRS- if no IRS occurred within <em>3</em> months after HAART initiation.

METHODS

Mycobacteria-specific [purified protein derivative (PPD), ESAT-6, 85B] Th1 cells producing interferon (IFN)-gamma quantified by ELISpot, in vitro production of 25 cytokines/chemokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) supernatants quantified by chemiluminescence.

RESULTS

Seven patients (<em>3</em>7%) experienced IRS (IRS+). Mycobacteria-specific (PPD) Th1 IFN-gamma-producing cells increased sharply during IRS (median, 2970 spot forming cells/10 PBMC), but not the cytomegalovirus-specific responses tested as control. Only three IRS+ patients had low ESAT-6- but no 85B-specific responses. IRS- patients did not develop acute PPD-specific responses except in one case. In addition, at time of IRS a peak of PPD-specific Th1 cytokines/chemokines [interleukin (IL)-2, IL-12, IFN-gamma, IP10 and monokine-induced by IFN-gamma] without Th2 cytokines, and a peak of non-specific inflammatory cytokines/chemokines (TNF-alpha, IL-6, IL-1beta, IL-10, RANTES and MCP-1) occurred. These findings were independent from CD4 cell count, viral loads or time of HAART initiation.

CONCLUSIONS

An acute exacerbation of Th1 responses against mycobacterial antigens appears to cause IRS in patients co-infected with HIV and TB. This key event provides new evidence valuable for the diagnosis and treatment of IRS.

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2<em>3</em>57, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2<em>3</em>57 reduced by 50% the release of tumor necrosis factor-<em>alpha</em> (TNF<em>alpha</em>) at 10 to 22 nM, the release of intracellular interleukin (IL)-1<em>alpha</em> at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of <em>interferon</em>-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNF<em>alpha</em>. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2<em>3</em>57, using assays for DNA degradation, annexin V, and caspase-<em>3</em>/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNF<em>alpha</em> and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNF<em>alpha</em> RNA by ITF2<em>3</em>57. Oral administration of 1.0 to 10 mg/kg ITF2<em>3</em>57 to mice reduced LPS-induced serum TNF<em>alpha</em> and IFNgamma by more than 50%. Anti-CD<em>3</em>-induced cytokines were not suppressed by ITF2<em>3</em>57 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2<em>3</em>57 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2<em>3</em>57 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.

Tumor recurrence after resection of hepatocellular carcinoma (HCC) can occur early (<2 years) or late (>2 years) as metastases or de novo tumors. <em>Interferon</em> (IFN) has the potential for chemoprevention against hepatitis C virus (HCV)-related cirrhosis. A predetermined group of 150 HCV RNA-positive patients undergoing resection of early- to intermediate-stage HCC was stratified into 80 HCV-pure (hepatitis B anticore antibody [anti-HBc]-negative) and 70 mixed HCV+hepatitis B virus (HBV) (anti-HBc-positive) groups, then randomized to IFN-<em>alpha</em> (<em>3</em> million units <em>3</em> times every week for 48 weeks [n = 76]) versus control (n = 74). The primary end point was recurrence-free survival (RFS); secondary end points were disease-specific and overall survival. Intention-to-treat and subgroup analysis on adherent patients were conducted. Treatment effects on early/late recurrences were assessed using multiple Cox regression analysis. No patient experienced life-threatening adverse events. There were 28 adherent patients (<em>3</em>7%). After 45 months of median follow-up, overall survival was 58.5%, and no significant difference in RFS was detectable between the two study arms (24.<em>3</em>% vs. 5.8%; P = .49). HCC recurred in 100 patients (48 IFN-treated, 52 controls), with a 50% reduction in late recurrence rate in the treatment arm. HCC multiplicity and vascular invasion were significantly related to recurrence (P = .01 and .000<em>3</em>). After viral status stratification, while no treatment effect was apparent in the mixed HCV+HBV population and on early recurrences (72 events), there was a significant benefit on late recurrences (28 events) in HCV-pure patients adherent to treatment (HR: 0.<em>3</em>; 95% CI: 0.09-0.9; P = .04). In conclusion, IFN does not affect overall prevention of HCC recurrence after resection, but it may reduce late recurrence in HCV-pure patients receiving effective treatment.

