Replication-deficient adenoviral (rAd5) vaccines containing codon-optimized E1, E2, E4, and E7 genes of canine oral papillomavirus (COPV) were tested singly or in combination to determine which vaccines could protect against mucosal challenge with COPV. In three studies, groups of 4-6 beagle dogs were immunized subcutaneously (s.c.) with 10(11) rAd5 at 8-10 weeks and 4-6 weeks prior to challenge with infectious COPV particles at multiple oral mucosal sites. Control dogs were immunized with equivalent doses of rAd5 expressing human papillomavirus (HPV) type 16 L1 (rAd5-HPV-16 L1). In the first study, complete protection from COPV-induced papillomas was achieved by immunization with rAd5 vaccine combinations expressing either E1 + E2 or E1 + E2 + E4 + E7; whereas two of six dogs immunized with rAd5-E4 + rAd5-E7 and six of six rAd5-HPV16-L1-immunized control dogs developed oral papillomas. In two subsequent studies, rAd5-E1 and rAd5-E2 vaccines were tested singly or in combination to assess levels of protective immunity to COPV challenge. Subcutaneous immunization with either one or two doses of rAd5 expressing the COPV E1 and E2 genes could protect>> 90% of challenged dogs from wart formation. In contrast, all eight dogs immunized with rAd5-HPV-16 L1 developed papillomas at multiple sites. Protection was accompanied by significant IFN-gamma responses to COPV E1 and E2 peptides. Partial protection was conferred by two immunizations with either rAd5-E1 (6 of 9 protected) or rAd5-E2 (8 of 9 protected). These data indicate that rAd5 expressing papillomavirus E1 and E2 proteins can induce strong protective responses even in outbred populations under practical immunization conditions.

To pursue the capacity of monkey dendritic cells (DC) to be modified by adenoviral vectors and present the encoded antigens, we generated DC from blood monocytes and infected them with recombinant adenoviruses encoding GFP reporter and SIVgag or nef genes. Recombinant, E1- and E3-deleted, adenoviruses could transfect immature DC to >90% efficiency. When differentiated in the presence of a maturation stimulus, the infected cells were identical to control uninfected DC in surface markers and potent stimulatory activity for the mixed leukocyte reaction. Recombinant adeno-SIVgag was comparable to vaccinia-gag in stimulating IFN-gamma-secreting CD8(+) T cells from PBMC of macaques vaccinated with SIV(mac239) Deltanef and challenged with pathogenic SIV or chimeric SIV/HIV. Small numbers of adeno-SIVgag-infected DC were sufficient to trigger specific ELISPOT responses by CD8(+) T cells from these animals. Some CD4(+) IFN-gamma-secreting cells were also found in the three of eight vaccinated animals with the highest CD8(+) responses. T cells from control animals did not respond to DC transfected with adeno-gag. Therefore recombinant adenoviruses efficiently transfect monkey DC in a nonperturbing fashion, and these DC efficiently present antigens to SIVgag immune CD8(+) T cells. These findings will allow autologous DC, expressing SIV genes with high efficiency, to be tested in vivo to achieve strong specific T cell immunity.

ISG15 is a ubiquitin-like modifier that is expressed in response to type 1 interferon signaling (IFN-α/β) and plays a role in antiviral responses. The core E1, E2, and E3 enzymes for ISG15 are Ube1L, UbcH8, and Herc5, respectively, and these are all also induced at the transcriptional level by IFN-α/β. We recently showed that Herc5 associates with polysomes and modifies target proteins in a cotranslational manner. Here, we describe the expression of the core conjugating enzymes in human cells, the detection of ISG15 conjugates, and the methods for fractionation of Herc5 with polysomes.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol component of green tea, has recently been identified as an inhibitor of hepatitis C virus (HCV) entry. Here, we examined whether EGCG can enhance hepatocyte-mediated intracellular innate immunity against HCV. HCV dsRNAs (Core, E1-P7, NS-3'NTR and NS5A) induced interferon-λ1 (IFN-λ1) expression in human hepatocytes. These HCV dsRNAs also induced the expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I) and several antiviral IFN-stimulated genes (ISGs) expression. Although EGCG treatment of hepatocytes alone had little effect on TLR3 and RIG-I signaling pathways, EGCG significantly enhanced HCV dsRNAs-induced the expression of IFN-λ1, TLR3, RIG-I and antiviral ISGs in hepatocytes. Furthermore, treatment of HCV-infected hepatocytes with EGCG and HCV dsRNAs inhibited viral replication. Given that EGCG has the ability to enhance HCV dsRNAs-induced intracellular antiviral innate immunity against HCV, suggesting the potential application of EGCG as a new anti-HCV agent for HCV therapy.

