Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a major periodontal pathogen. TLR2 plays a central role in the response to P. gingivalis infection in vivo. In its absence there is a weak inflammatory response; however, bacteria are cleared rapidly compared with wild-type mice. We examined the role of the TLR adaptor proteins MyD88 and TLR/IL-1R-domain-containing adaptor-inducing IFN-beta in the inflammatory response to P. gingivalis in vivo and in the ability to clear the bacterial infection. Proinflammatory cytokine production in response to P. gingivalis infection depends on TLR2, but it does not require MyD88 or TLR/IL-1R-domain-containing adaptor-inducing IFN-beta In contrast, the generation of intracellular toxic oxygen species and the ultimate clearance of P. gingivalis infection depend critically on MyD88, independent of TLR2. Thus, robust cytokine production and bacterial clearance are independent events mediated by distinct signaling pathways following infection with P. gingivalis. less...

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS. (HEPATOLOGY 2010.). less...

The present study demonstrates that cationic liposomes should be selected based on these findings for optimization of DNA-based therapies using lipoplexes. These results suggest that different intracellular trafficking derived from the type of liposomes determines the recognition of pDNA by ZBP1 after uptake of lipoplexes by the macrophages, followed by the release of type I IFNs and inflammatory cytokines A confocal microscopic study using fluorescently labeled pDNA complexes showed that the complexes that released a lot of cytokines showed an enhanced distribution of pDNA-derived fluorescence into the cytosol. The level of cytokine production and the increase in the Zbp1 mRNA varied depending on the type of cationic liposome used. A good correlation was observed between the cytokine level and the Zbp1 mRNA. Then, the release of the cytokines, the mRNA expression of Z-DNA binding protein-1 (Zbp1), a cytosolic double-stranded DNA sensor, and the cellular uptake of pDNA were examined in a macrophage-like cell line, RAW264.7. The production of IFN-beta, TNF-alpha and IL-6 by lipoplex was confirmed to be induced independently of the interaction between CpG DNA and TLR9 in macrophages from TLR9-knockout mice. To gain more insight into the CpG motif- and TLR9-independent induction of type I IFNs and proinflammatory cytokines by lipoplex, macrophage activation was evaluated in vitro using various cationic liposomes complexed with pDNA containing no CpG motifs. Moreover, recent studies have demonstrated that lipoplexes induce not only proinflammatory cytokines but also another type of cytokine, type I interferons (IFNs), irrespective of the frequency of CpG motifs in DNA and the expression of TLR9. This nonspecific cytokine production is problematic in nonviral gene therapy. It is well-known that lipoplexes induce a large amount of proinflammatory cytokines because unmethylated CpG dinucleotides (CpG motifs) abundantly present in pDNA are recognized by Toll-like receptor-9 (TLR9) expressed in immune cells such as macrophages and dendritic cells. To improve the transfection efficiency of plasmid DNA (pDNA) into cells, various types of cationic liposome have been used to prepare pDNA/cationic liposome complexes (lipoplexes). less...

Using yeast two-hybrid screening, we found three TRAF proteins TRAF1, 2 and 6, bound the N-terminal region of the TLR3/4 adaptor TICAM-1 (TRIF). TRAF2, a newly identified TICAM-1-binding protein, bound the PxQxS motif (aa 333-338) of TICAM-1 using mutagenesis by alanine substitutions. TICAM-1 is known to induce the activation of NF-kappaB and IRF-3, which leads to activation of the interferon (IFN)-beta promoter, an activity that is conserved in the N+TIR fragment (aa 1-533). By mutation of the two distinct binding sites for TRAF2 and TRAF6 in N+TIR TICAM-1, the induction of IFN-beta was completely abrogated Although the TRAF2 site single mutation only marginally affected TICAM-1-mediated type I IFN induction, it further impaired the function of the TRAF6 site mutant. Moreover, double point mutations of the TRAF2 and TRAF6 binding motifs in TICAM-1 N+TIR reduced the activation of IRF-3 and NF-kappaB, the critical transcription factors for IFN-beta expression Furthermore, TRAF2/6 functioned as an E3 ligase to induce K63-mediated ubiquitination on N+TIR which was abrogated in the mutant lacking the TRAF2/6 sites in parallel with IFN-inducing activity. Confocal microscopy analysis indicated that TRAF2 and TRAF6 merged with oligomerized (i.e. activated) TICAM-1 N+TIR. However, TRAF3, which is another TRAF family member essential for TLR3-mediated type-I IFN signaling, still assembled in the mutant lacking the TRAF2/6 sites. Our data suggest that the binding of TRAF2 and TRAF6 to TICAM-1 cooperatively activates the IFN-inducing pathway through ubiquitination of TICAM-1, a modification which occurs unrelated to TRAF3 recruitment in the TICAM-1 signaling complex. TRAF2/6 may participate in TICAM-1-mediated IFN-beta induction besides TRAF3. less...