Promonocytic (U1) and T lymphocytic (ACH-2) cell lines chronically infected with human immunodeficiency virus type 1 (HIV-1) constitutively express low levels of virus, but expression can be induced by phorbol esters and cytokines. Whereas ACH-2 cells produce infectious virions, U1 cells produce defective, noninfectious particles. Although <em>3</em>'-azido-<em>3</em>'-deoxythimidine (AZT) prevented acute HIV infection of susceptible cells, it did not prevent the induction of HIV expression in the infected cell lines. In contrast, <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>) inhibited the release of reverse transcriptase and viral antigens into the culture supernatant after phorbol ester stimulation of both cell lines. Further, IFN-<em>alpha</em> suppressed the production or release (or both) of whole HIV virions, but had no effect on the amount of cell-associated viral proteins. Also, after phorbol ester stimulation of ACH-2 cells, IFN-<em>alpha</em> reduced the number of infectious viral particles secreted into the culture supernatant, but had no effect on the infectivity of cell-associated virus. These findings lend support to the combined use of antiviral agents that have action at both the early (AZT) and the late (IFN-<em>alpha</em>) stages of HIV replication.

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>)-based therapies. However, the underlying mechanism of IFN-<em>alpha</em> therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-<em>alpha</em>, we created a HEK29<em>3</em>-based cell culture system to inducibly express individual <em>interferon</em>-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-<em>alpha</em> and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their <em>3</em>'-5' exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-l-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.

Type I <em>interferons</em>, also referred to as IFN-<em>alpha</em>/beta, form the first line of defense against viral infections. Major IFN-<em>alpha</em>/beta producers in the periphery are the plasmacytoid dendritic cells (pDCs). Constitutive expression of the IFN regulatory factor (IRF)-7 enables pDCs to rapidly synthesize large amounts of IFN-<em>alpha</em>/beta after viral infection. In the central nervous system (CNS), pDCs are considered to be absent from the parenchyma, and little is known about the cells producing IFN-<em>alpha</em>/beta. The study presented here aimed to identify the cells producing IFN-<em>alpha</em>/beta in the CNS in vivo after infection by neurotropic viruses such as Theiler's virus and La Crosse virus. No cells with high constitutive expression of IRF-7 were detected in the CNS of uninfected mice, suggesting the absence of cells equivalent to pDCs. Upon viral infection, IFN-beta and some subtypes of IFN-<em>alpha</em>, but not IFN-epsilon or IFN-kappa, were transcriptionally up-regulated. IFN-<em>alpha</em>/beta was predominantly produced by scattered parenchymal cells and much less by cells of inflammatory foci. Interestingly, in addition to some macrophages and ependymal cells, neurons turned out to be important producers of both IFN-<em>alpha</em> and IFN-beta. However, only <em>3</em>% of the infected neurons produced IFN-<em>alpha</em>/beta, suggesting that some restriction to IFN-<em>alpha</em>/beta production existed in these cells. All CNS cell types analyzed, including neurons, were able to respond to type I IFN by producing Mx or IRF-7. Our data show that, in vivo, neurons take an active part to the antiviral defense by being both IFN-<em>alpha</em>/beta producers and responders.