The aim of this study was to investigate whether resolvin E1 (RvE1) protects against hepatic fibrosis in a murine model of liver fibrosis induced by Schistosoma japonicum infection. A total of 30 pathogen-free Kunming mice were randomly and equally divided into three groups: Control (uninfected, untreated), model (infected, untreated) and RvE1 intervention (infected, RvE1-treated; 100 ng daily). The mice were infected with Schistosoma japonicum by inoculating the abdominal skin with 20±2 cercariae to induce models of liver fibrosis. The area and numbers of the granulomas in the livers were assessed through histopathology after 70 days of treatment. The levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ were evaluated in the serum by enzyme-linked immunosorbent assay (ELISA). The expression levels of TNF-α were detected in the hepatic tissue by reverse transcription-polymerase chain reaction and western blot analysis. The activity levels of alanine aminotransferase and aspartate aminotransferase were determined in the serum by ELISA. The expression levels of laminin (LN), hyaluronic acid (HA), procollagen type III (PC-III) and type IV collagen (IV-C) were detected in the serum by radioimmunoassays. The results revealed that the mean area of the granulomas was smaller in the RvE1 intervention group compared with that in the model group. Following RvE1 treatment, the serum levels of TNF-α were lower than those in the model group, while the serum levels of IFN-γ were higher compared with those in the model group. The expression levels of TNF-α were lower in the hepatic tissue following RvE1 treatment compared with those in the model group. The indicators of liver fibrosis, the levels of LN, HA, PC-III and IV-C in the serum, were lower following RvE1 treatment than those in the model group. In conclusion, RvE1 treatment may reduce the growth of granulomas, thereby slowing the process of hepatic fibrosis, and this effect may be the result of anti-inflammatory and immune system adjustment.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world including Africa, Indian Ocean Islands, India and South-East Asia. The appearance of many unusual severe manifestations including neurological disorders was reported in post resurgence epidemics with implication of novel East Central South African (ECSA) genotype with E1:A226V mutation. The molecular mechanism of CHIKV neuropathogenesis is not yet understood and very little is known about the host-pathogen interactions. In the present study replication kinetics and innate immune response of ECSA genotype of CHIKV with and without A226V mutation were determined in mouse neuroblastoma cell line (N2a). The 226V mutant strain was more replication competent in N2a cells with a peak titer of 10(8)PFU/ml compared to 10(6)PFU/ml for A226 virus. Besides, the 226V mutant virus showed relatively less induction of antiviral genes i.e. IFN-β, OAS-3, MX-2, ISG-15 and Toll like receptors 3 and 7 as compared to non mutant strain (A226). Further pretreatment of N2a cells either with Poly I: C, IFN-β or TNF-α resulted in inhibition of CHIKV replication hence confirming the role of TLR mediated innate immune response in CHIKV pathogenesis. Differential regulation of TLRs and associated down stream antiviral genes might have attributed for increased pathogenesis of the 226V mutant novel ECSA genotype of CHIKV during the recent epidemics.

Because interferon-gamma (IFN-gamma) selectively inhibits collagen gene expression, we hypothesized that expression of IFN-gamma cDNA in fibroblasts might be a useful strategy to inhibit the development of fibrosis. A replication-deficient E1-, E3- adenovirus vector encoding murine IFN-gamma (AdCM VmIFN gamma) was constructed. Infection of murine fibroblasts with AdCM VmIFN gamma in vitro was well tolerated. The results showed that IN F-gamma mRNA was expressed in infected cells, and as much as 17.7 ng of mIFN-gamma/10(6) cells was secreted into culture supernatants. Steady-state levels of alpha 1 (I) procollagen mRNA were decreased by 90% in infected cells compared to uninfected cells. The inhibition of collagen mRNA expression was partially abrogated with a neutralizing anti-mIFN-gamma antibody. Secretion of total collagen by AdCM VmIFN gamma-infected fibroblasts was decreased by 60% compared to uninfected cells. Induction cells. Induction of cytokine responsive gene-2 expression in AdCM VmIFN gamma-infected cells demonstrated that suppression of collagen production was a selective response. The results suggest a novel strategy of cytokine gene transfer and expression for the treatment of fibrotic lung diseases.