Host cells respond to viral infections by synthesizing and producing antiviral molecules such as type I interferons (IFN). The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple proteins expressed during the lytic replication cycle that alter the host's antiviral response. Considering that in KS lesions and primary effusion lymphoma cells KSHV is latent in the vast majority of cells, we were interested in determining whether latently-expressed viral proteins have the ability to modulate IFN synthesis. The latency-associated nuclear antigen (LANA) is a large nuclear protein that plays a role in the establishment and maintenance of latent KSHV episome in the nucleus of infected cells. LANA is also described to modulate the cellular transcription. Here we report that LANA inhibits IFN-beta transcription and synthesis by competing with the binding of interferon regulatory factor-3 (IRF3) to the IFN-beta promoter. Using mutants of LANA, we have identified the central acidic repeated region as the domain essential for interfering with the binding of IRF3 to the Positive Regulatory Domain (PRD)-I-III of the IFN-beta promoter In addition, the nuclear localization of LANA proved essential for IFN-beta inhibition Thus, LANA interferes with the formation of IFN-beta enhanceosome by competing with the fixation of IRF3 and as well as by inhibiting the expression of CREB-Binding Protein (CBP). The ability of LANA to inhibit IFNB gene expression highlights a new role for this protein in cellular gene modulation and immune evasion strategies. less...

Background: The objective of this study was to correlate the detection of neutralizing antibodies (NAbs) by the cytopathic effect (CPE) assay, with the expression of myxovirus resistance protein A (MxA), and the ratio between matrix metalloproteinase 9 (MMP-9) and its tissular inhibitor (TIMP-1), in order to evaluate their usefulness as markers of interferon beta (IFN-beta) bioavailability Methods: Pairs of blood and serum samples were collected from 50 patients with multiple sclerosis (MS) during 2 years of IFN-beta treatment Expression of MxA, MMP-9 and TIMP-1 were analysed by quantitative PCR, and NAbs were measured by CPE assay. Results: During the study, 60% of patients presented NAbs. The number of serum samples that were NAbs+ was significantly increased amongst patients with relapses (41/92 vs. 33/108, P = 0.04). With one serum sample and with a NAb titre >100 tenfold reduction unit (TRU), 66.7% of patients with MS suffered from relapses, 41.7% suffered from progression, and 75% was not an optimal clinical responder. We did not find any significant difference in MxA. We found that 62.5% of patients with MS patients whose ratio was increased twofold after 2 years suffered from relapses, 37.5% suffered from progression, and 68.7% was not an optimal clinical responder. Conclusion: The early detection of NAbs by CPE assay and the finding of only one serum sample with a NAb titre >100 TRU seem to be markers of low bioavailability of IFN-beta, whilst a twofold decrease in the MMP-9/TIMP-1 ratio by quantitative PCR assay seems to be a marker of high bioavailability of IFN-beta. less...

Human metapneumovirus (hMPV), is a major cause of upper and lower respiratory tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory tract infection with hMPV, we demonstrate the accumulation of IFN-gamma producing CD8+ T cells in the airways and lungs at day 7 post-infection, associated with cytotoxic T lymphocytes (CTL) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th-1 cytokines IFN-gamma and IL-12, and anti-viral (IFN-beta) cytokines, as well as chemokines Mip-1alpha, Mip-1beta, Mig, IP-10, CX3CL1. There was also a moderate increase in Th2-type cytokines cytokines IL-4 and IL-10 compared to uninfected mice. At twenty-one days post-infection, a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response between the two related pathogens. less...

OBJECTIVE: Interferon-beta, (IFN-beta) has been used in the treatment of cancers Inhibition of the enzyme cyclooxygenase (COX) with celecoxib had a significantly suppressive effect on tumor growth, angiogenesis, and metastasis in a variety of tumors. The aim of this study was to elucidate the antiglioma effect of combined treatment with IFN-beta and celecoxib in U87 glioma model. METHODS: The in vitro effects of IFN-beta (50-1,000 IU/mL) and celecoxib (50-250 microM) alone or combination of both on the proliferation and apoptosis of U87 cells were tested using MTT assay, FACS analysis and DNA condensation. To determine the in vivo effect, nude mice bearing intracerebral U87 xenograft inoculation were treated with IFN-beta intraperitoneally (2x10(5) IU/day for 15 days), celecoxib orally (5, 10 mg/kg) or their combination. RESULTS: IFN-beta or celecoxib showed an inhibitory effect on the proliferation of U87 cells. When U87 cells were treated with IFN-beta and celecoxib combination, it seemed that IFN-beta interrupted the antiproliferative and apoptotic activity of celecoxib. No additive effect was observed on the survival of the tumor bearing mice by the combination of IFN-beta and celecoxib. CONCLUSION: These results suggest that IFN-beta seems to inhibit the antiglioma effect of celecoxib, therefore combination of IFN-beta and celecoxib may be undesirable in the treatment of glioma. less...

IFN-beta is anticipated to have an important function in mucosal tolerance, as it is one of the major cytokines produced by plasmacytoid dendritic cells, and has recently been suggested as central to the maintenance of mucosal homeostasis. Here, we have investigated whether oral tolerance is dependent on endogenous IFN-beta by feeding low-dose self-antigen myelin basic protein to IFN-beta(-/-) mice with subsequent induction of experimental autoimmune encephalomyelitis (EAE). Our study shows that oral tolerance was readily induced in IFN-beta(-/-) mice compared with their wild-type littermates (IFN-beta(+/+)) The non-self-antigen ovalbumin induced oral tolerance in both groups. These data indicate that endogenous IFN-beta is not required for induction of oral tolerance, whereas delivery of recombinant IFN-beta results in significant reduction in clinical score of EAE Oral tolerance induction was associated with lower production of antigen-specific IFN-gamma, no shift toward antigen-specific Th2, Th17 or TGF-beta response was observed. Oral tolerance in IFN-beta(-/-) mice was also associated with the induction of regulatory and memory T cells in the mucosal-associated immune organs, however this was not a prerequisite for establishment of oral tolerance.Immunology and Cell Biology advance online publication, 12 January 2010; doi:10.1038/icb.2009.111. less...

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days. less...