The Ebola virus (EBOV) VP<em>3</em>5 protein antagonizes the early antiviral <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) response. We previously demonstrated that VP<em>3</em>5 inhibits the virus-induced activation of the IFN-beta promoter by blocking the phosphorylation of IFN-regulatory factor <em>3</em> (IRF-<em>3</em>), a transcription factor that is crucial for the induction of IFN-<em>alpha</em>/beta expression. Furthermore, VP<em>3</em>5 blocks IFN-beta promoter activation induced by any of several components of the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA-5)-activated signaling pathways including RIG-I, IFN-beta promoter stimulator 1 (IPS-1), TANK-binding kinase 1 (TBK-1), and IkappaB kinase epsilon (IKKepsilon). These results suggested that VP<em>3</em>5 may target the IRF kinases TBK-1 and IKKepsilon. Coimmunoprecipitation experiments now demonstrate physical interactions of VP<em>3</em>5 with IKKepsilon and TBK-1, and the use of an IKKepsilon deletion construct further demonstrates that the amino-terminal kinase domain of IKKepsilon is sufficient for interactions with either IRF-<em>3</em> or VP<em>3</em>5. In vitro, either IKKepsilon or TBK-1 phosphorylates not only IRF-<em>3</em> but also VP<em>3</em>5. Moreover, VP<em>3</em>5 overexpression impairs IKKepsilon-IRF-<em>3</em>, IKKepsilon-IRF-7, and IKKepsilon-IPS-1 interactions. Finally, lysates from cells overexpressing IKKepsilon contain kinase activity that can phosphorylate IRF-<em>3</em> in vitro. When VP<em>3</em>5 is expressed in the IKKepsilon-expressing cells, this kinase activity is suppressed. These data suggest that VP<em>3</em>5 exerts its IFN-antagonist function, at least in part, by blocking necessary interactions between the kinases IKKepsilon and TBK-1 and their normal interaction partners, including their substrates, IRF-<em>3</em> and IRF-7.

The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of -<em>3</em>.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-kappaB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-kappaB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-kappaB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5'-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to -5.8 and -7.2 kb revealed 2-<em>3</em>-fold and 4-5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from -<em>3</em>.8 to -7.2 kb identified five putative NF-kappaB cis-regulatory transcription factor binding sites upstream of -4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-kappaB motif at -5.8 kb is required for cytokine-induced promoter activity, while the sites at -5.2, -5.5, and -6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-<em>alpha</em> or interleukin-1beta, but not <em>interferon</em>-gamma, exhibited inducible DNA binding activity for NF-kappaB. These data indicate that NF-kappaB activation is required for cytokine induction of the human iNOS gene and identifies four NF-kappaB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-<em>alpha</em> and interleukin-1beta.

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-<em>3</em>/TRAM-1/SRC-<em>3</em>. In this report, we describe the biochemical purification of SRC-1 and SRC-<em>3</em> protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-<em>3</em>, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-<em>3</em>-IKK complex. We demonstrated that SRC-<em>3</em> was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-<em>3</em> was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-<em>3</em> phosphorylation in vivo and translocation of SRC-<em>3</em> from cytoplasm to nucleus in response to tumor necrosis factor <em>alpha</em> occurred in cells, suggesting control of subcellular localization of SRC-<em>3</em> by phosphorylation. Finally, the hypothesis that SRC-<em>3</em> is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of <em>interferon</em> regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-<em>3</em> null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-<em>3</em> to be a regulated event that plays an important role but also substantiate the role of SRC-<em>3</em> in multiple signaling pathways.

OBJECTIVE

To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals.

METHODS

Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor <em>alpha</em> [TNF<em>alpha</em>]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = <em>3</em>). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNF<em>alpha</em> and IL-6 was measured using Luminex bead array technology.

RESULTS

In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4.

CONCLUSIONS

TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.