Hepatitis C virus (HCV) has emerged as the major pathogen of liver disease worldwide. The mechanisms of HCV infection and interaction with a host are poorly understood. What exactly is required for efficient control of HCV infection is largely unknown. Standard treatment combining interferon-alpha (IFN-alpha) and ribavirine is effective in about 50% of the treated patients, however associated with significant toxicity and cost. Therefore, the development of new drugs or vaccines is urgently needed. An efficient vaccine against HCV infection requires induction of broad cellular and humoral immune responses against several viral proteins. We have engineered the combined vaccine candidate mT+mE1, an inclusion of multiple epitopes from HCV NS3, core (C) and E1 proteins. mT contains multiple conserved CD4(+) and CD8(+) T cell epitopes from HCV NS3 and C proteins. mE1 is based on eight dominant neutralizing epitopes of E1 protein from six HCV genotypes. In current study, we showed that immunization with mT+mE1 induced high titers of IgG, IgG1 and IgG2a antibodies to mE1, and high level of NS3- or C-specific CTLs. Furthermore, mT+mE1 elicited a Th1-biased immune response with secretion of high amounts of IFN-gamma, compared with mT alone. Prophylactic as well as therapeutic administration of mT+mE1 in BALB/c mice led to protecting mice against SP2/0 tumor cells expressing HCV NS3 protein. These results suggested that mT+mE1 elicited strong humoral immune responses and multiple specific cellular immune responses. The vaccine candidate is now being tested in pre-clinical trials.

Most studies on E1-deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV-1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV-1, would also be highly desirable. In this study, different immunization protocols using chimpanzee-derived adenoviral (AdC) vectors expressing Gag of HIV-1 clade B given in heterologous prime-boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8(+) T-cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag-specific CD8(+) T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine-induced cells which could be detected in the GT as well as systemic compartments. Antigen-specific CD8(+) T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN-γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee-derived (simian) adenovirus vectors is a suitable strategy to induce a long-lived genital CD8(+) T-cell response.

The hepatitis C virus (HCV) genotype 4 is spreading among southern European intravenous drug users, who are frequently co-infected with human immunodeficiency virus type 1 (HIV-1). Response to interferon (IFN) alpha-based therapies in HIV-1 positive patients co-infected with HCV genotype 4 is poor, similar to that obtained for HCV genotype 1 and much lower than for HCV genotypes 2 and 3. The lack of sequence data related to HCV of genotype 4 prompted us to sequence the complete genome of two genotype 4 variants isolated from two HIV-1 co-infected patients (24 and 25). Our aim was to investigate the evolutionary relationships of the former variants with other genotypes and/or genotype 4 subtypes. Sequence alignments and phylogenetic analysis from genomic regions 5'NC, core-E1 and NS5B revealed that the variants isolated from patients 24 and 25 (both subtyped 4c/4d by INNO-LIPA II HCV) belong to subtypes 4d and 4a, respectively. When looking at the complete genome sequence one of the variants showed a new genotype 4 subtype. Interestingly, sequence length differences in the interferon sensitivity determining region coding regions were observed when compared with sequences from other genotypes. Similarly, when the catalytic efficiency of the NS3/4 protease from patients 24 and 25 samples were determined, they displayed 70.6+/-7.7 and 23.5+/-3.4%, respectively, of the activity shown by genotype 1 NS3/4 proteases. Overall, pairwise comparison and phylogenetic analysis of nucleotide sequences of the complete genome or the different protein-encoding regions showed that genotype 4 sequences were more closely related to genotype 1 sequences. The description of new HCV genome variants may help our understanding of the HCV biology as well as the role of different genotypes in HCV treatment and therapy response.