Localized and systemic cytokine production in virus-infected cells play an important role in the outcome of viral infection and pathogenicity. Activation of the <em>interferon</em> regulatory factors (IRF) in turn is a critical mediator of cytokine gene transcription. Recent studies have focused on the 55-kDa IRF-<em>3</em> gene product as a direct transcriptional regulator of type 1 <em>interferon</em> (IFN-<em>alpha</em> and IFN-beta) activation in response to virus infection. Virus infection induces phosphorylation of IRF-<em>3</em> on specific C-terminal serine residues and permits cytoplasmic-to-nuclear translocation of IRF-<em>3</em>, activation of DNA binding and transactivation potential, and association with the CBP/p<em>3</em>00 coactivator. We previously generated constitutively active [IRF-<em>3</em>(5D)] and dominant-negative forms of IRF-<em>3</em> that control IFN-beta and IFN-<em>alpha</em> gene expression. In an effort to characterize the range of immunoregulatory genes controlled by IRF-<em>3</em>, we now demonstrate that endogenous human RANTES gene transcription is directly induced in tetracycline-inducible IRF-<em>3</em>(5D)-expressing cells or paramyxovirus-infected cells. We also show that a dominant-negative IRF-<em>3</em> mutant inhibits virus-induced expression of the RANTES promoter. Specific mutagenesis of overlapping ISRE-like sites located between nucleotides -12<em>3</em> and -96 in the RANTES promoter reduces virus-induced and IRF-<em>3</em>-dependent activation. These studies broaden the range of IRF-<em>3</em> immunoregulatory target genes to include at least one member of the chemokine superfamily.

OBJECTIVE

Treatment duration in patients with chronic hepatitis C in the era of standard <em>interferon</em>-<em>alpha</em> plus ribavirin was tailored according to hepatitis C virus (HCV) genotype: patients infected with HCV-1 were treated for 48 weeks, patients infected with HCV-2/<em>3</em> for 24 weeks. The aim of the present study was to investigate this schedule for HCV-2/<em>3</em> infected patients in the era of pegylated <em>interferon</em>-<em>alpha</em> plus ribavirin.

METHODS

Patients chronically infected with HCV-2 (n=42) or HCV-<em>3</em> (n=182) were treated with peg<em>interferon</em> alfa-2b 1.5 microg/kg subcutaneously once weekly plus ribavirin 800-1400 mg/day based on body weight for 24 weeks.

RESULTS

The end of treatment (EOT) and sustained virologic response (SVR) was higher in patients infected with HCV-2 (100 and 9<em>3</em>%, respectively) than in patients infected with HCV-<em>3</em> (9<em>3</em> and 79%, respectively). Baseline viremia (P=0.020), treatment duration >16 weeks (P<0.001) and steatosis (<5%, P=0.015) were significant independent predictors of SVR. Adverse events resulted in discontinuation in 5% and dose reduction in 22% of patients.

CONCLUSIONS

Treatment for 24 weeks with peg<em>interferon</em> alfa-2b and ribavirin is sufficient in HCV 2 or <em>3</em> infected patients. The lower SVR in patients infected with HCV-<em>3</em> compared with HCV-2 infected patients may be related to higher levels of steatosis in this population.

Previous studies have shown the implication of beta-defensins in host defense of the human body. The human beta-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human beta-defensin, called human beta-defensin <em>3</em> (hBD-<em>3</em>; cDNA sequence, Genbank accession no. AF295<em>3</em>70), based on bioinformatics and functional genomic analysis. Expression of hBD-<em>3</em> is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), hBD-<em>3</em> expression is increased particularly after stimulation by <em>interferon</em>-gamma. Synthetic hBD-<em>3</em> exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-<em>3</em> activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human beta-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC<em>3</em> cytidine deaminases have been detected in vitro and in vivo, and APOBEC<em>3</em>G (A<em>3</em>G) and APOBEC<em>3</em>F (A<em>3</em>F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC<em>3</em>B (A<em>3</em>B), APOBEC<em>3</em>C (A<em>3</em>C), A<em>3</em>F, and A<em>3</em>G. In primary human hepatocytes, <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A<em>3</em>G, A<em>3</em>F, and A<em>3</em>B reached expression levels of 10%, <em>3</em>%, and <em>3</em>%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A<em>3</em>B(L) inhibited HBV replication in vitro as efficiently as A<em>3</em>G or A<em>3</em>F, whereas the truncated splice variant A<em>3</em>B(S) and A<em>3</em>C had no effect. A<em>3</em>B(L) and A<em>3</em>B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A<em>3</em>G and A<em>3</em>F. Moreover, A<em>3</em>G, A<em>3</em>F, and A<em>3</em>B(L), but not A<em>3</em>B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A<em>3</em>B(L), A<em>3</em>F, and most markedly A<em>3</em>G, which are expressed in liver and up-regulated by IFN-<em>alpha</em> in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Rabies virus (RV) of the Rhabdoviridae family grows in <em>alpha</em>/beta <em>interferon</em> (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12<em>3</em><em>3</em><em>3</em>-12<em>3</em>4<em>3</em>, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-beta gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD DeltaPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P<em>3</em>, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-beta gene expression. In contrast to wt RV, the IFN-inducing SAD DeltaPLP caused S<em>3</em>86 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor <em>3</em> (IRF-<em>3</em>). Phosphorylation of IRF-<em>3</em> by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-<em>3</em> by an upstream kinase.

OBJECTIVE

CD4(+) lymphocytes constitutively expressing the IL-2-receptor <em>alpha</em>-chain (CD25) regulate the activation of CD4 and CD8 autoreactive T-cells by suppressing their proliferation and effector function. The aim of this study is to: (1) measure the percentage of CD4(+)CD25(+) T-cells (T-regs) in patients with autoimmune liver disease at presentation and during remission, (2) correlate their frequency with disease activity, (<em>3</em>) determine their ability to expand and (4) to inhibit <em>interferon</em>-gamma (IFNgamma) production by CD4(+)CD25- T-cells.

METHODS

41 patients were studied. Percentage of T-regs was determined on peripheral blood mononuclear cells (PBMCs) by triple-colour flow cytometry; their ability to expand by exposing PBMCs to a T-cell expander (CD<em>3</em>/CD28 Dynabeads); their immunoregulatory function by measuring their ability to suppress IFNgamma production by CD4(+)CD25(-) T-cells.

RESULTS

T-regs were significantly less in patients than in controls, and at diagnosis than during remission. Their percentage was inversely correlated with titres of anti-liver kidney microsomal and soluble liver antigen autoantibodies. T-regs ability to expand was significantly lower in patients than in controls, but that to suppress IFNgamma production by CD4(+)CD25(-) T-cells was maintained.

CONCLUSIONS

Decreased T-regs numbers and ability to expand may favour the emergence of liver-targeted autoimmunity, despite preserved suppressor function. Treatment should aim at increasing T-regs number.