MS patients' peripheral blood mononuclear cells (MNC) spontaneously produced more prostaglandin E1&2 (PGE) in vitro than did OND or normal control MNC correlating with lower levels of NK activity and endogenous interferon (IFN) production while there were no differences between OND and normal controls. MS neat CSF contained significantly higher levels of measurable PGE with concomitantly lower NK activity and % IFN-positive cells than was seen in OND CSF. The PGE producing cells were depleted in the nylon wool-passed (NWP) and enriched in the nylon wool-adherent (NWAd) populations in direct correlation with depletion and enrichment of esterase-positive monocyte/macrophages, respectively, suggesting that at least some of the PGE-producing cells were adherent monocyte/macrophages. At 24 and 48 h after stimulation with PHA, MS unfractionated MNC produced significantly more PGE than OND and normal MNC; at 24 and 72 h, MS NWAd cells produced significantly more PGE than normals and ONDs. MS and ONDs produced the same amount of PGE at 48 h in the stimulated NWAd fraction.

Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-1 beta are known modulators of bone remodeling in vitro and in vivo. The same cytokines induce the production of nitric oxide (NO) in various cell types, including osteoblasts and osteoclasts, and NO has recently been implicated in the regulation of bone resorption. We investigated the relationship between NO levels and cell viability in MC3T3-E1, a well-characterized osteoblastic cell line. NO donors at high concentrations >> or = 0.5 mM) produce a significant cytotoxic effect over a 48 h period. Various combinations of the three cytokines strongly promote endogenous NO production, and high NO levels are correlated with the loss of cell viability. Although TNF-alpha produces NO-independent cytotoxicity, NO greatly enhances this cytotoxic effect. Human and mouse TNF-alpha differ in their cytotoxic effects, and human TNF-alpha induces lower levels of NO production. In cocultures of RAW 264.7 mouse macrophages stimulated with lipopolysaccharide and IFN-gamma, and untreated MC3T3-E1 osteoblasts, addition of anti-TNF-alpha antibody and inhibition of NO synthesis have additive, protective effects on osteoblast viability. NO cytotoxicity involves an apoptotic mechanism. Our results underline the importance of NO and TNF-alpha as cytotoxic mediators in the osseous microenvironment and might explain the observed deficiency of bone formation in inflammatory sites.

Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation." Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin-proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases.

Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN.

The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.

Murine collagen-induced arthritis (CIA) is characterized by pannus formation, cell infiltration, and cartilage erosion, and shares histologic and immunologic features with rheumatoid arthritis. Numerous cytokines are reportedly associated with RA and/or CIA; however, their mechanistic role is not clear. To determine the role of IL-12 in CIA, DBA/1 LacJ mice were administered 3 x 10(8) plaque-forming units of mIL-12 i.p. in a nonreplicating adenoviral vector (AdIL-12) on day 25 following primary type II collagen immunization. Our studies demonstrated that systemic transient overexpression of IL-12 accelerated disease progression and augmented the arthritis severity relative to mice expressing a replication-deficient, E1-deleted Ad5 construct. A likely mechanism for this increase in pathology was the increase in the expression of cytokines and chemokines known to play a proinflammatory role in disease. In particular, levels of murine IFN-gamma were significantly increased in mice overexpressing AdIL-12 relative to the replication-deficient, E1-deleted Ad5 construct. Interestingly, the C-X-C chemokine murine macrophage inflammatory protein-2, as well as the C-C chemokines murine monocyte chemoattractant protein-1 and murine macrophage inflammatory protein-1alpha were up-regulated by AdIL-12 relative to controls. In an additional set of studies, neutralization of endogenous IL-12 in CIA mice was shown to delay disease onset and attenuate disease severity. IFN-gamma levels in the mice receiving anti-IL-12 were significantly decreased in joint homogenates. These studies demonstrate that IL-12 is an important cytokine involved in controlling the production of chemokines/cytokines leading to the evolution of experimental arthritis.

OBJECTIVE

Patients infected with genotype 2b hepatitis C virus (HCV) generally can achieve favorable responses to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV). However, a proportion of patients show poorer responses and the correlation between viral sequence variation and treatment outcome remains unclear.

METHODS

The pretreatment complete open reading frame (ORF) sequences of genotype 2b HCV determined by direct sequencing were investigated for correlation with the final outcome in a total of 60 patients.

RESULTS

In this study group, 87.5% (14/16) of non-sustained virological response (non-SVR) patients (n = 16) were relapsers. Compared to sustained virological response (SVR) patients (n = 44), non-SVR patients were older and could not achieve prompt viral clearance after the therapy induction. Comparing each viral protein between the two groups, viral sequences were more diverse in SVR patients and that diversity was found primarily in the E1, p7, and NS5A proteins. In searching for specific viral regions associated with the final outcome, several regions in E2, p7, NS2, NS5A, and NS5B were extracted. Among these regions, part of the interferon sensitivity determining region (ISDR) was included. In these regions, amino acid substitutions were associated with the final outcome in an incremental manner, depending upon the number of substitutions.