Chronic myeloid leukemia (CML) is genetically characterized by the presence of the reciprocal translocation t(9;22)(q<em>3</em>4;q11), resulting in a BCR/ABL gene fusion on the derivative chromosome 22 called the Philadelphia (Ph) chromosome. In 2-10% of the cases, this chimeric gene is generated by variant rearrangements, involving 9q<em>3</em>4, 22q11, and one or several other genomic regions. All chromosomes have been described as participating in these variants, but there is a marked breakpoint clustering to chromosome bands 1p<em>3</em>6, <em>3</em>p21, 5q1<em>3</em>, 6p21, 9q22, 11q1<em>3</em>, 12p1<em>3</em>, 17p1<em>3</em>, 17q21, 17q25, 19q1<em>3</em>, 21q22, 22q12, and 22q1<em>3</em>. Despite their genetically complex nature, available data indicate that variant rearrangements do not confer any specific phenotypic or prognostic impact as compared to CML with a standard Ph chromosome. In most instances, the t(9;22), or a variant thereof, is the sole chromosomal anomaly during the chronic phase (CP) of the disease, whereas additional genetic changes are demonstrable in 60-80% of cases in blast crisis (BC). The secondary chromosomal aberrations are clearly nonrandom, with the most common chromosomal abnormalities being +8 (<em>3</em>4% of cases with additional changes), +Ph (<em>3</em>0%), i(17q) (20%), +19 (1<em>3</em>%), -Y (8% of males), +21 (7%), +17 (5%), and monosomy 7 (5%). We suggest that all these aberrations, occurring in >5% of CML with secondary changes, should be denoted major route abnormalities. Chromosome segments often involved in structural rearrangements include 1q, <em>3</em>q21, <em>3</em>q26, 7p, 9p, 11q2<em>3</em>, 12p1<em>3</em>, 1<em>3</em>q11-14, 17p11, 17q10, 21q22, and 22q10. No clear-cut differences as regards type and prevalence of additional aberrations seem to exist between CML with standard t(9;22) and CML with variants, except for slightly lower frequencies of the most common changes in the latter group. The temporal order of the secondary changes varies, but the preferred pathway appears to start with i(17q), followed by +8 and +Ph, and then +19. Molecular genetic abnormalities preceding, or occurring during, BC include overexpression of the BCR/ABL transcript, upregulation of the EVI1 gene, increased telomerase activity, and mutations of the tumor suppressor genes RB1, TP5<em>3</em>, and CDKN2A. The cytogenetic evolution patterns vary significantly in relation to treatment given during CP. For example, +8 is more common after busulfan than hydroxyurea therapy, and the secondary changes seen after <em>interferon</em>-<em>alpha</em> treatment or bone marrow transplantation are often unusual, seemingly random, and occasionally transient. Apart from the strong phenotypic impact of addition of acute myeloid leukemia/myelodysplasia-associated translocations and inversions, such as inv(<em>3</em>)(q21q26), t(<em>3</em>;21)(q26;q22), and t(15;17)(q22;q12-21), in CML BC, only a few significant differences between myeloid and lymphoid BC are discerned, with i(17q) and TP5<em>3</em> mutations being more common in myeloid BC and monosomy 7, hypodiploidy, and CDKN2A deletions being more frequent in lymphoid BC. The prognostic significance of the secondary genetic changes is not uniform, although abnormalities involving chromosome 17, e.g., i(17q), have repeatedly been shown to be ominous. However, the clinical impact of additional cytogenetic and molecular genetic aberrations is most likely modified by the treatment modalities used.

<em>Alpha</em>/beta <em>interferons</em> (IFN-<em>alpha</em>/beta) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-<em>alpha</em>/beta responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV(KUN)), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-beta promoter-driven transcription and that a single amino acid substitution in NS2A (Ala<em>3</em>0 to Pro [A<em>3</em>0P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-122<em>3</em>5). Here we show that incorporation of the A<em>3</em>0P mutation into the WNV(KUN) genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-<em>alpha</em>/beta in infected human A549 cells than that detected following wild-type WNV(KUN) infection. Consequently, replication of the WNV(KUN)NS2A/A<em>3</em>0P mutant virus in these cells known to be high producers of IFN-<em>alpha</em>/beta was abortive. In contrast, both the mutant and the wild-type WNV(KUN) produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-<em>alpha</em>/beta production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in <em>3</em>-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-<em>alpha</em>/betagamma receptor knockout mice, suggesting that the A<em>3</em>0P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV(KUN) and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

With about <em>3</em>50 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within <em>3</em> hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-<em>alpha</em>, IL-1beta), but does not induce an <em>interferon</em> response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1<em>alpha</em> and HNF 4<em>alpha</em>, two transcription factors essential for HBV gene expression and replication.

CONCLUSIONS

Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.

STING (stimulator of <em>interferon</em> genes) is known to control the induction of innate immune genes in response to the recognition of cytosolic DNA species, including the genomes of viruses such as herpes simplex virus 1 (HSV-1). However, while STING is essential for protection of the host against numerous DNA pathogens, sustained STING activity can lead to lethal inflammatory disease. It is known that STING utilizes <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) and nuclear factor κB (NF-κB) pathways to exert its effects, although the signal transduction mechanisms remain to be clarified fully. Here we demonstrate that in addition to the activation of these pathways, potent induction of the Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) pathway was similarly observed in response to STING activation by double-stranded DNA (dsDNA). Furthermore, TANK-binding kinase 1 (TBK1) associated with STING was found to facilitate dsDNA-mediated canonical activation of NF-κB as well as IRF<em>3</em> to promote proinflammatory gene transcription. The triggering of NF-κB function was noted to require TRAF6 activation. Our findings detail a novel dsDNA-mediated NF-κB activation pathway facilitated through a STING-TRAF6-TBK1 axis and suggest a target for therapeutic intervention to plausibly stimulate antiviral activity or, alternatively, avert dsDNA-mediated inflammatory disease.

OBJECTIVE

The IKK complex, which is composed of two catalytic subunits, IKKα and IKKβ, has been suggested to be essential for the activation of canonical NF-κB signaling in response to various stimuli, including cytokines (e.g., interleukin-1α [IL-1α] and tumor necrosis factor alpha [TNF-α]), Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide [LPS]), and dsRNAs derived from viruses, or a synthetic analog. STING has been identified as a critical signaling molecule required for the detection of cytosolic dsDNAs derived from pathogens and viruses. However, little is known about how cytosolic dsDNA triggers NF-κB signaling. In the present study, we demonstrate that TBK1, identified as an IKK-related kinase, may predominantly control the activation of NF-κB in response to dsDNA signaling via STING through the IKKαβ activation loop. Thus, our results establish TBK1 as a downstream kinase controlling dsDNA-mediated IRF<em>3</em> and NF-κB signaling dependent on STING.

OBJECTIVE

Rituximab has been reported to have little activity in small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL) and to be associated with significant infusion-related toxicity. This study sought to decrease the initial toxicity and optimize the pharmacokinetics with an alternative treatment schedule.

METHODS

Thirty three patients with SLL/CLL received dose 1 of rituximab (100 mg) over 4 hours. In cohort I (n = <em>3</em>; 250 mg/m(2)) and cohort II (n = 7; <em>3</em>75 mg/m(2)) rituximab was administered on day <em>3</em> and thereafter three times weekly for 4 weeks using a standard administration schedule. Cohort III (n = 2<em>3</em>; <em>3</em>75 mg/m(2)) administered rituximab similar to cohort II for the first two treatments and then over 1 hour thereafter.

RESULTS

A total of <em>3</em><em>3</em> CLL/SLL patients were enrolled; only one patient discontinued therapy because of infusion-related toxicity. Thirteen patients developed transient hypoxemia, hypotension, or dyspnea that were associated with significant changes in baseline interleukin-6, interleukin-8, tumor necrosis factor alpha, and interferon gamma compared with those not experiencing such reactions. Infusion-related toxicity occurred more commonly in older (median age 7<em>3</em> v 62 years; P =.02) patients with no other pretreatment clinical or laboratory features predicting occurrence of these events. The overall response rate was 45% (<em>3</em>% CR, 42% PR; 95% CI 28% to 64%). Median response duration for these 15 patients was 10 months (95% CI, 6.8-1<em>3</em>.2; range, <em>3</em> to 17+).

CONCLUSIONS

Rituximab administered thrice weekly for 4 weeks demonstrates clinical efficacy and acceptable toxicity. Initial infusion-related events seem to be cytokine mediated and resolve by the third infusion making rapid administration possible. Future combination studies of rituximab with other therapies in CLL seem warranted.

To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-<em>alpha</em>, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-<em>alpha</em>, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-<em>alpha</em>, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added <em>3</em> d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for <em>interferons</em> in the therapy of HIV infection.