CONCLUSIONS

Viral sequences are more diverse in SVR patients than non-SVR patients receiving PEG-IFN/RBV therapy for genotype-2b HCV infection. Through systematic comparison of viral sequences, several specific regions, including part of the ISDR, were extracted as having significant correlation with the final outcome.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG>> GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

Current therapeutic options for hepatitis C are limited, especially for genotype 1. For genotypes 2 and 3, pegylated interferon in combination with ribavirin, can lead to a sustained virological response in up to 80% of patients. Unfortunately, adverse effects of IFN and ribavirin are a major problem and the list of contraindications for HCV therapy is long, including decompensated cirrhosis of the liver and psychiatric disorders. Therefore, alternative therapeutic approaches are needed. New delivery options for IFN and ribavirin are aimed at optimising efficiency and reducing adverse effects. Recent progress in the molecular virology of HCV has identified new targets for antiviral intervention. Inhibition of HCV gene expression and replication as well as immunotherapeutic concepts aimed at enhancing the cellular immune response against HCV are being explored. Solution of the crystal structures of HCV key enzymes led to the design of specific inhibitors including compounds active against the well characterised NS3 serine protease and RNA-dependent RNA polymerase which are currently in the early phase clinical investigation. New strategies for inhibiting HCV gene expression include the use of antisense oligodeoxynucleotides and ribozymes. Immunomodulation by agents such as inosine monophosphate dehydrogenase inhibitors, thymosin-alpha 1, histamine or amantadine are being studied in combination with IFN and/or ribavirin. Immunotherapeutic vaccination with recombinant HCV E1 protein improved host immunity against HCV and thus seems to be a promising new option.

Interactions between leukocytes and sinusoidal endothelial cells are known to be involved in the pathogenesis of acute liver injury. Various adhesion molecules and chemokines play key roles in these cell-to-cell interactions, and the expression of these adhesion molecules and the production of chemokines are regulated by inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interferon-gamma (IFN-gamma). We have shown that the expression of intercellular adhesion molecule-1 (ICAM-1) on cultured rat sinusoidal endothelial cells stimulated with TNF-alpha increases in a dose-dependent manner. The number of neutrophils that adhered to sinusoidal endothelial cells pretreated with TNF-alpha also increased in a dose-dependent manner and significantly decreased upon incubation with an anti-ICAM-1 antibody. In endotoxin-induced rat liver injury, the number of neutrophils infiltrating the sinusoids increased after serum TNF-alpha, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) reached their peak levels. In addition, the level of ICAM-1 expression on sinusoidal endothelial cells greatly increased from 8 h after exposure to endotoxin, and these cells were adhered to neutrophils that expressed both LFA-1 and Mac-1. Moreover, lipo-prostaglandin E1 (PGE1) reduced the extent of liver injury, and also reduced the number of neutrophils that infiltrated the liver, was reduced the production of MIP-2 and CINC, but not that of TNF-alpha, in rats injected with endotoxin.

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.

Proinflammatory cytokines, a combination of IL-1beta, TNF-alpha, and IFN-gamma, caused mRNA expression of GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, and of inducible nitric oxide synthase (iNOS) in a well-characterized osteoblastic clone MC3T3-E1 cell line. We found the expression of the GTP-CH gene in osteoblasts for the first time. The expression of GTP-CH and iNOS mRNAs was found to be maximal at 3 and 9 h, respectively. The expression of both genes elicited increases in BH4 and NO levels. Pharmacological studies using 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP-CH activity, showed that BH4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA. The results using an inhibitor of nuclear factor (NF)-kappaB and activating protein-1 (AP-1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF-kappaB and AP-1. In MC3T3-E1 cells BH4 and sepiapterin, producing BH4, could protect against apoptosis, i.e. the degradation of nuclear DNA in the cells, induced by NO derived from S-nitroso-N-acetyl-D-L-penicillamine. These results suggest that the induction of BH4 together with NO by proinflammatory cytokines could protect against NO-induced apoptosis in MC3T3-E1 cells.

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.